_[111111UllO-

lmmunogenetics 34: 121-128, 1991

genetics

© Springer-Verlag 1991

Interaction of CD4 with HLA class II antigens and HIV gpl20 Dominique Piatier-Tonneau, Louis-Noel Gastinel, Francois Amblard, Marianne Wojcik, Pierre Vaigot, and Charles Auffray Institut d'EmbryologieCellulaireet Mol6culairedu CNRSet du Coll~gede France, 49 bis, avenuede la Belle Gabrielle, F-94130Nogent-sur-Marne, France Received December 12, 1990; revised version received February 11, 1991

Abstract. We have developed a cellular adhesion assay in which B lymphocytes expressing HLA class II antigens form rosettes with COS cells expressing high levels of cell surface CD4 upon transient transfection with a CDM8CD4 plasmid construct. The assay is specific, quantitative, and overcomes the difficulties encountered with a previously described system using an SV40 viral vector. Rosette formation was inhibited by a series of CD4- and HLA-DR-specific antibodies, as well as by human immunodeficiency virus (HIV) gp 120, and a synthetic peptide derived from part of its binding site for CD4 (amino acid residues 414-434), but not by a variety of other effectors, including several soluble CD4 derivatives. The comparison of this pattern of inhibition with those observed in other systems further emphasizes the great similarity, but incomplete identity, in the CD4 binding sites for HLA class II antigens and HIV gpl20, and supports a model in which CD4 is considered as an allosteric servomodulator of T-cell adhesion and function which probably is induced to interact with HLA class II antigens when associated with the T c r / C D 3 complex.

Introduction The T lymphocyte cell surface glycoprotein CD4 has been the subject of extensive studies aimed at understanding its role in T-cell differentiation and development, in regulation of immune responses in physiological and pathological conditions (reviewed in Bierer et al. 1989; Parnes 1989; Robey and Axel 1990), and because it is the prominent receptor for the human immunodeficiency virus (HIV) (reviewed in Sattentau and Weiss 1988). Initially, CD4 was considered as a marker for the helper/inducer T lymphocyte subset, CD8 being asso-

Address correspondence and offprint requests to." D. Piatier-Tonneau.

ciated with the reciprocal cytolytic/supressor subset. Subsequently, experimental evidence has indicated a closer relationship with the major histocompatibility complex (MHC) restricting element of the T-cell responses, CD4 being associated with HLA class II restricted responses and CD8 with those restricted by class I antigens. Based on these observations, it was suggested that CD4 and CD8 might serve as adhesion receptors for the class II and class I antigens, thus facilitating antigen recognition through the T c r / C D 3 complex, particularly those of low affinity for the antigenic peptide/MHC complex that they recognize (Marrack et al. 1983; Gay et al. 1987). In this context, CD4 and CD8 were considered as accessory molecules. More recent evidence has indicated that CD4 also function to transduce intracellular signals as revealed in T-cell activation assays in the absence of class II antigens. In most cases, CD4 ligands, such as monoclonal antibodies (mAb), induced negative signals (Blanchard et al. 1987; Janeway et al. 1988), and rarely T-cell activation (Carrel et al. 1988). This led to the proposal of a co-receptor model of CD4 function (Janeway 1989). The multiple facets of CD4 function rely on molecular interactions with HLA class II antigens, the T c r / C D 3 complex (Mittler et al. 1989), and the intracellular T cell-specific protein tyrosine kinase p56 ~ck (Turner et al. 1990). In the case of HLA class lI antigens, this has been suggested indirectly in numerous studies (reviewed in Janeway 1989), and it is widely considered that CD4-dependent cellular adhesion of B lymphocytes to cells expressing CD4 upon transfection is direct evidence for CD4-class II interaction reflecting their properties as reciprocal adhesion receptors (Doyle and Strominger 1987). Similar studies and conclusions have been obtained in the case of CD8-class I interactions (Norment et al. 1988). Further definition of CD4 function and the molecular bases of interaction with its multiple ligands is necessary in order to facilitate understanding of immune responses, the design of immunomodulators and soluble CD4

