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Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much of the early analysis focused on human LAG-3, which was initially described as a CD4-like Ig superfamily protein (7). Both CD4 and LAG-3 have four extracellular Ig-like domains with conserved structural motifs throughout D1 to D4 domains (7, 8). Unlike CD4, LAG-3 is only expressed on activated T cells and a subpopulation of NK cells (7, 8, 9, 10). More recent studies in mice have shown that LAG-3 is also expressed on a subpopulation of 
T cells and CD4+CD25+ regulatory T cells (11). LAG-3 binds to MHC class II molecules, but with a much higher affinity than CD4 (11, 12, 13). Blocking of LAG-3-MHC class II interaction up-regulates CD4+ T cell activation, suggesting that LAG-3 might act as a negative competitor for CD4 (12, 13). Adhesion assays using human LAG-3-expressing systems suggested that oligomerization of LAG-3 might be crucial for LAG-3-MHC class II interaction (14). However, no biochemical data supporting LAG-3 oligomerization have been presented. A soluble human LAG-3-Ig fusion protein has been shown to induce dendritic cell maturation through MHC class II-dependent signal transduction (15, 16). Although studies have suggested that LAG-3 associates with the TCR:CD3 complex and negatively regulates TCR-induced signal transduction, the mechanism by which this occurs remains elusive (1). However, recent data from our laboratory have shown that the inhibitory effect of LAG-3 on CD4-dependent T cell function is dependent on a conserved KIEELE motif in the cytoplasmic domain (3, 4).
LAG-3 is expressed as a type I transmembrane protein on the cell surface (7). Interestingly, a soluble form of LAG-3 (sLAG-3) was detected by ELISA in human serum (17). In a recent study it was shown that serum samples from active tuberculosis patients had lower soluble LAG-3 level than those from uninfected individuals (18). In addition, patients with a favorable prognosis after treatment had higher serum soluble LAG-3 levels compared with patients with a poor clinical prognosis, suggesting that soluble LAG-3 may be biologically active and could act as a potential diagnostic indicator (18). However, it is still unclear how sLAG-3 is generated. Data have been presented suggesting that it is generated by an alternative splicing event (2); however, this has not been verified, and murine LAG-3 RNA occurs as a single length species (11).
These observations have added to the growing realization that very little is known about the biochemical makeup of this important regulatory T cell protein. In this study we addressed this and asked the following questions. First, does murine LAG-3 associate with any other proteins on the cell surface of T cells? Second, does LAG-3 oligomerize? Third, is murine LAG-3 generated in a soluble form?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LAG-3 constructs were produced using recombinant PCR as previously described (3, 4). All LAG-3 constructs were cloned into a murine stem cell virus-based retroviral vector, MSCV-IRES-GFP (pMIG). Details of primers and strategy will be provided on request (nianyu.li@stjude.org). The CD4+ 3A9 T cell hybridoma (hen egg lysozyme 48–63 specific; H-2Ak restricted) (19) and a CD4 loss variant (3A9.CD4–) (20) T cell hybridoma were transduced as previously described (11). Cells were sorted on a MoFlow (DakoCytomation, Fort Collins, CO) for uniform GFP expression.
Antibodies
The following Abs were used for immunoprecipitation and/or Western blotting: rat anti-LAG-3 mAb (C9B7W, specific for the D2 domain; BD Pharmingen, San Diego, CA), anti-CD4 mAb (GK1.5, specific for the D1 domain; BD Pharmingen), mouse anti-FLAG mAb (M2; Sigma-Aldrich, St. Louis, MO), mouse anti-Myc mAb (9E10; Sigma-Aldrich), rabbit anti-LAG-3.D1 (generated against a D1 loop peptide (DSGQPTPIPALDLHQGMPSPRQPAPGRYTKLH) by Covance Immunology Service, Princeton, NJ), rabbit-anti-LAG-3.CY (generated against a COOH-terminal peptide (QPFPAQRKIEELERELETEMGQEKLH) by Covance), and rabbit anti-murine CD4.D1/D2 (provided by K. Karjalainen, Istituto di Ricerca in Biomedicina, Bellinzona, Switzerland).
