Modulators of Immune Responses

The present invention relates to a specific sphingomyelinase for use in inhibiting immune response. Another aspect of the invention relates to a pharmaceutical composition comprising said sphingomyelinase for use in treating an autoimmune disorder or a pro-inflammatory response. In addition, another pharmaceutical composition comprising a specific inhibitor of said sphingomyelinase for use in treating an immunodeficiency disorder is comprised. The invention also relates to a diagnostic composition comprising a binding molecule which specifically binds said sphingomyelinase for use in diagnosing an immunodeficiency disorder, an autoimmune disorder or a pro-inflammatory response. The invention further relates to the use of said diagnostic composition for diagnosing an immunodeficiency disorder, an autoimmune disorder or a pro-inflammatory response. Furthermore, the present invention relates an antigen presenting cell that is inhibited in TLR signalling by said sphingomyelinase. In one aspect of the invention, said antigen presenting cell is a macrophage, a dendritic cell or a B cell. The present invention further relates to a non-human animal, wherein the activity and/or the expression of said sphingomyelinase is reduced to boost the immune system of said non-human animal. In addition, the invention relates to a method for producing antibodies against an antigen comprising the administration of said antigen to said non non-human animal. Finally, the invention relates to said method for producing antibodies, wherein said non-human animal is a knock out mouse for the gene encoding said sphingomyelinase, wherein said antibodies are high affinity antibodies.

The immune system is a remarkably effective structure that incorporates specificity, inducibility and adaptation. However, dysfunction of this defence mechanism results in the occurrence of several severe diseases such as immunodeficiency disorders, autoimmune disorders or harmful pro-inflammatory responses. Important factors of the innate immune system are Pattern recognition receptors (PRRs) which have evolved to sense invading pathogens by certain pathogen associated molecular patterns (PAMPs). While a set of intracellular recognition machineries are employed to detect intracellular invasion, Toll-like receptors (TLRs) populate the plasma membrane and endosomes of immune cells to recognize extracellular and phagocytosed pathogens (Medzhitov; 2008; Nature; 454; 428-35, Takeuchi; 2010; Cell; 140; 805-20, Yoneyama; 2010; Rev Med Virol; 20; 4-22). 10 TLRs in humans have been identified to date that recognize ligands from different invaders, such as lipopolysaccharide (LPS), non-methylated CpG-DNA, flagellin and lipoteichoic acid (O'Neill; 2008; Immunol Rev; 226; 10-8, Takeda; 2005; Int Immunol; 17; 1-14). PAMP encounter by a TLR induces receptor dimerization and receptor protein complex formation to elicit an innate immune response (Monie; 2009; Trends Biochem Sci; 34; 553-61 ). Importantly, several ligand bound TLR structures have been described recently and have shed light into the molecular mechanisms of PAMP recognition by the innate immune system (Kang; 201 1 ; Annu Rev Biochem; 80; 917-41 ). While a number of cell types have been reported to harbor distinct TLRs, immune cells and within those the antigen presenting cells (APCs) have the largest repertoire of TLRs to mount efficient early phase immune responses (Takeda and Akira; 2005; Int Immunol; 17; 1 -14). Given their central role in the regulation of immune responses and implications in cancer, tissue repair and autoimmune disease, TLR immune modulators are attractive goals for therapeutic intervention (Basith; 201 1 ; Expert Opin Ther Pat; 21 ; 927-44).

Stimulation of TLRs in APCs induces a variety of events. Whereas macrophages initially produce pro-inflammatory cytokines such as TNF, IL-6 and IL-12, plasmacytoid dendritic cells secrete large amounts of type I interferon upon encounter of pathogenic nucleic acids (Moresco; 201 1 ; Curr Biol; 21 ; R488-93). However, next to releasing cytokines, interferons and chemokines, APCs also play an important role in the phagocytic uptake, recognition, destruction, release and presentation of invaders to the adaptive immune system (Iwasaki; 2010; Science; 327; 291-5). Thus, employing phagocytosis and TLRs, APCs combine the two important aspects of antigen quality screening and subsequent presentation to T- cells - only if the endocytosed material has been proven by TLRs to be pathogenic (Blander; 2006; Nat Immunol; 7; 1029-35). As a consequence, MHCII presentation of pathogen derived peptides leads to the activation of na^'i^'veT-cells at the bridge between innate and adaptive immunity (Iwasaki; 2010; Science; 327; 291-5).

TLRs are transmembrane proteins and pathogen encounter leads not only to the assembly of signalosome multiprotein complexes but also to receptor endocytosis and recycling (Monie; 2009; Trends Biochem Sci; 34; 553-61 ). These massive changes in a TLR microenvironment require permanent restructuring of local membrane lipid rafts to allow phagocytotic vacuole formation, receptor oligomerization and phago-lysosomal fusion (Fessler; 201 1 ; J Immunol; 187; 1529- 35, Gombos; 2006; Immunol Lett; 104; 59-69, Lingwood; 2010; Science; 327; 46-50). These processes are important not only for the destruction of pathogens but also to provide distinct endosomal signalling platforms for the activation of the proinflammatory NFKB and the antiviral IFN pathways (Kagan; 2008; Nat Immunol; 9; 361 -8). In particular, the endosomal TLRs 7 and 9 signal from the late endolysosome and use MyD88 as adaptor protein to induce IRF7, whereas TLR3 signals from another endosomal compartment and uses the adaptor protein TRIF to activate IRF3 (Doyle; 2002; Immunity; 17; 251-63, Honda, Yanai; 2005; Nature; 434; 772-7).

While lipid associated proteins such as Raftlin and GDI 4 have been described to aid in the reorganization of receptor rafts (Schmitz; 2002; Curr Opin Lipidol; 13; 513-21 ) and endocytosis (Watanabe; 201 1 ; J Biol Chem; 286; 10702-1 1 ) there are dedicated enzymes that convert and modify lipids to fulfill a distinct set of functions. Sphingomyelin is one central lipid integral to the plasma and intracellular membranes. Sphingomyelinases (SMases) are the group of enzymes that consume sphingomyelin and produce the cleavage products phosphorylcholine and ceramide (Seto; 2004; Protein Sci; 13; 3172-86). This reaction can be triggered in cellular responses, in particular the stimulation of the receptors CD95, CD40 or TNFR mediate the activation of neutral, plasma membrane associated SMases and endosomal, acidic aSMases (Sharma; 1999; Cell Res; 9; 1 -10). The local accumulation of ceramide, either as integral membrane raft component to foster receptor clustering or as an intracellular, second messenger has been linked to a variety of biological functions, such as the regulation of senescence, growth arrest and apoptosis (Ogretmen; 2004; Nat Rev Cancer; 4; 604-16, Stancevic; 2010; FEBS Lett; 584: 1728-40). Mutations in the gene encoding acidic sphingomyelinase (ASM/Smpd1 ) have been associated with Niemann-Pick disease type A and B (Levran; 1991 ; Proc Natl Acad Sci U S A; 88; 3748-52), in which intracellular lipids accumulate, causing lysosomal storage disorder, hepatosplenomegaly and severe neurological problems in early childhood (Schuchman; 2009; Int J Clin Pharmacol Ther; 47 Suppl 1 ; S48-57). Additionally, ASM has been linked to a variety of infection processes (Truman; 201 1 ; Cell Mol Life Sci; 68; 3293-305). ASM derived ceramide membrane rafts are essential for the uptake of certain bacteria (Grassme; 2003; Nat Med; 9; 322-30, Grassme; 1997; Cell; 91 ; 605-15) and are the cause for increased inflammation and infection susceptibility in a mouse model of cystic fibrosis (Teichgraber; 2008; Nat Med; 14; 382-91 ). Moreover, ASM deficient mice have impaired growth restriction of Listeria monocytogenes but, interestingly, no alterations in cytokine responses (Utermohlen; 2003; J Immunol; 170; 2621-8). In support of this, a direct link of ASM derived ceramide to inflammatory cytokine signalling remains elusive and could not be observed in mice (Manthey; 1998; Cytokine; 10; 654-61 , Kuno; 1994; Int Immunol; 6; 1269-72). Due to its diverse implications, ASM has been suggested as a drug target for the therapy of cystic fibrosis and, in particular due to its well described anti- apoptotic effects, for the treatment of certain cancers (Ogretmen; 2004; Nat Rev Cancer; 4; 604-16, Wojewodka; 2011 ; J Lipids; 201 1 ; 674968). Generally, the reorganization of cholesterol and lipid composition at receptor rafts by interfering agents has been shown to be critical for the regulation of immune signalling, cytokine induction and MHC presentation (Fessler; 201 1 ; J Immunol; 187; 1529-35, Anderson; 2000; Nat Immunol; 1 ; 156-62). While it has been controversial whether ceramide could itself act as antagonist for TLR4 (MacKichan; 1999; J Biol Chem; 274; 1767-75, Fischer; 2007; Cell Microbiol; 9; 1239-51) it has been established already more than a decade ago that cellular ceramide levels are sensitive to LPS stimulation (MacKichan; 1999; J Biol Chem; 274; 1767-75). More recently, subcellular lipidomics of LPS activated RAW264.7 macrophages has shed light into the distinct, massive reorganization processes in the lipid composition of subcellular compartments that take place within minutes in an immune response (Andreyev; 2010; J Lipid Res; 51 ; 2785-97). However, it is important to note that ceramide metabolism includes more than 200 individual types of physiologically relevant ceramides with structural modifications that might play very different roles in distinct cellular processes. Hence, ceramide modulating enzymes are believed to act on very defined subcellular localizations with certain substrate specificity to influence specific cellular reactions (Hannun; 201 1 ; J Biol Chem; 286; 27855-62). While it is evident that sphingolipids are important in a variety of cellular processes (Bollinger; 2005; Biochim Biophys Acta; 1746; 284-94) and while several studies have suggested regulatory functions of ceramides on inflammatory responses (Boland; 1998; J Biol Chem; 273; 15494-500, Jozefowski; 2010; J Immunol; 185; 6960-73, Rozenova; 2010; J Biol Chem; 285; 21 103-13) up to now no sphingolipid modulating enzyme that would interact with and support the transmembrane TLRs in their function has been described.

The body's immune system is a very powerful weapon against pathogens, but dysfuncion can cause an underactivity or over-reactivity of this defence machinery which, in some instances, leads to severe diseases such as immunodeficiency disorders, autoimmune disorders or increased pro-inflammatory responses, immunodeficiency (or immune deficiency) is a state in which the immune system's ability to fight infectious disease or the growth of cancerous cells is compromised or entirely absent. Immunosuppression, the targeted reduction of the activation or efficacy of the immune system, is an option for the treatment of autoimmune disorders or pro-inflammatory responses. Immunosuppressant drugs known in the art are known to often cause various adverse reactions. A commonly used immunosuppressant drug is cyclosporine A, which is, e.g., often used to treat severe cases of rheumatoid arthritis (Matsuda; 2000; Immunopharmacology; 47; 1 19-125, Schreiber; 1991 ; Science; 251 ; 283-287), although it has many and sometimes severe side effects (de Mattos, 2000, Am J Kidney Dis, 35, 333-346).

The identification of modulators of immune reactions is an attractive goal for therapeutic intervention. Thus, the technical problem underlying the present invention is the provision of novel means and methods for the medical intervention of immunological disorders like autoimmune diseases, pro-inflammatory responses or immunodeficiency disorders.

The technical problem is solved by provision of the embodiments provided herein and as described by the claims. Accordingly, the present invention relates to a polypeptide for use in inhibiting immune response which is selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NOs: 1 or 3 or the nucleic acid sequence comprising nucleic acid residues 61 to 843 in SEQ ID NO: 1 or the nucleic acid sequence comprising nucleic acid residues 63 to 843 in SEQ ID NO: 3;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NOs: 2 or 4 or an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 2 or an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 4;

(c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence as depicted in SEQ ID NOs: 2 or 4 or of a functional fragment thereof, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine; or having an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 2 or an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 4;

(d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine;

(e) a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine; and

(f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d). The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that a lipid modifying enzyme, an acidic sphingomyelinase, is an interactor of TLRs 4,7,8 and 9. In context of the present invention, this acidic sphingomyelinase was termed LiMIT for "Lipid Metabolizing Interactor of TLRs". The LiMIT polypeptide is known in the art as "Acid sphingomyelinase-!ike phosphodiesterase Asm3b/Smpdl3b (UniprotKB / SwissProt ID P58242, Smpdl3b_MOUSE)" (see, e.g., Perosa; 2006; Blood; 107; 1070-1077, Munger; 2009; Genes Dev; 23; 2521-2536, Visel; 2003; Nucleic Acids Research; 32; D552-D556, Hansen; 2008; Genome Res; 18; 1670- 1679, Magdaleno; 2006; PLOA Biology; 4; 597-500) but up to now, it has never been connected with the modulation of immune responses. Murine LiMIT is a protein of about 456 amino acid length and human LiMIT is a protein of about 455 amino acid length. In addition, LiMIT harbours a signal peptide in the N-terminus (amino acids 1- 17 in murine LiMIT as depicted in SEQ ID NO: 4, and amino acids 1-18 in human LiMIT as depicted in SEQ ID NO: 2) that suggested membrane targeting and organelle trafficking, supporting the observation that LiMIT colocalizes and interacts with endosomal TLRs. Indeed, as shown in the appended illustrative examples, LiMIT is an endosomal factor that is able to modulate the processing and appearance of endosomal vesicles and could therefore be able to influence endosomal TLR signalling platforms and downstream signalling.

LiMIT contains a metallophosphatase domain (amino acids 21 to 281 in murine LiMIT and amino acids 21 to 281 in human LiMIT) with high similarity to the sphingomyelinase domain of a homologues protein of LiMIT, the sphingomyelinase ASM/Smpd1. Orthologs of LiMIT exist in organisms from human to insects/flies with high conservation of patterns especially in the N-terminal catalytic part of the protein. In mouse, LiMIT belongs to a family with two isoforms of ASML3a/Smpdl3a as closest homologs and ASM/Smpd1 as most distantly related protein. The metallophosphatase domain corresponds to the catalytic domain of LiMIT. In addition, LiMIT is a glycosylated and secreted protein.

In context of the present invention, it was surprisingly identified that LiMIT associates with TLRs, in particular with the TLRs 4, 7, 8, and 9 and negatively regulates TLR- mediated signalling. For example, as described herein and documented in the appended examples, depletion of LiMIT by shRNA leads to upregulation of MAPK14/p38 activation and increased Ι Β degradation. Furthermore, in context of the present invention it was unexpectedly identified that LiMIT is required to effectively control pro-inflammatory responses. For example, as discussed herein and documented in the examples, LiMIT was shown to control pro-inflammatory responses to LPS, imiquimod and CpG-DNA in vitro and to LPS and E. Coli in vivo. Moreover, with respect to the present invention, LiMIT was shown to regulate the strength of T cell responses. For instance, as described herein and documented in the appended examples, LiMIT deficient mice showed a strong increase in TLR- dependent T cell responses in vivo. Accordingly, with respect to the present invention it was shown that LiMIT is a negative regulator of TLR dependent cytokine responses. Thus, the present invention describes for the first time that the acidic sphingomyelinase LiMIT inhibits immune responses. In addition, in context of the present invention it was surprisingly shown that the sphingolipid metabolism and its surrounding machinery play a yet unanticipated but important role in innate immunity.

The innate immune system senses pathogens by recognizing certain conserved molecular patterns (PAMPs). While cytoplasmic receptors spot intracellular infections, Toll-like receptors (TLRs) are membrane associated sensors that detect extracellular and phagocytosed microbes to initiate pro-inflammatory responses and the presentation of pathogen derived peptides to effectively trigger adaptive immunity.

In the past years it has become evident that TLRs do not function alone and in particular TLR4 - the best studied receptor of this family - is an example of TLRs working in multiprotein complexes that are dynamic molecular machines mediating ligand recognition, phagocytosis, receptor trafficking and signal transduction (Monie; 2009; Trends Biochem Sci; 34; 553-61 , Fitzgerald; 2004; Microbes Infect; 6; 1361-7, Akashi-Takamura; 2008; Curr Opin Immunol; 20; 420-5). As TLRs have to transduce their signals through cellular membranes, their dynamics include not only receptor dimerization but require fast membrane dynamics in order to switch the pathogen recognition and destruction machinery on and off (Monie; 2009; Trends Biochem Sci; 34; 553-61 , Fessler; 201 1 ; J Immunol; 187; 1529-35). In this light it is surprising that up to now no lipid modifying enzyme has been identified that could catalyze the massive, physical changes that occur at an endosomal TLR microenvironment. An aspect of this invention is the identification of the acidic sphingomyelinase LiMIT as cofactor and interactor of the MYD88 dependent TLRs 4, 7, 8 and 9.

Sphingomyelinases consume membrane integral sphingomyelin to release phosphorylcholine and ceramide, the latter being implied in a variety of biological functions (Bollinger; 2005; Biochim Biophys Acta; 1746; 284-94). The activity of sphingomyelinases can be induced by certain stress stimuli, such as CD95 or TNF and the derived ceramide has often been linked to the induction of apoptosis and a variety of stress responses (Sharma; 1999; Cell Res; 9; 1-10). Functionally, ceramide has been suggested to act either as soluble second messenger or as modulator of local membrane rafts (Stancevic; 2010; FEBS Lett; 584; 1728-40, Boland; 1998; J Biol Chem; 273; 15494-500). Unfortunately, the complexity of a huge variety of ceramide types that are modulated, processed and created in local microdomains by a specific set of enzymes makes it difficult to understand the detailed function of this class of lipids in distinct, local signalling processes (Hannun; 201 1 ; J Biol Chem; 286; 27855-62) and for the same reasons a detailed understanding of the specific sphingolipid rearrangements by LiMIT will remain the task for dedicated lipidomics analysis.

LiMIT is a glycosylated, endosomal sphingomyelinase (SMase) that, surprisingly functions as a negative regulator of TLR signalling. It might appear surprising that an endosomal SMase would also regulate TLR4 function, but in fact, endosomal signalling of internalized TLR4 has been shown previously (Kagan; 2008; Nat Immunol; 9; 361 -8). As described herein and documented in the appended examples, LiMIT inhibited TNF, IL6, IL12, TGFp and 1L1 β cytokine responses and promoted the anti-inflammatory cytokine IL-10 (see, e.g., Figure 4), suggesting that LiMIT not only randomly promoted cytokine secretion, but specifically functions as an anti-inflammatory element. This is supported, as documented in the appended examples, e.g. by phospho-proteomics analysis of TLR4 induced macrophages which led to a characterization of LiMIT inhibited inflammatory signalling pathways (see, e.g., Figure 5E). While LiMIT was shown, in context of the invention, to be dispensable for ligand phagocytosis (e.g. for the phagocytic uptake of CpG-DNA as depicted in Figure 5A,B), it was observed that LiMIT induces repression especially of the p38 and c-Jun branches, which (co)regulate the expression of pro-inflammatory cytokines such as IL-6 but also influence on TLR containing phagosome maturation and MHCII antigen presentation (Blander; 2006; Nat Immunol; 7; 1029-35). In fact, as documented in the appended examples, transcriptomic analysis of LiMIT depleted macrophages showed a specific upregulation of MHC antigens and, furthermore, LPS/ovalbumin immunization experiments revealed higher activation of T cells in the absence of LiMIT. Accordingliy, LiMIT affects CD4⁺ adaptive immune responses. Hence, as described herein and shown in the appended examples, LiMIT not only regulates T cell responses via the upregulation of a multitude of cytokines (IL- 12p40/IL-23, IL-6, !L1 β and TGFp) leading to increased responses of the Th17 cytokine IL-17 (see, e.g., Figure 6F) (Yang; 201 1 ; Nat Immunol; 12; 247-254) but also by enhanced MHC presentation.

Ceramides do have local effects on receptor clustering (Bollinger; 2005; Biochim Biophys Acta; 1746; 284-94), phagosome maturation (Li; 1999; J Biol Chem; 274; 21 121 -7) and antigen presentation (Gombos; 2006; Immunol Lett; 104; 59-69). However, as mentioned above, up to now no sphingolipid modulating enzyme that would interact with and support the transmembrane TLRs in their function has been described. LiMIT represents the missing link between lipid cleavage and TLR- mediated pro-inflammatory singling. In addition, in context of the present invention, it was surprisingly found that LiMIT is not only a sphingomyelinase that is involved in inhibiting immune response but it is also regulated by sphingomyelin and ceramides. As shown in the appended illustrative examples, LiMIT transcript levels are responsive to the addition of the substrate sphingomyelin and cleavage product ceramide. In contrast, transcript levels of other sphingomyelinasen such as the LiMIT homologue ASM are unaffected by either stimulus.