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D. Piatier-Tonneau et al.: CD4 interaction with HLA class II and H1V gpl20

derivatives to be used as antiviral therapeutic agents in acquired immune deficiency syndrome (AIDS). In previous work, based on the similarity of tetrapeptide sequences present in CD4 and HLA class II/3 chains with the fibronectin cell attachment site RGDS (amino acid one-letter code), we designed synthetic dodecapeptides which have immunosuppressive properties towards in vitro proliferation of T4 cells and their ability to deliver T helper function to B lymphocytes (Mazerolles et al. 1988). Synergistic action of the DR-12 peptide (amino acids 35-46 of the/3 chain) and the T4-12 peptide (amino acids 50-61 of the CD4 V1 domain) together with DRspecific or CD4-specific antibodies at suboptimal doses suggested that they could interfere with CD4-class II interaction. Subsequently, we have demonstrated that the same peptides inhibit conjugate formation between CD4 T lymphocytes and B lymphocytes expressing class II antigens (Mazerolles et al. 1990). This latter study provided evidence that CD4-class II interaction is not directly involved in adhesion forces but might rather act as a regulator of adhesion through the CD2/CD58 (LFA-3) and CDlla-CD18/CD54 (LFA-1/ICAM-1) pathways. In this report, we provide a critical assessment of CD4-dependent cellular adhesion assays and of the role of CD4 as an adhesion receptor and we discuss a model in which CD4 is considered as an allosteric servomodulator of T lymphocyte adhesion, activation and function.

had been cyclized. Two HLA-DR derived peptides corresponded to

amino acid sequences of the/31 domain 29-53 and 35-46 designated as DR-12 in previous studies (Mazerolles et al. 1988, 1990). Seven peptides corresponded to gpl20 sequences, overlapping amino acids of the LAV Bru isolate 414-434, 418-434, 421-436, 428-445, 432-448, 49-65, and of the ARV2 isolate (amino acids 49-65); two were derived from gp41 sequences (amino acids 827-843 and 829-836). HIV derived peptides were kindly provided by the Agence Nationale de Recherches sur le SIDA (ANRS; Paris, France).

CD4 expression. CV1 cells seeded in 12-well culture plates (Costar Cambridge, MA) were infected with recombinant SV40-CD4 virus stocks according to the method described by Doyle and Strominger (1987), and assayed 42 h later. COS-7 cells were transfected with the recombinant CD4-CDM8 construct, obtained by cloning the 1.8 kilobase (kb) Eco RI-Bam HI CD4 cDNA, isolated and kindly provided by P. Maddon (Maddon et al. 1985), into the eukaryotic vector CDM8 (kindly provided by B. Seed). Transfections were performed according to the methods described by Seed and Aruffo (1987) with slight modifications. Briefly, 50% confluent cells in 100 mm Petri dishes were incubated for 4 h at 37°C in 4 ml of transfection medium Dubelco's modified Eagle's medium (DMEM) supplemented with 10 % Nu-serum (Collaborative-Research), 2 mM glutamine, 400 gg/ml diethylaminoethanol DEAE-dex~ran, 100 /xM chloroquine and 1 ~tg/ml of plasmid DNA, followed by treatment with 10% dimethylsulfoxyde DMSO m phosphate-buffered saline (PBS) for 1 min. One day after transfection the cells were trypsinized, plated either in new Petri dishes or in 12-well culture plates (Costar), and assayed one or two days later. Flow cytometry assay. Two or three days after transfection cells were detached from plates in PBS-1 mM ethylenediaminetetraacetate (EDTA), stained by indirect immunofluorescence (IDIF) using OKT4 (2,5 ~tg/ml) and a fiuorescein conjugated mouse immunoglobulin-specific goat antibody (Nordic, Tilberg, The Netherlands) and analyzed by flow cytometry with a FACS 440 (Becton Dickinson). Negative controls were performed using Leu2a (CD8-specific).