Surface biotinylation and cross-linking
Cell surface proteins were biotinylated or cross-linked as described previously (21, 22). In brief, cells (2 x 107) were washed three times in HBSS (Mediatech, Holly Hill, FL) and labeled with either 1 mg/ml NHS-SS-biotin or 1 mg/ml BS-3 (Pierce, Rockford, IL) for 30 min on ice. Excess biotin or BS-3 was quenched with 25 mM lysine/HBSS. Cells were then washed three times with HBSS before lysis in 1% Nonidet P-40 (Sigma-Aldrich).
Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting were performed as previously described (20, 22). In brief, cells were lysed with lysis buffer containing 1% Nonidet P-40 (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 2 mM Pefabloc (Roche, Indianapolis, IN), pH 7.4) on ice for 30 min, followed by centrifugation at 15,000 x g for 10 min. For plasma membrane preparation, cells were resuspended in ice-cold plasma membrane preparation buffer (10 mM HEPES, 10 mM NaCl, 1 mM KCl, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 2 mM Pefabloc, pH 7.4), incubated for 5 min on ice, and then disrupted with a Dounce homogenizer (30 strokes). The homogenate was centrifuged at 1,000 x g to pellet nuclei and unbroken cells. The supernatant was centrifuged for an additional 10 min at 15,000 x g. The resulting pellet was washed twice with the plasma membrane preparation buffer and then solubilized with 1% Nonidet P-40 lysis buffer. Lysates or eluted proteins from immunoprecipitates were resolved by SDS-PAGE (Invitrogen Life Technologies, Carlsbad, CA), and blots were probed as detailed. Blots were developed using ECL (Amersham Biosciences, Piscataway, NJ) and autoradiography. In some experiments, densitometric analysis was performed on Western blots using ImageQuant (Amersham Biosciences).
T cell activation in vitro and in vivo
Splenocytes (2 x 106 cells/ml in complete MEM modified for suspension cultures) from OTII (OVA-specific; H-2Ak-restricted) TCR transgenic mice were activated with OVA peptide 326–339 (10 µM). Splenocytes (1 x 107) and culture medium (1 ml) were collected constitutively for 6 days. For in vivo activation assays, C57BL/6 mice were given 100 µg of staphylococcal enterotoxin B (SEB) i.v (5). Mouse sera were collected on the days indicated and analyzed for sLAG-3 by ELISA. All animal experiments were performed in an American Association for Assessment of Laboratory Animal Care-accredited, specific pathogen-free facility following national, state and institutional guidelines. Animal protocols were approved by the St. Jude institutional animal care and use committee.
sLAG-3 purification and ELISA
For purification of sLAG-3, 1 x 109 LAG-3 transduced 3A9 cells were incubated with serum-free RPMI 1640 (Mediatech) for 2 days. The culture medium was harvested and applied to a C9B7W anti-LAG-3 mAb immunoaffinity column. The sLAG-3 was eluted with 50 mM glycine and 150 mM NaCl, pH 2.5, and dialyzed against PBS overnight. Purity was confirmed by SDS-PAGE and Coomassie Blue staining. Protein concentration was determined with the BCA protein determination kit (Pierce).
ELISA for sLAG-3
C9B7W (5 µg/ml) mAb was coated on 96-well, flat-bottom microtiter plates (Dynatech Laboratories, Franklin, MA) in carbonate buffer (50 mM Na2CO3, pH 10.4) at 37°C for 1 h. The plates were washed three times with PBS-Tween 20 (0.05%) and then blocked with 0.5% FBS in carbonate buffer at 4°C overnight. The plates were washed, and serum or cell culture medium was added. After 1-h incubation at 37°C, the plates were washed, then probed with rabbit anti-LAG-3.D1 antiserum (1/200 dilution, 37°C, 1 h). This was followed by three washes and another 1-h incubation with an HRP-conjugated, anti-rabbit Ig secondary Ab (1/2000 dilution; Amersham Biosciences). Plates were developed with tetramethylbenzidine substrate solution (Pierce), and the reaction was stopped by adding 50 µl of 1 N H2SO4 to each well. Absorbance was measured with a spectrophotometer (Molecular Devices, Sunnyvale, CA). The LAG-3 concentration was calculated using a purified sLAG-3 standard curve.