Furthermore, as documented in the appended examples, a de novo formation of endosome like structures was observed when LiMIT was overexpressed, supporting that LiMIT has the ability to alter intracellular membrane structures to modulate p38/JNK signalling at endosomal TLR signalosome microdomains. This has two major consequences: LiMIT deficiency produces not only increased inflammation in response to TLR ligands or pathogenic challenge but also triggers a more efficient activation of T cells. The innate-adaptive synapse between antigen presenting cells and naive T cells is a crucial step of the immune system to ensure that a strong antibody response to an antigen is only elicited if it has been identified as pathogen by the presence of a TLR stimulating PAMP. Only in that case phagosomes will mature accordingly, initiate specific signalling platforms and induce MHCII antigen presentation (Blander; 2007; Cell Microbiol; 9; 290-9). In this way the organism prevents self recognition, ensures self tolerance and prevents autoimmune reactions (Iwasaki; 2010; Science; 327; 291-5, Reis; 2004; Curr Opin Immunol; 16; 21-5). This is a clear and present danger as endosomal TLRs on antigen presenting cells can not entirely differentiate between self and non-self DNA and are a source for nucleic acid caused autoimmune diseases such as lupus (Krieg; 2007; Immunol Rev; 220; 251-69). Hence, as suggested with respect to the present invention, the restrictive role of LiMIT could be an important mechanism to prevent hyperinflammation and, most importantly, to restrict presentation of self-antigens and maintain self-tolerance and, thereof, autoimmune disease. Hence, according to the present invention, targeting LiMIT in chronic infections or immunization processes could promote the immunological effects on pathogen clearance or antibody production whereas LiMIT agonists or recombinant LiMIT enzyme could help to reduce chronic inflammation and autoimmune disease.

Accordingly, the present invention relates to the polypeptide LiMIT for use in inhibiting immune response which is defined by the following sequences:

(a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NOs: 1 or 3 or the nucleic acid sequence comprising nucleic acid residues 61 to 843 in SEQ ID NO: 1 or the nucleic acid sequence comprising nucleic acid residues 63 to 843 in SEQ ID NO: 3;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NOs: 2 or 4 or an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 2 or an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 4;

(c) a polypeptide encoded by a nucleic acid molecule encoding a polypeptide having an amino acid sequence as depicted in SEQ ID NOs: 2 or 4 or of a functional fragment thereof, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine; or having an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 2 or an amino acid sequence comprising amino acids 21 to 281 in SEQ ID NO: 4;

(d) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of a nucleic acid molecule as defined in (a) or (c) and encoding a functional polypeptide; or a functional fragment thereof, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine;

(e) a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to the polypeptide of any one of (a) to (d), whereby said polypeptide is functional; or a functional fragment thereof, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine; and

(f) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code to the nucleotide sequence of a nucleic acid molecule as defined in (a), (c), and (d).

The immune system protects the organisms with layered defense mechanisms of increasing specificity. Invading pathogens subsequently activate the innate immune system which provides an immediate, but non-specific response. Innate immune systems are found in all animals and also in plants (Litman; 2005; Nat Rev Immunol; 5; 866-79). If pathogens successfully evade the response of the innate immune system, vertebrates possess a second layer of protection, the adaptive immune system, which is activated by the innate immune response. The adaptive immune response is antigen specific and allows vertebrates for a stronger immune response as well as immunological memory (Pancer; 2006; Ann Rev Immunol; 24; 497-518).

In context of the invention it is described that LiMIT plays an unanticipated but important role in innate as well as adaptive immunity. Accordingly, a pharmaceutical composition comprising LiMIT can be used for inhibiting immune response which includes both the innate and the adaptive immune response. Since only vertebrates possess an adaptive immunity, it is preferred to use vertebrate LiMIT to inhibit immune response; it is mostly preferred to use human LiMIT to inhibit immune response.

As suggested by the domain and homology analysis and described above, LiMIT is an enzyme with sphingomyelinase activity, mediating the cleavage of membrane integral sphingomyelin into phosphorylcholine and ceramide (cf. Seto; 2004; Protein Sci; 13; 3172-86). In addition, in context of the invention it was shown that the N- terminal catalytic (metallophosphatase) domain of LiMIT has highest sphingomyelinase activity whereas full length LiMIT has slightly reduced substrate conversion.

Therefore, one embodiment of the invention relates to the LiMIT polypeptide for use in inhibiting immune response or a functional fragment thereof, wherein said functional fragment consists either of the catalytic domain of a polypeptide having the amino acid sequence of SEQ ID NOs: 2 or 4; or of the catalytic domain of a polypeptide having at least 80% homology to SEQ ID NOs: 2 or 4; wherein said functional fragment is functional, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine; and wherein the amino acid sequence of said catalytic domain consists of:

(i) the amino acids 21 to 281 in SEQ ID NO: 2;

(ii) the amino acids 21 to 281 in SEQ ID NO: 4;

(iii) the amino acids that correspond to the amino acids 21 to 281 in SEQ ID NO: 2 in a polypeptide having at least 80% homology to SEQ ID NO: 2; or

(iv) the amino acids that correspond to the amino acids 21 to 281 in SEQ ID NO: 4 in a polypeptide having at least 80% homology to SEQ ID NO: 4.

In one aspect, the invention relates to the LiMIT polypeptide for use in inhibiting immune response or a functional fragment thereof, wherein said functional fragment consists either of the catalytic domain of a polypeptide having the amino acid sequence of SEQ ID NOs: 2 or 4; or of the catalytic domain of a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NOs: 2 or 4; wherein said functional fragment is functional, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine; and wherein the amino acid sequence of said catalytic domain consists of:

(i) the amino acids 21 to 281 in SEQ ID NO: 2;

(ii) the amino acids 21 to 281 in SEQ ID NO: 4;

(iii) the amino acids that correspond to the amino acids 21 to 281 in SEQ ID NO: 2 in a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 2; or

(iv) the amino acids that correspond to the amino acids 21 to 281 in SEQ ID NO: 4 in a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 4.

Proteins involved in host defence against pathogens are often upregulated after pathogen encounter. As shown in the appended illustrative examples, transcript and protein levels of LiMIT are sensitive to TLR stimuli such as CpG-DNA. In addition, in context of the invention it is described that the regulatory effects of LiMIT on cytokine levels are attributed directly to TLR induced signalling events. In this respect, LiMIT acts not on the early phagocytic uptake of TLR ligand but later to modulate proinflammatory TLR signalling in the acidified endosome.

As described herein, LiMIT acts as a negative regulator of pro-inflammatory TLR signalling in antigen presenting cells such as macrophages. For example, as shown in the appended illustrative examples, after stimulation with TLR lingans (such as LPS) several nodes of pro-inflammatory signalling, including c-Jun, Erk, c-myc and p38, revealed higher phosphorylation states in LiMIT depleted antigen presenting cells (e.g. macrophages). In addition, as shown in the appended examples by using antigen presenting cells, LiMIT regulates p38 activation which has been implied as a regulatory mechanism in TLR signalling that not only affects cytokine secretion but also phagosome maturation and antigen presentation (Blander; 2006; Nat Immunol; 7; 1029-35).

Accordingly, another embodiment of the present invention relates to the LiMIT polypeptide for use in inhibiting immune response or a functional fragment thereof, wherein said polypeptide or functional fragment thereof inhibits TLR signalling in an antigen presenting cell. Antigen presenting cells such as macrophages, dendritic cells or B cells have a large repertoire of TLRs. Accordingly, in one embodiment of the invention said antigen presenting cell is a macrophage, a dendritic cell or a B cell.

The inhibition of TLR signalling may be measured by the following method. Beforehand it must be excluded that the polypeptide to be analyzed (such as LiMIT) is not endotoxin contaminated which, by itself, would trigger TLR activation. This can be done by using the Limulus Adebocyte Lysate (LAL) test (Lonza). The Limulus Adebocyte Lysate test (or QCL-1000™ assay) is a fast, quantitative, endpoint assay for the detection of, e.g., gram negative bacterial endotoxin. Samples are mixed with the LAL reagent and chromogenic substrate reagent over a short incubation period (16 minutes), and read on any spectrophotometer or plate reader which is capable of measuring 405-410 nm.

Subsequently, the TLR dampening response can be assayed by one of the following assays:

a) seed immune cells (e.g. mouse RAW264.7 macrophages/human THP-1 macrophages, primary antigen presenting cells from mice or human PBMCs) on ELISA plates (1 x10⁵/well), stimulate said immune cells with one concentration of TLR ligand LPS (l ung/ml) or endosomal TLR ligand CpG-DNA (1 μΜ) for 16h and titrate increasing amounts of the polypeptide to be analyzed (e.g. LiMIT) to the reaction 30min prior to stimulation (1 ng/ml to 10pg/ml). Assay for TLR induced immune responses using as readout i) ELISA for the pro-inflammatory cytokines IL-6, TNF and ii) qRT-PCR for the pro-inflammatory cytokines IL-6, TNF.

b) seed immune cells (mouse RAW264.7 macrophages/human THP-1 macrophages, primary antigen presenting cells from mice or human PBMCs) on ELISA plates (1x10⁵/well), stimulate said immune cells with a titration of the TLR ligands LPS (0,5ng-1 pg/ml) and CpG-DNA (0,2μΜ - 10μΜ) and ad 1 pg/ml of the polypeptide to be analyzed (e.g. LiMIT) to each well 30min prior to stimulation. After 16h measure TLR induced immune responses using as readout i) ELISA for the pro-inflammatory cytokines IL-6, TNF and ii) qRT-PCR for the pro-inflammatory cytokines IL-6, TNF. The detected ability of a polypeptide to inhibit TLR signalling which is measured by the above-described method may be compared to a standard or reference value. The reference value may be a positive control which may be measured by using a purified full length protein of LiMIT (e.g., consisting of SEQ ID NO: 4). Furthermore, inhibitory effects as control can be exerted by adding inhibitors of endosome maturation and acidification such as chloroquine and z-FA-FMK. Another reference value may be a negative control which may be detected by performing the above-described assay without adding the LiMIT polypeptide or by addition of a catalytically inactive polypeptide.

It is described in context of the invention that LiMIT not only randomly promoted cytokine secretion, but specifically functions as an anti-inflammatory element. For example, as described herein and documented in the appended examples, LiMIT inhibits TNF, IL6, IL12, TGFp and IL1 β cytokine responses and promotes the antiinflammatory cytokine IL-10. As mentioned above, it is described herein that LiMIT inhibits TLR-mediated inflammatory signalling in antigen presenting cells such as macrophages. For instance, the appended examples show that LiMIT inhibits inflammatory signalling pathways in TLR4 induced macrophages.

Therefore, a further embodiment of this invention relates to the LiMIT polypeptide for use in inhibiting immune response or a functional fragment thereof, wherein said polypeptide or said functional fragment thereof inhibits a macrophage-mediated proinflammatory response.

Stimulation of TLRs can cause immune responses. As described herein and documented by the appended examples, expression of LiMIT is sensitive to TLR stimuli. In addition, in context of the present invention it was shown that LiMIT is an anti-inflammatory factor that restricts TLR induced inflammation, antigen presentation, and subsequent T cell activation. Accordingly, LiMIT is a negative regulator of immune responses such as pro-inflammatory responses.

Furthermore, as described herein, different LiMIT transcript levels correlate with the degree of severity of an immune response. In particular, enhanced levels of LiMIT expression lead to a less intense immune response whereas reduced expression levels of LiMIT result in an enhanced immune response. Therefore, enhancing the level of LiMIT may be useful for treating diseases that are associated with an overactive immune system whereas reducing the level of LiMIT may be useful for treating diseases that are associated with an underactive immune system. Accordingly, another embodiment of the present invention relates to a pharmaceutical composition for use in treating a pro-inflammatory response and/or an autoimmune disorder, comprising the polypeptide LiMIT for use in inhibiting immune response or a functional fragment thereof and/or a nucleic acid molecule encoding said LiMIT polypeptide or said functional fragment thereof. In one aspect of the invention said pharmaceutical composition for use in treating a pro-inflammatory response and/or an autoimmune disorder comprises an agonist of LiMIT and/or a nucleic acid molecule encoding an agonist of LiMIT.

Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. Medicaments comprising agonists or (recombinant) LiMIT enzyme could help to reduce chronic inflammation and autoimmune diseases.

Accordingly, one embodiment of the invention relates to said pharmaceutical composition of the invention comprising LiMIT for use in treating an autoimmune disorder, wherein said autoimmune disorder is selected from the group consisting of:

(I) Systemic lupus erythematosus;

(ii) Rheumatoid arthritis;

(iii) Multiple sclerosis;

(iv) Idiopathic thrombocytopenic purpura;

(v) Sjogren's syndrome;

(vi) Diabetes;

(vii) Vasculitis;

(viii) Crohn's disease; and

(iv) Psoriasis.

As mentioned above, LiMIT is a specific, negative regulator of inflammation. Furthermore, it is described herein that LiMIT is a specific negative regulator of the inflammatory response of the host against pathogens. Accordingly, one embodiment of the invention relates to said pharmaceutical composition of the invention comprising LiMIT for use in treating a pro-inflammatory response, wherein said proinflammatory response is selected from the group consisting of:

(i) inflammation; (ii) hypersensitivity reaction; and

(iii) sepsis.

A pro-inflammatory response as used herein is known in the art and relates to any kind of inflammation in the complex biological response of tissues to harmful stimuli, such as pathogens, damaged cells, allergens or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue.

In response to an infection, a cascade of signals leads to the recruitment of inflammatory cells, particularly innate immune cells such as neutrophils and macrophages (Chen; 2010;, Nature Rev; 10;, 826-837). These cells, in turn, phagocytose infectious agents and produce additional cytokines and chemokines that lead to the activation of lymphocytes and adaptive immune responses. Simiiar to the eradication of pathogens, the inflammatory response is also crucial for tissue and wound repair. Inflammation as a result of trauma, ischaemia-reperfusion injury or chemically induced injury typically occurs in the absence of any microorganisms and has therefore been termed 'sterile inflammation' (Chen; 2010; , Nature Rev;, 10;, 826-837). Similar to microbially induced inflammation, sterile inflammation is marked by the recruitment of neutrophils and macrophages and the production of proinflammatory cytokines and chemokines, notably tumour necrosis factor (TNF) and interleukin 1 (IL1 ) (Chen;2010;, Nature Rev; 10;, 826-837). In context of the present invention, a pro-inflammatory response also includes all kinds of hyper-inflammatory illnesses such as hypersensitivity (also called "hypersensitivity reaction" or "hyperreaction") which refers to undesirable (hyper)reactions of the immune system resulting from exposure to allergens, irritants or pathogens. These reactions may be damaging, uncomfortable, or occasionally fatal.

Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the activation of immune cells followed by the release of various mediators and increased movement of leukocytes from the blood into the injured tissues. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells which are present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Sepsis is a serious medical condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome or SIRS) and the presence of a known or suspected infection. The body may develop this inflammatory response to microbes in the blood, urine, lungs, skin, or other tissues. Invasion of bacteria to otherwise sterile sites like the peritoneal cavity leads to the immediate initiation of an inflammatory response. Integral to this response are oxygen radicals that are primarily generated to kill microbes, but can also damage host structures through the peroxidation of membrane phospholipids (Hampton; 1998; Blood; 92; 3007-3017).

Accordingly, with respect to the present invention, it is envisaged to use the pharmaceutical composition of the invention comprising LiMIT for treating all kinds of inflammatory conditions including an sterile inflammation, an inflammatory hyperreaction or hypersensitivity reaction resulting from exposure to allergens or irritants, a hyperreaction or hypersensitivity reaction to pathogen infection (such as bacterial or viral infection); acute and chronic inflammation; hyperinflammation and sepsis. One particular embodiment of the invention relates to the pharmaceutical composition of the invention comprising LiMIT for use in treating a hypersensitivity reaction, wherein said hypersensitivity reaction is a Graft-versus-host disorder or Contact dermatitis.

Said pharmaceutical composition of the invention comprising LiMIT may also be useful to treat diseases of the metabolism such as Niemann-Pick disease, since dysfunction of LiMIT may be involved in such diseases. For example, as shown in the appended examples, a mutant of LiMIT (H135A) designed based on homology to a point mutation of the LiMIT homologue ASM that has been found in patients with Niemann-Pick disease (cf. Seto; 2004; Protein Sci; 13; 3172-86) showed reduced catalytic activity.

In addition to said pharmaceutical composition of the invention comprising LiMIT, another pharmaceutical composition comprising an inhibitor/antagonist of LiMIT is comprised by the present invention. Said antagonist or inhibitor may prevent, reduce, inhibit or inactivate the physiological activity of LiMIT, e.g., upon binding of said inhibitor/antagonist to LiMIT. A preferred inhibitor/antagonist to be used in this respect is an antagonistic antibody. Since LiMIT is a secreted protein, it can be found extraceilularly and is, therefore, readily available for such an antagonistic antibody. A detailed description of the inhibitors or antagonists to be applied in context of the present invention including (antagonistic) antibodies is provided herein below.

Said pharmaceutical composition of the invention comprising an inhibitor/antagonist of LiMIT may be useful in treating immunodeficiency disorders, since, as described above, reduced levels of LiMIT expression lead to an enhanced immune response. Accordingly, a pharmaceutical composition targeting LiMIT could reduce the level of LiMIT and therefore could treat immunodeficiency disorders. Accordingly, a pharmaceutical composition comprising an inhibitor/antagonist of LiMIT for use in treating an immunodeficiency disorder is described herein. In one aspect of the invention said immunodeficiency disorder is an acquired immunodeficiency disorder or a combined immunodeficiency disorder.

Combined immunodeficiency disorders affect both, B and T cell responses (Kumar and Clark, "Clinical Medicine", third edition (1994), Bailliere Tindall). These can stem from a variety of defective mechanisms in lymphocyte function, but tend to have rather similar clinical features, combining the opportunist infections of cell-mediated immunodeficiency with those of antibody deficiency. Severe combined immunodeficiency (SCID), also known as "Alymphocytosis," "Glanzmann-Riniker syndrome", "Severe mixed immunodeficiency syndrome", and "Thymic alymphoplasia" is a genetic disorder in which both "arms" (B cells and T cells) of the adaptive immune system are impaired due to a defect in one of several possible genes. SCID is a severe form of heritable immunodeficiency. It is envisaged in the context of the invention to use said pharmaceutical composition comprising an inhibitor/antagonist of LiMIT in treating SCID.

Acquired immunodeficiencies are commonly known but often less precisely defined in terms of immunological mechanisms. These immunodeficiencies also include immunosuppression resulting from specific diseases that affect immune competence, such as the acquired immunodeficiency syndrome (AIDS) resulting from HIV infection. The pathophysiology of AIDS is complex (Guss; 1994; J Emerg Med; 12; 375-84). HIV causes a depletion of CD4⁺ T helper lymphocytes leading to an inability of the immune system to fight against infections or kill cancerous cells. Although new T cells are continuously produced by the thymus to replace the ones lost, the regenerative capacity of the thymus is slowly destroyed by direct infection of its thymocytes by HIV. Eventually, the minimal number of CD4⁺ T cells necessary to maintain a sufficient immune response is lost, leading to AIDS. One aspect of the present invention relates to said pharmaceutical composition comprising an inhibitor/antagonist of LiMIT for use in treating an acquired immunodeficiency disorder like the acquired immunodeficiency syndrome (AIDS).

