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International Immunology, Vol. 11, No. 11, 1745-1752, November 1999
© 1999 Japanese Society for Immunology

The MHC class II ligand lymphocyte activation gene-3 is co-distributed with CD8 and CD3–TCR molecules after their engagement by mAb or peptide–MHC class I complexes

Sigrid Hannier and Frédéric Triebel

Laboratoire d'Immunologie Cellulaire, Institut Gustave-Roussy, 39, rue Camille Desmoulins, 94805 Villejuif and Laboratoire d'Immunologie des tumeurs, Faculté de Pharmacie, Université Paris XI, 5 rue Jean-Baptiste Clément, 92296 Chatenay-Malabry, France

Correspondence to: F. Triebel, Institut Gustave-Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Previous studies indicated that signaling through lymphocyte activation gene-3 (LAG-3), a MHC class II ligand, induced by multivalent anti-receptor antibodies led to unresponsiveness to TCR stimulation. Here, lateral distribution of the LAG-3 molecules and its topological relationship (mutual proximity) to the TCR, CD8, CD4, and MHC class I and II molecules were studied in the plasma membrane of activated human T cells in co-capping experiments and conventional fluorescence microscopy. Following TCR engagement by either TCR-specific mAb or MHC–peptide complex recognition in T–B cell conjugates, LAG-3 was found to be specifically associated with the CD3–TCR complex. Similarly, following CD8 engagement LAG-3 and CD8 were co-distributed on the cell surface while only a low percentage of CD4-capped cells displayed LAG-3 co-caps. In addition, LAG-3 was found to be associated with MHC class II (i.e. DR, DP and DQ) and partially with MHC class I molecules. The supramolecular assemblies described here between LAG-3, CD3, CD8 and MHC class II molecules may result from an organization in raft microdomains, a phenomenon known to regulate early events of T cell activation.

Keywords: MHC, T cell, TCR


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Lymphocyte activation is a central event in the generation of an immune response, resulting in clonal expansion of immunocompetent cells and their acquisition of effector functions (1). Activation is accompanied by the modification of the expression of several genes that encode molecules involved in cell proliferation, effector functions and cell–cell interactions, which are defined as very early, early and late activation molecules according to their expression kinetics. T cell activation depends on the complex integration of signals that are delivered by multiple antigen receptors that are serially triggered by physiological membrane-associated ligands on the antigen-presenting cell (APC) (i.e. peptide bound to MHC molecules). Most receptor-proximal activation events have nonetheless been identified using multivalent anti-receptor antibodies, eliminating the need to use the more complex APC, which are thought to trigger the same biochemical pathways.

The co-receptor molecules, CD4 and CD8, as well as other surface antigens, such as CD38, CD40 ligand or CD28 (1–4), potentiate signal one delivered by the TCR and increase lymphocyte sensitivity to the antigen. Thus, these molecules are crucial for triggering the immune response in vivo when the antigen concentration is low or when the antigen is a weak agonist. Such potentiation is generally thought to result from the ability of the TCR and the TCR-associated receptors to cluster at the contacts between T cells and APC during antigen-specific interactions. Indeed, some anti-TCR antibodies are very potent at activating T cells even though they do not efficiently cross-link the TCR, e.g. by promoting a closer association between CD4 and TCR (5).

Over the years, several molecules have been found to be associated to CD3–TCR complexes. Some molecules are constitutively expressed antigens such as the CD4 (5,6) and CD8 (7) co-receptors, CD5 (8), the adaptator protein termed TCR interacting molecule (TRIM) (9) or the glycosylphosphatidylinositol-anchored membrane protein termed thymic shared antigen-1 (TSA-1) (10). Other are activation antigens such as CTLA-4 (11) and H4 (12). Whereas the CD4 and CD8 co-receptors have generally been shown to potentiate T cell activation, TSA-1 (13), CTLA-4 (14) and CD5 (15) inhibit T cell responses.