Materials and methods mAbs, recombinant proteins and synthetic peptides. A panel of mAbs specific for different epitopes of the CD4 molecule were kindly provided by P. Rat (Johnson Pharmaceutical Research Institute, Raritan, NY): OKT4, OKT4A, OKT4B, OKT4C, OKT4D, OKT4E, and OKT4F; by G. Cordier (U 80 INSERM, Lyon, France): B14, and by C. Mawas (U 119 INSERM, Marseille, France): 13B8.2; MT151 was from Boehringer Mannheim (Meylan, France), Leu3a from Becton Dickinson (Mountain View, CA). D 1.12, specific for HLA-DR, was kindly provided by R. Accolla. W6-32, specific for HLA-class I (Barnstable et al. 1978), Leu2a (Becton Dickinson), specific for CD8, 84.H. 10 specific for CD54 (kindly provided by D. Olive), and 25.3.1, specific for CD 1la (Imrnunotech, Marseille, France) were used as controls. Recombinant purified soluble gp 120 was obtained from Dr. J. P. Lecoq and Dr. M. P. Kitny (Socitt6 Transg~ne, Strasbourg, France) and from Dr. H. Holmes (Medical British Council, Cambridge). Recombinant soluble CD4 containing the whole extracellular portion (Deen et al. 1988) was kindly provided by Dr. A. Truneh (Smith, Kline, and French Laboratories, Philadelphia, PA) and a chimaeric soluble CD4-immunoglobulin molecule (Traunecker et al. 1989) H-y3-CD4, comprising the V1-V2 domains of CD4 and part of the human IgG3 heavy chain, was a gift from Dr. A. Traunecker (Basel Institute for Immunology, Switzerland). Peptides were synthesized according to the solid phase method of Merrifield and were further purified by high pressure liguid chromatography (HPLC) (Neosystem, Strasbourg, France). Seven peptides overlapped the V1 domain of CD4 corresponding to amino acid sequences 19-42, 19-31, 31-42, and 50-61, designated as T4-12 in previous studies (Mazerolles et al. 1988, 1990), 74-92, 81-92, including two benzylated residues according to Lifson and co-workers (1988), and 44-57 which

B lymphocyte adhesion to infected CV1 and transfected COS cells. Adhesion of the HLA-class II expressing Burkitt lymphoma line Raji and its HLA class II negative mutant RJ2.2.5 (Accolla 1983) to CV1 cells infected with SV40, SV40-HA, and SV40-CD4 virus stocks was performed as described by Doyle and Strominger (1987). Briefly, 5.1053.106B lymphocytes labeled with 3SS-methionine were incubated for 1 h at 37°C with the CV1 cell monolayers. After washing, the cells were removed from the dishes by trypsinization and radioactivity was counted. The results were expressed as the number of B lymphocytes which have been bound in duplicate wells. B lymphocyte adhesion to transfected COS-7 cells plated in 12-well dishes (Costar) was performed 48 or 72 h after transfection. 5.106 B cells in 0.5 ml RPMI containing 2% fetal calf serum (FCS), 2 mM glutamine were incubated for 1 h at 37°C with the COS cell monolayer in each well, washed five times by dropping 1 ml medium into the wells as described (Clayton et al. 1989). Cells were washed once more with PBS, fixed with 3.7 % paraformaldehyde for 10 min, and permeabilized with 0.1% Triton-X100. Immunoperoxydase staining of the CD4-expressing COS cells was performed with 13B8.2 (5 ~tg/ml), after inhibition of the endogenous peroxydases with 0.3% H202. Subsequent incubations of cells with a biotinylated mouse immunoglobuIin-specific sheep antibody (Amersham, Les Ulis, France), an avidin-biotin-peroxydase complex (Amersham) and H202 in presence of diaminobenzidin (Sigma St. Louis, MO) were achieved. Rosettes were observed under light microscopy and quantified as the percentage of CD4 expressing COS ceils which had bound five B cells or more. Inhibition of rosettes was performed by preincubating cells for 30 min at 37°C with various concentrations of inhibitors: CD4-, HLA class II-specific and control antibodies, purified gpl20, recombinant soluble CD4 molecules and synthetic peptides

D. Piatier-Tonneau et al.: CD4 interaction with HLA class II and HIV gpl20 derived form them. Rosettes were allowed to form as described above in the presence of the inhibitors.