Another subgroup of immunodeficiency disorders are acquired neutrophil function disorders. Neutrophils are the most frequently occurring leukocytes in the body (50%- 65%) and have been shown to be capable of cross-presenting exogenous antigens, i.e. to be antigen presenting cells (Beauvillain; 2007; Blood; 110, 2965-73). An important infective cause of acquired neutrophil dysfunction is influenza, which causes a specific transient impairment of phagosome-lysosome fusion. This is the main reason for the high risk of staphylococcal pneumonia in influenza epidemics (Kumar and Clark, "Clinical Medicine", third edition (1994), Bailliere Tindall). Neutrophil and macrophage function can also be impaired in abnormal metabolic states such as Diabetes mellitus and Hypophosphataemia. Additionally, in alcoholic cirrhosis, inhibitors of endogenous chemotactic factors for neutrophils causing immunodeficiency have been found (Kumar and Clark, "Clinical Medicine", third edition (1994), Bailliere Tindall). In context of the invention, it is envisaged to use said pharmaceutical composition comprising an inhibitor/antagonist of LiMIT for treating an acquired immunodeficiency disorder like an acquired neutrophil dysfunction. Said acquired neutrophil dysfunction may be a diabetes-mediated acquired neutrophil dysfunction, an alcoholic cirrhosis-mediated acquired neutrophil dysfunction or an influenza-mediated acquired neutrophil dysfunction.

Cancerogenesis is the result of neoplastic cells on the one hand and host defence on the other. Cancer cells have mechanisms of evading normal defence mechanisms. However, neoplastic cells can only cause tumoural diseases or cancer if the host's defence mechanisms fail to control their growth. Accordingly, the present invention relates to said pharmaceutical composition comprising an inhibitor/antagonist of LiMIT for use in treating a tumoural disease or cancer. In one preferred aspect of the invention, said cancer is blood cancer, such as Myeloid leukaemia or Hodgkin's disease. In addition, in further aspects of the invention, said cancer is lung cancer, skin cancer, breast cancer, prostate cancer, brain cancer, liver cancer, cancer of the throat, adrenal cancer, alveolar cancer, bladder cancer, uterine cancer (such as cervical cancer), bowel cancer, gastric cancer, oesophageal cancer, pancreatic cancer or testicular cancer. Since LiMIT seems to be expressed in the colon (Novartis BioGPS: http://biogps.org/#goto=welcome, non-confirmed data), another preferred aspect of the invention is to treat colon carcinoma by using said pharmaceutical composition comprising an inhibitor/antagonist of LiMIT.

Another aspect of the invention relates to the pharmaceutical composition comprising and inhibitor/antagonist of LiMIT in order to enhance immune response to promote pathogen clearance. Inducing a stronger immune response in chronic infections or immunization processes could promote the immunological effects on pathogen clearance.

It is also envisaged to use LiMIT to reduce transplant rejection in autologous or allogeneic transplantation procedures. For example, organs, tissues, or cells intended for use in transplantation (e.g., xenotransplantation or homotransplantation) can be contacted ex vivo with LiMIT, e.g., to reduce the immune response of the recipient. It is also described herein that recipients of an organ, tissue, or cell transplant can be treated with LiMIT. Such treatment could commence prior to, during, or after the transplant.

In context of the present invention, said "pharmaceutical composition(s)" are medicaments. The pharmaceutical compositions of the invention may be in solid or, preferably, in liquid form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (an) aerosol(s) or, preferably, (a) solution(s). Furthermore, it is envisaged that the medicaments of the invention may comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.

The pharmaceutical compositions of the invention may be administered by different ways, e.g., parenteral, subcutaneous, intraperitoneal, topical, intrabronchial, intrapulmonary, intranasal or by infusion or injection. Administration of the suitable pharmaceutical compositions may also be effected, if desired for local treatment, by intralesional administration. Parenteral administrations include intraperitoneal, intramuscular, intradermal, subcutaneous intravenous or intraarterial administration. The pharmaceutical compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, e.g. a specifically effected organ.

However, one aspect of the present invention relates to the treatment of an autoimmune disorder or a pro-inflammatory response. Therein, fast and direct delivery of LiMIT, a functional fragment thereof or an agonist of LiMIT to the target cells, (e.g. macrophages, dendritic cells or B cells) has to be ensured. Another aspect of the invention relates to the treatment of an immunodeficiency disorder. Therein, fast and direct delivery of the antagonist/inhibitor of LiMIT to the target cells has to be ensured. Accordingly, in both aspects, it is preferred that said pharmaceutical compositions are administered by infusion intravenously. In general, biologicals are administrated intravenously to reduce destroying of the biological by gastric juice.

These pharmaceutical compositions of the invention can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosage for any single patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. However, a phase 1 trial dealing with the use of an acidic sphingomyelinase in an enzyme replacement therapy was conducted at the Mount Sinai Medical Center in New York City from December 2006 to April 2009. The primary trial objectives of this study were to evaluate the safety and pharmacokinetics of single doses of the LiMIT homologue ASM in adults with Niemann-Pick disease B. Single doses of 0.03, 0.1 , 0.3, 0.6, and 1 .0 mg per kg body weight (mg/kg) of recombinant ASM were infused sequentially by dose cohort. Dose-related clinical and lab adverse events began at 0.3 mg/kg and the maximum tolerated dose of recombinant ASM was 0.6 mg/kg. Since LiMIT and ASM are homologues sphingomyelinases, similar doses of LiMIT (or a LiMIT agonist) may be applied to reach effective inhibition of immune response in patients. It is also conceivable to apply similar doses of an antagonist/inhibitor of LiMIT to reach effective activation of immune response in patients.

Accordingly, if infused sequentially by dose cohort, doses of LiMIT (or an agonist an antagonist/inhibitor of LiMIT) should be in the range of 0.03 mg to 1 mg/kg, e.g. between 0.3 mg/kg and 0.6 mg/kg. If the regimen is not a sequential infusion, pharmaceutically active matter may be present in amounts between 0.01 and 20 mg/kg per dose, e.g. between 0.1 mg to 10 mg/kg, e.g. between 0.5 mg to 5 mg/kg. Yet, doses below or above the indicated exemplary ranges are also envisioned, especially considering the aforementioned factors. However, the exact concentration will have to be determined in pharmacokinetics and pharmacodynamics studies following the code of federal regulations of the FDA.

The pharmaceutical compositions of the invention may further comprise pharmaceutical carriers, excipients and/or diluents. Suitable pharmaceutical carriers, excipients and diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. Suitable carriers may comprise any material which, when combined with LiMIT, a functional fragments thereof or an antagonist/inhibitor of LiMIT, retains the biological activity of LiMIT, a functional fragment thereof or the antagonist/inhibitor of LiMIT, respectively (see Remington's Pharmaceutical Sciences; 1980; 16th edition; Osol. A. Ed). Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present including, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical compositions of the present invention may comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. In context of the present invention, it is envisaged that the pharmaceutical compositions of the invention may comprise further biologically active agents, depending on the intended use of the pharmaceutical composition. These further biologically active agents may be e.g. antibodies, antibody fragments, hormones, growth factors, enzymes, binding molecules, cytokines, chemokines, nucleic acid molecules and drugs. Further biologically active agents could also be lipids, especially sphingomyelin substrates which are already complexed with LiMIT and might mediate Limit anti-inflammatory effects. The pharmaceutical composition of the invention containing LiMIT (or a LiMIT agonist) is preferably to be co-administered with other known substances that inhibit immune responses such as conventional anti-inflammatory drugs. The pharmaceutical composition of the invention containing an antagonist/inhibitor of LiMIT is preferably to be co-administered with known substances that prevent infections, such as conventional antibiotic, antifungal or antiviral drugs.

In most cases, an early stage diagnosis of immunodeficiency disorders, autoimmune disorders or pro-inflammatory responses improves their chance of recovery. Therefore, novel strategies to diagnose malfunction of the immune system is a crucial step to maximize the chance of recovery.

It is described in context of the present invention that different LiMIT transcript or protein levels correlate with the degree of severity of an immune response. In particular, a high level of LiMIT expression correlates with a reduced immune response and a low expression level of LiMIT correlates with an enhanced immune response. Therefore, a high level of LiMIT may be indicative for an underactive immune system whereas a low level of LiMIT may be indicative for an overactive immune system. Accordingly, detecting the level of LiMIT may be useful to diagnose both, underactivity and overactivity of the immune system. Therefore, detecting the level of LiMIT may be useful to diagnose an immunodeficiency disorder as well as an autoimmune disorder or a pro-inflammatory response.

Accordingly, one embodiment of the present invention relates to a diagnostic composition for use in diagnosing at least one medical condition selected from the group consisting of:

(i) autoimmune disorder; (ii) pro-inflammatory response; and

(ii) immunodeficiency disorder,

wherein said diagnostic composition comprises a binding molecule which specifically binds to the LiMIT polypeptide for use in inhibiting immune response or to a functional fragment thereof, and/or to a nucleic acid molecule encoding said LiMIT polypeptide or said functional fragment thereof.

Thus, in one embodiment, the present invention relates to the use of a diagnostic composition for diagnosing at least one medical condition selected from the group consisting of:

(i) autoimmune disorder;

(ii) pro-inflammatory response; and

(ii) immunodeficiency disorder,

wherein said diagnostic composition comprises a binding molecule which specifically binds to the LiMIT polypeptide for use in inhibiting immune response or to a functional fragment thereof, and/or to a nucleic acid molecule encoding said LiMIT polypeptide or said functional fragment thereof.

In one aspect of the present invention said diagnostic composition is for use in diagnosing in vitro. One embodiment of the invention relates to said diagnostic composition comprising a binding molecule which specifically binds to LiMIT, wherein said binding molecule is a polynucleotide which hybridizes to a nucleic acid molecule encoding the LiMIT polypeptide for use in inhibiting immune response or a functional fragment thereof. In a further embodiment said polynucleotide binds to a nucleic acid molecule encoding LiMIT or a functional fragment thereof under stringent conditions. In another embodiment said polynucleotide binds to a nucleic acid molecule encoding LiMIT or a functional fragment thereof under non-stringent conditions. A definition of the term "hybridizes under stringent (or non-stringent) conditions" is given herein below.

As mentioned above, a low level of LiMIT may be indicative for an overactive immune system. Therefore, the determination of the level of LiMIT may be useful to diagnose autoimmune disorders or pro-inflammatory responses. Accordingly, one embodiment of the invention relates to said diagnostic composition of the invention for use in diagnosing an autoimmune disorder, wherein said autoimmune disorder is selected from the group consisting of:

(i) Systemic lupus erythematosus;

(ii) Rheumatoid arthritis;

(iii) Multiple sclerosis;

(iv) Idiopathic thrombocytopenic purpura;

(v) Sjogren's syndrome;

(vi) Diabetes;

(vii) Vasculitis;

(viii) Crohn's disease; and

(iv) Psoriasis.

Additionally, another embodiment of the invention relates to said diagnostic composition of the invention for use in diagnosing a pro-inflammatory response, wherein said pro-inflammatory response is selected from the group consisting of:

(i) inflammation;

(ii) hypersensitivity reaction; and

(iii) sepsis.

With respect to the present invention, it is envisaged to diagnose all kinds of inflammatory conditions including an sterile inflammation, an inflammatory hyperreaction or hypersensitivity reaction resulting from exposure to allergens or irritants, a hyperreaction or hypersensitivity reaction to pathogen infection (such as bacterial or viral infection); acute and chronic inflammation; hyperinflammation and sepsis. One particular embodiment of the invention relates to the diagnostic composition of the invention, wherein said hypersensitivity reaction is a Graft-versus- host disorder or Contact dermatitis.

As mentioned above, a high level of LiMIT expression correlates with a reduced immune response. Therefore, a high level of LiMIT may be indicative for an underactive immune system. Thus, detecting the level of LiMIT may be useful to diagnose an immunodeficiency disorder. Accordingly, the present invention relates to the above-described diagnostic composition comprising a binding molecule which specifically binds to the LiMIT polypeptide/nucleic acid molecule for use in diagnosing an immunodeficiency disorder. Said immunodeficiency disorder may be an acquired immunodeficiency disorder or a combined immunodeficiency disorder. It is envisaged in the context of the invention to use said diagnostic composition to diagnose the combined immunodeficiency disorder SCID. It is further described in the present invention to use said diagnostic composition for diagnosing an acquired immunodeficiency disorder. In one aspect of the invention said acquired immunodeficiency disorder is an acquired neutrophil dysfunction. Said acquired neutrophil dysfunction may be a diabetes-mediated acquired neutrophil dysfunction, an alcoholic cirrhosis-mediated acquired neutrophil dysfunction or an influenza- mediated acquired neutrophil dysfunction.

Furthermore, one embodiment of the present invention relates to the above- described diagnostic composition comprising a binding molecule which specifically binds to the LifvliT poiypeptide/nucieic acid molecule for use in diagnosing an immunodeficiency disorder, wherein said immunodeficiency disorder is a tumoural disease or cancer. In one preferred aspect of the invention, said cancer is blood cancer, such as Myeloid leukaemia or Hodgkin's disease. In further aspects of the invention, said cancer is lung cancer, skin cancer, breast cancer, prostate cancer, brain cancer, liver cancer, cancer of the throat, adrenal cancer, alveolar cancer, bladder cancer, uterine cancer (such as cervical cancer), bowel cancer, gastric cancer, oesophageal cancer, pancreatic cancer or testicular cancer. Since LiMIT seems to be expressed in the colon (Novartis BioGPS, http://biogps.org/#goto=welcome) non-confirmed data), one preferred aspect of the invention is to diagnose colon carcinoma by using said diagnostic composition of the invention.

Another aspect of the invention relates to said diagnostic composition of the invention for use in diagnosing a disease of the metabolism. It is envisaged that said disease of the metabolism is Niemann-Pick disease. Detecting LiMIT may be useful to diagnose a disease of the metabolism since dysfunction of LiMIT may be involved in such diseases. In this respect it may be useful to apply a binding molecule, preferably an antibody, which specifically recognizes point mutations, since, as shown in the appended examples, a mutant of LiMIT (H135A) designed based on homology to a point mutation of the LiMIT homologue ASM that has been found in patients with Niemann-Pick disease (cf. Seto; Protein Sci; 13; 3172-86) showed reduced catalytic activity.

It is described herein and exemplified in the appended examples that LiMIT regulates cytokine levels and that these regulatory effects of LiMIT on cytokine levels are attributed directly to TLR induced signalling events in antigen presenting cells. Furthermore, the appended examples document that LiMIT is a specific, negative regulator of TLR dependent signalling in antigen presenting cells, such as macrophages.

Therefore, a further embodiment of the invention relates to an antigen presenting cell, which is inhibited in TLR signalling by the LiMIT polypeptide for use in inhibiting immune response or by a functional fragment thereof, and/or by the nucleic acid molecule, encoding either said LiMIT polypeptide or said functional fragment thereof. The invention further relates to said antigen presenting cell, which is inhibited in TLR signalling by an agonist of LiMIT or by a functional fragment thereof, and/or by a nucleic acid molecule, encoding either said agonist of LiMIT or said functional fragment thereof. One embodiment of the invention relates to the above-described antigen presenting cell which is inhibited in TLR signalling by LiMIT (or a functional fragment thereof), wherein said antigen presenting cell is a macrophage, a dendritic cell or a B cell. Macrophages or dendritic cells which are inhibited in TLR signalling and, therefore, would be inhibited in cytokine secretion would induce less inflammation and would less activate T cells. Accordingly, these cells would be useful in blood and blood products which could be used, e.g., for blood transfusion. In addition, LiMIT could be included as soluble factor in mother^'s milk or synthetic mother^'s milk products to inhibit pro-inflammatory signalling. B cells, such as plasma cells, which are inhibited in TLR signalling by LiMIT would be inhibited in antibody production. These cells may be useful for patients receiving dialyses or for autoimmune patients.

As mentioned above, in context of the present invention it is described that LiMIT is a specific, negative regulator of immune responses in vivo. As discussed above and exemplified by the appended examples by using, e.g., knockdown macrophages or knockdown mice, it was shown that low activity of LiMIT, such as low LiMIT expression level, leads to an increased immune reaction. Therefore, reducing the activity and/or expression level of LiMIT may be a useful tool to increase an immune reaction, increasing the immune reaction of a non-human animal may be advantageous, in particular for the production of antibodies.

As described herein, reducing the activity and/or expression of the LiMIT polypeptide for use in inhibiting immune response leads to a boosted (i.e. increased) immune response. Accordingly, one embodiment of the present invention relates to a non- human animal, wherein the activity and/or expression of the LiMIT polypeptide for use in inhibiting immune response is reduced, wherein the immune system of said non-human animal is boosted. In a further embodiment of the invention said expression is reduced by RNA interference (e.g. dsRNA, RNAi, siRNA, shRNA, miRNA, or stRNA), DNA anti-sense oligonucleotides or genetic modification. In a further embodiment of the invention said non-human animal is one non-human animal selected from the group consisting of mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, camel, and primate.

As mentioned above, increasing the immune reaction of a non-human animal may be advantageous for the production of antibodies. Thus it Is envisaged with respect to the present invention to use the above-described non-human animal wherein the activity and/or expression of LiMIT is reduced for producing antibodies. Accordingly, one embodiment of the invention relates to a method for producing antibodies against an antigen comprising the administration of said antigen to said non-human animal. Preferably, said antibodies are high affinity antibodies. In addition, a further embodiment of the invention relates to the above-described method for producing antibodies wherein said non-human animal is a knock out mouse for the gene encoding the LiMIT polypeptide for use in inhibiting immune response. Preferably, the antibodies produced by using the LiMIT knock out mouse are high affinity antibodies. The present invention further relates to a method for producing antibodies against an antigen comprising the administration of a specific antagonist or inhibitor which targets LiMIT and the administration of said antigen to a non-human animal. In addition, it is envisaged to administer an antagonist or inhibitor of LiMIT to a human in order to increase antibody production. Accordingly, a LiMIT antagonist or inhibitor may be used as an adjuvant or to increase an adjuvant effect in a patient. As described herein and exemplified by the appended examples, LiMIT deficient mice show higher inflammation upon TLR challenge and better T cell proliferation in ovalbumin immunizations. TLR activation works just like an adjuvans for vaccination/immunization. Hence it is expected that a higher LPS adjuvant effect mediated by LiMIT deficiency in an immunization model leads not only to increased T cell activation but, subsequently to enhanced affinity maturation, hypermutation and isotype class switch. Such a high affinity antibody producing mouse would be of high value to the pharmaceutical industry, especially to produce antibodies against highly conserved antigens/epitopes that would not allow efficient antibody production due to self tolerance.

After immunization of a LiMIT deficient mouse, the production of high affinity antibodies can be tested, for example, by a classical immunization model in wild-type and LiMIT^"'^" mice using adjuvant a) LPS or b) Freunds Adjuvant and antigen a) ovalbumin or b) Hen egg lysozyme. After 3 consecutive injections (1 initial injection with complete Freund adjuvant and antigen, 2 boosts with Freunds incomplete adjuvant) mouse sera will be harvested and analyzed for antigen affinity in a dilution series ELISA using antigen coated plates. Additionally, the amount of antigen specific isotypes will be determined by ELISA using antigen coated plates and secondary isotype specific anti-mouse antibodies. Therefore, it is envisaged in context of the present invention to use LiMIT knock out (LiMIT^"'^") mice as a tool to produce high affinity antibodies.

As used herein, the terms "nucleic acid sequence(s)", "nucleic acid molecule(s)" or "polynucleotide(s)" relate to the sequence of bases comprising purine- and pyrimidine bases which are comprised by nucleic acid molecules, whereby said bases represent the primary structure of a nucleic acid molecule. Nucleic acid sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and mixed polymers, both sense and antisense strands, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Exemplary nucleic acid molecule encoding LiMIT are known in the art and also disclosed herein.

As used herein, the term "polypeptide" relates to a peptide, a protein, or a polypeptide which encompasses amino acid chains of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such proteins/polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention as well as other than the 20 gene-encoded amino acids, such as selenocysteine. Peptides, oligopeptides and proteins may be termed polypeptides. The terms polypeptide and protein are often used interchangeably herein. The term polypeptide also refers to, and does not exclude, modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation and the like. Such modifications are well described in the art.