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Results |
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We first asked whether there were any T cell surface molecules that associate with LAG-3. Initially, we used a murine T cell hybridoma 3A9 that was transduced with murine LAG-3 (11). Immunoblot analysis of surface biotinylated proteins immunoprecipitated with an anti-LAG-3 mAb (D2-specific, C9B7W) revealed two bands, at 
70 kDa (corresponding to the predicted size of LAG-3) and 54 kDa (p54; Fig. 1A). The p54 band was also observed on three other LAG-3-transduced cell lines: a CD4-deficient T cell hybridoma, 3T3 fibroblasts, and Chinese hamster ovary cells (Fig. 1A).
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We examined this further by ectopically expressing LAG-3 molecules that were FLAG-tagged at either the N (LAG-3.FLNH2) or the C (LAG-3.FLCOOH) terminus. The data clearly show that the anti-FLAG Ab pulled down both full-length LAG-3 and the p54 fragment from cells transduced with LAG-3.FLNH2, whereas full-length LAG-3 and the p16 fragment were immunoprecipitated from cells transduced with LAG-3.FLCOOH (Fig. 1, D and E). As expected, addition of the FLAG tag to the C terminus increased the size of the p16 fragment to 17 kDa (Fig. 1E).
Taken together, these results confirm that the p54 fragment is a COOH-terminally truncated version of LAG-3, which most likely, given its size, contains most or all of the extracellular domains. Furthermore, our results also confirm that the p16 fragment is the remaining COOH-terminal LAG-3 tail. As might be anticipated, the sizes of the two fragments appear to equal the size of the full-length LAG-3 molecule (70 kDa).
LAG-3 oligomers are expressed on the T cell surface
Given its size and membrane association, it is probable that the p16, not the p54, fragment contains the transmembrane domain. If so, how does p54 remain membrane associated? It could dimerize with either the full-length LAG-3 or another membrane-associated molecule. The latter seemed less likely, because no other LAG-3-associated molecule was identified in our surface biotinylation experiments, although we cannot rule out the possibility that it associates with a molecule that cannot be biotinylated due to a lack of exposed lysines. Previous studies have suggested that human LAG-3 is expressed as a dimer (14), although no biochemical support for this hypothesis has been presented. Thus, we tested whether murine LAG-3 is expressed as a dimer by using the membrane-impermeable, homobifunctional (sulfo-NHS ester) cross-linker BS-3. T cells were treated with BS-3, and lysates were immunoprecipitated with the anti-LAG-3 mAb. Western blot analysis with the anti-LAG-3.D1 antiserum revealed a 140-kDa band corresponding to the size of a LAG-3 dimer. Additional bands of 200 kDa or more were also detected, suggesting that LAG-3 may be expressed as an oligomer (Fig. 2A). Clearly not all the LAG-3 was cross-linked in these experiments. There are two possibilities. First, a proportion of LAG-3 may be expressed as a monomer. Second, all LAG-3 is oligomerized, but cross-linking is either inefficient or suboptimal. Additional approaches will be required to resolve this issue.
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LAG-3 is released as a soluble fragment
Several studies have shown that sLAG-3 is found in human serum (2, 17, 18). Thus, we tested whether the p54 truncated murine LAG-3 fragment was released from T cells as a soluble protein. Immunoblot analysis clearly showed the presence of sLAG-3 in the tissue culture medium of LAG-3-expressing T cell hybridomas (Fig. 3A); sLAG-3 was also generated from the 3T3 and Chinese hamster ovary LAG-3-expressing cell lines (data not shown). The Mr of sLAG-3 was equivalent to that of the truncated p54 fragment described above. Subsequent experiments demonstrated that only T cells expressing the N-terminally FLAG-tagged LAG-3 (LAG-3.FLNH2) produced sLAG-3 that could be revealed by anti-FLAG immunoblot (Fig. 3B). To confirm that the truncated p54 fragment was released into the culture medium and to assess the speed of this cleavage event, pulse-chase analysis was performed. T cells were surface-biotinylated and incubated at 37°C, and samples were collected at different time points (Fig. 3, C–E). Whole-cell lysates and supernatant were immunoprecipitated with the anti-LAG-3 mAb and probed with either streptavidin (Fig. 3C) or anti-LAG-3.D1 (Fig. 3D) antiserum. This analysis revealed three interesting points. First, densitometric analysis revealed an exponential decline in surface-biotinylated LAG-3, which inferred a cleavage process that followed first-order kinetics (Fig. 3E). The cleavage rate had a t1/2 of 5.4 h, and all detectable biotinylated LAG-3 was released by 24 h. This clearly shows that cleavage is not an immediate event and thus explains why all p70 molecules are not cleared. Second, all the surface-biotinylated LAG-3 was ultimately released into the culture medium, as shown by the progressive decline of biotinylated LAG-3 and the progressive increase in sLAG-3 in the medium. Note that the total amount of LAG-3 did not change as this cleaved LAG-3 was replaced with newly synthesized molecules (Fig. 3D, upper panel). Third, there appeared to be a progressive decline in both the full-length and truncated LAG-3 bands, suggesting that cleavage may be stoichiometric. Collectively, these results suggest that LAG-3 is cleaved and subsequently shed into the medium. Given that these cells were transduced with a fully spliced murine LAG-3 cDNA, it is likely that these fragments are generated as a consequence of proteolytic cleavage, rather than an alternative splicing event.
