Cell, Vol. 64, 511-520, February8, 1991,Copyright© 1991 by Cell Press

Requirement for Association of p56 with CD4 in Antigen-Specific Signal Transduction in T Cells Nicolas Glaichenhaus,* Nilabh Shastri,* Dan R. Littman,t and Julia M. Turnert$ * Department of Molecular and Cell Biology University of California Berkeley, California 94720 Howard Hughes Medical Institute and Department of Microbiology and Immunology University of California San Francisco, California 94143

Summary The T cell-specific tranamembrane glycoprotein CD4 interacts with class II MHC molecules via its external domain and is associated with the tyrosine kinsse p56 Ick via a cysteine motif in its cytoplasmic domain. We have assessed the ability of CD4 to synergize with the antigen-specific T cell receptor (TCR) for induction of transmembrane signals that result in lymphokine production. Mutant CD4 molecules were Introduced into T cells that lacked endogenous CD4 but expressed TCRs specific for lysozyme peptidss or the superantigen SEA bound to A b or A bm12 class II MHC molecules. With either Iigand, T cell actlvetion occurred only when CD4 was associated with p56 ~ck. These results demonstrate that residues within the cytoplasmic domain of CD4 are required for its coreceptor function in TCR-medlated signal traneduction and strongly support the notion that the association of CD4 with p56 ~ck is critical in this process. Introduction The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells and by a majority of immature thymocytes (reviewed in Littman, 1987; Parnes, 1989). Both molecules are members of the immunoglobulin supergene family but differ considerably in their primary sequences. While CD4 is apparently monomeric, CD8 can exist as aa homodimers or al~ heterodimers. The extracellular domains of CD4 and CD8a bind to monomorphic reg!ons of class II and class I major histocompatibility complex (MHC) molecules, respectively (Doyle and Strominger, 1987; Norment et al., 1988). Both CD4 and CD8a are associated with the lymphocyte-specific cytoplasmic tyrosine kinase p5rJck (Rudd et al., 1988; Veillette et al., 1988). CD4 and CD8a therefore have the potential both to mediate adhesion and to transduce transmembrane signals in developing and mature T cells. A majority of immature thymocytes express both CD4 and CD6. During intrathymic maturation, these CD4+CD8+ cells undergo a process of selection in which po:rPresentaddress:Departmentof Pathology,Universityof Cambridge, TennisCourt Road, CambridgeCB2 1QP,England.

tentially harmful cells with receptors specific for self-antigens complexed to self-MHC molecules are deleted, while cells that have the potential to interact with foreign antigens complexed to self-MHC molecules are positively selected (von Boehmer and Kisielow, 1990). During this process, reactivity of the T cell receptor (TCR) with either class I or class II MHC molecules results in the differentiation of the selected cells to either CD4-8 + (generally cytotoxic or suppressor cells) or CD4+8- (generally helper cells) phenotypes, respectively (Teh et al., 1988; Kaye et al., 1989). A variety of studies suggest that intracellular signals generated during CD4- or CD8-dependent TCRligand interactions in the thymus influence both positive and negative selection as well as the eventual CD4/CD8 phenotype of mature T cells (Ramsdell and Fowlkes, 1989; Zuniga-Pflucker et al., 1990; Sha et al., 1988; Kisielow et al., 1988; Kaye et al., 1989). The CD4 and CD8 glycoproteins also play an important role in the activation of mature T lymphocytes. While these molecules appear to stabilize the interactions of T cells with antigen-presenting or target cells (Marrack et al., 1983; Biddison et al., 1984), there is also accumulating evidence that they participate actively in transmembrane signal transduction. Antibodies against CD4 or CD8 block T cell activation via the TCR (Wassmer et al., 1985; Bank and Chess, 1985); a subset of CD4 molecules is associated with the TCR in activated and resting cells (Saizawa et al., 1987;Anderson et al., 1988); and antibodymediated cross-linking of CD4 or CD8(z to the TCR complex enhances T cell activation (Emmrich et al., 1986). Further evidence suggesting a signaling function for these molecules has come from gene transfer experiments in which the CD8a gene was introduced into T cells, which required this molecule for optimal activation (Gabert et al., 1987; Ballhausen et al., 1988). In one of these systems, tailless forms of CD8a were functionally impaired, perhaps due to their inability to associate with p56Ick (Zamoyska et al., 1989; Letourneur et al., 1990). These data have led to the concept that CD4 and CD8 function as coreceptors in collaboration with the TCR to transduce an activation signal across the plasma membrane of T cells. The amino acid residues in the cytoplasmic domains of CD4 and CD8a that determine their association with p561ok have recently been identified (Shaw et al., 1989, 1990; Turner et al., 1990). This information made it possible to test directly the functional significance of the physical association of CD4 with p56k~k in antigen-specific T cell activation. In the current study, we have generated transfectants bearing receptors that recognize either a specific peptide or the superantigen staphylococcal enterotoxin A (SEA) bound to class II MHC molecules. The activation of these T cells depends on the cell surface expression of the transfected CD4 gene product. By testing a series of mutant CD4 molecules that differ in their cytoplasmic tail residues, we show that T cell activation occurred only when p56~ckwas bound to CD4. Our results