As mentioned herein, the term "LiMIT" relates to the acid sphingomyelinase-like phosphodiesterase 3b precursor (Smpdl3b) protein/gene, preferably of murine, and more preferably of human origin. In context of the present invention, the term "LiMIT" further relates to "the polypeptide to be used for inhibiting immune response", "the polypeptide for use in inhibiting immune response" and "the polypeptide LiMIT for use in inhibiting immune response". In addition "LiMIT" relates to the "polypeptide to be used in context of the (present) invention". In addition, "LiMIT" refers to both, the LiMIT polypeptide and the nucleic acid molecule encoding the LiMIT polypeptide. In human there are two transcript variants of LiMIT; transcript variant 2 is 246 nucleotides longer at the C-terminus than transcript variant 1 , otherwise the sequences are identical. The nucleic acid and amino acid sequences of human transcript variant 1 and murine LiMIT are given in the appended sequence listing (SEQ ID NOs: 1 to 4). These sequences correspond to the experiments documented in the appended Examples. Yet, in addition to human transcript variant 1 and murine LiMIT, it is also envisaged that the transcript variant 2 of human LiMIT or LiMIT of other organisms, e.g. of mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, primate or fruit fly, be assessed in context of this invention. The term "LiMIT" also relates to a functional fragment of the LiMIT polypeptide, e.g. a functional fragment of a polypeptide having the amino acid sequence given in SEQ ID NOs: 2 or 4. As described herein below, a functional fragment of the LiMIT polypeptide relates to a fragment of LiMIT having a sufficient length to have LiMIT activity, e.g. to have the ability to convert sphingomyelin into phosphorylcholine. As described herein above and below, the ability to convert sphingomyelin into phosphorylcholine can be assayed, e.g., with the Amplex Red Sphingomyelinase kit (Invitrogen, Cat. No: A12220).

It is of note that the nucleic acid and amino acid sequences of LiMIT given herein are not limiting. Accordingly, the term "LiMIT" also encompasses LiMIT polypeptides/nucleic acid molecules having amino acid or nucleic acid sequences being homologous to or a functional fragment of the amino acid or nucleotide sequences shown herein, e.g. those of human LiMIT transcript variant 1. Definitions of the terms "homologous", "functional" and "functional fragment" as used herein are given herein below. The term "LiMIT" also relates to specific agonists of LiMIT. The term "agonist", as used herein, is meant to refer to an agent that mimics or up- regulates (e.g., potentiates or supplements) the activity/functionality of a protein of interest (such as LiMIT). In addition, the term "agonist" may also relate to an agent that facilitates or promotes (e.g., potentiates or supplements) an interaction between a polypeptide (e.g. LiMIT) and another polypeptide, such as Toll-like receptors, or between LiMIT and a non-proteinous molecule (e.g., a sphingolipid, ceramide, cytokine, nucleic acids, small molecules, ions etc). Double positive ions are required to activate LiMIT. Zn^÷+ ions, for example, seem to be promising LiMIT activating ions. An agonist can be a wild-type protein or derivative thereof having at least one function of the wild-type protein. An agonist of LiMIT can also be a small molecule that up-regulates the expression of the gene of LiMIT or which increases at least one activity of LiMIT. An agonist of LiMIT can also be a protein or small molecule which increases the interaction of LiMIT with a target molecule.

LiMIT may be obtained by purifying it from cells expressing/comprising the LiMIT polypeptide or a functional fragment thereof. These cells may overexpress LiMIT or a functional fragment thereof or may contain LiMIT or a functional fragment thereof endogenously. These cells may be prokaryotic cells such as E. coli or eukaryotic cells like insect cells. The production of LiMIT may be carried out following the instructions of the Qiaexpressionist

(http://www.qiagen.com/literature/render.aspx?id=128, Qiagen) for bacterial protein purification or by using BD Bioscience Baculo Gold instruction (Cat.No. 560138) for insect cell protein production and purification. As exemplified in the appended examples, LiMIT may be produced by purifying recombinant LiMIT from BL21 bacteria via HIS-tag purification following the instructions of the Qiaexpressionist. LiMIT or a functional fragment thereof may also be synthetically or chemically produced. Therefore solid phase peptide synthesis (SPPS) may be applied.

The terms "activity" or "functionality" are often used interchangeably herein and define whether a polypeptide is functional. A polypeptide is "functional" means, in context of the invention, that the polypeptide has the ability to carry out a specific "function". Accordingly, the terms "activity" or "functionality" relate to the ability of a specific protein to carry out a specific function. For instance, in context of the present invention, a function of LiMIT comprises the ability to convert sphingomyelin into phosphorylcholine. A possible method to assay for the functionality of LiMIT is to measure the ability to convert sphingomyelin into phosphorylcholine. One specific test to assay for the ability of LiMIT to convert sphingomyelin into phosphorylcholine is, for example, provided by Invitrogen with the Amplex Red Sphingomyelinase kit (Cat. No: A12220).

In this assay the sphingomyelinase activity can be measured in vitro by using a fluorescence microplate reader. In this assay, the purified protein of interest, e.g. LiMIT, (or a lysate wherein the protein of interest, e.g. LiMIT, is overexpressed) hydrolyses sphingomyelin into ceramide and phosphorylcholine. Exogenous addition of alkaline phosphatase mediates the conversion of phosphorylcholine to choline, which is further processed by exogenously added choline oxidase into Betaine and H2O2. Subsequently, H2O2 reacts in the presence of horseradish peroxidase with the Amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine) in a 1 :1 stochiometric reaction to produce the fluorescent product Resorufin which can be measured at an fluorescence emission at 571-585nm.

The functionality/activity of the polypeptide of interest that was measured by the above-described method may be compared to a standard or reference value of LiMIT activity. The reference value may be a positive control which may be detected by using a purified catalytic domain of LiMIT (e.g., consisting of amino acids 21 to 281 of SEQ ID NO: 4) or a purified full length protein of LiMIT (e.g., consisting of SEQ ID NO: 4) or a commercially available recombinant sphingomyelinase (E.g. from Bacillus cereus). Another reference value may be a negative control which may be detected by performing the above-described assay without adding said polypeptide of interest or by using a catalytically inactive variant of LiMIT.

As used herein, a "functional fragment" of a protein which displays a specific biological activity relates to fragments of said protein having a sufficient length to display said activity. Accordingly, a functional fragment of a protein showing e.g. a specific (enzymatic) activity may relate to a polypeptide which corresponds to a fragment of said protein which is still capable of showing said (enzymatic) activity. For example, a functional fragment of LiMIT in the context of the enzymatic activity of LiMIT may correspond to the catalytic domain of LiMIT which is about 50% of the human or mouse full length sequences or LiMIT (SEQ ID NOs: 2 and 4). Methods for determining whether a certain fragment of a protein is a functional fragment are known in the art. For example, a test for determining whether a fragment of LiMIT is functional, i.e., is still capable of converting sphingomyelin into phosphorylcholine is, for example, provided by Invitrogen with the Amplex Red Sphingomyelinase kit (Cat. No: A12220), as described herein above. Preferably, a functional fragment of LiMIT has substantially the same biological activity as LiMIT itself. Furthermore, a person skilled in the art will be aware that the (biological) activity/functionality as described herein often correlates with the expression level, preferably the protein or mRNA level. The term "expression" as used herein refers to the expression of a nucleic acid molecule encoding a polypeptide, whereas "activity" refers to the functionality/activity of said polypeptide, which can be determined as outlined herein. The explanations given herein in respect of the activity/functionality of "LiMIT" also apply, mutatis mutandis, to "(a) functional fragment(s)" of the full length LiMIT. In other words, a "functional fragment of LiMIT" has also the activity/functionality of the full length LiMIT as defined herein. Accordingly, also inhibitors/antagonists of functional fragments of LiMIT are described and provided herein. As mentioned, methods/assays for determining the activity/functionality of LiMIT or a "functional fragment of LiMIT" are well known in the art and also described herein. Preferably, the functional fragment is at least 40% more preferably at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of the amino acid sequence of the human or mouse full length sequences of LiMIT. In context of the present invention, "homologous" or "percent homology" means that amino acid or nucleotide sequences have identities of at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% to the sequences shown herein, e.g. those of human LiMIT transcript variant 1 , wherein the higher identity values are preferred upon the lower ones.

In accordance with the present invention, the term "identity/identities" or "percent identity/identities" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% or 70% identity, preferably, 70-95% identity, more preferably at least 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequences of, e.g., SEQ ID NOs: 1 or 3, or with the amino acid sequences of, e.g., SEQ ID NOs: 2 or 4, and being functional, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection.

Preferably the described identity exists over a region that is at least about 100 to 200 amino acids or nucleotides in length. It is more preferred that the described identity exists over a region that is about 200 to 400 amino acids or nucleotides in length. In case of nucleotide sequences, the described identity most preferably exists over a region that is at least 500, at least 600 or at least 700 nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson; 1994; Nucl Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; Comp App Biosci; 6; 237-245), as known in the art.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e., gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul; 1997; Nucl Acids Res 25; 3389-3402, Altschul; 1993; J Mol Evol; 36; 290-300, Altschul; 1990; J Mol Biol 215; 403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 1 1 , an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, and an expectation (E) of 10. The BLOSUM62 scoring matrix (Henikoff; 1989; PNAS; 89; 10915) uses alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

In addition, the present invention relates to a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule being degenerate as a result of the genetic code. When used in accordance with the present invention, the term "being degenerate as a result of the genetic code" means that due to the redundancy of the genetic code different nucleotide sequences code for the same amino acid.

In order to determine whether an amino acid residue or nucleotide residue in a amino acid or nucleic acid sequence corresponds to a certain position in the amino acid sequence of, e.g., SEQ iD NO: 2 or nucleotide sequence of e.g. SEQ ID NOs: 1 , the skilled person can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs.

For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool

Ri AST ίΆίτςΓΓίϋί ' I Qoy- |_nr rif A!terhu!' 1 ο,ο ,- ^[ _O t ^■ A Ste^ ni- I QQA- in - nit \ rap be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul; 1997; loc. cit., Altschul; 1993; loc. cit, Altschul; 1990; loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

% sequence identity x % maximum BLAST score

100 and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may Identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson; 1994; NucI Acids Res; 2; 4673-4680) or FASTDB (Brutlag; 1990; Comp App Biosci 6; 237-245), as known in the art.

The terms "hybridization", "hybridizes" or "hybridizing" as used herein relate to complementary (antisense) molecules, which specifically interact with/hybridizes to one or more nucleic acid molecules encoding LiMIT. Highly mutated LiMIT complementary constructs, which are not capable of hybridizing to LiMIT encoding nucleic acid molecules are not to be employed in the context of the present invention. The person skilled in the art can easily deduce whether a complementary construct specifically hybridizes to LiMIT encoding sequences. These tests comprise, but are not limited to hybridization assays, RNAse protection assays, Northern Blots, Northwestern blots, nuclear magnetic resonance and fluorescence binding assays, dot blots, micro- and macroarrays and quantitative PGR. In addition, such a screening may not be restricted to LiMIT mRNA molecules, but may also include LiMIT mRNA/protein (RNP) complexes (Hermann; 2000; Angew Chem Int Ed Engl; 39; 1890-1904, DeJong; 2002; Curr Trop Med Chem; 2; 289-302). Furthermore, functional tests including Western blots, immunohistochemistry, immunoprecipitation assays, and bioassays based on LiMIT-responsive promoters are envisaged for testing whether a particular complementary construct is capable of specifically interacting with/hybridizing to the LiMIT encoding nucleic acid molecules.

In addition, the terms "hybridization", "hybridizes" or "hybridizing" as used herein further relate to hybridizations under stringent or non-stringent conditions. Said hybridization conditions may be established according to conventional protocols, described, e.g., in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001 ), Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford, Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usuaiiy require stringent hybridization and washing conditions such as 0.1 x SSC, 0.1 % SDS at 65°C. Non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6 x SSC, 1 % SDS at 65°C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions. Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may comprise complementary nucleic acid sequences of the nucleic acid molecules which code for LiMIT or functional fragments of LiMIT. These hybridizing nucleic acid molecule fragments may have a length of between 20 and 200 nucleotides Furthermore, also the complementary nucleic acid molecule, the complementary fragments and (allelic) variants of the nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules are included in the present invention. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an anti-parallel configuration. A hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed). In context of the present invention, the terms "complementary" or "complementarity" refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. A complementary polynucleotide of a specified nucleotide sequence is an antisense polynucleotide of said specified nucleotide sequence. For example, the sequence "A- G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single- stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.

The term "hybridizing (sequences)" preferably refers to the sequences which display a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, particularly preferred at least 95%, more particularly preferred at least 96%, even more particularly preferred at least 97% and most preferably at least 98% or up to 100% identity with a complementary nucleic acid sequence of the nucleic acid sequence encoding LiMIT or a functional fragment thereof and being a functional polypeptide, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine as described herein and tests are described for assaying said function (like, e.g., measuring the sphingomyelinase activity by using the Invitrogen Amplex Red Sphingomyelinase kit; Cat. No: A12220 as described herein; see also Example 7). Moreover, the term "hybridizing (sequences)" preferably refers to complementary nucleic acid sequences of the nucleic acid sequences encoding amino acid molecules having a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, particularly preferred at least 95%, more particularly preferred at least 96%, even more particularly preferred at least 97% and most preferably at least 98% or up to 100% identity with an amino acid sequence of LiMIT (e.g. SEQ ID NOs: 2 or 4) or a functional fragment thereof and being a functional polypeptide, wherein the function comprises the ability to convert sphingomyelin into phosphorylcholine as described herein above.

The terms "antagonist", "inhibitor" or "inhibitor/antagonist" are often used interchangeably herein. These terms are known in the art and relate to a compound/substance capable of fully or partially preventing or reducing the physiologic function or activity of (a) specific protein(s). Therefore, in the context of the present invention, said inhibitor/antagonist may prevent or reduce or inhibit or inactivate the physiological activity of LiMIT, e.g., upon binding of said compound/substance to LiMIT. Binding of an inhibitor/antagonist to a given protein, e.g. LiMIT, may prevent the binding of an endogenous activating molecule binding to said protein. The term "inhibitor/antagonist" also encompasses competitive antagonists, (reversible) non-competitive antagonists or irreversible antagonist, as described, inter alia, in Mutschler, "Arzneimittelwirkungen" (1986), Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, Germany. As described herein, a preferred inhibitor of LiMIT is an antibody which specifically binds to LiMIT, preferably an antagonistic antibody. As used herein, the term "antagonistic antibody" describes an antibody that is capable of inhibiting and/or neutralising the (biological) activity of a specific target protein, such as LiMIT. For instance, an antagonistic antibody may block the binding or substantially reduce the binding of LiMIT to other molecules, such as to sphingomyelin or ceramide and thereby inhibiting the function of LiMIT i.e., the ability to convert sphingomyelin into phosphorylcholine. This decreased functionality can be measured, for example, by the herein described method to assay the function of LiMIT such as by the Amplex Red Sphingomyelinase kit (Invitrogen, Cat. No: A12220). In addition, binding of an antagonistic antibody to the catalytic domain of LiMIT may lead to an inhibition of LiMIT (enzymatic) function. Methods for the use and production of antagonistic antibodies are well known in the art and are described, e.g., in Tartaglia; 1992; J Biol Chem; 267; 4304-4307, Tartaglia; 1993; Cell; 73; 213-216 and WO 94/09137. It is envisaged that the antagonistic antibody preferably and specifically binds LiMIT or fragments thereof. Antagonistic antibodies that target LiMIT may be produced, for example, by performing screening techniques using a human Fab phagemid library (see, inter alia, Huag; 2006; J Leuko Biol; 80; 905-914 and Hoet; 2005; 23; 344-348). In addition, the production of antagonistic antibodies by phage display is described in Molek; 2011 ; Molecules; 16; 857-887. The functionality of an antagonistic antibody, i.e. the ability of an antibody to act antagonistic, can be assayed as described in the art. Possible methods to assay the functionality of an antagonistic antibody comprise, for example, phage ELISA, cell binding assays and blocking assays as described in, for example, Huag; 2006; J Leuko Biol; 80; 905-914, Hoet; 2005; 23; 344-348, Jostock; 2004; 289; 65-80, and Baumann; 2010; J Exp Med; 207; 2689-701. In addition, biomarker studies can be performed to test whether LiMIT inhibition causes modulation/increase of immune readouts such as IL6 and TNF (Guadagnoli; 201 1 ; Blood; 117; 6856-65). As mentioned above, a pharmaceutical composition comprising an antagonistic antibody would be very useful for targeting LiMIT since, as shown in the appended examples, LiMIT is a secreted protein and therefore, can be found extracellulaly and is, thus, readily available for an antagonistic antibody. In addition thereto, however, in the context of the present invention, an "antagonist" or "inhibitor" of LiMIT may also be capable of preventing the function of a given protein, such as LiMIT, by preventing/reducing the expression of the nucleic acid molecule encoding for said protein. Thus, an inhibitor/antagonist of LiMIT may lead to a decreased expression level of LiMIT (e.g. decreased level of LiMIT mRNA or LiMIT protein) which is reflected in a decreased functionality of LiMIT, wherein the function of LiMIT comprises the ability to convert sphingomyelin into phosphorylcholine. This decreased functionality can be measured, for example, by the herein described method to assay the function of LiMIT such as by the Amplex Red Sphingomyelinase kit (Invitrogen, Cat. No: A12220). An inhibitor of LiMIT, in the context of the present invention, accordingly, may also encompass transcriptional repressors of LiMIT expression that are capable of reducing the level of LiMIT. As described herein in detail, the decreased expression and/or activity of LiMIT by an inhibitor/antagonist of LiMIT results in an increased immune reaction. As used herein, a specific antagonist or inhibitor of LiMIT targets the LiMIT polypeptide or a functional fragment thereof and/or the nucleic acid molecule encoding LiMIT or a functional fragment thereof. It is envisaged that antisense molecules such as interfering RNA and DNA antisense oligonucleotides inhibit the expression or function of LiMIT, in particular of mammalian LiMIT and interact with LiMIT as expressed by the coding regions (e.g. SEQ ID NOs: 1 and 3) as well as with LiMIT as expressed by isoforms and variants of said LiMIT. Said isoforms or variants may, inter alia, comprise transcription variants, allelic variants or splice variants. The term "variant" means in this context that the LiMIT nucleotide sequence and the encoded LiMIT amino acid sequence, respectively, differs from the distinct sequences shown in the appended SEQ ID NOs 1 to 4, by mutations, e.g. deletion, additions, substitutions, inversions etc. Furthermore, it is also envisaged that the antisense molecules to be used in accordance with the present invention against LiMIT expression or function interfere specifically with regulatory sequences of LiMIT. Particular antisense molecules which can be applied in context of the present invention are interfering RNAs. The term "RNA interference" or "inhibiting RNA" (RNAi/iRNA) describes the use of double-stranded RNA to target specific RNAs for degradation, thereby silencing their expression. Preferred inhibiting RNA molecules may be selected from the group consisting of double-stranded RNA (dsRNA), RNAi, siRNA, shRNA and stRNA. dsRNA matching a gene sequence is synthesized in vitro and introduced into a cell. The dsRNA may also be introduced into a cell in form of a vector expressing a target gene sequence in sense and antisense orientation, for example in form of a hairpin mRNA. The sense and antisense sequences may also be expressed from separate vectors, whereby the individual antisense and sense molecules form double-stranded RNA upon their expression. It is known in the art that in some occasions the expression of a sequence in sense orientation or even of a promoter sequence suffices to give rise to dsRNA and subsequently to siRNA due to internal amplification mechanisms in a cell. Accordingly, all means and methods which result in a decrease in activity (which may be reflected in a lower expression of LiMIT), in particular by taking advantage of LiMIT-specific siRNAs (i.e. siRNAs that target specifically LiMIT mRNA or a functional fragment thereof) are to be used in accordance with the present invention. For example sense constructs, antisense constructs, hairpin constructs, sense and antisense molecules and combinations thereof can be used to generate/introduce these siRNAs. The dsRNA feeds into a natural, but only partially understood process including the highly conserved nuclease dicer which cleaves dsRNA precursor molecules into siRNAs. The generation and preparation of siRNA(s) as well as the method for inhibiting the expression of a target gene is, inter alia, described in WO 02/055693, Wei; 2000; Dev Biol; 15; 239-255, La Count; 2000; Biochem Paras; 111 ; 67-76, Baker; 2000; Curr Biol; 10; 1071-1074, Svoboda; 2000; Development; 127; 4147-4156 or Marie; 2000; Curr Biol; 10; 289-292. These siRNAs built then the sequence specific part of an RNA-induced silencing complex (RISC), a multicomplex nuclease that destroys messenger RNAs homologous to the silencing trigger. Elbashir; 2001 ; EMBO J; 20; 6877-6888 showed that duplexes of 21 nucleotide RNAs may be used in cell culture to interfere with gene expression in mammalian cells. It is already known that RNAi is mediated very efficiently by siRNA in mammalian ceils but the generation of stable cell lines or non-human transgenic animals was limited. However, new generations of vectors may be employed in order to stably express, e.g. short hairpin RNAs (shRNAs). Stable expression of siRNAs in mammalian cells is inter alia shown in Brummelkamp; 2002; Science; 296; 550-553. Also Paul; 2002; Nat Biotechnol; 20; 505-508 documented the effective expression of small interfering RNA in human cells. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells was also shown by Yu; 2002; PNAS; 99; 6047-6052. The shRNA approach for gene silencing is well known in the art and may comprise the use of st (small temporal) RNAs; see, inter alia, Paddison; 2002; Genes Dev; 16; 948-958. These approaches may be vector-based, e.g. the pSUPER vector, or RNA poll E I vectors may be employed as illustrated, inter alia, in Yu; 2002; loc. cit. , Miyagishi; 2002; loc. cit. or Brummelkamp; 2002; loc. cit.