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We next ask which domains of LAG-3 were responsible for cleavage. Given that the LAG-3-related molecule CD4 is not cleaved and released into the medium, we used a panel of LAG-3:CD4 chimeras to determine the site of cleavage (Fig. 4A). Our initial analysis suggested that that the LAG-3 D1, D2, transmembrane, and cytoplasmic domains were not required for cleavage (Fig. 4, B and C). This was most graphically seen by the presence or absence of soluble protein in the medium. For constructs that contained the LAG-3:D1 domain, there appeared to be a correlation between the presence of a truncated product and the release of soluble protein into the supernatant (Fig. 4B, lanes 2–4). For constructs that lacked the cytoplasmic domain or had the much smaller CD4 cytoplasmic domain, this correlation could not be readily evaluated, because the truncated and full-length proteins almost comigrated (Fig. 4B, last three lanes).
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Does LAG-3 oligomerize via the D1 domain?
Previous studies have suggested that human LAG-3 dimerizes via the D1 domain (14). However, this conclusion was based on indirect functional analysis of LAG-3 point mutations rather than a direct biochemical examination of dimerization. Thus, we asked whether our constructs could be used to determine whether murine LAG-3 dimerizes via the D1 domain. Initially, we noticed that the cleavable LAG-3:CD4 chimeras, which contained a CD4 rather than a LAG-3 D1 domain, lacked the lower truncated fragment that was seen with wild-type LAG-3 (Fig. 4C). This issue was examined more directly by comparing wild-type LAG-3 with CD4D1:LAG-3D2-CY, which differ only in the D1 domain. Because truncated LAG-3 must be membrane associated for it to be visualized in these experiments, any disruption of dimerization would prevent its immunoprecipitation and detection by Western blot analysis. As clearly shown, a lower truncated fragment is associated with wild-type LAG-3, but not with CD4D1:LAG-3D2-CY, even though both generate a soluble product (Fig. 4F). These data suggest that the soluble CD4D1:LAG-3D2-CP fragment cannot remain membrane associated, inferring a role for the LAG-3 D1 domain in LAG-3 dimerization. Although these data are consistent with previous suggestions (14), we cannot completely rule out the possibility that the presence of the CD4.D1 domain disrupts LAG-3 dimerization via a different domain.
Soluble LAG-3 is released by activated T cells and is found in sera
To date we have shown that transduced cell lines will generate sLAG-3. Does this also occur with normal T cells in vitro and in vivo? Splenocytes from OTII TCR transgenic mice were stimulated in vitro with OVA326–339 peptide. Cells and culture medium were collected daily, and the presence of full-length, truncated, and sLAG-3 forms was assessed. Western blot analysis showed that LAG-3 was not detectable in resting, naive T cells (Fig. 5A). However, LAG-3 was expressed by day 1, peaked by day 2, and started to diminish 5–6 days poststimulation. In contrast, both truncated and sLAG-3 were detectable by day 2, but continued to increase beyond day 6, suggesting that the regulation of expression and cleavage may be different or the latter delayed. This was also evident when the sLAG-3 concentration was determined by ELISA, with up to 700 ng/ml produced (Fig. 5B). It should be noted that the Mr of the sLAG-3 generated by splenic T cells was comparable to that generated by T cell hybridomas, suggesting that the cleavage mechanism is the same. Generation of the p16 LAG-3 cytoplasmic tail fragment correlated with the production of sLAG-3 (Fig. 5A). However, the amount of this fragment relative to that found in T cell hybridomas appeared to be less, suggesting that it might be more rapidly degraded in normal T cells.
