OF T CELLS

FIELD OF THE INVENTION:

The present invention relates to methods for screening drugs for reducing CD28 costimulation of T cells in a subject in need thereof.

BACKGROUND OF THE INVENTION:

T cell proliferation is the normal component of the immune reaction toward an antigen

(e.g. a pathogen antigen). However in certain circumstances T cell proliferation appears deleterious. For example, organ transplantation elicits a complex series of immunologic processes that are generally categorized as inflammation, immunity, tissue repair and structural reinforcement of damaged tissues. Typically T cell proliferation leads to inflammation by the secretion of proinflammatory cytokines, e.g., interleukin-2 (IL-2). Accordingly, the skilled man in the art has tried to develop immunosuppressive agents. In the "two-signal model" of T cell activation, the first signal is delivered via the T cell antigen receptor (TCR) following recognition of antigenic peptides bound to class I and II molecules of the major histocompatibility complex (MHC). This signal confers antigen specificity on T cell responses. The second "costimulatory" signal is delivered by the CD28 molecule upon binding of the CD80 and CD86 ligands on antigen presenting cells (APC). CD28 delivers signals via its cytoplasmic tail that has no intrinsic catalytic activity, but possesses several protein-protein interaction motifs enabling it to associate with effector molecules. By synergizing with the TCR, CD28 activates the classical NF-κΒ signaling pathway and leads to the transcription of genes involved in T cell survival and proliferation. TCR- CD28-induced NF-KB activation is thought to be triggered by the phosphorylation of the Carmal (Cardl l) adaptor by protein kinase C (PKC) family kinases, primarily PKC-Θ. This results in a conformational change in Carmal allowing it to recruit the ΙκΒ kinase complex and to stimulate the NF-κΒ signaling pathway. Accordingly, the selective inhibition of the agonist signal delivered to the T cell by CD28, for example by means of specific blocking of the CD28/CD80/CD86 interaction, has been explored in the prior art to prevent T lymphocyte activation, and thus to promote the induction of tolerance in the case of organ transplantation and also in the case of the treatment of autoimmune diseases. SUMMARY OF THE INVENTION:

The present invention relates to methods for screening drugs for reducing costimulation of T cells in a subject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

Using an N-ethyl-N-nitrosourea- mutagenesis screen, the inventors identified a mutation in the Rltpr gene. They demonstrated that Rltpr is a lymphoid cell-specific, actin- uncapping protein essential for CD28 costimulation and regulatory T cell development. TCR- CD28 engagement at the immune synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr forms. However, the connection between CD28 and protein kinase C-θ and Carmal, two key effectors of CD28 costimulation, was abrogated in T cells with the Rltpr mutation and CD28 costimulation did not occur. The findings provide a more complete model of CD28 costimulation in which Rltpr plays a key role. Accordingly, the present invention relates to a method for screening a plurality of test substances useful for reducing CD28 costimulation of T cells in a subject in need thereof comprising the steps consisting of al) testing each of the test substances for its ability to bind to a RLTR polypeptide and a2) positively selecting the test substances that are able to bind to the RLTR polypeptide.

As used herein the term "Rltpr" has its general meaning in the art and refers to RGD motif, leucine rich repeats, tropomodulin domain and proline-rich containing. The human exemplary amino sequence is SEQ ID NO: 1(RLTPR-001 ENST00000334583 http://www.ensembl.org/Homo_sapiens/Info/Index) and the murine exemplary amino sequence is SEQ ID NO: 2.

SEQ ID NO: l :

MAQTPDGISCELRGEITRFLWPKEVELLLKTWLPGEGAVQNHVLALLRWRAY

LLHTTCLPLRVDCTFSYLEVQAMALQETPPQVTFELESLRELVLEFPGVAALE

QLAQHVAAAIK VFPRSTLGKLFRRPTPASMLARLERSSPSESTDPCSPCGGFL

ETYEALCDYNGFPFREEIQWDVDTIYHRQGCRHFSLGDFSHLGSRDLALSVAA

LSYNLWFRCLSCVDMKLSLEVSEQILHMMSQSSHLEELVLETCSLRGDFVRRL

AQALAGHSSSGLRELSLAGNLLDDRGMTALSRHLERCPGALRRLSLAQTGLTP

RGMRALGRALATNAAFDSTLTHLDLSGNPGALGASEDSGGLYSFLSRPNVLSF LNLAGTDTALDTVRGCSVGGWMTGRADWRAGRGGLGPPAGVANSLPPQLF AAVSRGCCTSLTHLDASRNVFSRTKSRAAPAALQLFLSRARTLRHLGLAGCKL PPDALRALLDGLALNTHLRDLHLDLSACELRSAGAQVIQDLVCDAGAVSSLD LADNGFGSDMVTLVLAIGRSRSLRHVALGRNFNVRCKETLDDVLHRIVQLMQ DDDCPLQSLSVAESRLKLGASVLLRALATNPNLTALDISGNAMGDAGAKLLA KALRVNSRLRSVVWDRNHTSALGLLDVAQALEQNHSLKAMPLPLNDVAQAQ RSRPELTARAVHQIQACLLRNNRADPASSDHTTRLQPLGLVSDPSEQEVNELC QSVQEHVELLGCGAGPQGEAAVRQAEDAIQNANFSLSILPILYEAGSSPSHHW QLGQKLEGLLRQVGEVCRQDIQDFTQATLDTARSLCPQMLQGSSWREQLEGV LAGSRGLPELLPEQLLQDAFTRLRDMRLSITGTLAESIVAQALAGLSAARDQL VESLAQQATVTMPPALPAPDGGEPSLLEPGELEGLFFPEEKEEEKEKDDSPPQK WPELSHGLHLVPFIHSAAEEAEPEPELAAPGEDAEPQAGPSARGSPSPAAPGPP AGPLPRMDLPLAGQPLRHPTRARPRPRRQHHHRPPPGGPQVPPALPQEGNGLS ARVDEGVEEFFSKRLIQQDRLWAPEEDPATEGGATPVPRTLRKKLGTLFAFK PRSTRGPRTDLETSPGAAPRTRKTTFGDLLRPPTRPSRGEELGGAEGDTSSPDP AGRSRPRYTRDSKAYSMILLPAEEEATLGARPDKRRPLERGETELAPSFEQRV QVMLQRIGVSRGSGGAEGKRKQSKDGEIKKAGSDGDIMDSSTEAPPI SIKSRTHSVSADPSCRPGPGSQGPESATWKTLGQQLNAELRSRGWGQQDGPGPPSPG QSPSPCRTSPSPDSLGLPEDPCLGPRNEDGQLRPRPLSAGRRAVSVHEDQLQAPAERPL RLQRSPVLKRRPKLEAPPSPSLGSGLGTEPLPPQPTEPSSPERSPPSPATDQRGGGPNP

SEQ ID NO:2: Mouse Rltpr mRNA sequence: Accession#: HF678090 http://www.ebi.ac.uk/ena/data/view/HF678090