A microRNA (miRNA) is a short RNA molecule found in eukaryotic cells. A microRNA molecule has very few nucleotides (an average of 22) compared with other RNAs. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts, usually resulting in translational repression or target degradation and gene silencing (Bartel; 2009; Cell; 136; 215-233, Bartel; 2004; Cell; 116; 281-97. The human genome may encode over 1000 miRNAs (Homo sapiens miRNAs in the miRBase at Manchester University, Bentwich; 2005; Nat Genet; 37; 766-70), which may target about 60% of mammalian genes (Lewis; 2005; Cell; 120; 15-20, Friedman; 2009; Genome Res; 19; 92-105), and are abundant in many human cell types (Lim; 2003; Genes Dev; 17; 991-1008). In context of the present invention, LiMIT targeting miRNAs may be identified, purified or synthesized and applied to inhibit the expression of LiMIT.

Methods to deduce and construct siRNAs are known in the art and are described in Elbashir; 2002; Methods; 26; 199-213, at the internet web sites of commercial vendors of siRNA, e.g. Qiagen GmbH

(https://www1.qiagen.com/GeneGlobe/Default.aspx); Dharmacon (www.dharmacon.com); Xeragon Inc. (http://www.dharmacon.com/Default.aspx), and Ambion (www.ambion.com), or at the web site of the research group of Tom TuschI (http://www.rockefeller.edu/labheads/tuschl/sima. html). In addition, programs are available online to deduce siRNAs from a given mRNA sequence (e.g. http:/7www.ambion.com/techiib/misc/siRNA_finder.htmi or http://katahdin.cshl.org:9331/RNAi/html/rnai.html). Uridine residues in the 2-nt 3' overhang can be replaced by 2'deoxythymidine without loss of activity, which significantly reduces costs of RNA synthesis and may also enhance resistance of siRNA duplexes when applied to mammalian cells (Elbashir; 2001 ; loc. cit). The siRNAs may also be sythesized enzymatically using T7 or other RNA polymerases (Donze; 2002; Nucleic Acids Res; 30; e46). Short RNA duplexes that mediate effective RNA interference (esiRNA) may also be produced by hydrolysis with Escherichia coli RNase III (Yang; 2002; PNAS; 99; 9942-9947). Furthermore, expression vectors have been developed to express double stranded siRNAs connected by small hairpin RNA loops in eukaryotic cells (e.g. Brummelkamp; 2002; Science; 296; 550-553). All of these constructs may be developed with the help of the programs named above. In addition, commercially available sequence prediction tools incorporated in sequence analysis programs or sold separately, e.g. the siRNA Design Tool offered by www.oligoEngine.com (Seattle, WA) may be used for siRNA sequence prediction.

Based on the teaching provided herein, a skilled person in the art is easily in the position not only to prepare such interfering RNAs but also to assess whether an interfering RNA is capable of antagonizing/inhibiting LiMIT. It is envisaged that the herein described interfering RNAs lead to a degradation of LiMIT mRNA and thus to a decreased protein level of LiMIT. Accordingly, specific interfering RNAs can be used in accordance with the present invention as inhibitors of LiMIT expression (and function). siRNAs and shRNAs which may be applied in context of the invention are shown in SEQ ID NOs: 5 to 8 and SEQ ID NOs: 9 and 10, respectively, and their use is described in the appended Examples. In particular, siRNAs and shRNAs which may, for example, be applied in context of the present invention are: siRNAs:

Thermo scientific, siGENOME SMARTpool M-040463-01-0005, Mouse SMPDL3B,

NM_133888, 5 nmol;

Target sequences in the smart pool:

D-040463-Ο : CAAAAGAGGUGCCUUCUAU (SEQ ID NO: 5)

D-040463-02: CGAGAGAGCUUCAAUGAGG (SEQ ID NO: 6)

D-040463-03: AAUCAUGACUUCCACCCUA (SEQ ID NO: 7)

D-040463-04: GCACGUACUGGAGGUGUUA (SEQ ID NO: 8) shRNAs (stable, lentiviral integration):

1. TRCN0000099681 ; NM_133888, shRNA Mm; Lentiviral; pLKO.1 ; RMM3981- 98062179; Hairpin sequence for TRCN0000099681 :

CCGGGCAACGTTATTACGTCTATAACTCGAGTTATAGACGTAATAACGTTGCTTT TTG

Mature Sense for TRCN0000099681 : GCAACGTTATTACGTCTATAA

Mature Antisense for TRCN0000099681 : TTATAGACGTAATAACGTTGC

(SEQ ID NO: 9)

2. TRCN0000099683; NM_133888; shRNA Mm; Lentiviral; pLKO.1 RMM3981 - 98062195 Hairpin sequence for TRCN0000099683:

CCGGCCCAACTACACCGTATCCAAACTCGAGTTTGGATACGGTGTAGTTGGGTT TTTG

Mature Sense for TRCN0000099683: CCCAACTACACCGTATCCAAA

Mature Antisense for TRCN0000099683: TTTGGATACGGTGTAGTTGGG

(SEQ ID NO: 10) It is also envisaged that expression or function of LiMIT is inhibited by genetic modification such as the knockout of a gene, in particular a gene encoding LiMIT. In context of the present invention, genetic modification also relates to (targeted) mutagenesis resulting in, e.g., loss-of function mutants.

The inhibitor/antagonist of LiMIT expression or function may also comprise an aptamer. In the context of the present invention, the term "aptamer" comprises nucleic acids such as RNA, ssDNA (ss = single stranded), modified RNA, modified ssDNA or PNAs which bind a plurality of target sequences having a high specificity and affinity. Aptamers are well known in the art and, inter alia, described in Famulok; 1998; Curr Op Chem Biol; 2; 320-327. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (Gold; 1995; Ann Rev Biochem; 64; 763-797).

LiMIT is a secreted protein and therefore can be found extracellularly. However, both the extracellular and the (endosomal) intracellular LiMIT protein may exhibit immune inhibitory function. Accordingly, the antagonist/inhibitor of LiMIT expression or function may also comprise extracellular and/or intracellular binding partners of LiMIT. As used herein, the term "extracellular binding partner" relates to extracellular molecules capable of preventing or reducing LiMIT activity. In line with this, the term "intracellular binding partner" relates to intracellular molecules capable of preventing or reducing LiMIT activity. Such intracellular binding partners of LiMIT, inter alia, may relate to endogenous inhibitor/repressor proteins of LiMIT. In one aspect of the invention the intracellular binding partner is an intracellular antibody. Intracellular antibodies are known in the art and can be used to modulate or inhibit the functional activity of the target molecule. This therapeutic approach is based on intracellular expression of recombinant antibody fragments, either Fab or single chain Fv, targeted to the desired cell compartment using appropriate targeting sequences (Teillaud; 1999; Pathol Biol; 47; 771-775).

Antagonists/inhibitors of LiMIT function may be deduced by methods in the art. Such methods may comprise, but are not limited to methods where a collection of substances is tested for interaction with LiMIT or with (a) fragment(s) thereof and where substances which test positive for interaction in a corresponding readout system are further tested in vivo, in vitro or in silico for their inhibiting effects on LiMIT expression or function.

Said "test for LiMIT interaction" of the above described method may be carried out by specific immunological, molecular biological and/or biochemical assays which are well known in the art and which comprise, e.g., homogenous and heterogenous assays. The natural endogenous ligand(s) of LiMIT remain(s) to be identified. Yet, LiMIT ligands capable of inhibiting LiMIT function may be identified by screening large compound libraries based on their capacity to interact with the LiMIT protein. In a preferred embodiment, such antagonists or inhibitors of LiMIT function are capable of binding the catalytic domain of LiMIT. Other specific inhibitors may be lipid mimics. One example for a lipid mimic is FTY-720, which is a sphingolipid mimetic, and a specific interactor of S1 P1 receptors, see, e.g., Baumruker, 2007; Etal, txp. piriioti mvesug. urugs, ι ο, o -a. i iuu iei s cuuio inhibitor of LiMI I may be a chemical compound that was described to inhibit acidic Sphingomyelinases, e.g. antidepressants like Chloropromazine. It is envisaged, that said inhibitors/antagonists of LiMIT are used for boosting the immune system in a mammal, preferably, in a human.

The term "antibody" includes a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson; 1994; J Immunol Methods 175; 267-273; Yarmush; 1992; J Biochem Biophys Methods 25; 85-97. The term "antibody" includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind an antigen (such as LiMIT or a functional fragment thereof), comprising or alternatively consisting of, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward; 1989; Nature 341 ; 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Antibody fragments or derivatives further comprise F(ab')2, Fv or scFv fragments. Various procedures are known in the art and may be used for the production of such antibodies and/or fragments (see, for example, Harlow (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York).

Although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); See, e.g., Bird; 1988; Science 242; 423-426; and Huston; 1988; Proc Natl Acad Sci USA 85; 5879- 5883). Such single chain antibodies are included by reference to the term "antibody". Fragments can be prepared, for example, by recombinant techniques or enzymatic or chemical cleavage of intact antibodies. Accordingly, the term "antibody" also comprises fragments thereof which still specifically bindsbind to LiMIT or a fragment thereof. Techniques described for the production of single chain antibodies are described, e.g., in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994) and US

A Q-lfi 77fi Further terhnini IPQ fnr †hp> nrnHi ir.tinn nf sinnlp r.hain antibodies can be adapted to produce single chain antibodies to LiMIT. Accordingly, single chain antibodies are also included by the term "antibody."

Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those skilled in the art and described in the scientific and patent literature, (see, e.g., Coligan, Current Protocols in Immunology; Wiley/Greene, N.Y. (1991 ); Stites (eds.) Basic and Clinical Immunology (7th ed.) Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, Monoclonal Antigodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York). Antibodies can also be generated in vitro, e.g., by using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals (see, e.g., Hoogenboom; 1997; Trends Biotechnol 15; 62-70; Katz; 1997; Annu Rev Biophys Biomol Struct; 26; 27-45. ).

The ability of an antibody to bind to a protein (such as LiMIT) may be determined by using any of a variety of procedures familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the protein may be detected by using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays and Western Blots.

The term "antibody" also comprises variants thereof which still specifically bind to a target protein (such as LiMIT). The term "antibody variant," refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such variant necessarily has less than 100% sequence identity or similarity with the amino acid sequence, has at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. The antibody variants can be produced, e.g., by peptidomimetics.

The antibody useful in context of the present invention can be, for example, polyclonal or monoclonal. The term "monoclonal antibody" as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For preparation of monoclonal antibodies, several techniques which provide antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler; 1975; Nature; 256; 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor; 1983; Immunology Today; 4; 72) and the EBV- hybridoma technique (Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein; 1975; Nature 256; 495-497, or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson; 1991 ; Nature; 352; 624-628, as well as in Marks; 1991 ; J Mol Biol; 222; 581-597.

The term "polyclonal antibody" as used herein, refers to an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-iymphocyte in the presence of several other B-lymphocytes which produced non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.

The term "antibody" further comprises diclonal and oligoclonal antibodies. The term "diclonal antibody" refers to a preparation of at least two antibodies to a target protein (such as LiMIT). Typically, the different antibodies bind different epitopes. The term "oligoclonal antibody" refers to a preparation of 3 to 100 different antibodies to a target protein (such as LiMIT). Typically, the antibodies in such a preparation bind to a range of different epitopes.

The term "antibody" also relates to bifunctional antibodies. The term "bifunctional antibody" or "bispecific antibody" as used herein refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments (see, e.g., Songsivilai; 1990; Clin Exp Immunol; 79; 315- 321 , Kostelny; 1992; J Immunol; 148; 1547-1553). In addition, bispecific antibodies may be formed as "diabodies" (Holliger; 1993; Proc Nat Acad Sci USA; 90; 6444- 6448) or as "Janusins" (Traunecker; 1991 ; EMBO J; 10; 3655-3659 and Traunecker; 1992; !nt J Cancer Supp!; 7; 51-52). The term "antibody" also relates to a "trifunctional antibody". The term "antibody" further comprises fully-human antibodies. The term "fully-human antibody" as used herein refers to an antibody which comprises human immunogiobulin protein sequences only. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Similarly, "(fully-)mouse antibody" or "(fully-)murine antibody" refers to an antibody which comprises mouse (murine) immunoglobulin protein sequences only. Alternatively, a "fu!ly-human antibody" may contain rat carbohydrate chains if produced in a rat, in a rat cell, in a hybridoma derived from a rat cell. The term "(fully-) rat antibody" refers to an antibody that comprises rat immunoglobulin sequences only. Fully-human antibodies may be produced, for example, by phage display which is a widely used screening technology which enables production and screening of fully human antibodies. Accordingly, also phage antibodies can be used in context of this invention. Phage display methods are described, for example, in US 5,403,484, US 5,969,108 and US 5,885,793. Another technology which enables development of fully-human antibodies involves a modification of mouse hybridoma technology. Mice are made transgenic to contain the human immunoglobulin locus in exchange for their own mouse genes (see, for example, US 5,877,397). Also (fully-)mouse or (fully-) rat antibodies may be used in context of the present invention.

The term "antibody" as used herein also comprises chimeric antibodies. The term "chimeric antibody" refers to an antibody which comprises a variable region of a human or non-human species fused or chimerized with an antibody region (e.g., constant region) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

The term "antibody" also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. The term "recombinant human antibody" includes all human sequence antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal. The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies.

The term "antibody" also relates to humanized antibodies. "Humanized" forms of non- human (e.g. murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may comprise residues, which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see: Jones; 1986; Nature; 321 ; 522-525, Reichmann; 1988; Nature; 332; 323-327 and Presta; 1992; Curr Op Struct Biol; 2; 593-596. Also, transgenic animals may be used to express humanized antibodies to LiMIT. A popular method for humanization of antibodies involves CDR grafting, where a functional antigen-binding site from a non-human 'donor' antibody is grafted onto a human 'acceptor' antibody. CDR grafting methods are known in the art and described, for example, in US 5,225,539, US 5,693,761 and US 6,407,213. Another related method is the production of humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci which are capable of undergoing gene rearrangement and gene conversion (see, for example, US 7, 129,084).

Accordingly, in context of the present invention, the term "antibody" relates to full immunoglobulin molecules as well as to parts of such immunoglobulin molecules. Furthermore, the term relates, as discussed above, to modified and/or altered antibody molecules. The term also relates to recombinantly or synthetically generated/synthesized antibodies. The term also relates to intact antibodies as well as to antibody fragments thereof, like, separated light and heavy chains, Fab, Fab/c, Fv, scFv, di-scFv, sdAb, Fab', F(ab')2. The term "antibody" also comprises bifunctional antibodies, trifunctional antibodies, fully-human antibodies, chimeric antibodies, humanized antibodies and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.

Accordingly, the terms "antibody" or "antibodies" as employed herein also comprise antibody variants, antibody fragments and the like. As mentioned, techniques for the production of antibodies are well known in the art and summarized, e.g., in Petering; New Biotech; 201 1 ; 28; 588-544. In addition, several techniques for the production of antibodies are described, e.g. in Harlow "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

With respect to the herein described pharmaceutical composition comprising an inhibitor/antagonist of LiMIT, it is envisaged to use an antagonistic antibody to target LiMIT. Accordingly, the term "antibody" as used herein also relates to an antagonistic antibody. A more detailed description of antagonistic antibodies is provided in the context of inhibitors/antagonists of LiMIT, above. However, all definitions and examples discussed herein also apply for antagonistic antibodies mutatis mutandis. With respect to the herein described diagnostic composition comprising a specific binding molecule which specifically binds to LiMIT, it is preferred that said binding molecule is an antibody. Antibodies can be used for the monitoring of the presence, absence, amount, and identity of LiMIT, in particular in diagnosis. It is also envisaged in context of the invention to use antibodies that specifically recognize post- translational modifications, such as phosphorylation or glycosylation of LiMIT, or specific point-mutations within LiMIT. In addition, it is described in context of the present invention to use a non-human animal wherein the activity and/or expression of LiMIT is reduced for use in producing antibodies, preferably, high affinity antibodies. The term "high affinity" for an antibody refers to an equilibrium association constant (K_a) of at least about 10⁷ M^"1 , at least about 10⁸ M^"1, at least about 10⁹ M^"1, at least about 10¹⁰ M^"1 , at least about 10^{1 1} M^"1, or at least about 10¹² M^"1 or greater, e.g., up to 10¹³ M^"1 or 10¹⁴ M^"1 or greater. However, "high affinity" binding can vary among antibody isotypes. The term "K_a", as used herein, is intended to refer to the equilibrium association constant of a particular antibody-antigen interaction. This constant has units of 1/M. "High affinity antibody" describes antibodies that have undergone extensive hypermutation, affinity maturation and proper isotype switching to applicable isotypes such as preferably IgG. A LiMIT knock out mouse would produce a much higher pool of antibodies specific for LiMIT that have these features. In the case of a "normal" immunogenic antigen, it is an advantage to be able to select the best antibody. However, in the case of a not very immunogenic antigen, reduced self tolerance might lead to the production of antibodies that would not be made in conventional animals / mice. The phrase "specifically bind(s)" or "bind(s) specifically" when referring to a binding molecule refers to a binding molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule, such as LiMIT. The phrases "specifically binds to" refers to a binding reaction which is determinative of the presence of a target protein (such as LiMIT) in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding molecules bind preferentially to a particular target protein (e.g. LiMIT) and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions may require a binding molecule that is selected for its specificity for a particular target protein. A variety of assay formats may be used to select binding molecules that are specifically reactive with a particular target protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore and Western blot may be used to identify binding molecules that specifically react with LiMIT. Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background. Given that the binding molecule is an antibody, the

proteins and other biologies. Typically, the antibody binds with an association constant (K_a) of at least about 1 x 10⁶ M^"1 or 10⁷ M^"1, or about 10⁸ M^"1 to 10⁹ M^'1, or about 10¹⁰ M^"1 to 10^{1 1} M^"1 or higher, and binds to the predetermined antigen (e.g. LiMIT) with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.

The phrases "an antibody recognizing LiMIT", "an antibody specific for LiMIT", and "an antibody targeting LiMIT" are used interchangeably herein with the term "an antibody which binds specifically to LiMIT".

The "diagnostic composition" of the invention comprises suitable means for detection. Uaiiy Uii lU ί E , such an antibody which specifically binds to LiMIT or to a functional fragment thereof or polynucleotide(s) having the complementary sequence of a nucleic acid molecule encoding LiMIT or a functional fragment thereof. It is described in context of the present invention that said binding molecule recognizes specific point mutations within LiMIT or specific post-translational modifications of LiMIT. Suitable binding molecules, such a phosphospecifc or point mutation specific antibodies are well known in the art. These binding molecules may be suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of well-known carriers include glass, polystyrene, polyvinyl ion, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. A particular solid phase may also be the membranes used in Western or Northern blots. It is evident that not only the binding molecule but also the target molecule (in context of this invention, LiMIT) may be bound to the solid phase and may be tested with the binding molecules provided in liquid phase.

