LAP, a lymphocyte activation gene-3 (LAG-3)-associated protein that binds to a repeated EP motif in the intracellular region of LAG-3, may participate in the down-regulation of the CD3/TCR activation pathway
2001; Wiley; Volume: 31; Issue: 10 Linguagem: Inglês
10.1002/1521-4141(2001010)31
ISSN1521-4141
AutoresNathalie Iouzalen, Susanne Andreae, Sigrid Hannier, Frédéric Triebel,
Tópico(s)T-cell and B-cell Immunology
ResumoEuropean Journal of ImmunologyVolume 31, Issue 10 p. 2885-2891 ArticleFree Access LAP, a lymphocyte activation gene-3 (LAG-3)-associated protein that binds to a repeated EP motif in the intracellular region of LAG-3, may participate in the down-regulation of the CD3/TCR activation pathway Nathalie Iouzalen, Nathalie Iouzalen Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, FranceSearch for more papers by this authorSusanne Andreae, Susanne Andreae Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, FranceSearch for more papers by this authorSigrid Hannier, Sigrid Hannier Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, FranceSearch for more papers by this authorFrédéric Triebel, Corresponding Author Frédéric Triebel Frederic.Triebel@cep.u-psud.fr Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, France Laboratoire d'Immunologie Cellulaire, Institut Gustave-Roussy, Villejuif, FranceLaboratoire d'Immunologie des Tumeurs, Université Paris-Sud, 5 rue Jean-Baptiste Clément, F-92296 Chatenay Malabry, France Fax: +33-1-4211-5268Search for more papers by this author Nathalie Iouzalen, Nathalie Iouzalen Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, FranceSearch for more papers by this authorSusanne Andreae, Susanne Andreae Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, FranceSearch for more papers by this authorSigrid Hannier, Sigrid Hannier Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, FranceSearch for more papers by this authorFrédéric Triebel, Corresponding Author Frédéric Triebel Frederic.Triebel@cep.u-psud.fr Laboratoire d'Immunologie des Tumeurs, Université Paris-Sud, Chatenay Malabry, France Laboratoire d'Immunologie Cellulaire, Institut Gustave-Roussy, Villejuif, FranceLaboratoire d'Immunologie des Tumeurs, Université Paris-Sud, 5 rue Jean-Baptiste Clément, F-92296 Chatenay Malabry, France Fax: +33-1-4211-5268Search for more papers by this author First published: 28 September 2001 https://doi.org/10.1002/1521-4141(2001010)31:10 3.0.CO;2-2Citations: 44 The first two authors contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract The threshold, extent and termination of TCR activation is controlled in part by inhibitory co-receptors expressed on activated T cells. The lymphocyte activation gene product (LAG-3), a ligand for MHC class II molecules co-caps with the CD3/TCR complex and inhibits cell proliferation and cytokine secretion in response to CD3 signaling. We first investigated whether LAG-3 is localized in activated T cells in detergent-resistant membrane rafts enriched in glycosphingolipids and cholesterol. We showed that both LAG-3 and MHC class II are present in the cell fraction of glycosphingolipid-rich complexes (GSL complexes) before the assembly of the immunological synapse by CD3/TCR complex cross-linking. Using the LAG-3 intracytoplasmic region as bait in the yeast two-hybrid cloning system, we next identified a novel protein termed LAP for LAG-3-associated protein. LAP is encoded by a 1.8-kb RNA message in lymphocytes and encodes a 45-kDa protein that is expressed in most tissues. We showed that LAP binds specifically in vitro and in vivo to the Glu-Pro (EP) repeated motif present in the LAG-3 intracytoplasmic region. LAP also binds to the EP motif of another functionally important receptor, the PDGFR. Thus, LAP is a candidate molecule for a new type of signal transduction and/or coupling of clustered rafts to the microtubule networks that could explain how negative signaling of co-receptors may occur through molecules devoid of any immunoreceptor tyrosine-based inhibitory motif consensus sequence. Abbreviations: ITIM: Immunoreceptor tyrosine-based inhibitory motif LAG: Lymphocyte activation gene product LAP: LAG-3-associated protein IC: Intracytoplasmic