1 MAQT PDDI SC ELRGE I TRFL WPKEAELLLK TWLPQEGAEQ SH I LALLRWR AYLLHTCLPL

61 RVDCTFSYLE VQAMALQET P PRVTFELE SL PELVLEFPCV AALEQLAQHV AAAI KKVFPR

12 1 STLGKLFRKP T PS SLLARLE RSHPLE ST I P S S PCGGFLET YEALCDYNGF PFREE I QWDV

1 8 1 DT I YHRQGCR HFCLGDFSHF GSRDLALSVA ALSYNLWFRR LSCEDMKLSL EVSEQ I LHMT

2 4 1 SQS SYLEELV LEACGLRGDF VRRLAQALAG HFNSGLRELS LSGNLLDDRG MAALSRHLEH

3 0 1 CPGALRRLSL AQTGLT PRGM RALGRALATN ATFDSTLTHL DLSGNPGALG PSQDSGGLYT

3 61 FLSRPNVLAY LNLAGTDATL GTLFTALAGG CCS SLTHLEA SRNI FSRMKS QAAPAALQRF

42 1 LGGTRMLRHL GLAGCKLPPE ALRALLEGLA LNTQ I HDLHL DLSACELRSV GAQVI QDLVC 4 8 1 DAGALS SLDL SDNGFGSDMV TLVLAI GRSR SLKHVALGRN FNVRCKETLD DVLHRIAQLM 541 QDDDCPLQSL SVAESRLKQG ASILIRALGT NPKLTALDIS GNAIGDAGAK MLAKALRVNT

601 RLRSVIWDRN NTSALGLLDV AQALEQNHSL KSMPLPLNDV TQAHRSRPEL TTRAVHQIQA

661 CLWRNNQVDS TSDLKPCLQP LGLISDHSEQ EVNELCQSVQ EHMELLGCGA GPQGEVAVHQ

721 AEDAIQNANF SLSILPILYE AGRSPSHHWQ LQQKLESLLG QVGEICRQDI QDFTQTTLDT

781 TRSLCPQMLQ TPGWRKQLEG VLVGSGGLPE LLPEHLLQDA FSRLRDMRLS ITGTLAESIV

841 AQALAGLHAA RDRLVERLTQ QAPVTMAPAV PPLGGNELSP LETGGLEELF FPTEKEEERE

901 KDESSSWKWL EPSNCFHLVS SLHGAAEEAE RDPELAAPGE DAEPQAGPSA RGSPSPAAPG

961 PPAGPLPRMD LPPAGQPLRH PTRARPRPRR QHHHRPPPGG PQVPPALLQE GNGLTARVDE

1021 GVEEFFSKRL IQQDHFWAPE EDPATEGGAT PVPRTLRKKL GTLFAFKKPR STRGPRPDLE

1081 SPGAAARAR KSTLGDLLRP PARPGRGEEP GGAEGGTSSP DPARRNRPRY TRESKAYSMI

1141 LLPAEEEAAV GTRPDKRRPL ERGDTELAPS FEQRVQVMLQ RIGVSRASGG AESKRKQSKD

1201 GEIKKAGSDG DIMDSSTETP PISIKSRTHS VSADPSCRPG PGGQGPESAT WKTLGQQLNA

1261 ELRGRGWGQQ DGPGPPSPCP SPSPRRTSPA PDILSLPEDP CLGPRNEDGQ LRPRPLSAGR

1321 RAVSVHEDQL QAPAERPLRL QRSPVLKRRP KLEAPPSPSL GSGLGSKPLP PYPTEPSSPE

1381 RSPPSPATDQ RGGGPNP* 1397

The term "polypeptide" means herein a polymer of amino acids having no specific length. Thus, peptides, oligopeptides and proteins are included in the definition of "polypeptide" and these terms are used interchangeably throughout the specification, as well as in the claims. The term "polypeptide" does not exclude post-translational modifications that include but are not limited to phosphorylation, acetylation, glycosylation and the like. Especially, the term includes all phosphorylated forms of the polypeptide (e.g. all phosphorylated forms of Rltpr). Also encompassed by this definition of "polypeptide" are homologs thereof. Two amino acid sequences are "substantially homologous" or "substantially similar" when greater than 80 %, preferably greater than 85 %, preferably greater than 90 % of the amino acids are identical, or greater than about 90 %, preferably greater than 95 %, are similar (functionally identical). The term "sequence identity" refers to the identity between two peptides. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of sequence identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acid sequences that are shared between these sequences. To determine the percent identity of two amino acids sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino add sequence or a first nucleic acid sequence for optimal alignment with the second amino acid sequence or second nucleic acid sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. In this comparison the sequences can be the same length or may be different in length. Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (J. Theor. Biol, 91 (2) pgs. 370-380 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Miol. Biol, 48(3) pgs. 443-453 (1972), by the search for similarity via the method of Pearson and Lipman, PNAS, USA, 85(5) pgs. 2444-2448 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetic Computer Group, 575, Science Drive, Madison, Wis.) or by inspection. The term "sequence similarity" means that amino acids can be modified while retaining the same function. It is known that amino acids are classified according to the nature of their side groups and some amino adds such as the basic amino acids can be interchanged for one another while their basic function is maintained. Accordingly, the term "Rltpr polypeptide" refers to the Rltpr protein or a fragment thereof that comprises the region around the amino acid residue at position 469 ins SEQ ID NO: l or the amino acid residue at 432 in SEQ ID NO:2 (both residues are in highlighted in bold). Typically a Rltpr polypeptide comprises the domain consisting of the eight leucine repeat regions (LRR) ranging from the amino acid residue at position 102 to the amino acid residue at position 677 in SEQ ID NO : 1 or ranging from the amino acid residue at position 65 to the amino acid residue at position 650 in SEQ ID NO : 2. Polypeptides of the invention may be produced by any technique known per se in the art, such as without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination(s).

Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions.

Alternatively, the polypeptides of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

A wide variety of host/expression vector combinations are employed in expressing the nucleic acids encoding for the polypeptides of the present invention. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col EI, pCRl, pBR322, pMal-C2, pET, pGEX, pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM989, as well as other phage DNA such as Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2 microns plasmid or derivatives of the 2 microns plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or the expression control sequences; and the like.

Consequently, mammalian and typically human cells, as well as bacterial, yeast, fungi, insect, nematode and plant cells an used in the present invention and may be transfected by the nucleic acid or recombinant vector as defined herein. Examples of suitable cells include, but are not limited to, VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines such as ATCC No. CCL61, COS cells such as COS-7 cells and ATCC No. CRL 1650 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361, A549, PC12, K562 cells, 293T cells, Sf9 cells such as ATCC No. CRL1711 and Cvl cells such as ATCC No. CCL70. Other suitable cells that can be used in the present invention include, but are not limited to, prokaryotic host cells strains such as Escherichia coli, (e.g., strain DH5-[alpha]), Bacillus subtilis, Salmonella typhimurium, or strains of the genera of Pseudomonas, Streptomyces and Staphylococcus. Further suitable cells that can be used in the present invention include yeast cells such as those of Saccharomyces such as Saccharomyces cerevisiae.

Typically, step al) consists in generating physical values which illustrate or not the ability of said test substance to bind the Rltpr polypeptide. The "physical values" that are referred to above may be of various kinds depending of the binding assay that is performed, but notably encompass light absorbance values, radioactive signals and intensity value of fluorescence signal. The test substances may be assayed for binding to Rltpr polypeptide by method known in the art. Many different competitive binding assay format(s) can be used. For example, the binding assays include, but are not limited to, competitive assay systems using techniques such western blots, radioimmuno assays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York).