Lymphocyte activation gene-3 (LAG-3) is a T and NK cell-specific activation antigen (16) which, like CD4, is a ligand for MHC class II molecules (17,18). LAG-3 signaling induced by multivalent anti-receptor antibodies leads to unresponsiveness to TCR stimulation with inhibition of both proliferation and cytokine secretion, and down-regulation of TCR expression (19). LAG-3 cross-linking inhibits calcium response to CD3 stimulation only in conditions where LAG-3 and CD3 are co-engaged. Unexpectedly, this MHC class II ligand LAG-3 is expressed at higher levels on CD8+ than on CD4+ T cells (20). This difference in expression patterns between the two T cell subsets may be due to the fact that LAG-3 is nested in the CD4 locus (21) and that its promotor may be partially silenced when the CD4 gene is activated by proximal or distal enhancers (22). Here we studied LAG-3 association with CD3–TCR complexes and TCR co-receptors after their engagement by using mAb or after antigen recognition that allowed serial triggering of the TCR. We also tested whether LAG-3 co-clustered with MHC molecules following aggregation of the latter proteins with specific mAb.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Antibodies
The following mouse mAb were used at saturating concentrations. Protein A-affinity-purified OKT3 (IgG2a) specific for CD3{varepsilon}, OKT4 (IgG2b) specific for CD4, OKT8 (IgG2a) specific for CD8, D1.12 (IgG2a) specific for HLA-DR, W6.32 (IgG2a) specific for MHC class I and C4C8D9 (IgG1) specific for LFA-1 were used as ascitic fluids. 17B4 (IgG1) mAb recognized the LAG-3.1 epitope on the extra loop of the first IgSF domain of LAG-3 (17). 13B8-2 (IgG1) specific for CD4, BMA031 (IgG2b) specific for TCR, I3 (IgG2a) specific for MHC class II molecules were purchased from Immunotech (Marseilles, France). BRAFB6 (IgG2b) specific for HLA-DP was obtained from Novocastra (Newcastle, UK) and TU169 (IgG2a) specific for HLA-DQ obtained from PharMingen (San Diego, CA). Goat anti-mouse mAb (Texas Red-conjugated anti-IgG1 or anti-IgG2a, and FITC-conjugated anti-IgG2a or anti-IgG2b) were from Southern Biotechnology Associates (Birmingham, AL).

T cells
Peripheral blood mononuclear cells were isolated from heparinized venous blood of healthy volunteer donors by Ficoll-Paque (Pharmacia, Piscataway, NJ) density gradient centrifugation method. T cells were stimulated by 1 µg/ml phytohemagglutinin (PHA-P; Murex Diagnostics, Chatillon, France) for 3 days or for 20 days with re-stimulation every week by addition of 1 µg/ml PHA and IL-2 at 10 IU/ml (Roussel Uclaf, Romainville, France) plus IL-12 at 2 ng/ml (R & D Systems, Minneapolis, MN). The HLA-A2-restricted CD8+ cytolytic 11C2 clone was obtained after stimulation and cloning of TILs from a patient with a metastatic renal cell carcinoma (23). 11C2 recognizes a decapeptide at position 286–295 of HSP70-2 mutated at position 292 (SLFEGIDI*YT, asterisk indicates the F to I substitution). Cells were thawed and cultured for 1 day in RPMI 10% human AB serum with IL-2 at 10 IU/ml (Roussel Uclaf). The autologous Epstein–Barr virus (EBV)-transformed B cell line was cultured in RPMI 10% FCS.

Capping and immunofluorescence microscopy
Antibody-induced capping was performed as described (19). Briefly, PHA blasts were incubated in ice-cold RPMI/5% FCS at 5x106 cells/ml with dialysed mAb specific for a given T cell surface molecule for 20 min, washed and incubated for 20 min with 20 µg/ml of the appropriate FITC- or Texas Red-labeled goat anti-mouse subclass-specific Ig. Cells were then either fixed immediately or incubated at 37°C for 5–15 min to induce capping of the mAb-tagged cell surface receptors. Cells were washed and incubated for 20 min in ice-cold PBS containing 0.1% NaN3 with mAb specific for a second T cell surface molecule, followed by incubation with FITC- or Texas Red-labeled goat anti-mouse subclass-specific Ig.