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180 ng/ml sLAG-3 in serum, as determined by ELISA (Fig. 5C). As expected, no sLAG-3 was detected in serum from LAG-3–/– mice, and heterozygote LAG-3+/– mice had half the amount of sLAG-3 compared with wild-type C57BL/6 mice. Interestingly, sera from C57BL/6 and RAG-1–/– mice had the same amount of sLAG-3 despite the latter lacking T cells, suggesting that NK cells may be the primary source of sLAG-3 in unmanipulated mice. Because T cell activation results in an increase in sLAG-3 production, we asked whether activation in vivo would similarly result in an increase in serum sLAG-3. It has previously been shown that injection of mice with SEB resulted in an 2-fold increase in the number of TCR-V
8+ T cells by day 2, which subsequently returned to normal by day 4 (4). Interestingly, this was precisely mirrored by an 2-fold increase in the serum sLAG-3 concentration (Fig. 5D). These data support the suggestion that the serum sLAG-3 concentration may be used diagnostically as an indication of T cell activation due to infection or autoimmunity (18, 23, 24). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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54-kDa truncated form of LAG-3 (9). What might be the basis for this discrepancy? First, it is possible that Triebel and colleagues did not observe this band due to differences in their experimental system or reagents used. Second, these observations may reflect differences in murine and human LAG-3, although our experiments clearly showed that the human LAG-3 CP could also be cleaved when inserted into murine LAG-3. Third, it is conceivable that there are differences in the way LAG-3 is cleaved in human and murine cells. This seems unlikely because we have seen cleavage of LAG-3 in 293T cells, a human embryonic kidney cell line. Additional studies will be required to resolve these issues.
The source of sLAG-3 remains obscure. Our previous studies showed that a significant number of cells in the splenic red pulp expressed LAG-3 (11). To date, 
T cells, T cells, and NK cells appear to be the only source of LAG-3, with 10% of the latter constitutively LAG-3+ in resting mice. Given that RAG-1–/– mice had the same level of serum sLAG-3 as C57BL/6 mice, it is possible that NK cells are the source. However, this has yet to be confirmed directly, and additional studies will be required to resolve this important question.
Previous studies have suggested that human LAG-3 dimerizes via the D1 domain, although this was based on indirect functional analysis of LAG-3 point mutants rather than direct biochemical evidence (14). Such support is now provided by our experiments, which are consistent with LAG-3 dimerization via the D1 domain. However, this interaction appears to be relatively weak, because it could only be directly observed after cross-linking. Several studies have also suggested that CD4 dimerizes, but via a different domain, D4 (25). Curiously, no truncated version of CD4 was found associated with the cleavable form of CD4 as might have been anticipated (Fig. 4E). Although it is possible that the LAG-3 CP is disrupting this association, it is also conceivable that this dimerization is either very weak or only occurs with a low percentage of CD4 molecules.
Our data strongly suggest that proteolytic cleavage of LAG-3 occurs within the short CP region between the membrane-proximal D4 domain and the transmembrane domain. Importantly, cell surface cleavage could be conferred on CD4 by insertion of the 20-aa LAG-3 CP. Clearly, it will be important to determine the protease responsible for LAG-3 cleavage and how this process is regulated. Although this effort is ongoing, it does appear that it is not a random event of a protease-sensitive site, because lysis of cells at room temperature in the absence of protease inhibitors gives rise to a number of degradation products, but does not lead to an increase in the amount of truncated LAG-3 (data not shown).