For example, the BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed. For example, step a) may include the use of an optical biosensor allowing the detection of interactions between molecules in real time, without the need of labelled molecules. This technique is indeed based on the surface plasmon resonance (SPR) phenomenon. Briefly, the Rltpr polypeptide may be attached to a surface (such as a carboxymethyl dextran matrix). Then the test substance is incubated with the previously immobilised polypeptide. Then the binding including the binding level, or the absence of binding is detected. For this purpose, a light beam is directed towards the side of the surface area where the polypeptide is attached and is reflected by said surface. The SPR phenomenon causes a decrease in the intensity of the reflected light with a combination of angle and wavelength. The binding of the test substance to the polypeptide causes a change in the refraction index on the substrate surface, which change is detected as a change in the SPR signal. In another one embodiment of the invention, the screening method includes the use of affinity chromatography.

In some embodiment, the screening method of the invention further comprise the steps consisting of bl) testing each of the substances positively selected at step a2) for its ability to reduce the CD28 costimulation signalling in a T cell and b2) positively selecting the test substances that are able to reduce the CD28 costimulation in a T cell.

Typically, step bl) may be performed by any in vitro or in vivo assay that the skilled man in the art can settle for testing the ability of the test substance to reduce the CD28 costimulation signalling in a T cell.

For example, the proliferation of a T cell after CD28-TCR co-stimulation may be assayed in the presence of the test substance. Typically, the skilled man in the art may used the method described in the EXAMPLE. Briefly, T cells are activated with a combination of anti-CD3 antibodies and anti-CD28 antibodies in the presence of the test substance. The proliferation level is then determined and is compared to the level determined in the absence of the test substance. It is concluded that the test substance reduce the CD28 costimulation signalling when the proliferation level determined in the presence of the test substance is lower that the proliferation level determined in the absence of the test substance.

Altenatively, the screening method may also comprise a step consisting of determining the ability of the test substance to prevent CD28 synergizing with the TCR in assays measuring IL-2 production and IFN-γ production by primary T cells. It is concluded that the test substance is able to inhibit the CD28 co-stimulation signal when the IL-2 production and/or the IFN-γ production determined in the presence of the test substance is lower when the IL-2 production and/or the IFN-γ production determined in the absence of the test substance. In some embodiments, the screening method of the invention may further comprise a step consisting of providing an animal (e.g. a mouse) having T cells expressing a TCR specific for an antigen, administering the animal with an amount of antigen and with an amount of the test substance, determining the production of IL-2 and/or of IFN-γ by the T cells expressing the TCR specific for the antigen, comparing production of IL-2 and/or of IFN-γ determined at the preceding step with the production determined in an animal that was not administered with the test substance and positively selecting the test substance when the production of IL-2 and/or of IFN-γ determined when the animal was administered with the test substance is lower than the production of IL-2 and/or of IFN-γ determined when the animal was not administered with the test substance. Typically, OT-I mice expressing a TCR specific for the OVA257-264 peptide bound to H-2K as described in the EXAMPLE may be used.

In some embodiment, the test substance may be evaluated for its ability to induce the functional defects as observed for T cells deficient for CD28 or for T cells harbouring a Rltpr loss of function mutation such as observed for Rltpr ^BAS mouse. For example, the proliferation level determined in the presence of the test substance may be compared with the proliferation level of purified T cells from Rltpr ^Bas or Cd28-/- mice as described in the EXAMPLE. Then the test substance may be selected when the proliferation level determined in the presence of the test substance is above the same as the proliferation level determined for the purified T cells from Rltpr ^Bas or Cd28-/- mice.

In some embodiments, the screening method may further comprises the steps consisting of administering an animal (e.g. a mouse) with an amount of the test substance, then determining the frequency of thymic natural Treg cells (i.e. Foxp3+ Treg cells) in the animal, comparing the frequency with the frequency of thymic natural Tregs determined in an animal that was not administered with the test substance and positively selecting the test substance when the frequency determined in the animal administered with the test substance is lower than the frequency determined in the animal that was not administered with the test substance.

In some embodiments, the screening method may further comprises the steps consisting of administering an animal (e.g. a mouse) with an amount of the test substance, then determining the frequency of thymic natural Treg cells (i.e. Foxp3+ Treg cells) in the animal, comparing the frequency with the frequency of thymic natural Tregs determined in a CD28 deficient animal or in a Rltpr ^Bas animal and positively selecting the test substance when the frequency determined in the animal administered with the test substance above the same as determined for the CD28 deficient animal or for the Rltpr ^Bas animal. In some embodiments, the screening method may further comprises the steps consisting of administering an animal (e.g. a mouse) with an amount of the test substance, then determining the frequency of TFH cells (e.g. CXCR5+PD1+ TFH cells) in the animal, comparing the frequency with the frequency of TFH cells determined in an animal that was not administered with the test substance and positively selecting the test substance when the frequency determined in the animal administered with the test substance is lower than the frequency determined in the animal that was not administered with the test substance.

In some embodiments, the screening method may further comprises the steps consisting of administering an animal (e.g. a mouse) with an amount of the test substance, then determining the frequency of TFH cells (e.g. CXCR5+PD1+ TFH cells) in the animal, comparing the frequency with the frequency of TFH cells determined in a CD28 deficient animal or in a Rltpr ^Bas animal and positively selecting the test substance when the frequency determined in the animal administered with the test substance above the same as determined for the CD28 deficient animal or for the Rltpr ^Bas animal.

In some embodiment, the screening method of the invention further comprises a step consisting of determining the ability of the test substance to inhibit the recruitment of PKC-Θ and Carmal at the T cell immunological synapse. Said recruitment may be assayed by any method known in the art. For example, the assay described in the EXAMPLE may be performed. Briefly, CD4+ T cells expressing PKC-9-EGFP or Carmal-EGFP are imaged using planar bilayers containing I-Ek-MCC88-103, ICAM-1 and CD80 in presence of the test substance. Then the accumulation of either PKC-Θ or Carmal at the cSMAC is then evaluated and it is concluded that the test substance is able to inhibit the recruitment of PKC-Θ and Carmal at the T cell immunological synapse when no accumulation is determined.

The test substances that have been positively selected at the end of any one of the embodiments screening method which has been described previously in the present specification may be subjected to further selection steps in view of further assaying their in vivo properties on animal model for a disease of interest or eventually in human in the context of clinical trials. Indeed the test substances that may be selected at the end of any one of the embodiments of the in vitro screening may find various applications.

For example, the test substances may constitute drug candidate for transplantation. For example, the selected test substance may be assayed for their ability to prevent or suppress immune responses associated with rejection of a donor tissue, cell, graft, or organ transplant by a recipient subject. Graft-related diseases or disorders include graft versus host disease (GVDH), such as associated with bone marrow transplantation, and immune disorders resulting from or associated with rejection of organ, tissue, or cell graft transplantation (e.g., tissue or cell allografts or xenografts), including, e.g., grafts of skin, muscle, neurons, islets, organs, parenchymal cells of the liver, etc. With regard to a donor tissue, cell, graft or solid organ transplant in a recipient subject, it is believed that the selected substances may be assayed to prevent acute rejection of such transplant in the recipient and/or for long-term maintenance therapy to prevent rejection of such transplant in the recipient (e.g., inhibiting rejection of insulin-producing islet cell transplant from a donor in the subject recipient suffering from diabetes). Thus the selected test substance may be useful for preventing Host- Versus-Graft-Disease (HVGD) and Graft- Versus-Host-Disease (GVHD). The CTPS1 inhibitor may be administered to the subject before and/or after transplantation (e.g., at least one day before transplantation, from one to five days after transplantation, etc.). In some embodiments, the CTPS1 inhibitor may be administered to the subject on a periodic basis before and/or after transplantation.