Solid phase carriers are known to those in the art and may comprise polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes and the walls of wells of a reaction tray, plastic tubes or other test tubes. Suitable methods of immobilizing of binding molecules like, anticalins, antibody(y/ies), polynucleotide(s), aptamer(s), polypeptide(s), etc. on solid phases include but are not limited to ionic, hydrophobic, covalent interactions or (chemical) crosslinking and the like. Examples of immunoassays which can utilize said compounds of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Commonly used detection assays can comprise radioisotopic or non-radioisotopic methods. Examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay) and the Western and Northern blot assay. Furthermore, these detection methods comprise, inter alia, IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay). Furthermore, the diagnostic compounds of the present invention may be are employed in techniques like FRET (Fluorescence Resonance Energy Transfer) assays.

Appropriate labels and methods for labelling are known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include inter alia, fluorochromes (like fluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (like 32P, 33P, 35S or 1251), biotin, digoxygenin, colloidal metals, chemi- or bioluminescent compounds (like dioxetanes, luminol or acridiniums).

A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covalent coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immunoassays", Burden and von Knippenburg (Eds), Volume 15 (1985); "Basic methods in molecular biology", Davis LG, Dibmer MD, Battey Elsevier (1990); Mayer, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987); or in the series "Methods in Enzymoiogy", Academic Press, Inc. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions,

A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is mouse or a human.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment/treating" as used herein covers any treatment of a disease in a subject and includes: (a) preventing and ameliorating an immunodeficiency disorder, an autoimmune disorder, or a proinflammatory response from occurring in a subject which may be predisposed to the disease; (b) inhibiting these diseases, e.g. arresting its development like the inhibition of an inflammation; or (c) relieving the disease, e.g. causing regression of the disease, like the repression of an inflammation. In accordance with the present invention, the term "prevention" or "preventing" of an disease means the disease per se can be hindered of developing or to develop into an even worse situation. Accordingly, it is one aspect of the present invention that LiMIT or a LiMIT agonist can be employed in avoidance of an autoimmune disorder or a pro-inflammatory response. In line with this, a LiMIT inhibitor/antagonist can be employed in avoidance of an immunodeficiency disorder. In accordance with the present invention, LiMIT as well as LiMIT inhibitors/antagonists may also be employed before an immunodeficiency disorder, an autoimmune disorder, or a pro-inflammatory response develops. In addition, in accordance with the present invention, a LiMIT inhibitor/antagonist can also be employed in a method for producing antibodies in a non-human animal. Preferably, these antibodies are high affinity antibodies.

As disclosed and provided for herein, LiMIT, LiMIT agonists or LiMIT inhibitors/antagonists may also be employed in the amelioration and/or treatment of disorders wherein the diseased status has already developed, i.e. in the treatment of an existing inflammatory disorder. Accordingly, the term "treatment/treating" as used herein also relates to medical intervention of an already manifested disorder, like the treatment of an already defined and manifested immunodeficiency disorder, autoimmune disorder or pro-inflammatory response.

The term "level" in context of "level of LiMIT" as used herein is known in the art and relates to the level of LiMIT activity and/or LiMIT expression (e.g. level of LiMIT mRNA or LiMIT protein). The level of LiMIT expression may be reflected by the activity of LiMIT. An antagonist/inhibitor of LiMIT may lead to a decreased expression level of LiMIT (e.g. decreased level of LiMIT mRNA or LiMIT protein) which is reflected in a decreased activity of LiMIT. Another antagonist/inhibitor of LiMIT may directly decrease the activity of LiMIT, e. g. by interacting with the LiMIT protein. In this scenario, the expression of LiMIT may be unaffected whereas the activity is decreased. Accordingly, the term "level" as used herein also relates to the activity or functionality of a protein, e.g. LiMIT. In this respect, high level of LiMIT means high expression and high activity/functionality of LiMIT and low level of LiMIT means low expression and/or low activity/functionality of LiMIT. The activity/functionality of LiMIT (e.g. the ability of LiMIT to convert sphingomyelin into phosphorylcholine) can be measured/detected by the herein described methods to assay the function of LiMIT (e.g, by the Amplex Red Sphingomyelinase kit (Invitrogen, cat. No: A12220)). Additionally, in context of the present invention, an "enhanced", or "high" level of LiMIT means that the level of LiMIT is enhanced compared to a control. For example, the reason for an enhanced level of LiMIT compared to a control may be overexpression of LiMIT in a cell. In this respect, the control could be the same cell with the exception that LiMIT is not overexpressed. Another reason for an enhanced level of LiMIT compared to a control may be a pathological condition wherein LiMIT expression and/or activity is enhanced, for example, in particular cells, tissues or fluids of a subject. In this respect, the control could be the same cells, tissues or fluids of a healthy subject. In line with this, in context of the present invention, an "reduced", "low" or "less" level of LiMIT means that the level of LiMIT is reduced compared to a control. For example, the reason for a reduced level of LiMIT compared to a control may be the knockout of LiMIT in a cell or in an animal. In this respect, the control could be the same cell or animal, respectively, with the exception that LiMIT is not knocked out. Another reason for a reduced level of LiMIT compared to a control may be inhibition of LiMIT expression and/or activity by an inhibitor In a cell or animal. In this respect, the control could be the same cell or animal, respectively, with the exception that LiMIT expression and/or activity is not inhibited by an inhibitor. Another reason for a reduced level of LiMIT compared to a control may be a pathological condition wherein LiMIT expression and/or activity is reduced, for example, in particular cells, tissues or fluids of a subject. In this respect, the control could be the same cells, tissues or fluids of a healthy subject. In addition, in context of the invention, the level of LiMIT may also be reduced by genetic modification such as (targeted) mutagenesis resulting in loss-of function mutants. Genetic modification also relates to the deletion of a gene encoding LiMIT resulting in the generation of a knock-out mutant. Genetic modification further relates to partial deletion of a gene encoding LiMIT, which may result in a subject which has a reduced activity and/or expression of LiMIT. In line with this it is also envisaged in context of the invention that the level of LiMIT is enhanced by genetic modification such as (targeted) mutagenesis resulting in mutants with enhanced LiMIT expression or activity. In context of the present invention, the proof of principle that the polypeptide LiMIT inhibits immune response and may be used for the medical intervention of autoimmune diseases and/or pro-inflammatory responses and that targeting LiMIT may be used for the medical intervention of immunodeficiency disorders has been demonstrated by the appended examples and the supplementary notes herein above.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1. Ceramides modulate TLR signalling. (A,B) RAW264.7 cells were stimulated for 16h with 0,5μΜ Imiquimod (A) or 1 μΜ CpG-DNA (B). 2h prior stimulation cells were shifted to serum free medium and bovine brain Sphingomyelin or bovine brain Ceramide (A) or C2 and C-6 Ceramides or DMSO control (B) were added. The influence of lipids on proinflammatory cytokine induction by endosomal TLRs was analyzed by ELISA for IL-6. (C) Average sequence coverage and spectral counts of LiMIT peptides detected by MS in endosomal TLR TAP purification.

Figure 2. LiMIT is a glycosylated sphingomyelinase inducible by TLR stimuli. (A) Domain structure of LiMIT: In the N-terminus LiMIT harbours a signalling peptide (aa1 -17) and a catalytic, predicted Metallophosphatase domain (aa21-281 ). The asterisk represents the H135Y mutations which has been designed based on homology to a disease associated mutation in ASM. (B) 293T cells were transfected with UMIT-V5 or mock control. 48h later cells were harvested and half of the Iysates were digested with PNGaseF. LiMIT migration behavior and glycosylation status was analyzed by western blotting for V5. (C) RAW264.7 cells were stimulated with OOng/ml LPS in a 4h time course and endogenous LiMIT protein levels were analyzed by western blotting. (D) RAW macrophages were stimulated with 1 μΜ CpG- DNA for the indicated time and the transcript levels of ASM, ASML3a, LiMIT and TLR9 were measured by qRT-PCR. (E) E.Coli BL21 cells were transformed with the catalytic domain of LiMIT (aa18-281), full length WT LiMIT and LiMIT H135A. Bacteria were grown over night to full density, diluted cultures (shifted in fresh LB medium 1 : 100) were split and half were induced for protein expression by addition of IPTG. After incubation at 37°C for 3h bacteria were harvested. To analyze Sphingomyelinase activity equal amounts of bacterial lysates were added into the Amplex Red Sphingomyelinase reaction (Invitrogen) using sphingomyelin as exogenous substrate. As a control, recombinant SMase from B.subtilis was used (upper panel). Data in A-E are representative of at least two independent experiments.

Figure 3. LiMIT is an interactor and negative regulator of TLRs 4,7,8 and 9. (A) Myc- LiMIT was cotransfected into HelaS3 cells with V5-TLR3, -TLR7, -TLR8, -TLR9 or V5-IL1 R as a negative control. Colocalization was analyzed by confocal imaging. Representative areas of overlapping localization are shown magnified in the insets in the merge panel. As shown in Figure 3A, LiMIT clearly colocalizes with the MyD88 dependent TLRs 7,8 and 9, only little with TLR3 and not with IL1 R. (B,C) Myc-tagged LiMIT was double transfected in 293T cells with either one V5-tagged TLR or V5- Unc93B or V5-IL1 R as a negative control. 48 h after transfection, V5-tagged proteins were immunoprecipitated out of cell lysates using V5 agarose. Coprecipitation of LiMiT was analyzed by Western blotting for myc and V5. IB, immunobiot. RAW264.7 cells were lentiviraliy transduced with pLKO shRNA knockdown vectors for LiMIT or control. After selection of stable integrates by Puromycin knock down efficiency was analyzed by western blotting for endogenous LiMIT (D, upper panel) and qRT-PCR using 2 independent primer pairs (D, lower panel). (E) RAW264.7 shRNA stable cell lines were seeded on ELISA plates and stimulated for 8h with 100ng/ml LPS, 1 μΜ imiquimod or 1 μΜ CpG-DNA. The effect of LiMT depletion on pro-inflammatory cytokine secretion was analyzed by ELISA for IL-6. (F) RAW264.7 cells were electroporated with siRNA for LiMIT or Ctrl using AMAXA nucleofector. 24h later cells were seeded on ELISA plates and stimulated with the indicated amounts of imiquimod for 6h. The effect of LiMIT depletion on pro-inflammatory cytokine secretion was analyzed by ELISA for IL-6. (G) RAW264.7 Flip in cells carrying a stable, doxycyclin inducible genomic integration of LiMIT were seeded on ELISA plates and stimulated with the indicated amounts of LPS or CpG-DNA. 24 h prior to stimulation half of the cells were induced by Doxycyclin. After 8h cell supernatants were harvested and analyzed by IL-6 ELISA. Data in A-G are representative of three independent experiments.

Figure 4. LiMIT is a negative regulator of inflammation in vivo. (A) Targeting strategy for mouse LiMIT (left). lacZ, gene encoding β-galactosidase; PGK, promoter of the gene encoding phosphoglycerate kinase; neo, neomycin-resistance cassette. (B) BMDMs from WT or LiMIT -/- mice were lysed and the presence of endogenous LiMIT was analyzed by western blotting. (C-E) WT and LiMIT -/- mice were injected with 50pg LPS i.p. After 6h mice were sacrificed and the levels of the proinflammatory cytokines IL-6 (C) and TNF (D) in the serum were measured by ELISA and (E) peritoneal lavage was taken and total cells and neutrophils were counted. (F,G) WT and LiMIT -/- mice were injected i.p. with ix 10⁴ CFU of E.Coli. (F) After 16h mice were sacrificed and the peritoneal lavage was analyzed for the cytokines IL-10 and IL1 β by ELISA. (G) After 6h mice were sacrificed and the serum cytokine levels of IL-6, TNF, IL12p40, TGF and KC were analyzed by ELISA. Bars are means of each group ± SEM (^* p≤ 0,05) and are representative of two independent experiments, each performed with eight mice/group.

Figure 5. LiMIT alters intracellular organelle structures and suppresses TLR MAPK signalling. (A) Increasing amounts of fluorescent CpG-DNA were added to the medium of shCtrl or shLiMIT macrophages for 2h. Then cells were washed and the phagocytic uptake of DNA was measured by FACS. (B) WT or LiMIT -/- mice were injected with cy3-CpG DNA. After 1 h mice were sacrificed and F4/80⁺ macrophages in the PLF were analyzed for cy3-CpG fluorescence by FACS. (C, D) NIH3T3 cells were transfected with LiMIT-V5. After 24 h cells were fixed and stained for V5 and endogenous (C) EEA1 or (D) Lamp! Subcellular localization of LiMIT and endosomal markers was analyzed by deconvolution microscopy. While cells that do not overexpress LiMIT have no detectable local accumulation of endosomal markers, those cells that show aggregates of LiMIT overexpression concomitantly reveal a strong and visible de novo accumulation of Eea1 and Lampl (see Figures 5C and 5D). This suggests that LiMIT is an endosomal factor that is able to modulate the processing and appearance of endosomal vesicles and could therefore be able to influence endosomal TLR signalling platforms and downstream signalling. (E, F) RAW264.7 stable shRNA knock down cells for LiMIT or shControl were stimulated with 100ng/ml LPS (E) for 10min. Phosphopeptides were enriched and detected by MS. Differentially posttranslationally regulated proteins of the KEGG TLR pathway found are depicted in the graph. (F) in a 60min time course. The phosphorylation status of pp38 and the protein levels of ΙκΒα and tubulin (control) in the lysates were analyzed by western blotting.

Figure 6. LiMIT is a negative regulator of immunization induced, TLR dependent T- cell responses. (A, B) RAW264.7 stable LiMIT shRNA or shControl macrophages were stimulated in biological triplicates with 100ng/ml LPS for 2h. (A) Cells were harvested, RNA was isolated and transcript levels were compared by affimetrix microarray analysis. Data were analyzed with MEV software and highly differentially regulated transcripts were summarized in the heat map. (B) qRT-PCR with two independent primer pairs for H2-Aa, normalized to the transcript levels of the housekeeping gene cyclophilin B. (C-F) WT and Limit^"'^" mice were immunized with ovalbumin and LPS. After 10 days, spleen cells of WT and Limit^"'^" mice were harvested. (C) Splenocytes of immunized mice were restimulated with 100 pg/ml of Ovalbumin in vitro and proliferation was quantified by H₃Thymidine incorporation. (^* p < 0,05). (D) Splenocytes were restimulated with 1 pg/ml of the OVA peptide SIINFEKL for 16h and secretion of IFNv was measured by ELISA (^* p < 0,05). (E, F) Splenocytes were stimulated in vitro with anti-CD3 for three days, proliferation was quantified by H₃Thymidine incorporation and levels of IL-17 in the supernatant were measured by ELISA. Bars are means of each group ± SEM (* p < 0,05) and are representative of two independent experiments, each performed with eight mice/group.

Figure 7. LiMIT bioinformatical analysis. (A) Intersection of proteins found in all endosomal TLR pulldowns with the nature Lipid Maps Proteomic Database of 1083 murine lipid associated proteins. Numbers in squares represent average peptides found in specific TLR pulldowns. (B) Amino acid sequence of LiMIT (Genbank database accession no. NM_133888) with domains identified by SMART domain analysis: signal peptide (aa1 -17), metallophosphatase domain (aa21-281 ). (C,D) Phylogenetic tree of LiMIT showing (C) conservation of the LiMIT protein from insects to human and (D) homology based analysis of LiMIT related proteins in mouse. (E) Sequence alignment of LiMIT orthologs.

Figure 8. (A) RAW264.7 cells were stimulated with 100 g/ml Sphingomyelin, 50Mg/ml Ceramide, 10μΜ Imiquimod, 2μΜ CpG-DNA or l Opg/ml Poiy(l:C) for 2h. The transcript levels of LiMIT or ASM as control were analyzed by qRT-PCR. (B) RAW264.7 macrophages lentivirally tranduced with two independent shRNAs for LiMIT or control were cultivated in serum free medium and stimulated with 1 μΜ CpG- DNA or 1 μΜ imiquimod for 4h, harvested and RNA was isolated. Relative abundance of TNF and IL-6 transcript levels were analyzed by qRT-PCR. (C) RAW264.7 FRT TR cells were tested for doxycyclin inducible, stable integration of WT or D93A LiMIT- HA. Cells were cultivated in the presence or absence of doxycyclin for 24 h prior to iysis and western biotting for the HA epitope. Data in A-C are representative of at least two independent experiments.

Figure 9. (A,B) WT and LiMIT -/- mice were injected with 50^g LPS I. p. After 6h mice were sacrificed. (A-B) Serum and peritoneal lavage were taken and (A) the levels of the pro-inflammatory cytokine IL-6 in the serum was measured by ELISA and (B) the amount of macrophages in the peritoneal lavage fluid was counted. (C) WT and LiMIT -/- mice were injected with 50pg CpG-DNA i.p. After 1 h mice were sacrificed and peritoneal lavage was taken and the percentage of Gr1⁺, F4/80^" cells was quantified by FACS analysis. Data in A-C are representative of at least two independent experiments. Bars are means of each group ± SEM (^* p < 0,05).

Figure 10. Heat map of LPS induced changes in RAW264.7 macrophages carrying shControl. RAW264.7 stable LiMIT shRNA or shControl macrophages were cultivated in serum free medium and stimulated with 100ng/ml LPS for 2h in biological triplicates. RNA was isolated and transcript abundance analyzed using affimetrix microarrays. Depicted is a heat map of the LPS induced changes in transcript levels of the shRNA control cell line as analyzed by multi experiment viewer (MEV). The Examples illustrate the invention. Example 1 :

MATERIALS AND METHODS

Reagents. ELISA kits were purchased from BD. All synthetic TLR ligands, Poly(l:C), imiquimod, CpG-DNA-ODN1826 and LPS (£. coli K12) were obtained from InvivoGen. Cy3-labeled oligonucleotides were synthesized at Microsynth. Protein G- Sepharose was purchased from GE Healthcare, and streptavidin beads were purchased from Thermo Fisher Scientific. Sphingolipids were obtained from Avanti Polar Lipids. Mouse anti-V5 antibody was obtained from Invitrogen. Rabbit antimyc was purchased from Sigma-Aldrich mouse anti-Lampl was purchased from vstressgen ana mouse ann-tea i was purcnasea ΤΓΌΓΤΪ BU. r*ou anuuOuy was obtained from Abeam, and GR-1 antibodies were obtained from BD. Antibodies to CD44 and CD62L (all rat a-mouse) were from BD Bioscience, CD4 (rat a-mouse) Beckman Coulter, CD69 hamster a-mouse from Pharmingen. Anti-collagen ELISA, Mouse Th1/Th2 10plex FlowCytomix Multiplex as well as IL-23 and IL-22 Flow cytomix simplex were obtained from Bender Medsystems and used according to manufacturer^'s protocols (Cat. No BMS810FFRTU).

All secondary antibodies were purchased from Invitrogen (Alexa Fluor 488, 546, 594, 680, and 800 nm). RT-PCR reagents were bought from GeneXPress, and buffers were bought from Sigma-Aldrich. The Amplex red Sphingomyelinase kit was from invitrogen (Cat. No: A12220). The LiMIT polyclonal antibody was made as follows: recombinant LiMIT (aa1-281 ) was purified from BL21 bacteria via HIS-tag purification following the instruction of the Qiaexpressionist. Recombinant protein was shipped to Charles River for immunization of rabbits. Rabbit sera were tested and purified against the recombinant LiMIT protein.

MS and data analysis. Proteomic TAP-tag approach and MS analysis have been described earlier (Baumann; 2010; J Exp Med; 207; 2689-2701 ).