Results

Adhesion of B lymphocytes to CD4 expressing cells. As expression of CD4 in simian kidney epithelial CV1 cells upon infection with a SV40-CD4 construct was reported to mediate adhesion of B lymphocytes expressing HLA class II antigens (Doyle and Strominger 1987), we sought to use this assay in order to map the sequence responsible for interaction with the class II antigens. In a first series of five experiments, in which we compared the ability of 35S-methionine labeled Raji cells and their HLA class II negative mutant RJ.2.2.5 to bind to CV1 cells infected either with SV40 or with the SV40-CD4 construct, we found no evidence for a significant quantitative difference between the two constructs and the two cell lines (Fig. la).

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Similar negative results were obtained in a second series of five experiments in which a control SV40haemagglutinin (SV40-HA) construct was included as a reference sialic receptor for cell adhesion (Fig. lb), even though a greater number of labeled B lymphocytes were used in the assay. In addition, we often observed greater binding of the RJ.2.2.5 cells to CV1 cells whether they were infected with SV40 and/or SV40-CD4 construct or not. We were led to conclude that this assay, while occasionally giving rise to specific rosetting of the B lymphocytes visible under the microscope, could not be used in a reproducible and quantitative manner as previously reported. As this COS-CD4

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Fig. 3. Rosette formation between CD4-expressing COS cells and B lymphocytes expressing HLA class II molecules, Raji, or not, RJ2.2.5. After rosette formation, COS cells were stained with an indirect immunoperoxydase method using the 13B8.2 CD4-specific antibody. Clusters of small round Raji cells forming rosettes (arrows) are present on stained COS cells.

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D. Piatier-Tonneau et al.: CD4 interaction with HLA class II and HIV gp120

appeared to us as a c o n s e q u e n c e o f the use o f a lytic S V 4 0 infection cycle (see Discussion), w e used another transient expression assay based on the use o f the plasmid vector C D M 8 for transfection (Seed and A r u f f o 1987). Transfection of C O S - 7 cells with the C D M 8 - C D 4 construct resulted in expression o f high levels o f C D 4 detected by flow c y t o m e t r y at the cell surface on 5 - 1 2 % o f the cells using the O K T 4 C D 4 - s p e c i f i c m o n o c l o n a l antibody. F l u o r e s c e n c e intensity was in the range o f 1 0 - 1 0 0 arbitrary units, as c o m p a r e d to 0 . 8 - 2 for the T cell line C E M , and 2 - 7 for an H e L a cell line stably transfected with C D 4 (Fig. 2). A d h e s i o n o f Raji cells o c c u r r e d only to C D 4 positive C O S cells but not to H e L a - T 4 or C E M cells, w h i l e no

conjugates w e r e o b s e r v e d with R J . 2 . 2 . 5 cells (Fig. 3, and data not shown). Rosettes w e r e quantitated as the n u m b e r o f C D 4 positive C O S cells, visualized by i m m u n o p e r o x ydase staining with the 13B8.2 C D 4 - s p e c i f i c antibody, w h i c h h a v e retained five or m o r e B lymphocytes: 40 + 4 % rosettes w e r e o b s e r v e d with Raji cells, none with R J . 2 . 2 . 5 cells (Fig. 4a).

Inhibition of CD4-class H dependent adhesion. Rosettes w e r e specifically inhibited by O K T 4 A (CD4-specific) and D1-12 ( H L A - D R - s p e c i f i c ) antibodies, while no inhibition was o b s e r v e d with Leu2a (CD8-specific), W 6 - 3 2 (class I-specific), 8 4 . H . 1 0 (CD54-specific), and 25.3.1 ( C D l l a - s p e c i f i c ) antibodies (Fig. 4a), establishing that