GSL: Glycosphingolipid-rich 1 Introduction The LAG-3 protein 1, 2, associated with CD3/TCR complexes 3, 4, interacts with the non-polymorphic regions of MHC class II molecules 2, 5, 6 on antigen-presenting cells (APC). LAG-3 signaling may play a negative regulatory role in T cell activation 3. In particular, triggering this negative regulatory receptor with mAb cross-linking results in the inhibition of TCR-induced calcium fluxes, down-modulation of the CD3/TCR complexes expression and finally leads to functional non-responsiveness 3. It was thus proposed that MHC class II-induced changes in the formation of the LAG-3/TCR complexes may play a role in negatively regulating the CD3/TCR activation pathway 3. Glycosphingolipid-rich (GSL) complexes, characterized by low density and resistance to non-ionic detergents at 4°C, are derived from membrane structures called glycosphingolipid-rich membrane domains or rafts (GSL rafts) 7–9. GSL rafts are discrete domains in the cell membrane network, predominantly located in the plasma membrane. They provide clustering sites for molecules involved in signal transduction and are required for coupling the engagement of the TCR with downstream signaling events in the early stages of T lymphocyte activation 10. Antigen recognition by the TCR is facilitated by the assembly of an immunological synapse, several micrometers in diameter, at the contact zone with the APC. Clustering of raftsis an essential feature in the formation of an immunological synapse. This process can take hours to complete and is associated with actin and microtubule networks toward the contact site 11. After receptor cross-linking, TCR raft residency increases and they become partly insoluble in detergent. We have previously shown that engagement of the CD3/ TCR complexes with an mAb or serial triggering of the TCR via antigen recognition resulted in LAG-3 being co-clustered with the CD3/TCR complexes 4. Similarly, following CD8 or MHC class II engagement, LAG-3 was found to be co-distributed with the engaged molecule 4. We thus hypothesized that the supramolecular assemblies between LAG-3, CD3, CD8 and MHC class II molecules may result from the organization within raft microdomains 4. As an inhibitory co-receptor expressed on activated T cells, LAG-3 may signal through its short intracytoplasmic (IC) region to finely regulate the balance between a positive signal mediated through the TCR that initiates the response and a negative signal that controls the threshold, extent and termination of TCR activation. However, the cytoplasmic domain of either hLAG-3 or mLAG-3 6 does not contain any immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus sequence as are usually found in various inhibitory receptors nor any CxC p56lck binding motifs, in contrast to CD4. Thus, the pathways involved in LAG-3-dependent TCR signaling regulation are totally unknown. These findings induced us to directly clone proteins expressed in activated T cells that would specifically bind to the IC region of hLAG-3. Using the yeast two-hybrid system and the LAG-3 IC region as bait, we identified a novel protein, termed LAP for LAG-3-associated protein, that binds to the Glu-Pro (EP) repeated motifs present within the LAG-3 IC region C terminus. 2 Results and discussion 2.1 LAG-3 and MHC class II are expressed in GSL complexes on the surface of human activated T cells GSL complexes (raft microdomains) were isolated in a low-density fraction, at the interface between the 35% and 5% fractions of a discontinuous sucrose gradient, as described by Montixi et al. 9. Twelve fractions of the gradient were analyzed by Western blotting. LAG-3, DR-α and p56lck were detected in fraction 9, representing the GSL complex isolates, and were no longer detected after addition of 0.2% saponin (cholesterol depletion leading to raft disruption) to 1% Triton X-100 (data not shown). CD45, a phosphotyrosine phosphatase known to be excluded from raft microdomains, was used as a negative control. Thus, LAG-3 is present in raft microdomains before engagement of the TCR by specific mAb or peptide/MHC complexes. In addition, we found that MHC class II (DR-α) molecules were also