For analysis of antigen recognition-induced caps during T–B cell conjugate interactions, the EBV B cells were loaded for 1 h in RPMI/5% FCS at 37°C with the mutated HSP70-2 decapeptide at 2x10–5 M. The 11C2 cytotoxic T lymphocyte (CTL) clone and autologous EBV B cells were then incubated at a 1:3 T:B cell ratio for 30 min. Samples of cells (2x105) were removed and plated on poly-L-lysine-coated glass microslides (Menzel-Glaser, Saarbrucken, Germany) and cells were fixed 10 min with freshly prepared PBS/4% formaldehyde. Cells were then labeled as described for antibody-induced capping.

Finally, cells were mounted on slides using Immu-mount (Shandon, Pittsburgh, PA) as an anti-fade solution. To generate optical sections of fluorescently labeled cells, an imaging system consisting of a Provis AX70 microscope (Olympus France, Rungis, France) equipped with a x100 oil immersion objective lens carrying a piezoelectric Z-axis focus device, a CCD camera (Photometrics Sensys, Tucson, AZ) and a set of computer controlled excitation filters was used. The light haze inherent to fluorescent signals was deblurred mathematically using the Exhaustive Photon Reassignment software (Scanalytics, Billerica, MA) (24).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
TCR-induced co-capping of LAG-3 molecules
We have recently shown that lateral association of LAG-3 and CD3{varepsilon} occurs in mAb-induced CD3-capped T cells (19). This finding does not necessarily imply that LAG-3 would co-cap with the TCR. Indeed, functional uncoupling of some TCR{alpha}ß and CD3 chains occurs in activated T cells (25,26). Also, anti-CD3{varepsilon} mAb were found to be inefficient at inducing co-capping of the CD8 co-receptor while anti-TCR{alpha}ß mAb did induce CD8–TCR co-capping (7). We thus studied whether LAG-3 may associate with TCR molecules on the surface of human activated T lymphocytes. TCR molecules on PHA blasts were collected into caps by cross-linking with specific mAb and the capped cells were examined by indirect immunofluorescence microscopy to determine whether LAG-3 was co-collected with the TCR caps. Conventional fluorescence microscopy images were acquired and deblurred by using a deconvolution process with exhaustive photon reassignment. After warming the labeled cells for 5 min at 37°C, TCR molecules were collected into a cap (Fig. 1AGo). A LAG-3 uniform staining was observed on TCR-uncapped cells (not shown) whereas TCR-capped cells displayed a significant co-distribution of LAG-3 with the TCR caps (Fig. 1AGo). By contrast, no co-capping was found with LFA-1 cell surface receptors on TCR-capped cells (Fig. 1BGo), as shown before with mAb-capped murine T cells (5,12). Data from several experiments are summarized in Table 1Go. Conversely, when the co-capping experiment was done the other way around, no co-capping of the TCR was observed on LAG-3-capped cells (not shown). Overall, LAG-3 was found to be specifically co-distributed with the CD3–TCR complex after mAb-induced TCR engagement on human activated T cells. We also tested two activated NK cell lines positive for CD16, a receptor known to be closely associated with CD3{zeta} chains (27). Capping of CD16 molecules did not induce any co-capping of LAG-3 (data not shown).



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  		Fig. 1. LAG-3 and TCR co-cap after TCR engagement with mAb. TCR-polarized distribution on PHA blasts was induced by cross-linking the TCR molecules with BMA 031 mAb (IgG2b) plus FITC-labeled anti-IgG2b goat anti-mouse for 5 min at 37°C. LAG-3 (A), LFA-1 (B) and CD4 (C) distribution was analyzed on the surface of TCR-capped activated T cells with 17B4 (IgG1), C4C8D9 (IgG1) and 13B8-2 (IgG1) plus the corresponding Texas Red-labeled goat anti-mouse subclass-specific Ig. An optical section of two examples of cells for each case is shown.