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D. Piatier-Tonneauet al. : CD4 interaction with HLA class II and HIV gpl20 adhesion was dependent on expression of CD4 and I-ILA class II antigens, possibly reflecting a direct interaction of these two cell surface glycoproteins. Further characterization of the CD4 regions crucial for rosette formation was performed by testing a panel of CD4-specific antibodies, recombinant soluble CD4 and gpl20 molecules, as well as peptides derived from them. From a panel of 11 CD4-specific antibodies, only OKT4 had no blocking activity even at high concentration (12.5 gg/ml), OKT4B and OKT4D lost their ability to block conjugate formation at 1 gg/ml, while the others produced complete or strong inhibition at 1 gg/ml (Fig. 4b). Seven peptides derived from the V1 domain of CD4 and two peptides derived from the/31 domain of HLADR, including the DR-12 and T4-12 peptides shown to inhibit conjugate formation between T and B lymphocytes (Mazerolles et al. 1990), failed to inhibit the rosetting assay (data not shown). HIV gpl20 was found to be as strong an inhibitor of the rosettes as the blocking CD4-specific antibodies. Inhibition in this case was suppressed by prior incubation of HIV gpl20 with 100 nM of two different recombinant soluble CD4 derivatives, sCD4 (Deen et al. 1988) and chimaeric H-3,3-CD4 (Traunecker et al. 1989). Efficiency of dimeric H-73-CD4 was slightly greater than that of monomeric sCD4. Both recombinant CD4s did not interfere with cellular adhesion even at high concentration (2 ~tM; Fig. 4c). Among nine peptides derived from gp 120 and gp41 sequences, five of which encompass a binding site of gp120 to CD4 (Lasky et al. 1987; Cordonnier et al. 1989), only peptide 414-434 was able to partially block rosette formation (Fig. 4c). Peptides 49-65 from either LAV BRU or ARV2 isolates, which contain a CD4 RADS-like reverse sequence, SDAK and SDAR respectively, as well as gp41 peptides 827-843 and 829-836, as described by Golding and co-workers (1988) to share HLA-class I1/3 chain homology, had no effect on rosette formation. All the results accumulated indicated that the rosetting assay could be used as a quantitative measure of putative molecular interactions between HLA class II antigens and CD4.

Discussion CD4: to be or not to be an adhesion molecule. Critical assessment o f adhesion assays dependent on expression o f CD4. The role of CD4 and HLA class II antigens as

reciprocal adhesion receptor, suggested indirectly in a number of studies using specific antibodies, gained strong support when it was reported that expression of CD4 in simian kidney epithelial ceils when infected with an SV40-CD4 construct induced adhesion of B lymphocytes only if they expressed HLA class II antigens (Doyle and

125

Strominger 1987). Our extensive efforts to use this assay in a quantitative manner led to negative results, and we failed to reproduce the results of the initial report, whether CV1 cells were incubated with a saturating number of B lymphocytes (3.106) or not (5.105). Instead, we observed no significant difference in the binding of B lymphocytes to CV1 cells infected with SV40 alone or the SV40-CD4 construct, whether they expressed class II antigens or not. Microscopic examination however revealed in some cases the presence of specific rosettes as initially reported. Our experience with this assay led us to suggest the following possibilities to explain the failure to use this assay in a reproducible and quantitative manner. First, one has to consider that expression of CD4 relies on infection with a viral stock that contains both wild type SV40 and SV40-CD4. Amplification of these viral stocks results in overrepresentation of wild type SV40, and possible recombination events with the SV40-CD4 construct, which overall lead to reduction of CD4 expression efficiency. These problems were not circumvented by using primary viral stocks. Second, the time window during which optimal CD4 expression is obtained, i.e., between 48 and 72 hours, is very short and also close to the time when cell lysis starts to occur, leading to gross membrane alterations (which can trigger non specific adhesion) and cell detachment. Third, labeling of the B lymphocytes with 35Smethionine in order to quantitate binding proved unreliable because of high background. This is due in large part to the fact that extensive washing does not allow the removal of many labeled B lymphocytes from the edges of the wells. Fourth, the raw data in the original report indicated a signal to background ratio of 2-3, and their quantitation in subsequent experiments relied in subtraction of the high background and normalization by comparison to the results obtained with an haemagglutinin-SV40 construct acting as a sialic acid receptor. This results in over representation of the differences observed, and in our experiments this appears to be misleading, since in many cases adhesion of the RJ.2.2.5 cells was higher than that of the Raji parental cell line. It appears that induction of rosette formation relies on the expression of a very high level of CD4 at the cell membrane, which cannot be obtained consistently in this assay. Indeed, in parallel studies of CD8-class I interactions Norment and co-workers (1988) have described the need to use stably transfected hamster ovary CHO cells, expressing extremely high levels of CD8 (20-80 times higher than normal T cells), to induce CD8-class I dependent cellular adhesion. Moreover, they used as a negative control a CHO line expressing a relatively low level of CD8 (still 2-4 times that of T cells). Close examination of their results indicate that they consider a signal to background