present in raft microdomains on activated T cells (results not shown). The partitioning of MHC class II into the raft fraction has been reported to occur in the myelomonocytic THP-1 cells following their cross-linking with antibodies and to be mandatory for protein tyrosine kinase activation 12. In B cells, MHC class II were found to be constitutively present in rafts and this concentration of MHC class II molecules facilitates antigen presentation 13. The presence of LAG-3 in raft microdomains before engagement of the TCR argues for its close association with CD3/TCR complexes and explains, in part, our previous observations where LAG-3 was found to be co-clustered with CD3/TCR complexes and also with CD8 in co-capping experiments 4. One may not exclude that, in addition to the typical trans-interaction between LAG-3 on T cells and MHC class II on APC, a cis-interaction takes place between LAG-3 and MHC class II molecules co-clustered in raft microdomains. In this vein, we have previously reported significant co-clustering between LAG-3 and MHC class II molecules in co-capping experiments performed on activated T cells using a specific mAb 4. The role, if any, of such a cis-interaction between LAG-3 and MHC class II molecules is presently unclear. 2.2 Isolation of a novel human protein, LAP, interacting with LAG-3 To identify proteins that bind to the intracellular domain of human LAG-3 in vivo, we performed interaction screening using the yeast two-hybrid system. First, we verified that no LAG-3 construct in pLex or pLex/NLS displayed any lacZ reporter gene activity in yeast cells expressing pGAD without insert. This indicated that LAG-3 does not show any nonspecific binding to DNA sequences leading to GAL promoter activation. Then, we transformed strain L40 with pLex/NLS-hLAG-3/I to screen about 2×105 colonies of a human activated T cell cDNA library. Around 200 colonies that grew on histidine-free drop-out medium were selected, replated onto selective medium and assayed for β-galactosidase expression. From these, 13 showed reporter gene activities. To confirm the specificity of these interactions, the plasmid DNA from selected clones was isolated and used for transformation of the strain AMR70, which were then mated with strain L40 containing either the bait plasmid pLex/NLS-hLAG-3/I or a control plasmid (pLex-Lamin or pLex/NLS-RalB). We obtained three specific clones showing strong interaction with hLAG-3/I (signals appeared in less than 2 h) and not with Lamin or RalB. The inserts of these clones were submitted to restriction mapping and sequence analysis. The three cDNA were found to encode a unique partial (i.e. lacking the ATG translation initiation codon) sequence of 243 amino acids, termed LAP (not shown). This novel molecule has some homology with the C-terminal region of the TCP-10 protein previously cloned in human 14, 15 and mouse 16–18. TCP-10 is a T-complex responder (TCP) gene that may play a role in the transmission ratio distortion phenotype. A region of LAP is 56% identical to the 181 C-terminal residues of human TCP-10 protein and 66% identical to the 106 C-terminal residues of the murine TCP-10 protein. The 5′ end of the LAP cDNA was further extended by 5′RACE cloning starting from PHA-blasts mRNA. The nucleotide sequence data reported here will appear in the DDBJ/EMBL/GenBank nucleotide sequence database under the accession number AJ303006. Analysis of the LAP cDNA revealed a nucleotide (nt) sequence of 1353 bases that contains a single open reading frame (ORF) of 372 amino acids. This ORF starts at position 70 and ends with the translation stop codon, TGA, located at nt 1186. During the writing of this manuscript, a search of EMBL database showed that this LAP sequence is in fact 99% identical (9 nt mismatches including 4 in the coding region with a single amino acid difference at the C terminus) to the 3prime; end of the recently published CPAP (centrosomal P4.1-associated protein) molecule which is part of the γ-tubulin complex 19. These 9 nt mismatches were also found on several EST sequences, confirming the differences observed between CPAP and LAP. Sequence identity of TCP-10, CPAP and LAP proteins