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Results A Model System for Analysis of CD4 Function To study the ability of modified forms of the CD4 molecule to cooperate with the TCR upon antigen-specific stimulation, we generated two T cell lines having three essential features: they express TCRs of known antigen specificity, their activation is dependent on CD4, but they lack expression of this molecule at the cell surface. These cells were generated by transfection of TCR a and 13chain genes into the lymphoma cells 58c(-13- (Letourneur and Malissen, 1989). They lack expression of endogenous CD4, CD8, and TCR a and 13 chains but have all the other components required for cell surface expression of the TCR. The TCR o and 13chain genes were isolated from two murine CD4-positive, hen egg lysozyme-specific T cell hybrids: bm2T3.1 and BO4H9.1 (Shastri, 1989). Both hy-

brids express V133and are specific for synthetic peptides of hen egg lysozyme bound to either the A bml2or A b molecules, respectively (see Experimental Procedures). In addition, bm2T3.1 and BO4H.9.1 cells respond to SEA, a superantigen that is known to stimulate T cells bearing particular VI~, including VlY3(Marrack andKappler, 1990). The TCR ~ and 13chain genes isolated from bm2T3.1 and BO4H9.1 hybrids were coelectroporated with a selectable G418 resistance gene into the 58a-13- recipient cells. Flow cytometric analysis (Figure 1A, [a] and [c]) of two representative clones, 150 and 171, showed that the transfected TCR (~13chains of bm2"1-3.1 and BO4H9.1 cells were expressed on the cell surface in association with the endogenous CD3 polypeptides. The cells, however, remained CD4 negative. Upon co-culture with the appropriate antigen-presenting cells (APCs) and lysozyme peptides, 150 and 171 cells secreted only barely detectable levels of the lymphokine IL-2 (Figure 1B, left). Interestingly, the same results were obtained with SEA, in contrast to other reports involving superantigen-reactive cells that respond in the absence of

CD4-p56/ck Interaction in T Cell Activation 513

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Figure 2. Mutants Used in the Analysis of CD4 Function The amino acid sequence (residues 394-431) of the cytoplasmic domain of CD4 is shown. Residues altered by site-directed mutagenesis are boxed, and substituted residues of individual mutants are indicated. The relative degree of association between CD4 molecules and p56 ~ckis shown, ranging from no detectable p56 ~ckcoimmunoprecipitated ( - ) to amounts equivalent to those coimmunoprecipitated with wild-type CD4 ( + + + ) . Relative levels of IL-2 production by the transfectants are also indicated.