What is the role of LAG-3 cleavage? Does sLAG-3 have an important physiological function? Does the release of the p16 tail fragment contribute to LAG-3 signaling? These are clearly important questions that remain unresolved. There are three possible roles for sLAG-3 and LAG-3 cleavage. First, it may induce signaling in MHC class II+ cells. It has been shown that the human LAG-3:Ig fusion protein, which contains the entire extracellular domain, bound to MHC class II molecule and induced dendritic cell maturation by triggering MHC class II signaling (15, 16). It is not yet known whether LAG-3 has the same effect on murine DCs. Also, it is unclear whether LAG-3 bivalency is important for this effect. Given that LAG-3 is expressed as a dimer on the cell surface, it is possible that sLAG-3 is also a dimer. Whether sLAG-3 has the same effect on DC maturation remains to be determined. Second, because LAG-3 has a very high affinity for MHC class II molecules it may block MHC:CD4 or even the MHC:TCR interaction. Indeed, there is some support for this idea from studies using human LAG-3:Ig fusion protein. However, a LAG-3 mutant that contains a deletion of the KIEELE motif (LAG
K) has no effect on T cell function, yet generates the same amount of sLAG-3 as wild-type LAG-3 (4) (Fig, 4B). Third, it is also possible that the cleavage of LAG-3 is important for regulating cell surface expression of LAG-3. This might have the dual consequence of reducing LAG-3 signaling, by removing LAG-3 from the cell surface and generating a sLAG-3 product that might compete with membrane-associated LAG-3 for ligand binding. One might expect that these events would collectively reduce LAG-3 signaling and thus function. Therefore, regulation of LAG-3 cleavage might act as a rheostat for the regulation of LAG-3 function.
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Acknowledgments |
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Footnotes |
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1 This work was supported by the Pediatric Oncology Education Program (Grant CA23944; to S.M.M.), funds from the National Institutes of Health (Grant AI39480), a Cancer Center Support CORE grant (CA21765), and the American Lebanese Syrian Associated Charities (to D.A.A.V.). 
2 Address correspondence and reprint requests to Dr. Dario A. A. Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. E-mail address: dario.vignali{at}stjude.org
3 Abbreviations used in this paper: LAG-3, lymphocyte activation gene 3 (CD223); CP, connecting peptide; SEB, staphylococcal enterotoxin B; sLAG-3, soluble LAG-3.
Received for publication June 17, 2004. Accepted for publication September 28, 2004.
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References |
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production. FASEB J. 10:769.[Abstract]
on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells. Eur. J. Immunol. 27:2239.[Medline]
production are variably coregulated in different human T lymphocyte subpopulations. J. Immunol. 161:489.This article has been cited by other articles:
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  C. J. Workman, Y. Wang, K. C. El Kasmi, D. M. Pardoll, P. J. Murray, C. G. Drake, and D. A. A. Vignali LAG-3 Regulates Plasmacytoid Dendritic Cell Homeostasis J. Immunol., February 15, 2009; 182(4): 1885 - 1891. [Abstract] [Full Text] [PDF]
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B. Liang, C. Workman, J. Lee, C. Chew, B. M. Dale, L. Colonna, M. Flores, N. Li, E. Schweighoffer, S. Greenberg, et al. Regulatory T Cells Inhibit Dendritic Cells by Lymphocyte Activation Gene-3 Engagement of MHC Class II J. Immunol., May 1, 2008; 180(9): 5916 - 5926. [Abstract] [Full Text] [PDF]
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 J. K. Hansen, K. P. Demick, J. M. Mansfield, and K. T. Forest Conserved Regions from Neisseria gonorrhoeae Pilin Are Immunosilent and Not Immunosuppressive Infect. Immun., August 1, 2007; 75(8): 4138 - 4147. [Abstract] [Full Text] [PDF]
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 K. J. Garton, P. J. Gough, and E. W. Raines Emerging roles for ectodomain shedding in the regulation of inflammatory responses J. Leukoc. Biol., June 1, 2006; 79(6): 1105 - 1116. [Abstract] [Full Text] [PDF]
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)/CD4 (
) chimeras used. Human LAG-3 domains are in gray. D1-D4, Extracellular Ig domains D1 to D4, respectively; CP, CP region; TM, transmembrane domain; CY, cytoplasmic domain. Letters in the boxes indicate the amino acids at the NH2 (on the left) or COOH (on the right) terminus of the domains. B–F, Lysates or supernatant from the T cell hybridomas indicated were immunoprecipitated with either anti-LAG-3 mAb (B, D, and F) or anti-CD4 mAb (C and E), and blots were probed with anti-LAG-3.D1 antiserum (B, D, and F, third panel), anti-LAG-3.CY (F, second panel), streptavidin (F, first panel), or anti-CD4.D1/2 antiserum (C, E, and F, fourth panel).