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D. Piatier-Tonneauet al.: CD4 interaction with HLA class II and HIV gpl20

ratio of 2-3 (that is similar to the one reported in the SV40-CD4 assay) as indication of the absence of specific adhesion. In view of these observations, we designed a different assay based on transient expression of a CDM8-CD4 construct in COS-7 cells and the enumeration of the rosettes formed with CD4 positive cells by microscopic inspection. This assay is similar in design to that described by Clayton and co-workers (1989), but relies on a distinct readout system since these authors have encountered the same difficulties of high background in quantitating their assay using 51Cr labeling of the B lymphocytes as those discussed here. The key features of our assay that make it useful for the visualization of CD4-class II interaction are these: a reproducible high level of CD4 (up to 40-50 times higher than in the T cell line CEM) is expressed by the transfected cells in the absence of membrane alteration or cell lysis; extensive washing can be performed without disrupting the rosettes; quantification is based on post-binding staining of the fixed cells with an CD4-specific antibody and counting of the CD4 positive cells which have retained five or more B lymphocytes (usually 10-20).

Is CD4 an adhesion receptor ? Cellular adhesion has been used for a long time as a way to visualize molecular interaction, e.g., between carbohydrates which are the substrates of specific glycosidases or ligands of lectins (Carter et al. 1981). The studies discussed here suggest that CD4 and HLA-class II can function as reciprocal adhesion receptors. However, while bona fide adhesion receptors are characterized by the demonstration of direct molecular binding, this remains to be shown conclusively for CD4 and HLA class II in the absence of membrane interactions. Moreover, we have provided evidence that CD4-class II interaction does not contribute in a significant manner to the forces of cell adhesion during the formation of conjugates between CD4 T lymphocytes and B lymphocytes, which appear to rely mainly on the CD2/ CD58 and CD1 la-CD18/CD54 pathways (Mazerolles et al. 1990). In this respect, CD4 and HLA class II molecules cannot be considered as reciprocal adhesion receptors. Nevertheless, we consider the cellular adhesion assays discussed here a convenient but artificial way of revealing CD4-class II interaction and defining sequences involved in these interactions. CD4: allosteric servomodulator of T cell adhesion ? Inhibition of CD4-dependent rosette formation can be used to analyze CD4-class II interaction in the absence of the Tcr/CD3 complex. Only CD4 and HLA class II ligands that have a known strong affinity for the molecules they recognize could inhibit rosette formation. This is the case of most CD4-specific antibodies, the HLA-

DR-specific antibody tested and HIV gpl20. Notably, most CD4-specific antibodies which recognize epitopes which have been mapped in other studies to the V1 and/or V2 domain of CD4 were inhibitory, whereas OKT4 binding to the V4 membrane proximal domain failed to inhibit. This lack of inhibition of OKT4 is in total discrepancy with previous results (Clayton et al. 1989) describing a strong inhibitory effect of this antibody on rosettes obtained in a comparable cellular adhesion assay which differed only in the readout system. However, based on the reproducibility of the non inhibitory activity of OKT4 in our hands, this antibody was used as a negative control of inhibition in the subsequent experiments designed to study other molecules as inhibitors. The observation that anti-CD4 antibodies, which are potent inhibitors on rosette formation, exhibit either strong (Leu3a, OKT4A, and OKT4F) or moderate (OKT4C, OKT4E, B14, T151, and 13B8.2) HIV blocking activity, suggests that the CD4 binding sites for gp 120 and HLA class II antigens are overlapping but partly distinct: This is also supported by the weaker inhibition of OKT4D on rosette formation than on gpl20 binding. These results may also indicate that the CD4 binding site is more extended for class II than for gp120, as previously suggested (Clayton et al. 1989). Furthermore, it has also been suggested that MHC class II and gp120 binding sites were separable (Lamarre et al. 1989 a, b), since mutations of the V2 and V3 domains of CD4 inhibited IL-2 production and formation of cellular aggregates in a xenogeneic assay dependent on the expression upon transfection of human CD4 by a mouse T cell hybridoma bearing a T-cell receptor specific for the H-2D d mouse class I antigen and of HLA class II by the target cell, while some V1 mutations abolished gpl20 binding only. This assay and our rosette assay exhibit striking differences with regard to inhibition by CD4-specific antibodies, as evidenced by the lack of inhibition of OKT4C, OKT4E, and OKT4F on the xenogeneic assay contrasting with their potent blocking effect on rosettes. Conversely, OKT4 inhibited formation of the aggregates but had no effect in our assay. These differences probably reflect the fact that the xenogeneic assay is not solely dependent on CD4-class II but also on CD4-Tcr/CD3 interactions. Fluorescence energy transfer experiments have provided evidence for a close association of human CD4 and the mouse Tcr/CD3 complex in a similar hybridoma (Mittler et al. 1989). A large number of other effectors were tested in our rosette formation assay. With the exception of one peptide overlapping a gp 120 binding site for CD4, which partially blocked the assay, all were without effect including soluble recombinant CD4 derivatives and peptides derived from CD4, HLA-DR and other parts ofgpl20. According to the recent determination of the crystal structure of the first two domains of sCD4 (Ryu et al. 1990; Wang et al. 1990), none of the CD4 synthetic peptides we used might