CD4 expression (MacDonald et al., 1990; Fleischer and Schrezenmeier, 1988) (Figure 1B, right). However, when cell surface CD4 was expressed following retrovirus-mediated gene transfer, both 150 and 171 cells responded strongly to either the peptide-MHC or the SEA-MHC ligand (Figures 1A and 1B). In contrast, expression of CD8a did not enhance IL-2 secretion above that obtained with the parental CD4-negative cells (see below). The functional responsiveness of CD4-positive derivatives of 150 and 171 cells was not due to alterations in the surface density of the TCR-CD3 complex (Figure 1A) and was evident only for antigen-specific stimulation as both the CD4negative and CD4-positive derivatives responded strongly to stimulation by cross-linked anti-CD3s (500A2) or antiTCR (H57-597) monoclonal antibodies (MAbs) (data not shown). Thus, the ability to transduce TCR-mediated antigen-specific activation signals in both 150 and 171 cells required the expression of the CD4 molecule. Association of CD4 and p56 Ick in T Cells

Amino acid residues involved in the interaction of CD4 and CD8a with p561ckhave recently been identified using transient expression of mutant proteins in nonlymphoid HeLa and COS-7 cells (Shaw et al., 1989, 1990; Turner et al., 1990). The N-terminal region of p56~ck interacts noncovalently with the cytoplasmic domains of CD4 and CD8a via pairs of cysteine residues in each molecule. We questioned whether the same residues were required for the association of these proteins in T cells. In addition, it was of interest to determine whether other T cell-specific factors, though not required for stable association of CD4 or CD8a with p56Ick in nonlymphoid cells, might modify these interactions in T cells. To address these questions, wild-type CD4 or versions of CD4 bearing mutations in the cytoplasmic tail (Figure 2) (Turner et al., 1990) were introduced into 150 and 171 cells by retrovirus-mediated gene transfer. To obviate possible problems associated with the selection of individual

clones, bulk populations of infected cells expressing high surface levels of CD4 were selected by flow cytofluorometry following antibody staining. The resulting pools exhibited approximately similar levels of CD4 at the cell surface (Figures 3A and 3B). Thus, all the CD4 mutants tested were capable of being expressed at the cell surface. The ability of the CD4 molecules to associate with endogenous p56Ick in T cells was analyzed by immunoprecipitating CD4 from nonionic detergent lysates and by immunoblotting the immune complexes with an N-terminal p561Ck-specific antiserum (Veillette et al., 1988). Total amounts of endogenous p56Ick were determined by immunoblotting the whole lysates with the p56Ick antiserum and were found to be comparable in all cell lines (Figures 4A [ii] and 4B [ii]). CD4-p56/ck complexes were evident in both 150 and 171 cells when the wild-type CD4 molecules were expressed (Figures 4A [i] and 4B [i]). As expected, the tailless CD4 molecule (CD4-T1, Figure 2) failed to associate with p56jck. In contrast, a partially truncated form of CD4 having 28 of the 38 cytoplasmic residues including the cysteine motif (CD4-T3, residues 394-421) did associate with p56/ck, albeit to a slightly lesser degree (Figure 4). However, substitution of alanine for Cys-418 or both Cys-418 and Cys-420 (CD4-MCA1, -MCAI/2) completely prevented association with p56~ck, clearly demonstrating that these residues are required for the association of p56Ick with CD4 in T cells. When Cys-420 (CD4-MCA2) was replaced by alanine, a very low level of coimmunoprecipitated p56Ick was detectable. This finding differs slightly from that in COS-7 cells, in which there was no detectable association of CD4-MCA2 with p56~ck (Turner et al., 1990). Also, as seen earlier in COS-7 cells, substitution of leucine for Ser-406, Ser-427, or all three serine residues in the cytoplasmic tail (CD4-MS1, -MS3, -MSl/2/3) had either no or a minor effect on association with p56Ick. A further observation in both T cells and COS-7 cells

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fluorescence i n t e n s i t y (log. scale) Figure 3. SurfaceExpressionof Murine CD4, CD8cqand human CD4 in 150- and 171-DerivedT Cells Cells derivedfrom 150 (A) and 171 (B) T cells after gene transferwere stained with appropriate specific antibodies: MAb GKI.5 for cells expressing CD4-WT, -T1, -T3, -MCA1, -MCA2, -MCAI/2, -MS1, -MS3, -MSl/2/3, and -CD4/8~z;MAb 53-6.7for cells expressingmurineCD8a; and MAb OKT4for humanCD4, followedby FITC-conjugatedgoat antirat IgG. Cells incubatedwith the second antibodyalone representthe negative control profile, shown as a vertical line.