 

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  		Table 1. CD3 -associated LAG-3 molecules co-cap with TCR and CD8 molecules on human activated T cellsa
 
The other MHC class II ligand, CD4, has been shown to be physically associated with the TCR on a cloned murine T cell line (5,28). We tested whether TCR engagement by specific mAb may induce CD4 co-clustering on human PHA blasts (Fig. 1CGo). CD4+ TCR-capped activated T cells displayed few CD4 co-caps (17%), as compared to LAG-3 co-caps (75%) on LAG-3+ TCR-capped cells (Table 1Go).

LAG-3 is associated with CD8 following CD8 engagement by specific mAb
The CD3–TCR complex-associated LAG-3 molecules are MHC class II ligands (17). We analyzed whether LAG-3 was associated on PHA blasts with the two other MHC ligands associated with TCR molecules, i.e. the CD4 and CD8 co-receptors (Fig. 2Go). After mAb-induced capping of the co-receptor, 96% of LAG-3+ CD8-capped cells (Fig. 2AGo and Table 1Go) displayed LAG-3 co-aggregation whereas no CD8/LFA-1 co-cap was observed (Fig. 2BGo and Table 1Go). Interestingly, only 26% of LAG-3+ CD4-capped cells displayed LAG-3 co-caps (Fig. 2CGo and Table 1Go). Clustering of CD45 did not induce any co-clustering of the LAG-3 molecule (not shown).



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  		Fig. 2. LAG-3 is associated with CD8 molecules and, to a lesser extent, to CD4 molecules. CD8 and CD4 caps were induced by preincubating PHA blasts with OKT8 (IgG2a) (A and B) or OKT4 (IgG2b) (C) plus appropriate FITC-labeled goat anti-mouse subclass-specific Ig for 15 min at 37°C. Shown are LAG-3 (A) and LFA-1 (B) distribution on mAb-induced CD8-capped cells and LAG-3 distribution on mAb-induced CD4-capped cells (C). An optical section of two examples of cells for each case is shown.

 
LAG-3 is associated with CD3–TCR complexes following antigen recognition
Anti-CD3 mAb are high-affinity ligands and their incapacity to dissociate does not allow serial triggering of TCR molecules, as observed following MHC–peptide complex recognition (4). We thus assessed whether LAG-3 may associate with CD3–TCR complexes after antigen recognition-induced TCR engagement (Fig. 3Go). We analyzed CD3 caps on T–B cell conjugates. The 11C2 CD8+ CTL clone specific for a mutated HSP70 peptide expressed by the autologous renal adenocarcinoma cell line (23) was incubated with autologous EBV-transformed B cells pulsed with the peptide. After 30 min, some T–B cell conjugates were formed with clear CD3 caps (Fig. 3AGo). By contrast, no CD3 cap was observed on conjugates in the absence of the HSP70 decapeptide (Fig. 3BGo). On T cells activated in the presence of the peptide, LAG-3 co-capped with CD3 (Fig. 3AGo), whereas LFA-1 did not (Fig. 3CGo). Some LAG-3-capped conjugates did not display CD8 co-cap (Fig. 3DGo) but CD8 co-caps were observed on most of the conjugates (example in Fig. 3DGo'). Along the same line, most CD3-capped conjugates displayed CD8 co-caps (Fig. 3EGo) even if some of them were partial (Fig. 3EGo'). Similar results were obtained with another renal cell carcinoma-specific CTL (3B8) primed with an intestinal carboxyl esterase nonamer peptide presented by HLA-B7 (data not shown). Specific TCR engagement of these CTL by MHC class II– autologous tumor cells could not be observed in this assay system due to the formation of non-specific conjugates (data not shown). Altogether, these results showed that after antigen recognition, LAG-3 is co-distributed with the CD3–TCR–CD8 complex on human activated T cells.