is restricted to two conserved regions at the C terminus. One carries a leucine zipper which may form a series of heptad repeats involved in coiled-coil formations, and the second contains unusual glycine repeats 19. We then tested whether human LAP could bind to the murine LAG-3 IC region. We observed a weak interaction between these two heterologous proteins (Table 1) with a small activation of the HIS3 gene but no detectable LacZ activity. We next examined which region of human LAG-3 interacts with LAP. The binding of LAP with hLAG-3/IΔC and hLAG-3/EP constructs was tested in yeast cells and we found that LAP indeed binds specifically to the short C-terminal region of LAG-3 containing the EP-rich region (Table 1). Table 1. Interaction of LAP and LAG-3 proteinsa) fused to LexA BD fused to GAL4 AD LAG-3 regions – Lamin RalB LAP hLAG-3/l R457 to L503 – – – ++++ NLS-hLAG-3/l R457 to L503 – – – +++++ NLS-mLAG-3/l L456 to L507 – – – + NLS-hLAG-3/l▵C L457 to E481 – – – +/– hLAG-3/EP E478 to L503 – – – ++ NLS/hLAG-3/EP E478 to L503 – – – ++++ a) hLAG-3 and mLAG-3 IC regions were expressed as fusion proteins to the LexA DNA binding domain (LexA BD) in the pLex vector containing or not a nuclear localization sequence (NLS). The pGAD vector encoded the GAL4 activation domain (GAL4 AD) alone or fused to LAP or an unrelated protein (Lamin or RalB). Two procedures for interaction studies were performed: (1) co-transfection of yeast strain L40 with the two indicated plasmid combinations shown, (2) transformation of strain L40 with a pLex construct, which are then mated with AMR70 transformed with a pGAD construct. To demonstrate in vitro binding between LAP and hLAG-3 proteins, LAP linked to GST or GST alone were expressed in bacteria and bound to glutathione-Sepharose beads. Bound proteins were incubated with total cell lysates prepared from PHA-activated T lymphocytes. The results demonstrate that the LAG-3 protein was specifically precipitated from the T cell lysate when using affinity beads containing the LAP protein (Fig. 1A). The control GST beads did not precipitate any detectable LAG-3 protein from the T cell lysate. Therefore, LAG-3 binds specifically to the LAP protein in vitro, in agreement with the data obtained from the yeast two-hybrid screening procedure. The possibility that the interaction between LAP and LAG-3 proteins in both the yeast two-hybrid system and in T cell lysates required an additional adaptor protein was not ruled out. Thus, we used a direct binding assay in which the in vitro-translated LAG-3 protein was tested for interaction with beads bound to GST-LAP or GST alone. As shown in Fig. 1B, affinity beads containing the GST-LAP fusion protein pulled down the LAG-3 protein in a specific manner. This supports the existence of a specific direct physical interaction between LAP and LAG-3 proteins without the need for the presence of a third adaptor protein. Figure 1Open in figure viewerPowerPoint In vitro interaction of human LAP with hLAG-3. LAP binds specifically (A) to the natural hLAG-3 (70-kDa) protein present in whole cell lysate of PHA-activated human PBMC or (B) to a protein produced by in vitro translation of a hLAG-3 mRNA in a rabbit reticulocyte lysate. Overall, the interaction between LAP and hLAG-3 has been confirmed both in vivo and in vitro using recombinant LAP protein. In particular, we have shown that the LAP protein was able to bind LAG-3 in lysates of activated T cells. This interaction was specific and was also observed using in vitro translated recombinant LAG-3. 2.3 The C-terminal region of LAP binds the EP region of hLAG-3 To determine the region of the LAP protein that contains the LAG-3 binding site, we constructed deletion mutants of the LAP cDNA (Fig. 2A). The binding of these mutants with hLAG-3/I, hLAG-3/IΔC and hLAG-3/EP were tested, with RalB as a negative control. Deletion of the extreme C-terminal regions (mutant D3) already abolished some binding activity (Fig. 2C), while the shorter constructs (D1 and D2) did not bind to hLAG-3 at all. Thus, the binding site for LAP on the EP motifs is located in its C-terminal region. Given that this region in CPAP is thought to be involved in