The test substances may also constitute drug candidate for the treatment of autoimmune diseases. As used herein, an "autoimmune disease" is a disease or disorder arising from and directed at an individual's own tissues. Examples of autoimmune diseases include, but are not limited to Addison's Disease, Allergy, Alopecia Areata, Alzheimer's disease, Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, Ankylosing Spondylitis, Antiphospho lipid Syndrome (Hughes Syndrome), arthritis, Asthma, Atherosclerosis, Atherosclerotic plaque, autoimmune disease (e.g., lupus, RA, MS, Graves' disease, etc.), Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Autoimmune inner ear disease, Autoimmune Lymphoproliferative syndrome, Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Orchitis, Azoospermia, Behcet's Disease, Berger's Disease, Bullous Pemphigoid, Cardiomyopathy, Cardiovascular disease, Celiac Sprue/Coeliac disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic idiopathic polyneuritis, Chronic Inflammatory Demyelinating, Polyradicalneuropathy (CIPD), Chronic relapsing polyneuropathy (Guillain-Barre syndrome), Churg-Strauss Syndrome (CSS), Cicatricial Pemphigoid, Cold Agglutinin Disease (CAD), COPD, CREST syndrome, Crohn's disease, Dermatitis, Herpetiformus, Dermatomyositis, diabetes, Discoid Lupus, Eczema, Epidermolysis bullosa acquisita, Essential Mixed Cryoglobulinemia, Evan's Syndrome, Exopthalmos, Fibromyalgia, Goodpasture's Syndrome, Hashimoto's Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, immunoproliferative disease or disorder (e.g., psoriasis), Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, Insulin Dependent Diabetes Mellitus (IDDM), Interstitial lung disease, juvenile diabetes, Juvenile Arthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease, Lambert-Eaton Myasthenic Syndrome, Lichen Planus, lupus, Lupus Nephritis, Lymphoscytic Lypophisitis, Meniere's Disease, Miller Fish Syndrome/acute disseminated encephalo myeloradiculopathy, Mixed Connective Tissue Disease, Multiple Sclerosis (MS), muscular rheumatism, Myalgic encephalomyelitis (ME), Myasthenia Gravis, Ocular Inflammation, Pemphigus Foliaceus, Pemphigus Vulgaris, Pernicious Anaemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes (Whitaker's syndrome), Polymyalgia Rheumatica, Polymyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis/Autoimmune cholangiopathy, Psoriasis, Psoriatic arthritis, Raynaud's Phenomenon, Reiter's Syndrome/Reactive arthritis, Restenosis, Rheumatic Fever, rheumatic disease, Rheumatoid Arthritis, Sarcoidosis, Schmidt's syndrome, Scleroderma, Sjorgen's Syndrome, Stiff-Man Syndrome, Systemic Lupus Erythematosus (SLE), systemic scleroderma, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Thyroiditis, Type 1 diabetes, Type 2 diabetes, Ulcerative colitis, Uveitis, Vasculitis, Vitiligo, and Wegener's Granulomatosis. The test substance of may be selected from the group consisting of peptides, peptidomimetics, small organic molecules, or nucleic acids. For example the test substance according to the invention may be selected from a library of compounds previously synthetized, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthetized de novo. In a particular embodiment, the test substance may be selected form small organic molecules. As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. EXAMPLE:

Material & Methods

Mice. To facilitate the mapping of mutations abrogating the Lat ^Y136F lymphoproliferative disorder, mice homozygous for a Lat ^Y136F mutant allele were constructed on a pure B6 background using Bruce 4 embryonic stem cells. They showed the same phenotype as that of the Lat ^Y136F mice that were previously established on a 129 background (data not shown).

5C.C7, AND and OT-I TCR transgenic mice, Cd28^ , Cd3e ^{5/ 5} and Cd3e ^{5/ 5} x

/ / 17 27 29 51 52

Cd8(F ^~ Cd&6— mice have been described ¹ '' ^J1 ' . Mice were maintained in specific pathogen- free conditions and all experiments were done in accordance with French and European guidelines for animal care. ENU mutagenesis and screening of mutant mice. The ENU mutagenesis screen was performed in a B6. Lat ^Y136F background as previously described ⁵³ . Mutant mice were identified using blood samples collected from the retro-orbital sinus of sedated mice using heparinized microhematocrit tubes. Staining was performed on 30 μΐ of whole blood using Trucount tubes (BD Biosciences) and a combination of 8 antibodies consisting of PerCP- Cy5.5 conjugated anti-CD45.2 (104), APC conjugated anti-CD5 (PK136), PE-Cy7 conjugated anti-CD 19 (1D3/6D5), Pacific Blue conjugated anti-CD4 (RM4-5), Alexa-700 conjugated anti-CD8a (53.6.7), PE conjugated anti-CD62L (MEL-14), FITC conjugated CD44 (IM7) and Alexa-700 conjugated anti-IA/IE (M5/114.15.2) all from BD Biosciences. A lyse/no- wash procedure was performed using a BD FACS lysing solution (BD Biosciences). For each sample, a minimum of 30 000 CD45.2 ⁺ cells were analyzed. The absolute number of CD19 ⁺ B cells and of CD4 ⁺ and CD8 T cells per μΐ of blood was calculated using the formula: cell counts/bead counts x total Trucount beads/μΐ of blood.

Targeted sequencing and identification of genetic variation. The genetic variations induced by ENU-mutagenesis in the Basilic Lat ^Y136F mice were analyzed using targeted next- generation sequencing ⁵⁴ , adapted for a SOLiD™ 4 sequencer. Briefly, short fragments (-150 nucleotide-long) from paired-end libraries were prepared using focussed acoustic fragmentation (Covaris TM S2) and according to the preparation guide from Life Technologies (cms_081748.pdf). Two custom-designed AgilentTM SurePrint G3 Mouse DNA Capture lxlM Arrays were designed to cover the targeted 16 Mb region on chromosome 8 and used to perform the library enrichment according to AgilentTM protocol #G4458-90000. Templated beads were prepared using the SOLiD™ EZ Bead™ System (EZ Bead E80) and sequenced according to standard Applied Biosystems Life Technologies protocols. The sequence reads were analyzed with the BioScope™ software suite (cms_4448431.pdf). SNPs and Small InDels were identified using the Genome Variant Analyzer pipeline developed at TGML Sequencing Platform (GeVarA). Flow cytometry. Stained cells were analyzed using a BD LSR II system (BD

Biosciences). Data were analyzed with Flow Jo software (Tree Star) or FCS Express™ 4, Diva. Cell viability was evaluated using SYTOX Blue (Life Technologies). Antibodies used were directed to CD5 (53-7.3), CD4 (RM4-5), CD8a (53-6.7), CD69 (H1.2F3), CD24 (Ml/69), TCR (H57-597), CD28 (37.51) and CXCR5 (2G8) all from BD Biosciences; to CD25 (PC61.5), CD279 (PD1, 29F.1A12) all from BioLegend and to GITR (DTA-1), CD 122 (TM- Bl) and FoxP3 (FJK-16s) all from eBioscience. CellTrace™ CFSE proliferation kit was purchased from Molecular Probes™.