was that, compared with CD4, 25- to 50-fold less p56/ck coprecipitated with CD8a or with a chimeric CD4-CD8cz molecule comprising the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of CD8a (Figure 4). The reason for the apparent lower affinity of p561ok for the cytoplasmic tail of CD8a compared with that of CD4 is not clear, but may be related to either structural differences between CD8a and CD4, or the absence of the CD813 chain in these cells. We have also expressed human CD4 in 171 cells and found that it associated effectively with endogenous murine p56 Ick (Figure 4B). We conclude from these experiments that the cytoplasmic cysteine residues of CD4 are critical for association with p56/ck in T cells and that the requirements for this interaction are similar in T cells and nonlymphoid cells. Our ability to disrupt association with p56/ok by single point

mutations in CD4 allowed us to assess the requirement for CD4-p56 ~ck interactions in the activation of T cells, Ability of Mutant CD4 Molecules to Synergize with the TCR in T Cell Activation To determine the functional significance of the CD4p56 Ick association in T cell activation, 150 and 171 cells expressing wild-type or mutant forms of CD4 were assayed for their ability to secrete IL-2 in response to peptide-MHC or SEA-MHC stimulation (Figures 5 and 6). At all antigen concentrations tested, cells expressing tailless CD4 molecules secreted either barely detectable (171.CD4T1) or significantly lower (150.CD4-T1) levels of IL-2 than the corresponding cells expressing wild-type CD4. We also confirmed the previous finding that human CD4 can effectively replace murine CD4 in the antigen-specific activation of class II MHC-restricted murine T cells (von Hoegen etal., 1989). To determine whether the impaired function of the tailless CD4-T1 molecule was due to its inability to associate with p56Ick, mutants of CD4 bearing substitutions of one or both cytoplasmic cysteine residues were assayed for activity in 150 and 171 cells. As shown in Figures 5 and 6, substitution of either one (CD4-MCA1, CD4-MCA2) or both (CD4-MCA1/2) cytoplasmic cysteine residues caused dramatic loss of function, equivalent to removal of the entire cytoplasmic domain of CD4. Because these three CD4-MCA mutants did not associate with p5~ ck (Figure 4), these results show a direct relationship between the ability of CD4 to associate with p56 Ick and its ability to synergize with the TCR in T cell activation. Interestingly, the truncation mutant CD4-T3, which retains the cysteine motif required for interaction with p56~k, allowed only an intermediate functional response (Figures 5D and 6B). The 171.CD4-T3 cells secreted higher levels of IL-2 than ceils expressing tailless CD4, but lower levels of IL-2 than ceils expressing wild-type molecules. This could perhaps be due to a requirement for the missing C-terminal residues of CD4 in regulation of kinase activity or in the interaction of the CD4-p56 ~k complex with other factors. To assess the relationship between the functional activity of CD4 and its association with p56 tck further, we tested mutant CD4 molecules in which the cytoplasmic serine residues were substituted with leucinas (Figure 2). Substitution of leucine for Ser-406, Ser-427 (CD4-MS1, -MS3), and Ser-413 (data not shown), which had~no effect on association with p56~k, also had no effect on IL-2 production; cells expressing these mutant molecules gave responses equivalent to cells bearing wild-type CD4 (Figures 5 and 6). However, despite a minimal effect on association of CD4 with p56~k, substitution of all three cytoplasmic serines (CD4-MSl/2/3) significantly decreased the level of IL-2 production in response to both the peptide-MHC and SEA-MHC ligands (Figures 5 and 6). Since cytoplasmic serine residues are known to be involved in protein kinase C-mediated internalization of CD4 molecules (Acres st al., 1986), a possible explanation for the impaired function of CD4-MS1/2/3 could be the inability of this molecule to be internalized. Indeed, unlike cells expressing wild-type CD4, the phorbol ester PMA could not induce internalization of

CD4-p56 Ick Interaction in T Cell Activation

515

Figure 4. Analysis of Association of p56'ck with Mutant CD4 Molecules in the T Cell Transfectants Lysatss were prepared from derivatives of transfectants 150 (A) and 171 (B) expressing mutant CD4 molecules or CDSa. Protein samples (1 rag) of lysate were immunoprecipitated with MAb GKI.5 (anti-CD4) or MAb 53-6.7 (antiCD8a) as appropriate. Western blots of immune complexes (i) or whole-cell lysatss (ii) were probed with an antiserum specific for amino-terminal residues of p56/ck. Exposures were overnight with the exception of samples corresponding to CD8a-, CD4/8a-, and human CD4-expressing cells (36 hr).