centrosomal P4.1 protein binding 19, one may not exclude that the LAP protein, like CPAP, may participate to the formation of microtubule networks. LAP would then function to cluster rafts into the immunological synapse following TCR engagement, a phenomenon that requires the polarization of actin and microtubules 11. Figure 2Open in figure viewerPowerPoint Interactions tested in the two-hybrid system using co-transformation with two plasmids and mating of two yeast strains. (A) Three partial LAP proteins (D1, D2 and D3) lacking their C-terminal domain were cloned in frame with the GAL4 AD protein, using a partial 1104 bp LAP cDNA. (B) The EP-rich C-terminal region of the PDGF receptor (PDGFR) was fused with the LexA BD. (C) Results of the interactions in the two-hybrid system. 2.4 LAP binds to the IC region of the PDGF receptor containing an EP motif The PDGF receptor 20 has a long IC tail containing numerous motifs known to be involved in signaling. We noticed that a repeated EP motif not known to be involved in signal transduction was found in its C-terminal region (Fig. 2B). We therefore wondered whether the LAP protein could bind to this EP motif-containing segment and found that it actually binds to both the short IC region of hLAG-3 and the EP-motif (Fig. 2C). One may not exclude that LAP interacts with other membrane receptors containing the EP motif since we showed here that it also binds to the PDGFR intracellular region. Thus, this EP motif may well be a common transduction motif used by other functionally important receptors. 2.5 LAP is a 45-kDa protein expressed in all tested human cells To determine the size and expression of the LAP protein, total cell lysates were analyzed by Western blotting with a rabbit polyclonal serum raised against a LAP peptide with no sequence homology with TCP-10. Two bands at 30 and 45 kDa were detected in PBMC on activated T cells (Fig. 3). The 30-kDa band was shown to be nonspecific since it was also detected using the preimmune serum (Fig. 3). The 45-kDa band corresponds to LAP as it was no longer detected following preincubation of the immune serum containing the LAP peptide (10–6 M at 4°C for 1 h) (Fig. 3) while preincubation with a control peptide had no effect (data not shown). In addition, this 45-kDa band was found in cytoplasmic but not in nucleic T cell extracts (data not shown). These results clearly indicate that LAP is expressed as a 45-kDa cytoplasmic protein in PBMC and in activated T cells with a higher expression level in the latter cells. Figure 3Open in figure viewerPowerPoint The anti-LAP immune serum reveals a specific band at 45 kDa. Western blots were performed using 10 μl total cell lysates of PBMC (lanes 2, 4, 6) or PHA blasts (lanes 1, 3, 5). The blots were incubated in rabbit preimmune serum (lanes 1, 2), rabbit polyclonal antibody against LAP (lanes 3, 4) or the latter preincubated with 10–6 M LAP peptide (lanes 5, 6). The arrow indicates the LAP 45 kDa protein. Western blotting was also performed with total cell lysates of the Jurkat T cell line, two EBV-transformed B cell lines and a renal cell carcinoma cell line (RCC7). LAP is expressed in these cell lines as a 45-kDa protein with lower expression in PBMC (data not shown). LAP is thus expressed in T and non-T hematopoietic cell lines as well as in non-hematopoietic cell lines. In addition, we detected LAP in different untransformed human tissues, including the lung, liver, kidney, testes (no overexpression), pancreas and heart, but not in the spleen and brain (data not shown). 2.6 Two RNA species are derived from the LAP gene We first analyzed the LAP gene by digesting DNA from different cell lines and PBL, Southern blotting and hybridizing using the LAP cDNA as a probe. We found unique EcoRI (5.5 kb), HindIII (9 kb) and XhoI (>12 kb) fragments indicating that the LAP or CPAP gene is either present in the human genome as a single copy gene or represents two closely related genes (data not shown). Total and poly-A+ RNA samples of PHA-blasts were run on a denaturing agarose gel and analyzed by Northern blotting. The LAP RNA seemed to be rarely expressed, as it was only detected by using 15 μg poly-A+ RNA