Plasmids. All constructs were cloned using the gateway system. Murine and human TLR constructs (available from GenBank/EMBL/DDBJ under the following accession nos.: mTLR3, NMJ26166; mTLR4, NM_021297; mTLR7, NM_13321 1 ; mTLR8, NMJ 33212; mTLR9, NM_031 178; hTLR3, NM__003265; hTLR7, NM_016562; HTLR8, NM_138636; and hTLR9, NM_017442) were cloned from InvivoGen vectors (pUno-HA vectors)., IL-1 R (available from GenBank under accession no. NM_000877.2) and LiMIT/Smpdl3b (NMJ 33888) were amplified from a cDNA library (Takara Bio Inc.) using the following primers: LiMIT 5^{^}-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA GAC TGC CAT GAC GCT GCT CGG GTG GCT GAT ATT CCT G-3^' and LIMIT 3^'- GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TAA CAC CTC CAG TAC GTG CAG-51L-1 R 5^'-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC CAT GAA AGT GTT ACT CAG ACT TAT TTG-3; and IL-1 R 3^"-CCC GAG AGG CAC GTG AGC CTC TCT TTG GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT-5\ For site directed mutagenesis of LiMIT the following primers were usea: LIIVII ι Π Ι ΟΛ Ό - <^ Ι Ι ι υ un m i J ι ϋΛ ι ι ι ι ^^-o ana

LiMIT Η135Α 3 -GGG GTG AAA ATC AGC ATT TCC CAA AGC-5^' . LiMIT D28A 5^'- GTT CTG GCA CAT CTC CGC CCT GCA TCT GGA CCC C-3^'and LiMIT D28A 3^'- GGG GTC CAG ATG CAG GGC GGA GAT GTG CCA GAA C-5^'; LIMIT D93A 5^'- CATTCTC-TGGAC-AGGGGC-C-GAC-AC-AC-C-GC-AC-GTC— 3 and LiMIT D93A 3 - GAC GTG CGG TGT GTC GGC CCC TGT CCA GAG AAT G-5^';

Unc93B cDNA was a kind gift from Melanie Brinkmann/Hidde Ploegh and was re- cloned using the following gateway primers: 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA GAC TGC CAT GGA GGT GGA GCC TCC GCT CTA CCC TGT G- 3^' and 3- GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT CTG CTC CTC AGG CCC ATC GCC ACC-5^'. LIMIT catalytic domain fragment (aa1-281 ) was amplified using the internal primer 3 - GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCG GTA TGG TGG TGT CCA AAG AAC TGC CC-5\ All gateway entry clones for C-terminal tagging of full-length cDNAs were created using 20-25 bp of flanking regions and the following gateway primer sequences: sense a#B1

primer, 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA GAC TGC CAT G( NNN) 5-10-3 ; and antisense affB1 primer, 5^"-GGG GAC CAC TTT GTA CAA GAA vectors for protein expression were pTracer-V5 (Invitrogen), pcDNA6myc, and pCeMM CTAP(SG). Cell culture. Cell lines used were RAW264.7 murine macrophages,

Hek293, NIH3T3 and Hel_aS3 cells. Cells were cultured in DME (PAA) supplemented with 10% FCS (Invitrogen) and antibiotics (100 U/ml penicillin and 100 pg/ml streptomycin) at 37°C, 5% C02. In all types of stimulation experiments RAW macrophages have been washed twice and shifted to serum free medium 1 h prior stimulation to avoid lipid rescue by FCS derived sphingolipids. Raw FRT-TR were generated by two lentiviral transductions of Raw cells, first with increasing concentrations of VSV-G pseudotyped virus encoding the FRT LacZeo cassette (pHRSIN FRT LacZeo). Transfected cells were seeded in 24 well plates for selection with 200 ug/ul Zeocin. Colonies from the lowest titer yielding resistant clones were tested by Southern blot for single integration of provirus. Genomic DNA, digested with EcoRI, was probed with a radiolabeled Hindlll/EcoRV fragment of pFRT LacZeo (Invitrogen), spanning part of LacZ. Approximately 50% of tested clones carried a single copy while all others contained two. A second transduction with pLV6/TR lentivirus (Invitrogen), coding for the Tet repressor, yielded colonies which were selected with increasing concentrations of Blasticidin (max. 50 ug/ml) to isolate clones that expressed h igh levels of Tet repressor.

RT-PCR. RNA was isolated using the Qiagen RNAeasy kit. 100 ng RNA/samp!e was reversely transcribed using reagents/RT (Fermentas). cDNA was diluted 1 :20, and transcript abundance of murine IL-6 and TNF was analyzed using SYBR green (GeneXPress) and the following primers: IL-6 5^' -TTC CAT CCA GTT GCC TTC TT- 3^' ; IL-6 3^' -ATT TCC ACG ATT TCC CAG AG-5; TNF^' 5^' -CAA AAT TCG AGT GAC AAG CCT G-3-; TNF3 -GAG ATC CAT GCC GTT GG C-5^'; cyclophilin B 5 -CAG CAA GTT CCA TCG TGT CAT CAA GG-3^"; and cyclophilin B 3^*-GGA AGC GCT CAC CAT AGA TGC TC-5^" ; LiMIT S^'-GCA CGT GGC CAA TAA CAT AC -3^' and LiMIT 3^"-GCA CGT GGC CAA TAA CAT AC-5^' ; LiMIT-B 5^" -AAG TCT ATG CTG CTC TGG GAA-3^'and LiMIT-B 3^'-TGC CAC CTG GTT ATA GAT GC-5^'; ASM 5^*-AAC CCT GGC TAC CGA GTT TA-3^'and ASM 3^'- GCC TGG GTC AGA TTC AAG AT -5: Quantitative real-time PGR analysis was performed on a Rotor Gene 6000 (QIAGEN). Measured TNF and IL-6 cycles were normalized to the ones of cyclophilin B. In vivo experimental procedures. Heterozygous LiMIT +/- mice (B6N;B6N- Smpdl3b<tm1a(EUCOMM)Wtsi>/H) were purchased from the EMMA consortium (EUCOMM) and shipped from the MRC. LiMIT homozygous knockout was confirmed by genotyping using the following primers: LiMIT WT 5^'- GTG TAA GCC TTC TCC CCC AG-3^'; LiMIT WT 3^'- CAG AAA AAG TTC TAC GGA CCA GC -5^'; LiMIT MUT 3^'- TTG GTG ATA TCG TGG TAT CGT T -5^'. C57BL/6J mice were obtained from Charles River. Pathogen-free 9-11-wk-old female C57BL/6J and homozygous B6N;B6N-Smpd/36^{tm1 a(EUCOM )Wtsi}/H LiMIT -/- mice were used in all in vivo experiments, which were approved by the Animal Care and Use Committee of the Medical University of Vienna. Mice were injected i.p. with either 50pg LPS or 8nmol CpG-DNA in 200 μΙ of saline per mouse. After 6h, mice were sacrificed, and peritoneal lavage was taken. Cell counts were determined on each peritoneal lavage sample stained with Turks solution, and differential cell counts were performed on cytospin samples stained with Giemsa. For immunization mice were injected intraperitoneally with egg white equivalent to 250 pg ovalbumin (OVA), in either PBS alone or mixed with LPS (50pg). Spleen cells were harvested, passed through a nylon mesh and cultured at a density of 1 ^χ 10^ cells/ml in RPMI-1640 with I- glutamine plus 10% FCS, penicillin/streptomycin and p₂-mercaptoethanol and cultured for 3 days in the presence or absence of either ovalbumin or the SIINFEKL OVA peptide or stimulated with plate bound anti-CD3 mAb (145-2C1 1 ). Supernatants were collected for cytokine analysis after 48 h. Cells were then incubated for further 18 h in the presence of 1 pCi/well of [³H]thymidine to quantify proliferation.

Phagocytosis assay and FACS. For analysis of phagocytosis of fluorescent DNA, either RAW264.7 or peritoneal macrophages were plated at a density of 10⁶ on 6-well plates. 1 μΜ cy3-CpG-DNA was added to the cell culture medium for the indicated time points. 1 well of each cell type was shifted to 4°C before the addition of oligonucleotides and served as a phagocytosis negative control. After three washes with ice cold PBS, cells were harvested and resuspended In PBS, and cy3- positive/F4-80-positive cells were analyzed on a FACSCalibur flow cytometer (BD). A set of 20,000 gated cells was analyzed for their GMI (geometric mean intensity of Cy3 fluorescence). The phagocytoid index was calculated as follows: GMI sample χ amount of gated cells - GMI 4°C control χ amount of gated cells. For the analysis of phagocytosis in vivo, WT or LiMIT -/- were injected i.p. with 50pg cy3-CpG DNA (ODN 1826)/mouse. After 1 h mice were sacrificed, PLF was taken, cells stained with F4/80 and Gr1 antibodies and the uptake of fluorescent DNA represented by a GMI value was analyzed by FACS and Flowjo software.

Immunoprecipitation and Western blotting. 1.5 * 10⁹ RAW264.7 macrophages per endosomal TLR construct have been used for TAP. All steps have been performed as described in Burckstummer 2006, Nat.Methods, Nat Methods.10 3-9., despite the use of lysis buffer (50 mM Tris-HCI, pH 6, 150 mM NaCI, 1 mM EDTA, 7.5% glycerol, 25 mM NaF, 0.2% NP-40, 1 mM DTT, and 1 mM Na3V04, adjusted to pH 6.0 with HCI). Immunoprecipitation of tagged TLRs and LIMIT was performed as follows: 6 x 10⁶293T cells were seeded on 10-cm dishes and double transfected with 7.5 pg of plasmid DNA encoding LIMIT-myc and 7.5 ug of plasmid DNA encoding either the C-terminally V5-tagged human TLRS, TLR7, TLR8, or TLR9 or IL-1 R protein. 48 h later, cells were lysed with lysis buffer (1 % NP40, 20 mM Tris-Hcl, pH onli i Io K/o fo line ι icoH Affair _h i i ihoiinn

beads on a rotating wheel for 2 h, beads were washed four times with cold lysis buffer and taken in Laemmli buffer. Boiled eluates were run on SDS-PAGE and transferred on membranes by Western blotting. V5 and myc epitopes were visualized by Western blotting and detected using an Odyssey system (LI-COR Biosciences). PNGaseF treatments to deglycosylate overexpressed LiMIT were carried out following the instruction of the New England Biolabs manual. LiMIT and ASM secretion was analyzed by overexpression of C-terminally V5-tagged constructs in 293T cells. Compared was the detection of tagged proteins in lysates or cell culture medium that was cleared by high speed centrifugation (13000 RPM). To prevent LiMIT secretion Brefeldin A (Sigma) was added at a concentration of 5 pg/ml for 6h.

Sphingomyelinase assays were performed according to the guidelines for acidic SMases of the invitrogen amplex red SMase kit (Cat. No: A12220). In brief, BL21 bacteria were transformed with expression plasmids for LiMIT and grown to full density over night. Bacteria were shifted 1 : 100 in fresh LB medium and LiMIT protein expression was induced by addition of IPTG for 3h. Then, bacteria were harvested and equal amounts were lysed with lysis buffer (1 % NP40, 1 pg/ml Lysozyme, 20 mM Tris-Hcl, pH 6, 150 mM NaCI) and 20μΙ of bacterial lysate was added into a 10ΟμΙ Amplex red reaction.

Immunofluorescence analysis. For colocalization analysis, Hel_aS3 cells were plated on coverslips at a density of 3 105/well on a 6-well plate. 24 h later, cells were transfected with 1 pg human LIMIT-myc and 1 pg human TLR-V5 construct using Lipofectamine (QIAGEN). 24 h later, cells were washed and fixed with 4% PFA/PBS for 10 min at room temperature. After three washes with PBS, slides were permeabilized with 0.1 % Triton/PBS for 10 min. Then, coverslips were blocked with 3% BSA/PBS for 30 min, and incubations with primary and secondary antibody were each performed for 1 h in 3% BSA/PBS at room temperature. Primary antibodies were stained with the fluorescence coupled antibodies Alexa Fluor 488-goat anti- rabbit (A1 1008) and Alexa Fluor 594-goat anti-mouse (A 1005; both from Invitrogen). DNA was visualized by DAPI (Roth). After another three wash steps with PBS, coverslips were mounted on glass slides using ProLong Gold (Invitrogen). Stainings of endogenous proteins in RAW264.7 macrophages were performed equally, except that direct primary antibodies binding to the respective proteins were used. The localization of proteins was analyzed using a confocal microscope (LSM510; Carl Zeiss, Inc.), equipped with the 63* numerical aperture 1.4 Plan- Apochromat lens. DAPI was excited with the 405-nm laser line and the emitted fluorescent light was detected in the range of 420-470 nm. Green represents fluorescent signal collected in the range of 505-550 nm upon excitation with a 488- nm laser. However, as the appended Figures are in black-and-white, all results are shown in black-and-white. Red corresponds to a signal in the range of 575-620 nm and resulted from 561-nm excitation. As described above, as the appended Figures are in black-and-white, all results are shown in black-and-white. Confocal stacks with pinhole of 1 airy unit were recorded, with the section spacing of 150 nm. Threshold- based colocalization was performed on the three-dimensional datasets using the Definiens Software Suite (Definiens AG). Fixed thresholds were used to determine the green, red, and colocalizing population. For colocalization analysis of phagocytosed cy3-CpG-DNA with LIMIT in RAW264.7 macrophages, a confocal microscope (TCS SP5; Leica) with a 63* oil immersion objective (Leica) was used. Fluorochromes were excited using an argon laser at 488 nm and a HeNe laser at 568 nm for Texas red. Detector slits were configured to minimize any cross talk between the channels. Z stacks (optical sections) of the images were collected with an optical thickness of 0.2 mm. Images were processed using the LAS system software (Leica).

All other pictures were taken with a microscope (Eclipse 80i; Nikon) with a Plan-Fluor 40* objective (Nikon) with a numerical aperture of 0.75. All images were processed with Photoshop (Adobe) and assembled in Illustrator software (Adobe) with identical processing for all images in one experiment.

Gene Expression Profiling. Total RNA was isolated with the RNeasy Mini kit per manufacturer's instructions (Qiagen, Cat. No 74104). Total RNA (200 ng) was then used for GeneChip analysis. Preparation of terminal-labeled cDNA, hybridization to genome-wide human Gene Level 1.0 ST GeneChips (Affymetrix) (Cat. No 901 178) and scanning of the arrays were carried out according to manufacturer's protocols (https://www.affymetrix.com; (Tauber, Jais; Mol Cancer; 9; 200)). RMA signal extraction, normalization and filtering was performed as described (www.bioconductor.org/) (Tauber; 2010; Mol Cancer; 9; 200). A signal intensity filter was applied to remove genes of low overall intensity: The filtering criteria for the exemplary data sets required the expression level to be higher than 100 in at least 1 sample.

Example 2:

Ceramides modulate TLR signalling

It was tested whether sphingomyelin or ceramide may effect TLR signalling and the cytokine expression profile after incubating RAW264.7 macrophages with sphingolipids followed by stimulation with TLR ligands was analyzed. Lipids alone did not induce cytokine signalling and sphingomyelin pretreatment of imiquimod stimulated cells did not alter IL-6 levels. However, bovine brain ceramide doubled the IL-6 secretion after TLR7 stimulation (Figure 1A). To test the effect of more defined ceramides on TLR activation synthesized, soluble C2 and C6 ceramides were applied. Compared to stimulation without lipids, an increase of CpG-DNA induced IL- 6 levels when using C6 ceramides was observed, whereas C2 ceramides had the opposite effect (Figure 1 B). This suggests that different species of ceramides can have different pro- and anti-inflammatory effects on TLR signalling in response to DNA.

Example 3:

LiMIT is a protein that interacts with endosomal TLRs

It was looked whether lipid modifying enzymes can be found in the MS data from a previously described proteomic screen for interactors of endosomal TLRs (Baumann; 2010; J Exp Med; 207; 2689-701 ).To this aim proteins that were identified in all TLR pulldowns with the list of 1083 murine proteins from the Nature LIPID MAPS proteome database (http://www.lipidmaps.org/data/proteome/index. cgi?db=lmpd&form=introduction) were intersected and 1 1 proteins were identified (Figure 7A), including the previously described TLRS co-activator Raftlin (Watanabe, Tatematsu; J Biol Chem; 286; 10702-1 1 ) and the yet undescribed Acid sphingomyelinase-like phosphodiesterase Asm3b/Smpdl3b (UniprotKB/SwissProt ID P58242, Smpdl3b_MOUSE) which was termed LiMIT for "Lipid Metabolizing Interactor of TLRs". In this tandem affinity purification approach LiMIT was identified with 5 to 1 1 peptides in pulldowns of the endosomal TLRs 7, 8 and 9, corresponding to a total sequence coverage of 10 to 26% (Figure 1 C).

Example 4:

Sequence and domain structure of LiMIT

The sequence and domain structure of LiMIT was analyzed using bioinfomatic tools (http://smart.embl-heidelberg.de/ ). LiMIT is a protein of 456 amino acid length (Figure 7B), harbouring a signal peptide in the N-terminus (amino acids 1 -17) that suggested membrane targeting and organelle trafficking, supporting the observation that LiMIT colocalized and interacted with endosomal TLRs. Furthermore, a metallophosphatase domain (aa21-281 ) with high similarity to the Sphingomyelinase domain of ASM/Smpd1 was identified (Seto; 2004; Protein Sci; 13; 3172-86). Orthologs of LiMIT were found in organisms conserved from human to insects/flies (Figure 7C) with high conservation of patterns especially in the N-terminal catalytic part of the protein (Figure 7E). Interestingly, LiMIT belongs to a family with two isoforms of ASML3a/Smpdl3a as closest homologs and ASM/Smpd1 most distantly related in mouse (Figure 7D).

Example 5:

LiMIT is a glycosylated sphingomyelinase

As suggested by its signal peptide LiMIT could be a secreted and, thus, glycosylated protein, similar to ASM (Schissel; 1998; J Biol Chem; 273; 18250-9). In fact, previous MS based proteomic studies have identified LiMIT in seminal fluid and urine (Pilch; 2006; Genome Biol; 7; R40, Marimuthu; 201 1 ; J Proteome Res; 10; 2734-43). To test whether LiMIT is glycosylated a V5-tagged LiMIT was overexpressed in 293T cells and a visible shift in migration behavior on SDS page geis was observed when lysates were treated with the endoglycosidase PNGaseF, an enzyme releasing relinked oligosaccharides (Figure 2B). These data suggest that LiMIT is a putative sphingomyelinase that is posttranslationally glycosylated.

Example 6:

LiMIT expression is inducible by TLR stimuli

Proteins involved in host defence against pathogens are often upreguiated after pathogen encounter. Therefore it was tested whether LiMIT transcript and protein levels would be sensitive to TLR stimuli. LPS stimulation time courses were performed in RAW264.7 macrophages followed by western blotting for endogenous LiMIT and an increase in LiMIT protein levels over time was observed (Figure 2C). Using qRT-PCR the expression levels of LiMIT, its interactor TLR9 and the LiMIT homologs ASM3A and ASM were compared upon CpG-DNA stimulation after 2 and 4 hours in FCS depleted macrophages. Only LiMIT and TLR9 showed up to four fold upregulation whereas ASM and ASM3A were either not regulated or downregulated (Figure 2D). Moreover, LiMIT transcript levels were responsive to diverse TLR stimuli as well as to the addition of the putative substrate sphingomyelin and cleavage product ceramide, whereas ASM transcript levels were unaffected by either stimulus (Figure 8A). Hence, LiMIT is upreguiated during innate immune responses on both, transcript and protein level. Example 7:

LiMIT has Sphingomyelinase activity

The domain and homology analysis suggested that LiMIT is an enzyme with sphingomyelinase activity, mediating the cleavage of membrane integral sphingomyelin into phosphorylcholine and ceramide (Seto; 2004; Protein Sci; 13; 3172-86). As most bacteria do not harbor sphingolipids (An; 2011 ; Proc Natl Acad Sci U S A; 108 SuppI 1 ; 4666-71) and should therefore not provide substrate for sphingomyelinases bacterial expression was used to circumvent insolubility of the recombinant, hydrophobic protein and exogenous sphingomyelin substrate was added. LiMIT expression Plasmids were transformed into BL21 bacteria and the expression of LiMIT was induced by addition of IPTG. Equal amounts of bacteria were harvested and bacterial lysates were added to the Amplex Red Sphingomyelinase reaction for acidic SMases (Invitrogen) to determine the relative sphingomyelinase activity over time. In this enzymatic assay the sphingomyelinase cleavage product phosphorylcholine (Seto; 2004; Protein Sci; 13; 3172-86) was processed to choline by exogenously added alkaline phosphatase and further by choline oxidase to betaine and H₂O₂ which was quantified in a HRP induced colorimetric reaction. Recombinant SMase was added to the with sphingomyelin complexed bacterial lysate as positive control (Figure 2E, upper panel). It was found that the N-terminal catalytic domain of LiMIT (aa21 -281 ) had highest SMase activity whereas full length LiMIT showed slightly reduced substrate conversion, when expression was induced with IPTG (Figure 2E, lower panel). Interestingly, a mutant (H135A), designed based on homology to a point mutation of ASM that has been found in patients with Niemann-Pick disease (Seto; 2004; Protein Sci; 13; 3172-86) showed reduced catalytic activity. Taken together these data describe LiMIT as a glycosylated sphingomyelinase that is induced by TLR stimuli.