Plasmids and cell transfection. Full-length cDNA encoding Rltpr and Rltpr ^as were appended at their 3 ' end to sequences coding for a One-Strep-Tag (OST) or a Yellow Fluorescent Protein for Energy Transfer (YPET) reporter. The Rltpr-OST and Rltpr ^Ba -OST cDNA were cloned into the pMX-IRES-eGFP retroviral expression vector ⁵⁵ , whereas the Rltpr-YPET and Rltpr ⁸ ™ '-YPET were cloned into the pMSCV-IRES-hCD2t retroviral expression vector ⁵⁶ . PLAT-E cells were used to produce retroviral particles ⁵⁷ . 48 h after transfection, viral supernatants were harvested and used to transduce T cell hybridoma or briefly activated CD4 T cells from AND or 5C.C7 mice.

T cell isolation and stimulation. CD5 , CD4 or CD8 T cells were purified from pooled lymph nodes and spleens of WT, Rltpr ^Bas and Cd28 ^~ ' ^~ mice using mouse depletion Dynabeads untouched kits (Life Technologies) with a cell purity of over 90%. Purified cells were stimulated with plate-bound anti-CD3 antibody (145-2C11) with or without soluble anti- CD28 antibody (37-51) for 72 h. Alternatively, phorbol ester (PMA 10 ng/ml, Sigma- Aldrich) plus ionomycine (500 ng/ml, Sigma-Aldrich) were used to stimulate purified cells. Supernatants were collected at the indicated times and IL-2 and IFNy concentration were measured using BD™ Cytometric Bead Array or Milliplex Map mouse kit (Merck Millipore). Generation of antibodies against Rltpr. Polyclonal antisera against Rltpr were generated by immunizing rabbits with a glutathione S-transferase to which was appended a segment of Rltpr corresponding to aminocids 1147-1397.

Western blot and immunoblot analyses. Thymocytes and peripheral T cells were lysed by addition of lysis buffer (100 mM Tris pH 7.5, 274 mM NaCl, 1 mM EDTA, 10% glycerol, 0.2% n-Dodecyl- -Maltoside). Postnuclear supernatants were separated on 8% SDS- acrylamide gels, transferred to membrane and subjected to immunoblot analysis using antibodies against Rltpr (see above) or SLP-76 (4958, Cell Signaling). Membranes were incubated with anti-rabbit IgG (CF770, Biotium) and analyzed with an Odyssey Imaging System (LI-COR). The BI-141 T cell hybridoma ⁵⁸ was retroinfected with an empty virus or with viruses expressing Rltpr-OST or Rltpr ^Bas -OST. Cell lysates were immunoprecipitated with Strep-Tactin sepharose beads (IBA GmbH). Beads were eluted with D-Biotin (Sigma- Aldrich). Eluates were separated on 8% SDS-acrylamide gels and subjected to immunoblot analysis using polyclonal rabbit antibodies specific for Rltpr and for the alpha subunit of CP (AB6016; Millipore).

Imaging at T cell-APC interfaces. Image acquisition and analysis were performed as described ³⁸ . Briefly, T cell-APC interactions were imaged at 37°C. Every 20 seconds, 1 DIC and 21 fluorescence images that spanned 20 μιη in the z-plane at 1 μιη intervals were acquired. The acquisition and analysis software was Metamorph (Molecular Devices). The formation of a tight cell couple, time 0 in our analysis, was defined as either the first time point with a fully spread T cell-APC interface or 40 s after first membrane contact, whichever occurred first. A region of sensor accumulation was defined by an average fluorescence intensity of >135% of the background cellular fluorescence. To classify spatial accumulation features, six mutually exclusive interface patterns were used as defined by strict geometrical constraints ³⁸ . Imaging at T cell-planar bilayer interface. Planar bilayers construction, confocal

9, 59 microscopy imaging and image processing and data analysis have been previously defined

CD28 internalization assay. Cells were incubated for 30 min on ice with anti-CD28 antibody (1 Mg/ml) followed by biotinylated goat anti-mouse antibody (2 g/ml) for another 30 min on ice. Cells then were washed and warmed to 37°C for 5, 10, or 15 min to allow internalization and then treated with 0.1% NaN ₃ on ice. Cells were stained with PE- conjugated streptavidin, washed, and subjected to flow cytometry analysis (FACSCalibur; Becton Dickinson). The data were expressed as the percentage change in surface level CD28 over the time course.

Statistical analysis. The unpaired Student t test was used for statistical analyses with GraphPad Prism software. The p values were calculated by t test: *p, <0.05, **p, <0.01, ***/?,<0.001 and <0.0001. All data are presented as the arithmetic mean ± SEM or SD as indicated.

Results

The Basilic Lat ^{Y 6F} phenotype

We conducted an ENU- induced mutagenesis screen on C57BL/6J (B6) mice homozygous for the Lat ^Y136F mutation to identify mutations capable of abrogating their lymphoproliferative disorder. Compared to wild-type (WT) mice, the blood of Lat ^Yi36F mice contains large numbers of abnormal CD4 ⁺ T cells that show a CD44 ^high CD62L ^low T _H 2 effector phenotype, and normal numbers of B cells that show an activated phenotype resulting from the action of the T _R 2 effectors. By screening the blood of Lat ^Yi36F G3 offspring, we identified a mouse denoted Basilic Lat ^Yi36F , the blood of which contained 500- fold fewer CD44 ^high CD62L ^low CD4 ⁺ T cells. The B cells found in the blood of the Basilic Lat ^Y136F mouse had a resting phenotype, an observation consistent with the near absence of T _H 2 effectors. A similar phenotype was also observed in the spleen. Therefore, the Basilic mutation abrogated the expansion of pathogenic Lat ^Y136F CD4 ⁺ T cells and the ensuing exaggerated B cell activation.

Characterization of the Basilic mutation The Basilic mutation was fixed in the Lat background and inherited as a fully penetrant recessive trait. To map it, we screened F2 mice from Basilic Lat ^Yi36F B6 mice x C3HeB/FeJ (C3H) intercrosses. 12 homozygous Lat ^Yi36F mice with a Basilic phenotype and 10 homozygous Zat ^Y136F mice without a Basilic phenotype were genotyped with a panel of 64 simple sequence length polymorphism (SSLP) markers corresponding to 2 to 6 SSLP markers per chromosome. This analysis revealed a linkage to chromosome 8 with the highest lod score of 3.97 associated with a marker (D8mit242) corresponding to position 50,07 cM . High-resolution haplotype mapping revealed that the Basilic mutation was localized in a 16-megabase (Mb) interval of chromosome 8. These 16 Mb of DNA were captured from Basilic Lat ^Yi36F mice using specially designed oligonucleotide arrays and then sequenced by next-generation sequencing. After comparison with a B6 reference sequence 3 single nucleotide polymorphism (SNP) were located within the coding regions of 3 distinct genes. Further analysis, excluded two of the three candidate genes and left us with a single coding region-associated SNP that involved the RGD, leucine-rich repeat, tropomodulin and proline- rich containing (Rltpr) gene. Rltpr is also known as leucine-rich repeat (LRR) containing 16c (Lrrcl6c).

Sequencing a full-length Rltpr cDNA isolated from B6 thymocytes and determining the exon-intron structure of the B6 Rltpr gene led us to re-annotate the available mouse sequence (ENSMUSG00000050357; http://mouse.ensembl.org/). The Rltpr gene is composed of 39 exons and codes for a 1397 amino acid protein. Consistent with this analysis, immunoblots of thymic and peripheral T cells with Rltpr-specific rabbit polyclonal antibodies detected a single protein species of approximately 150 kilodaltons. The mutation identified in the Rltpr gene of Basilic Lat ^{Y 6F} mice is denoted as C57BL/6-i?/t/?r ^Bas (Rltpr ^3*8 in short). It affected exon 16 and corresponded to a T to C nucleotide transition at position 108,213,695.