while not being detected in total RNA samples (up to 20 μg, data not shown). Two faint bands hybridized with the labeled cDNA LAP probe, one with a size of 4.5 kb and a weaker one at 1.8 kb. As these two bands correspond exactly to the sizes of the 28 S and 18 S rRNA, the blot was then rehybridized with saturating amounts of ribosomal RNA (10 μg/ml) added to prevent any nonspecific binding of the probe to the remaining rRNA in the sample and the same result was obtained (data not shown). Since these two signals were only seen with highly purified poly-A+ RNA and not with total RNA samples containing a greater amount of rRNA, we concluded that LAP was specifically expressed as a 1.8-kb mRNA. The stronger 4.5-kb signal may correspond to CPAP which has been shown to be weakly expressed in most tissues and overexpressed in testis 19. Thus, LAP is a new human protein, expressed in all tested human cells and derived from a rare mRNA. It appears that LAP and CPAP are derived from either a single gene or two closely related genes which are strongly expressed in the testes for the CPAP mRNA (4.5 kb) and weakly expressed in other cells as two messages (4.5 and 1.8 kb) coding for CPAP and LAP, respectively. We could not determine whether the 9 nt differences detected in the C-terminal region are related to alleles of a single gene or to two different genes. 3 Concluding remarks The specific immunoprecipitation of LAG-3 by LAP-GST beads from activated T lymphocyte lysates suggests that the two overlapping 150-kDa CPAP 19 and 45-kDa LAP proteins have different functions, that is binding to γ-tubulin in the centrosome especially in testis cells (CPAP) or binding to EP motifs present on membrane expressed receptors (LAP). Given that EP motifs are rare in human proteins, it is possible that the specific binding of LAP on such motifs bears an important biological significance for signal transduction and/or coupling of clustered rafts to the microtubule networks. 4 Materials and methods 4.1 Plasmid construction The hLAG-3/I and mLAG-3/I fragments encode the full-length intracellular region of human LAG-3 and murine LAG-3, respectively. The hLAG-3/IΔC encodes the intracellular domain of human LAG-3 deleted of its 22 C-terminal amino acids (ΔC) whereas hLAG-3/EP codes only for the EP-rich region located at the end of the C-terminal part of hLAG-3. The PCR products were cloned into the twohybrid vectors pBMT116 (pLex) or a derivative containing an additional Nuclear Localization Sequence (pLex/NLS) 21 in frame with the LexA DNA binding protein yielding the following constructs: pLex-hLAG-3/I and pLex/NLS-hLAG-3/I (from R457 to L503); pLex/NLS-mLAG-3 (from L456 to L507); pLex-hLAG-3/IΔC and pLex/NLS-hLAG-3/IΔC (fromR457 to E481); pLex-hLAG-3/EP and pLex/NLS-hLAG-3/EP (from E478 to L503). 4.2 Two-hybrid screen and interaction analysis Yeast, medium and two-hybrid procedures were handled according to published methods 21, 22. For the two hybrid-screen, we used a human activated PBL librarycloned in the pGAD-1318 vector (Hybrigenics, Paris, France) which contains the activation domain of GAL4 under the control of the entire ADH1 strong yeast promoter. For library screening, yeast strain L40 which contains the LacZ and HIS3 reporter genes downstream of the binding sequence of LexA, was sequentially transformed with pLex/NLS-hLAG-3/I and 60 μg of the human activated T cell library using the lithium acetate method. Double transformants were plated on yeast drop-out medium lacking tryptophan, leucine and histidine, and were incubated at 30°C for 3 days. Positive colonies His+ were patched on selective plates for growth and were then replicated on Whatman 40 paper. The β-galactosidase activity was tested by a filter assay. For interaction studies, two methods were used: by co-transformation of strain L40 with pairs of pLex and pGAD vectors, or by mating the strain L40 expressing a pLex vector with the strain AMR70 containing a pGAD vector. In both cases, binding was tested for growth in histidine-deficient medium and for β-galactosidase activity. Signals described as being negative were not detected even after 3 days or 24 h for the HIS3 and LacZ reporter genes, respectively. No discrepancy was ever observed between the histidine auxotrophy and the β-galactosidase tests. 