Example 8:

Analysis of the interaction of LiMIT with TLRs on a subcellular level

It was addressed whether the observed interaction of LiMIT with TLRs (cf. Example 3) could be observed on a subcellular level by co-localization analysis. To test the subcellular proximity of LiMIT with TLRs confocal imaging of V5-tagged TLRs and myc-tagged LiMIT in transfected HelaS3 cells was performed. LiMIT clearly colocalized with the MyD88 dependent TLRs 7,8 and 9, only little with TLR3 and not with IL1 R (Figure 3A). Furthermore, co-immunoprecipitation of V5 tagged TLRs with myc-tagged LiMIT from double transfected 293T cells was performed (Figure 3B/C). LiMIT associated strongly with the MyD88 dependent TLRs 7,8 and 9, weakly with TLR3 (Figure 3B) and not with the endosomal control protein Unc93B. As CD14 had previously been shown to be a co-receptor not only for TLR4 but also for all endosomal TLRs (Baumann; 2010; J Exp Med; 207; 2689-701 , Lee; 2006; Immunity; 24; 53-63) we tested for and readily detected co-precipitation of LiMIT with TLR4, but not with the TIR-domain containing IL1 R receptor was detected (Figure 3C). Taken together these data show that LiMIT associates with the MyD88 dependent TLRs 4, 7, 8 and 9.

Example 9:

LiMIT has modulatory function on inflammatory responses in macrophages To elucidate whether LiMIT has a modulatory function on inflammatory responses lentiviral shRNA targeting LIMIT was used to deplete LIMIT in RAW264 7 macrophages. Cell lines stably carrying lentivirally transduced shRNAs for LIMIT or ctrl depletion were created. The efficiency of the depletion of endogenous LiMIT was tested by western blotting and qRT-PCR (Figure 3D). When stimulated with LPS, imiquimod, or CpG-DNA LiMIT knockdown macrophages showed a strong increase in IL-6 cytokine levels (Figure 3E), suggesting LiMIT as a negative regulator of proinflammatory responses. Similarly, the transcript levels of IL-6 and TNF were highly upregulated in LiMIT depleted cells (Figure 8B). To exclude secondary off-target effects caused by the random integration of the shRNA lentivirus or target unspecificity of the shRNA sequence, LiMIT depletion was repeated using siRNA against LiMIT or LaminA as control in RAW264.7 macrophages. 24 h after siRNA electroporation, cells were counted, replated and stimulated with imiquimod (Figure 3F). Strikingly, also reduction of LiMIT by siRNA increased the amount of IL-6 elicited by imiquimod treatment. Next it was addressed whether LiMIT overexpression would affect inflammatory cytokine levels. To this aim RAW264.7 FRT TR cells that carry an FRT recombination site for a single homologous integration of a gene of interest and a Tet repressor for Doxycyclin inducible expression were created. Following electroporation, recombination and selection of isogenic cells, doxycyclin inducible expression of LiMIT was tested by western blotting (Figure 8C). RAW264.7 FRT TR- LiMIT cells were seeded, LiMIT expression was induced with doxycyclin followed by stimulation with the TLR ligands LPS and CpG-DNA (Figure 3G). Opposite to the observed siRNA/shRNA loss of function phenotype, the induction of LiMIT expression lead to a severe decrease in IL-6 production. Taken together, these data suggest that LiMIT acts as a negative regulator of pro-inflammatory TLR signalling in macrophages.

Example 10:

Immunomodulatory function of LiMIT in vivo

To study the role of LiMIT in vivo mice with a homozygous deletion of LiMIT were obtained from EMMA (European Mouse Mutant Archive)(Figure 4A). The

homozygous knock out was confirmed by PGR (data not shown) and western blotting (Figure 4A). The TLR4 ligand LPS was injected WT or LiMIT -/- mice intraperitoneal^ (Figure 4B,C) or intravenously (Figure 9A) with 50 g LPS per mouse and the serum cytokine levels of TNF and IL-6 were measured after 6h. Intriguingly, compared to wild-type control mice, pro-inflammatory cytokines were significantly higher in the serum of mice deficient of LiMIT. Accordingly, when the cells present in the

peritoneal lavage fluid (PLF) of mice that were injected intraperitoneally with LPS for 6h were counted, LiMIT deficient mice contained a higher number of total cells, in particular significantly more neutrophils (Figure 4E), and slightly more macrophages (Figure 8B) in the peritoneum. Similarly, mice that were injected i.p. with CpG-DNA for 1 h revealed a strong increase in Gr1⁺ granulocytes in the peritoneal lavage fluid (Figure 9C), reflecting increased inflammation in the absence of LiMIT.

To test whether LiMIT may be involved in the host-response against pathogens an E. coli peritonitis model was used. LiMIT and wild-type control mice were injected intraperitoneally with bacteria and lavage cytokine levels were measured 16h later. A clear decrease in the anti-inflammatory cytokine IL-10 (Netea; 2004; J Immunol; 172; 3712-8) was observed in LiMIT deficient mice, whereas the levels of I L1 β, a cytokine involved in the recruitment of neutrophils (Rogers; 1994; J Immunol; 153; 2093-101 ), were strongly increased (Figure 4F). Next the early effect of E.Coli peritonitis on serum cytokines after 6h was tested. Interestingly, the serum levels of IL-6, KC (IL-8), TNF, IL12p40 as well as the activated T-cell regulatory cytokine TGFp were detectable in the WT but were significantly higher in LiMIT -/- mice (Figure 4G). These data show that LiMIT is a specific, negative regulator of TLR dependent inflammation in vivo. Alterations in TLR activation can often be attributed to enhanced phagocytic ligand uptake and, hence, increased presence of TLR stimulus in the endosome. However, neither RAW264.7 LiMIT shRNA macrophages that were exposed to different concentrations of fluorescent CpG-DNA (Figure 5A) nor peritoneal macrophages from mice that were injected intraperitoneally with fluorescent cy3-CpG 1 h before they were sacrificed (Figure 5B) showed significant differences in the phagocytic uptake of TLR ligand that could explain the increased TLR signalling induction in LiMIT deficient cells.

Exampie 11 :

LiMIT is an endosomai factor

It was analyzed whether LiMIT could modulate the endosomai compartments to influence TLR signalling and endosomai processing. Ceramide had been shown to ,

7\ Tr^ Ir^^^t^^l^A t-„„„^ 1

subcellular effect on endogenous protein markers for early (Eeal ) and late (Lampl ) endosomes was studied by deconvolution microscopy. While cells that did not overexpress LiMIT had no detectable local accumulation of endosomai markers, those cells that showed aggregates of LiMIT overexpression concomitantly revealed a strong and visible de novo accumulation of Eea1 and Lampl . This suggests that LiMIT is an endosomai factor that is able to modulate the processing and appearance of endosomai vesicles and could therefore be able to influence endosomai TLR signalling platforms and downstream signalling.

Example 12:

LiMIT is a negative regulator of TLR-mediated pro-inflammatory signalling

I ciuuiuaLc VV ! ICU IGI II ic i cyuicuui y ci icots ui i— 11 8 H I ui i uyiOiMi ic icvcis uuuiu uc attributed directly to the TLR induced signalling events the LPS induced posttranslational molecular changes in shRNA depleted and control cells were dissected by iTRAQ quantitative phosphoproteomics. To this aim, shLiMIT and shControl cells were stimulated for 10 minutes with LPS before cells were harvested. Equal amounts of tryptic peptides of each cell line were iTRAQ-labelled and the pooled sample was used for a global phosphopeptide enrichment using a modified immobilized metal-affinity chromatography (IMAC) protocol (Ficarro; 2009; Anal Chem; 81 ; 4566-75). After fractionation of eluted phosphopeptides each fraction was analyzed on a hybrid LTQ-Orbitrap Velos instrument and acquired data were processed and searched with phosphorylated serine, threonin or tyrosin as variable modification. A selective, LPS dependent modulation of phosphopeptide enrichment was found by analyzing proteins from the KEGG TLR pathway (Figure 5E). In particular, several nodes of proinflammatory signalling, including c-Jun, Erk, c-myc and p38, revealed higher phosphorylation states in LiMIT depleted macrophages. Interestingly, the activation of p38 has been implied as a regulatory mechanism in TLR signalling that not only affects cytokine secretion but also phagosome maturation and antigen presentation (Blander; 2006; Nat immunol; 7; 1029-35). To corroborate a role of p38 regulation by LiMIT LPS stimulation time courses and western blotting for pp38 in LiMIT and Ctrl shRNA cell lines were performed. A somewhat faster degradation of the NF B negative regulator Ι Β and higher phosphorylation of p38 in LiMIT deficient cells was found. Taken together these data suggest that LiMIT acts not on the early phagocytic uptake of TLR ligand but later to modulate proinflammatory TLR signalling in the acidified endosome.

Example 13:

LiMIT depletion leads to MHC antigen upregulation and T cell activation

To shed light on the specific targets of LiMIT regulation and the effects exerted on transcript regulation the LiMIT shRNA and control macrophage cell line were compared for differences in transcript abundance with or without LPS treatment in biological triplicates using microarrays. LPS treatment led to a significant change in transcript abundance in control cells with upregulation of candidate cytokines such as I L β or IL-6 (Figure 10). Intersection of transcripts between control and LiMIT depleted cells revealed a small subset of significantly, differentially regulated transcripts at the 2h time point (Figure 6A). includina most prominently the two Histocompatibility type II antigens A and E (H2-Ea and H2Aa). To confirm the specificity of MHC antigen upregulation in LiMIT depleted cells direct qRT-PCR for H2Aa was performed using two independent primer pairs and the specific induction of this antigen in LiMIT depleted cells was confirmed (Figure 6B).lt was tested whether the regulation of MHC by LiMIT could affect CD4⁺ adaptive immune responses. Bioinformatical analysis of LiMIT tissue expression suggested specificity for immune cells and within those especially antigen presenting cells (http://biogps.org/#goto=genereport&id=27293). To address, whether LiMIT TLR modulation in antigen presenting cells would have a regulatory effect on T-cell activation mice were immunized with the antigen ovalbumin. As a full blown adaptive immune response to an antigen with elaborate activation of T-cells requires not only the antigen but also sufficient induction of TLR signalling (Iwasaki and Medzhitov; 2010; Science; 327; 291-5) LPS was used as an adjuvans. 10 days after immunization mice were sacrificed and splenocytes were isolated. To measure antigen specific T cell responses in VVT or LiMIT -/- mice, splenocytes were restimulated with ovalbumin (Figure 6C) or the OVA peptide SIINFEKL (Figure 6D) and T-cell proliferation was measured by H₃thymidin incorporation (Figure 6C). A significantly increased proliferative response was detected in LiMIT -/- mice compared to WT mice. Moreover, when LPS/OVA immunized LiMIT -/- mice were re- stimulated with the SIINFEKL peptide, T-cells showed a significantly higher secretion of IFNy. Next, cytokine production of CD3 stimulated T cells was measured. Increased levels of the Th17 cytokines IL- 7 (Figure 6F) as well as IL22 (data not shown) were detected. Proliferation of T cells after CD3 stimulation was not different, suggesting increased Th17 polarization of T cells. This suggests that LiMIT deficient mice display enhanced antigen specific T cell responses after immunization with antigen using a TLR ligand as adjuvant. In summary, LiMIT is an anti-inflammatory factor that restricts TLR induced inflammation, antigen presentation and subsequent T-cell activation.

The present invention refers to the following nucleotide and amino acid sequences: SEQ ID No. 1 :

Nucleotide sequence of human LiMIT (transcript variant 1 )

atgaggctgctcgcctggctgattttcctggctaactggggaggtgccagggctgaaccagggaagttctggcacatc gctgacctgcaccttgaccctgactacaaggtatccaaagaccccttccaggtgtgcccatcagctggatcccagcca gtgcccgacgcaggcccctggggtgactacctctgtgattctccctgggccctcatcaactcctccatctatgccatgaa ggagattgagccagagccagacttcattctctggactggtgatgacacgcctcatgtgcccgatgagaaactgggag aggcagctgtactggaaattgtggaacgcctgaccaagctcatcagagaggtctttccagatactaaagtctatgctgc tttgggaaatcatgattttcaccccaaaaaccagttcccagctggaagtaacaacatctacaatcagatagcagaact atggaaaccctggcttagtaatgagtccatcgctctcttcaaaaaaggtgccttctactgtgagaagctgccgggtccca gcggggctgggcgaattgtggtcctcaacaccaatctgtactataccagcaatgcgctgacagcagacatggcggac cctggccagcagttccagtggctggaagatgtgctgaccgatgcatccaaagctggggacatggtgtacattgtcggc cacgtgcccccggggttctttgagaagacgcaaaacaaggcatggttccgggagggcttcaatgaaaaatacctga aggtggtccggaagcatcatcgcgtcatagcagggcagttcttcgggcaccaccacaccgacagctttcggatgctct atgatgatgcaggtgtccccataagcgccatgttcatcacacctggagtcaccccatggaaaaccacattacctggag tggtcaatggggccaacaatccagccatccgggtgttcgaatatgaccgagccacactgagcctgaaggacatggt gacctacttcatgaacctgagccaggcgaatgctcaggggacgccgcgctgggagctcgagtaccagctgaccga ggcctatggggtgccggacgccagcgcccactccatgcacacagtgctggaccgcatcgctggcgaccagagcac actgcagcgctactacgtctataactcagtcagctactctgctggggtctgcgacgaggcctgcagcatgcagcacgt gtgtgccatgcgccaggtggacattgacgcttacaccacctgtctgtatgcctctggcaccacgcccgtgccccagctc ccgctgctgctgatggccctgctgggcctgtgcacgctcgtgctgtga

SEQ !D No. 2:

Amino acid sequence of human LiMIT (transcript variant 1 )

iviRLLAWLiFLANVvGGARAEPGKFvVHiADLHLDPDYKVSKDPFQVCPSAGSQPVPD

AGPWGDYLCDSPWALINSSIYAMKEIEPEPDFILWTGDDTPHVPDEKLGEAAVLEIV

ERLTKLIREVFPDTKVYAALGNHDFHPKNQFPAGSNNIYNQIAELWKPWLSNESIALF

KKGAFYCEKLPGPSGAGRIWLNTNLYYTSNALTADMADPGQQFQWLEDVLTDASK

AGDMVYIVGHVPPGFFEKTQNKAWFREGFNEKYLKWRKHHRVIAGQFFGHHHTD

SFRMLYDDAGVPISAMFITPGVTPWKTTLPGWNGANNPAIRVFEYDRATLSLKDMV

TYFMNLSQANAQGTPRWELEYQLTEAYGVPDASAHSMHTVLDRIAGDQSTLQRYY

VYNSVSYSAGVCDEACSMQHVCA RQVDIDAYTTCLYASGTTPVPQLPLLLMALLG

LCTLVL ςρη i n M , -¾·

Nucleotide sequence of murine LiMIT

atgacgctgctcgggtggctgatattcctggccccctggggagtcgcaggggctcaactagggaggttctggcacatct ccgacctgcatctggaccccaactacaccgtatccaaagaccccctccaggtgtgcccgtcggccggctcccagcct gtgctaaatgctggcccctggggggactacctctgcgattctccttgggcccttatcaactcgtccttgtatgccatgaag gagattgaaccaaagcctgacttcattctctggacaggggacgacacaccgcacgtccccaatgagagtctaggag aggcagctgtgctggcaattgtggaacgcttgaccaacctcatcaaggaagtctttccagacactaaagtctatgctgc tctgggaaatcatgacttccaccctaagaaccagttcccagcacagagcaaccgcatctataaccaggtggcagag ctgtggagaccctggcttagtaacgaatcctacgctctcttcaaaagaggtgccttctattctgagaagttgccgggtccc agcagggcggggcgagttgtggtcctcaacaccaatctgtactacagcaacaacgagcagacagctggcatggct gaccccggcgagcagttccggtggctgggagatgtcctgagcaatgcatctcgggatggggagatggtgtatgttatt ggccacgtgcccccggggttctttgagaagacacagaacaaggcctggttccgagagagcttcaatgaggagtatct gaaggtgatccagaagcaccatcgggtcatagcagggcagttctttggacaccaccataccgacagcttccgaatgtt ctatgacaacacaggtgcccccataaacgtcatgtttctcacacccggggtcacaccgtggaagaccacattacctgg agtggtcgatggggccaacaatccagggatacgcattttcgagtatgatcgagccacactcaacttgaaggacttggt gacttacttcttgaacctgaggcaggcgaatgtacaagagaccccacggtgggagcaggagtaccgcctgacgga ggcctaccaggtgccggatgccagcgtcagciccaigcacacggcgctgacccgcattgccagtgagcctcacatc ctgcaacgttattacgtctataactcggtcagctacaaccatttgacctgtgaggacagctgccgcatcgagcacgtttgt gctatacaacacgtggctttcaacacttatgctacctgcttgcatggtcttggtgccaagctggtgcctggtttcctgctcat actgactctgctgccaagcctgcacgtactggaggtgitatga

SEQ ID No. 4:

Amino acid sequence murine LiMIT

MTLLGWLIFLAPWGVAGAQLGRFWHISDLHLDPNYTVSKDPLQVCPSAGSQPVLNA

GPWGDYLCDSPWALINSSLYAMKEIEPKPDFILWTGDDTPHVPNESLGEAAVLAIVE

RLTNLIKEVFPDTKVYAALGNHDFHPKNQFPAQSNRIYNQVAELWRPWLSNESYAL

FKRGAFYSEKLPGPSRAGRVWLNTNLYYSNNEQTAGMADPGEQFRWLGDVLSNA

SRDGEMVYVIGHVPPGFFEKTQNKAWFRESFNEEYLKVIQKHHRVIAGQFFGHHHT

DSFRMFYDNTGAPINVMFLTPGVTPWKTTLPGWDGANNPGIRIFEYDRATLNLKDL

VTYFLNLRQANVQETPRWEQEYRLTEAYQVPDASVSSMHTALTRIASEPHILQRYY

VYNSVSYNHLTCEDSCRIEHVCAIQHVAFNTYATCLHGLGAKLVPGFLLILTLLPSLH

VLEVL

SEQ ID No. 5:

Nucleotide sequence of siRNA-01 (Thermo scientific, siGENOME SMARTpool M- 040463-01 -0005, Mouse SMPDL3B, NM_133888, 5 nmol)

CAAAAGAGGUGCCUUCUAU SEQ ID No. 6:

Nucleotide sequence of siRNA-02 (Thermo scientific, siGENOME SMARTpool M- 040463-01-0005, Mouse SMPDL3B, NM_133888, 5 nmol)

CGAGAGAGCUUCAAUGAGG

SEQ ID No. 7:

Nucleotide sequence of siRNA-03 (Thermo scientific, siGENOME SMARTpool M- 040463-01 -0005, Mouse SMPDL3B, NM_133888, 5 nmol)

AAUCAUGACUUCCACCCUA

SEQ ID No. 8:

Nucleotide sequence of siRNA-04 (Thermo scientific, siGENOME SMARTpool M- 040463-01 -0005, Mouse SMPDL3B, NM_133888, 5 nmol)

GCACGUACUGGAGGUGUUA

SEQ ID No. 9:

Nucleotide sequence of shRNA-1 (TRCN0000099681 ; NM_133888; shRNA Mm Lentiviral pLKO.1 ; RMM3981-98062179; Hairpin sequence for TRCN0000099681 ) CCGGGCAACGTTATTACGTCTATAACTCGAGTTATAGACGTAATAACGTTGCTTT

TTG

SEQ ID No. 10:

Nucleotide sequence of shRNA-2 (TRCN0000099683; NM 133888; shRNA Mm; Lentiviral; pLKO.1 RMM3981-98062195; hairpin sequence for TRCN0000099683) CCGGCCCAACTACACCGTATCCAAACTCGAGTTTGGATACGGTGTAGTTGGGTT TTTG