Rltpr ^Bas is a partial loss of function mutation

The mouse genome contains two Rltpr paralogs that are denoted as Lrrcl6a and Lrrl6b ²⁰ . Orthologs of the Lrcc gene family can be found in vertebrates and invertebrates (Ensembl GeneTree ENSGT00390000014487). The vertebrate Lrcc gene family codes for cytosolic proteins that comprise concatenated LRRs, a capping protein (CP) interaction (CPI) motif and a region rich in proline residues. In its free form, CP has an affinity of 0.1-1 nM for the barbed ends of actin filaments. Upon binding to the CPI motif, the affinity of CP for actin filaments decreases by approximately 100-fold. It has thus been inferred that the whole vertebrate Lrcc gene family codes for actin-uncapping proteins that uncap actin filaments pre- capped by CP ^21"23 .

The eighth LRR found in Rltpr comprises the 12-residue long LRR consensus sequence ²⁴ . The non conservative L432P substitution found in Rltpr ^Bas likely disrupted the structure of the eighth LRR. This may account for the 1.5- and 2.5-fold decreased levels of Rltpr ^Bas observed in thymocytes and peripheral T cells, respectively, as compared to Rltpr. To verify the prediction that Rltpr is capable of constitutively associating with CP, Rltpr was fused to a One-Strep-Tag (OST) ²⁵ and expressed in a T cell hybridoma. Pull-down experiments demonstrated that Rltpr constitutively associated with CP. Importantly, an Rltpr ^Bas -OST fusion protein was also capable of associating with CP. Therefore, the Rltpr ^Bas mutation preserved the actin- uncapping function of Rltpr and thus corresponded to a partial loss of function mutation of the Rltpr gene. Rltpr expression

We analyzed Rltpr expression in thymus, spleen, bone marrow (BM), brain and testis by quantitative RT-PCR analysis. Rltpr was expressed in large amounts in adult thymus and spleen, smaller amounts in BM and was undetectable in brain and testis. The Rltpr ^Bas mutation was without effect on the pattern and levels of transcription (data not shown). The BioGPS gene expression atlas database (http://biogps.org/) confirmed that Rltpr expression was restricted to lymphoid tissues. In contrast, the Lrrcl6a and Lrrcl6b mouse paralogs were predominantly expressed innon-lymphoid tissues (http://biogps.org .

Quantitative RT-PCR analysis of thymocyte subsets showed that Rltpr expression was upregulated in preselection CD69 DP cells and down-regulated in post-positive selection CD69 ⁺ DP cells and in CD4 ⁺ and CD8 ⁺ single positive thymocytes. Peripheral CD4 ⁺ and CD8 ⁺ T cells also expressed substantial amounts of Rltpr, a pattern consistent with the Immgen database (www.immgen.org and immunoblot analysis. Therefore, Rltpr is an actin-uncapping protein that is predominantly expressed in T cells.

Segregating Rltpr from La? ^libt mimicks a CD28 deficiency

Mice with a compound Rltpr ^Bas Lat ^Y136F mutation showed a phenotype highly reminiscent of that of Lat ^Y136F mice deprived of CD80 and CD86 ligands ¹⁷ . This led us to test whether Rltpr belonged to the signaling pathway responsible for CD28 costimulation. For that purpose, we segregated the Rltpr ^as mutation from the Lat mutation and determined whether mice homozygous for the sole Rltpr ^Bas mutation - called Rltpr ^Bas mice - functionally mimicked a CD28 deficiency. Akin to CD28-deficient (Cd28 ^~ ' ^~ ) mice, Rltpr ^Bas mice showed a normal sequence of T cell development and their periphery was populated with CD4 ⁺ and CD8 ⁺ T cells that showed a normal phenotype. No measurable effect on negative and positive selection was observed when OT-I, H-Y, OT-II and AND TCR transgenes were bred onto Rltpr ^Bas . Importantly, the Rltpr ^Bas mutation was without effect on the expression of CD28 in peripheral T cells. Moreover the decrease in CD28 expression that is normally associated with positive selection in the thymus was fully recapitulated in Rltpr ^Bas mice. Therefore, Rltpr ^Bas did not affect the expression of CD28.

To compare the importance of Rltpr and CD28 during T cell activation, negatively purified T cells from Rltpr ^Bas , Cd28^ ^~ , and WT control mice were stimulated with graded doses of anti-CD3 antibody in the presence or absence of a fixed dose of anti-CD28 antibody. Rltpr ^Bas prevented CD28 synergizing with TCR-CD3 to the same extent as a lack of CD28. Both CD4 ⁺ and CD8 ⁺ T cells isolated from Rltpr ^B and Cd28^ mice and labeled for CFSE showed a reduced proliferation in response to anti-CD3 plus anti-CD28 antibody stimulation. The Rltpr ^Bas mutation also prevented CD28 synergizing with the TCR in assays measuring IL-2 production and IFN-γ production by primary T cells.

Similar to CD28-deficient CD4 ⁺ T cells ²⁶ , addition of exogenous IL-2 overcame the inefficient proliferation manifested by T cells purified from Rltpr ^Bas mice in response to CD3 plus CD28 stimulation. As observed for CD28-deficient T cells ²⁷ , T cells purified from Rltpr ^Ba mice also showed a dramatically reduced proliferation in mitogen (concanavalin A) responses.

To further support the view that Rltpr ^as and Cd28^ mice showed similar T cell functional defects, the Rltpr ^Bas allele was backcrossed onto OT-I mice expressing a TCR specific for the OVA257-264 peptide bound to H-2K ^{b 28} ' ²⁹ . OVA257-264 was capable of inducing OT-I Rltpr ^Bas CD8 ⁺ T cell proliferation to the same extent as OT-I Rltpr CD8 ⁺ T cells. In contrast, the presence of Rltpr ^Ba dramatically reduced the production of IL-2 and of IFN-γ by OT-I CD8 ⁺ T cells and the proliferation induced by the Q4 and T4 weak agonist variants of OVA257-264. Importantly, the Rltpr ^Ba mutation had an impact on OT-I proliferation similar to that resulting from the use of CD80/CD86-deficient APCs. Therefore, side-by-side comparison of Rltpr ^Bas and of Cd28 ^~ ' ^~ mice showed that Rltpr ^Bas by itself constituted a functional phenocopy of a Q 28 null mutation, suggesting that Rltpr constitutes a previously unrecognized and essential component of the CD28 signaling pathway.

Defective development of natural T _REG cells and of T _FH cells in Rltpr ^as mice

CD28 has an important cell-intrinsic role in the generation of thymic nTreg cells.