D. Piatier-Tonneau et al. : CD4 interaction with HLA class II and HIV gp120

restore appropriate structure for the C" edge and the C'C" turn of the gpl20 binding site. The case of sCD4 is of particular interest in view of its potential use as a drug in AIDS therapy (reviewed in Auffray et al. 1991). In this context the lack of inhibition of rosette formation in our assay and in the xenogeneic assay, as well as the fortunate lack of interference of sCD4 derivatives with the function of class II positive cells, even when linked to toxins (Chaudary et al. 1988; Till et al. 1988), appears paradoxical. A possible explanation is that structural differences between soluble and membrane forms of CD4 could be responsible for a differential ability to interact with class II molecules, which could also depend on association with the Tcr/CD3 complex. Since it has been observed that more than one CD4 molecule associates with one Tcr/CD3 complex (Chuck et al. 1990), it is plausible that this association mediates clustering of CD4 and conformational changes that induce cooperative binding to HLA class II molecules. This effect could be mimicked by the very high density of CD4 obtained by transfection. In physiological conditions, these associated forms of CD4 appear to play the most relevant role in down regulation of cell adhesion between T and B lymphocytes (Mazerolles et al. 1990). At present, the different functions of CD4 mediated by molecular interactions with HLA class II antigens, the Tcr/CD3 complex, and p56 l°k can be integrated in a model in which CD4 is not an adhesion receptor, or a simple signal transducer, but rather an allosteric servomodulator of T-cell adhesion, activation, and function that acts as a sensor of the interplay of the molecular interactions with its various ligands. Ultimately, it will be necessary to study these interactions in a fully reconstructed system in which both the Tcr/CD3 complex and the major adhesion pathways will be directly available for manipulation and study. Such studies will provide a better understanding of how the immune response is regulated in healthy and pathological conditions, and assays to aid in the design of specific drugs to suppress autoimmune and viral diseases.

We thank Mrs. Sandrine El Marhomy, Nancy Fr6chin, Caroline Knipp, and Michelle Martin for technical assistance; Fabienne LamirauIt and Christine R6my for typing the manuscript; Jack Strominger and Carolyn Doyle for providing advice, reagents, and laboratory facilities in using their SV40-CD4 assay; Alain Fischer, Rafick Sdkaly, Stephan Shaw, and their coileagues for many helpful discussions; Roberto Accolla, Genevieve Cordier, Harvery Holmes, Marie-Paule Ki6ny, Paul Maddon, Claude Mawas, Daniel Olive, Patrlcia Rao, Brian Seed, Andr6 Traunecker, and Alem Truneh for providing reagents used in this study; Marc Girard, Jeanne-Mane Leconte, and Jean-Pierre Lecoq for their encouragementand. This work was supported by CNRS and in part by grants from AMFAR, ANRS, ARC, DRET, and INSERM, The KorberPreis Foundation, Pasteur Vaccins and Rh6ne Poulenc Sant6 to C.A. Acknowledgments.

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