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  		Fig. 3. LAG-3 co-caps with TCR–CD3 complex after antigen recognition. The 11C2 CTL clone, specific for a mutated HSP70 peptide, was incubated with autologous EBV-transformed B cells for 30 min at 37°C. Shown are formed T–B cell conjugates in which B cells were previously pulsed with (A, C, D, D', E and E') or without (B) the peptide. TCR engagement by MHC–peptide complex recognition induced CD3{varepsilon} caps on T cells in conjugates (A, C, E and E'). (A and C) LAG-3 and LFA-1 distribution on CD3-capped cells respectively. (B) CD3 and LAG-3 uniform distribution in the absence of the HSP-70 peptide. (D and D') CD8 distribution on LAG-3-capped cells (two conjugates). (E and E') CD8 distribution on CD3-capped cells (two conjugates). B cells are shown on immunofluorescence microscopy by increasing goat anti-mouse subclass-specific Ig background.

 
LAG-3 co-caps with MHC class II
MHC class II molecules are expressed on human activated T cells. We tested whether LAG-3 and its ligands could associate on the same cell surface (Fig. 4Go). Activated peripheral blood lymphocytes were enriched in MHC class II+ T cells by culture with PHA for 20 days. mAb-induced MHC class II engagement led to LAG-3 co-clustering (Fig. 4AGo and Table 2Go). This was observed for all three subclasses, i.e. DR, DP or DQ molecules (Fig. 4AGo and Table 2Go). Table 2Go summarizes data obtained in several experiments and indicates that the majority of MHC class II-capped cells also had LAG-3 co-caps (an example in Fig. 4BGo). Few LAG-3 co-caps were also observed on MHC class I-capped cells (Fig. 4CGo and Table 2Go). Overall, these results clearly show that LAG-3 and MHC class II molecules co-clustered on the surface of activated T lymphocytes following mAb-induced capping.



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  		Fig. 4. LAG-3 is associated with MHC class II molecules. MHC class II-polarized distribution on PHA blasts was induced by cross-linking the MHC class II molecules or specifically DR, DP, DQ by indirect immunofluorescence with specific antibodies. LAG-3 (A) and LFA-1 (B) distribution on MHC II-capped cells. (C) LAG-3 distribution on MHC class I-capped cells.

 

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  		Table 2. CD3–TCR complex-associated LAG-3 molecules co-cap with MHC class II molecules on human activated T cellsa
 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Surface receptor patching and capping are initial events in signal transduction in lymphocytes during which numerous proteins migrate to a single pole of the cell after stimulation with antigen or other proliferative stimuli. In the present study, co-capping experiments revealed that LAG-3, a ligand for MHC class II, is co-distributed with CD3–TCR complex and CD8 molecules after their engagement by mAb as well as after antigen recognition. Unlike co-precipitation studies which rely on molecules being able to maintain associations after solubilization in detergents, co-capping studies identify either interactions which occur directly or indirectly between molecules in physiological conditions, or only a clustering of these molecules in raft microdomains which do not necessarily involve protein interactions.

Using different detergents, such as Brij96, CHAPS, Triton X100 or NP-40, we have been unable to detect any association between LAG-3 and either CD3{zeta} or DR molecules. It may therefore be speculated that the supramolecular assemblies described here between LAG-3 and both CD3/CD8 and MHC class II molecules are based on the residence of the relevant molecules within a limited area such as detergent insoluble sphingolipid-cholesterol-rich rafts. Such rafts are known to concentrate many proteins involved in early events of T cell activation, such as CD4, CD8, lck, fyn, cbl or ras (29–31). By contrast, CD3–TCR complexes are excluded from these raft microdomains and enter them only following TCR engagement to interact with MHC ligands such as CD4 (32). Similarly to CD4, LAG-3 may be concentrated in detergent-insoluble rafts, explaining why the recruitment of CD4 (5,12), CD8{alpha} (7) or LAG-3 (the present study) by the corresponding mAb did not lead to TCR–CD3 co-capping, while TCR engagement which is followed by the recruitment of rafts at its site leads to co-capping of these MHC ligands. CD45 is another molecule not included in raft microdomains (31) and its capping does not induce LAG-3 co-caps (the present study). Also, this hypothesis explains why co-immunoprecipitation experiments looking for LAG-3/CD3{zeta} or LAG-3/DR associations in the presence of different detergents were negative.