Cd28 ^~ ' ^~ mice or mice lacking both CD80 and CD86 have an approximately 80% reduction in the frequency of thymic nTreg cells with no changes in conventional T cell subsets ^30"32 . Rltpr ^Bas mice showed a reduction in thymic nTreg cells as severe as Cd28 ^~ ' ^~ mice and this reduction was not associated with changes in other intrathymic T cell subsets. As a result, the spleens of Rltpr ^Bas and Cd28 ^~ ' ^~ mice contained approximately 5-fold reduced numbers of Foxp3 ⁺ T _reg cells.

nTreg cells develop in the thymus in a two-step process. The first step is governed by TCR and CD28 signals and involves the differentiation of CD4 ⁺ single positive thymocytes into CD4 ⁺ CD25 ⁺ CD122 ⁺ GITR ⁺ Foxp3 nTreg cell progenitors. In the second step, upon stimulation via IL-2, these progenitors convert into CD4 CD25 CD122 ⁺ GITR Foxp3 ⁺ nTreg cells. CD28 is involved in the first stage of Treg differentiation ³² . Analysis of Rltpr ^Bas thymi revealed a similar reduction in the numbers of nTreg cell progenitors. T _FH cells constitute a CD4 ⁺ T _H lineage specialized in provision of help to B cells. CD28 costimulation has also been shown to be essential for T _FH cell development ³³ . Analysis of the spleen of Rltpr ^Bas mice, showed a reduction in CXCR5 PD1 ⁺ T _FH cells that was similar to that of Cd28 ^~ ' ^~ mice. Therefore, analysis of the development of nT _reg cells and of T _FH cells further indicated that i?V ^Bas and Cd28 ^~ '- mice showed the same T cell functional defects.

Rltpr and Rltpr ^Bas MCs colocalize with CD28 MCs

To determine the mechanism by which Rltpr ^Bas abrogates CD28-mediated costimulatory signals, we assessed the dynamic movement of Rltpr and Rltpr ^Bas at the IS with a high spatial resolution. For this purpose, we used glass-supported planar bilayers containing mobile ligands as APC substitutes. We first determined whether the Rltpr ^Bas mutation affected the generation of TCR and of CD28 MCs and their subsequent translocation to the cSMAC. The Rltpr ^as allele was first backcrossed onto AND mice that express a TCR specific for I-E ^k molecules bound to the MCC ₈ 8-i o3 peptide ²⁹ . AND Rltpr and AND Rltpr ^Bas CD4 ⁺ T cells were then subjected to retroviral infection to express a EGFP-tagged CD28 reporter ⁹ . AND Rltpr CD28-EGFP and AND Rltpr ^Bas CD28-EGFP CD4 ⁺ T cells were allowed to settle on planar bilayer membranes containing glycophosphatidylinostitol (GPI) anchored I-E ^k molecules loaded with MCC ₈₈ -io3, ICAM-1-GPI and CD80-GPI molecules. The movement of the AND TCR (tagged with a DyLight 649-labelled anti-TCR Fab) and of CD28-EGFP was imaged after T cell-planar bilayer contact by confocal microscopy. Rltpr ^Bas had no measurable effect on the generation of TCR and CD28 MCs and on their subsequent translocation to the cSMAC.

To visualize the dynamics of Rltpr and of Rltpr ^Bas following TCR-CD28 mediated T cell activation and determine whether such dynamics depended on CD28-CD80 engagement, full-length Rltpr and Rltpr ^Bas proteins were fused to a Yellow Fluorescent Protein for Energy Transfer (YPET) reporter ³⁴ and separately expressed in CD4 ⁺ AND T cells. AND Rltpr- YPET and AND Rltpr ^Bas -YPET CD4 ⁺ T cells expressing similar reporter levels were allowed to settle on planar bilayers. As previously observed for TCR-CD3 ³⁵ ' ³⁶ and CD28 ⁹ , Rltpr- YPET and Rltpr ^Bas -YPET MCs were generated at the T cell-bilayer interface as soon as a T cell attached and they subsequently translocated to the cSMAC. Rltpr- YPET and Rltpr ^Bas - YPET MCs constantly colocalized with CD28 MCs. Upon translocation to the cSMAC they segregated away from TCR MCs to occupy the CD28 ^hi CD3 ^l0 signaling cSMAC. Therefore, both Rltpr and Rltpr ^Bas generated clusters when T cells attached to the planar bilayer. Importantly, both Rltpr and Rltpr ^Bas MCs colocalized with CD28 MCs, suggesting that the lack of CD28-mediated costimulatory signals observed in the presence of Rltpr ^Bas was not due to the inability of Rltpr ^Bas to colocalize with CD28 MCs.

Rltpr and Rltpr ^Bas MCs form in a CD80 dependent manner

CD28 MC formation is completely dependent on binding of CD28 to CD80 ⁹ . Likewise, the formation of Rltpr- YPET and Rltpr ^Bas -YPET MCs only occurred when the planar bilayer membrane contained CD80 molecules. CD28, Rltpr and Rltpr ^Bas MC formation depended on the presence of CD80 and followed a similar spatiotemporal distribution at the IS. Rltpr and Rltpr ^Bas are thus likely part of the CD28 "signalosome". However, Rltpr and Rltpr ^Bas were not coprecipitated with CD28 in lysates from CD4 ⁺ T cells following stimulation with TCR plus CD28 antibodies or PMA, suggesting that the CD28- Rltpr colocalization observed at the IS resulted from an indirect association between CD28 and Rltpr. Rltpr is essential for CD28-induced PKC-Θ and CARMA1 translocation to the cSMAC Engagement of the TCR results in the generation of diacylglycerol (DAG) and suffices to promote transient and diffuse PKC-Θ recruitment at the T cell-APC interface ⁹ . However, the formation of discrete PKC-Θ MCs and their subsequent translocation to the cSMAC depends on CD28-CD80 binding ⁹ , and a similar trend has been observed for Carmal ¹² ' ³⁷ . Considering that CD28 colocalized with both Rltpr and Rltpr ^Bas and that CD28 costimulation was totally blocked in the presence of Rltpr ^Bas , we analyzed whether Rltpr ^Bas abrogated the capacity of CD28 to recruit PKC-Θ and Carmal at the IS. Accordingly, AND Rltpr and AND Rltpr ^Bas CD4 ⁺ T cells expressing PKC-9-EGFP or Carmal -EGFP were imaged using planar bilayers containing I-E ^k -MCC88 io3, ICAM-1 and CD80. In contrast to Rltpr, in the presence of Rltpr ^Bas there was no accumulation of either PKC-Θ or Carmal at the cSMAC. Therefore, although Rltpr and Rltpr ^Bas MCs were both capable of forming and of translocating to the cSMAC upon TCR stimulation and in a manner dependent on CD28-CD80 binding, the presence of Rltpr ^Bas prevented the accumulation of PKC-Θ and Carmal at the cSMAC. These results suggest that Rltpr plays an essential role in CD28-mediated costimulation based on its capacity to couple CD28 to PKC-Θ and Carmal .

Spatiotemporal distribution of Rltpr and Rltpr ^as at T cell-APC interface

Subcellular proximity during intracellular signaling is often indicative of functional connections. Therefore, we analyzed the spatiotemporal distribution of Rltpr and Rltpr ^Bas in live T cell-APC conjugates and compared it to that of 53 transmembrane and cytoplasmic molecules involved in T cell activation ³⁸ . Rltpr- YPET and Rltpr ^Bas -YPET CD4 ⁺ T cells were separately expressed in primary 5C.C7 CD4 ⁺ T cells that have a TCR specific for a moth cytochrome c peptide (MCC82 103) and H-2 I-E ^k . Upon tight conjugate-formation with MCC- pulsed mouse CH27 B lymphoma cells areas of Rltpr- YPET and Rltpr ^Bas -YPET enrichment were determined in three dimensions up to 7 minutes after conjugate formation and classified at 12 time points according to six subcellular spatial patterns (38).