4.3 Protein expression and purification LAP polypeptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli and immobilized on affinity matrix beads. Briefly, fresh overnight cultures of E. coli HB101 or XL-1 blue cells harboring the pGEX plasmid expressing GST or GST-LAP proteins were diluted 1:10 in Luria-Bertani (LB) broth supplemented with 20 μg/ml ampicillin and the cultures were grown for 3 h with 0.1 mM IPTG (Sigma, St. Louis, MO). Cell pellets were collected by centrifugation and lysed in Tris buffer containing 1% NP40 and anti-proteases. The soluble fraction was prepared by centrifugation at 10,000×g for 15 min at 4°C. The GST and recombinant GST fusion proteins were purified by coupling to glutathione-Sepharose 4B beads (Pharmacia, Uppsala,Sweden) by gentle mixing at 4°C for 40 min followed by extensive washing. The protein-bound affinity beads were analyzed and quantitated by Coomassie blue R-250 staining following SDS-PAGE analysis. 4.4 Preparation of cell lysates and in vitro binding assays Human PBMC were isolated from venous blood by Ficoll-Paque density gradient centrifugation. T lymphocytes were obtained by stimulating PBMC with 1 μg/ml of PHA-P (Wellcome, Beckenham, GB) at 37°C and 10% CO2 in complete culture medium (RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 4 mM L-glutamine, 1 mM pyruvate, 0.2 mM NaOH, 50,000 IU penicillin and 50 mg/ml streptomycin). After 3 days of culture, whole cell lysates were prepared in Tris cell lysis buffer containing 1% NP40 and anti-proteases. The hLAG-3 protein was synthesized in vitro using the T7-coupled rabbit reticulocyte lysate system (TNT, Promega, Madison, WI). Equal amounts of GST-LAP or control GST proteins immobilized on beads were incubated for 3 h at 4°C with direct whole cell lysates (after centrifugation of nuclei) or with the in vitro translated hLAG-3 protein in a binding buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin). Bound proteins were then extensively washed in PBS buffer and analyzed by Western blotting. 4.5 Cell lines and antibodies The Jurkat T cell line and the Epstein Barr Virus (EBV)-transformed B cell line were grown in complete 1640 RPMI culture medium at 37°C and 6% CO2. RCC7 (a renal cell carcinoma cell line, 23) were cultivated in complete DMEM medium at 37°C and 6% CO2. A polyclonal serum was raised against a peptide (SPREPLEPLNFPDPEYK) derived from the deduced amino acid sequence of LAP by immunizing rabbits with three injections of peptide-BSA (Neosystem, Strasbourg, France). 4.6 Western blot Cells (106) were washed and lysed at 4°C for 60 min in 100 μl Tris cell lysis buffer. Cell debris were removed by 10-min centrifugation at 10,000×g and the lysates heat-denatured in SDS sample buffer for 5 min. Total cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were saturated with 5% dry milk for 1 h at 37°C and incubated with primary antibody diluted 1:3,000 in TBS for 1.5 h with slow agitation. After incubating the membranes with the GAR-peroxidase secondary antibody, the signal was detected by enhancedchemiluminescence (ECL, Amersham, Buckinghamshire, GB). To determine the tissue distribution of LAP, a commercial Western blot containing 75 μg of total cellular protein from eight different human tissues (Chemicon, Temecula, USA) was used. Acknowledgements This work was supported by a grant from "La Ligue contre le Cancer (Comité des Hauts de Seine)" and from Serono International. N.I. and S.A. are recipientsof a fellowship from "La Ligue contre le Cancer (Comité du Val de Marne)". WILEY-VCH WILEY-VCH WILEY-VCH References 1 Triebel, F., Jitsukawa, S., Baixeras, E., Roman-Roman, S., Genevée, C., Viegas-Pequignot, E. and Hercend, T., LAG-3, a novel lymphocyte activation gene closely related to CD4. J. Exp. Med. 1990. 171: 1393– 1405. CrossrefCASPubMedWeb of Science®Google Scholar 2 Baixeras, E., Huard, B., Miossec, C., Jitsukawa, S., Martin, M., Hercend, T., Auffray, C., Triebel, F. and Piatier-Tonneau, D., Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J. 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