We observed that LAG-3 was preferentially associated with the CD8 rather than the CD4 co-receptor at the cell surface. Differences between the two T cell subsets have also been found when studying LAG-3 expression and its up-regulation with cytokines. The expression of LAG-3 is 7 times higher on CD8+ than CD4+ T cells (20). Up-regulation of LAG-3 expression by IL-2 plus IL-12 is higher on CD8+ than CD4+ T cells as well (22). This phenomenon could be explained by the sharing of some regulatory elements common to LAG-3 and CD4 genes on chromosome 12p13 where both genes are nested (21), regulatory elements which could partially silence the LAG-3 promotor when the CD4 promotor is activated (22). Unbalanced LAG-3 expression is unlikely to have introduced a bias in our analysis of LAG-3 association with co-receptors because we have chosen to analyze only cells with high LAG-3 expression levels. It is possible that a close association between LAG-3, CD3–TCR complexes and CD8 reflects a distinct and predominant functional role of LAG-3 on CD8+ T cells. In addition to the report of a negative regulatory role of LAG-3 on TCR signaling and to a LAG-3-mediated down-regulation of TCR expression (19), LAG-3-specific mAb, either as whole IgG or as Fab fragments, that inhibit LAG-3/MHC class II interactions were shown to induce prolonged activation and proliferation of CD4+ antigen-specific T cell clones following antigen recognition (33). These results suggest that LAG-3 exerts a negative regulatory role on antigen-dependent stimulation via its association with CD3–TCR complexes. However, LAG-3-specific mAb have not been found to date to alter the proliferation, cytotoxic or cytokine responses of CD8+ T cells (33 and unpublished results).

MHC class II are expressed preferentially on professional APC. Results of the present study suggest that a ligand of MHC class II, LAG-3, is engaged close to CD3–TCR complexes and CD8 co-receptors which together recognize MHC class I molecules on APC. The LAG-3/CD8 association on T cells may in part stabilize the MHC class I and class II associations that are identified in fluorescence energy transfer experiments on APC, such as B cells (34,35).

Interaction of TCR co-receptors with their MHC ligands results in ordered oligomerization, which is required for full TCR activation. The results of mutagenesis experiments have suggested that CD4 or CD8 oligomerization and lattice formation occur at the cell surface (36,37). A refined model of co-oligomerization of CD4, MHC class II and TCR has been proposed (38) where an MHC class II superdimer recognized by TCR promotes the dimerization of two CD4 molecules at their Ig-like D4 domain interface and then such CD4 dimers would further associate through specific loops (CDR3 and CC') of the D1 domain to form an oligomer. In turn, the dimerization and oligomerization of CD4 help to stabilize and cross-link the complex of TCR with its ligand (38). Similarly, oligomerization of LAG-3 on the cell surface may be required to form a stable MHC binding site, as suggested by the finding of three dominant negative mutations (R88A, D109E and R115A) in D1 which inhibit wild-type LAG-3 binding (39). Thus, as for CD4, there is a possible structural basis for the role of LAG-3 in TCR partial agonism and antagonism. The way LAG-3 interacts with TCR complexes, and undergoes subsequent dimerization and oligomerization may determine, in part, the fate of T cell activation.


   Acknowledgments
 
This work has been supported by `Association pour la Recherche contre le Cancer'. We thank Drs C. E. Demeure, N. Mooney and A. Trautmann for critical reading of the manuscript.


   Abbreviations
 
APC antigen-presenting cell
CTL cytotoxic T lymphocyte
EBV Epstein–Barr virus
LAG-3 lymphocyte activation gene-3
PHA phytohemagglutinin

   Notes
 
Transmitting editor: A. McMichael

Received 24 March 1999, accepted 15 July 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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