Rltpr and Rltpr ^Bas were unusual among the molecules analyzed to date in that they straddled four different subcellular distributions, suggestive of a protein linking various subcellular processes. First, in a sizable fraction of T cell-APC couples, Rltpr accumulated at the center of the T cell-APC interface in a manner similar to CD28. This accumulation was rapid and well sustained. In contrast, the central accumulation of Rltpr ^Bas was often delayed and more transient. Second, in a substantial fraction of conjugated T cells, both Rltpr and Rltpr ^Bas accumulated according to a 'lamellal' pattern, a F-actin-based structure that supports the activity of proteins linking proximal signaling and actin dynamics (K.T. Roybal and C.W., submitted). Third, most of the central accumulation of Rltpr ^Bas - but not of Rltpr - occurred in a large invagination forming at the center of the interface within 1 minute of contact and thought to recycle proximal TCR signaling components through receptor internalization ³⁹ . Fourth, Rltpr and Rltpr ^Bas often displayed an internal punctuate accumulation, a pattern that is probably associated with intracellular vesicles and has only been observed equally prominently with TCR complexes in which the clusters of arginine and lysine residues found in the CD3^ subunit were mutated ⁴⁰ . This internal punctuate accumulation is suggestive of a role of Rltpr in CD28 internalization. Such internal punctuate structures occurred in 93 ± 5 % of cell couples involving T cells expressing Rltpr ^Bas - YPET, as compared to 58 ± 10 % of those involving T cell expressing Rltpr- YPET (p< 0.001).

The proteins that showed the closest spatiotemporal distribution to Rltpr were Rpre, a sensor for the negatively charged polyphosphoinositides found at the cytosolic surface of the plasma membrane and of intracellular vesicles, and the adaptors Nek, Grb2, and Cin85. Both Nek and Cin85 have been shown to link antigen receptors to actin regulation and receptor internalization ⁴¹ . Therefore, these studies suggest that, in addition to its capacity to link CD28 to PKC9 and Carmal, Rltpr additionally controls CD28 internalization by coupling it to actin polymerization. CD28 has been shown to be rapidly internalized after ligation ⁴² ' ⁴³ . Intriguingly, T cells expressing Rltpr ^Bas showed an increase in antibody-induced CD28 internalization as compared with T cells expressing Rltpr. Therefore, by preserving the actin- uncapping activity but breaking the linkage of CD28 to PKC9 and Carmal, the Rltpr ^Bas mutation might reduce the "drag" through the cytosol and thus increase CD28 internalization over residency at the cell surface.

Discussion:

Using a sensitized ENU mutagenesis screen we demonstrated that the poorly characterized Rltpr gene product is essential for CD28 costimulation. Consistent with its involvement in CD28 function, Rltpr was primarily expressed in developing and mature T cells. We showed that Rltpr ^Bas did not affect the expression of CD28 at the T-cell surface and that TCR-CD28 engagement at the IS resulted in the spatial co localization of CD28 with both Rltpr and Rltpr ^Bas molecules. However, Rltpr ^Bas abrogated the connection between CD28 and PKC- Θ and Carmal . Consistent with that mechanistic defect, comparative analysis of antigen- induced CD4 ⁺ and CD8 ⁺ T cell activation and of nTreg and T _FH cell development in Rltpr ^Bas and Cd28 ^~ ' ^~ mice showed that the Rltpr ^Bas mutation constituted a functional phenocopy of a CD28 deficiency. These last findings reinforce in a unbiased manner the importance of CD28- PKC-9-Carmal signaling for the differentiation of nTreg and T _FH cells ⁴⁴ ' ⁴⁵ .

Many LRR-containing domains provide a structural framework for protein-protein interactions ²⁴ , and the non-conservative Rltpr ^Bas mutation that occurred in the eighth LRR likely prevented Rltpr from playing its role of intermediate between CD28 and PKC- Θ/Carmal . Owing to its large size, Rltpr is likely a multitask protein composed of distinct functional domains. Rltpr ^Bas was still capable of constitutively associating with CP, a finding consistent with the large distance that separate the Rltpr ^Ba mutation from the CPI motif. Actin polymerization in vivo occurs primarily at the barbed end of actin filament and mechanisms that control the number of free barbed ends are crucial for the regulation of actin filament assembly within cells. The presence of a CP at the barbed end of an actin filament inhibits the addition of actin subunits at that end. Therefore, by preventing the action of CP, actin- uncapping proteins such as Rltpr generate free barbed ends and enhance actin polymerization. pi 16, a Dictyostelium Lrcc-like protein, localizes in dynamic actin-rich cellular extensions including macropynocytic crown and the leading edge of locomoting cells ⁴⁶ . Dictyostelium cells lacking pi 16 showed a reduction in the formation of these structures. On that basis, it has been proposed that most Lrcc-family proteins act to promote actin polymerization and enhance processes such as cell movement and endocytosis ^21-23 ' ⁴⁶ ' ⁴⁷ . By demonstrating that the actin-uncapping protein Rltpr is essential for CD28 costimulation, the present study supports the view that actin dynamic regulation plays an important role in CD28-mediated costimulation ⁴² ' ⁴³ .

Using a system-scale approach ³⁸ , we showed that the pattern of distribution of Rltpr at the T cell-APC interface had the greatest similarity with that of the Nek and Cin85 adaptors. Nek and Cin85 have been shown to link antigen receptors to actin regulation and receptor internalization ⁴¹ . Interestingly, Cin85 primarily down-regulates the activity of receptor protein-tyrosine kinase via endocytosis and possesses actin-uncapping activity ²¹ . Reminiscent of our observations with Rltpr, CD2 is also prominently found in the large T cell invagination that forms at T cell-APC interface ³⁹ , and it associates with CP via Cin85 ⁴⁸ . The exacerbated occurrence of patterns associated with internalization (invagination and internal accumulation) in T cells expressing Rltpr ^Bas as compared to WT T cells is consistent with the view that the L432P mutation relieved Rltpr from long-lasting interactions with effectors such as PKC-Θ and Carmal that reside at the inner face of the T cell membrane. Congruent with hat view, T cells expressing Rltpr ^Bas showed a higher rate of antibody-mediated CD28 endocytosis as compared to WT T cells. Moreover, the central accumulation of Rltpr ^Bas -YPET was shorter than that of Rltpr- YPET. Therefore, the extent to which the CD28-Rltpr "nucleation center" resides at the T cell surface prior to endocytosis appears to depend on its ability to interact with PKC-Θ and Carmal . Interestingly, gene-expression profiling of whole skin samples from psoriatic patients showed that the Rltpr gene was downregulated ⁴⁹ , and a recent study demonstrated that polymorphisms with the human Rltpr gene were associated with ankylosing spondylitis ⁵⁰ , pointing to a possible role of Rltpr in T-cell dependent autoimmunity.

In conclusion, these genetic, functional and imaging studies have uncovered a role for Rltpr, a lymphoid lineage-specific actin-uncapping protein, as an essential player of the CD28 costimulatory pathway. They led us to modify the existing model of CD28 costimulation to a new more complete model in which Rltpr plays an essential role owing to its capacity to couple CD28 to PKC-Θ and Carmal . Whether CD28 engagement induces distinct signals or simply amplifies TCR signaling has been the object of controversy. It will be interesting to determine whether Rltpr constitutes the long-sought component specific for the CD28 signaling pathway.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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