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wild-type CD4 in their ability to synergize with the T cell receptor following antigen stimulation (Figures 5C, 5F, and 6). Thus, the relative significance of these two serine residues in phorbol-induced internalization of murine CD4 is similar to that previously reported for human CD4 (Shin et al., 1990). However, differences in the ability of CD4 mol-

surface CD4 molecules in cells expressing CD4-MS1/2/3 (Figure 7, top). The same result was found with the CD4MS1 mutant, but not with CD4-MS3, which was internalized as efficiently as the wild-type molecule following treatment with PMA (Figure 7, bottom). Yet, both CD4-MS1 and CD4MS3 mutants were comparable to each other and to the

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ecules to be internalized clearly do not correlate with their T cell activation function. Interestingly, CD8a was completely ineffective as a coreceptor for the TCRs of 150 and 171 cells in response to either the peptide-MHC or SEA-MHC ligand, despite the presence of class I MHC molecules on the APCs (Figures 5A, 5D, and Figure 6). This is probably due to the requirement for TCR and CD8(~ to bind to the same MHC molecule (Potter et al., 1989; Salter et al., 1990). We also found that chimeric CD4-CD8Q molecules failed to enhance the level of IL-2 production above CD4-negative levels (Figures 5C, 5F, and 6). As we did not observe even a basal level of adhesion-mediated IL-2 production, it is possible that the external CD4 domain of this molecule is impaired for interaction with MHC molecules. We ascertained that this finding was not due to an experimentally introduced mutation in the CD4 ectodomain, but it could result from a conformational aberration conferred by the CD8a transmembrane domain. Discussion Critical Role of CD4-p56 ~ck in T Cell Activation In this report, we provide direct evidence relating the coreceptor function of CD4 with its ability to associate with p56~k (see Figure 2 for a summary of these results). In both 150 and 171 T cells, activation with either the pep-

tide-MHC or the superantigen SEA-MHC ligand was dramatically enhanced by expression of the wild-type CD4, compared with expression of the tailless mutant. Within the cytoplasmic tail, mutation of one or both residues of the cysteine motif prevented the association of CD4 with p561ok and concomitantly led to a dramatic decrease in the level of IL-2 response upon T cell stimulation. Interestingly, the slight increase in responsiveness of 150.MCA-2 cells, when compared with 150.MCA-1 or 150.'1"1cells, correlated with the very small amount of p56Ick coimmunoprecipitated with CD4-MCA-2. In addition, all the mutants that retained the ability to bind p56 tck also secreted significantly high levels of IL-2. Although the possibility that CD4 interacts with a yet undefined protein via the same cysteine motif cannot be formally dismissed, our results strongly suggest that the association of CD4 with p56~ck is critical for generation of TCR-mediated transmembrane signals. Surprisingly, even though CD4-T3 and CD4-MS1/2/3 molecules bound p561ok almost as well as the wild-type CD4, their coreceptor activity in antigen-specific T cell activation was significantly impaired. Thissuggests that mere physical association between CD4 and p56~k is not the only requirement for their participation in T cell activation. One attractive possibility, which remains to be tested, is that deletion of ten C-terminal residues of CD4 or mutation of all three serine residues may prevent interaction with other factors that regulate the activity or substrate specificity of the associated p56~ck. Taken together, our results establish that the physical association of CD4 with p56~ckis a necessary, but perhaps not a sufficient, condition for antigen-specific T cell activation. Internalization of CD4 Is Not a Prerequisite for T Cell Activation In both murine and human T cells, treatment with either phorbol ester or antigen-MHC leads to internalization of

CD4-p56Ick Interactionin T Cell Activation 517

CD4 molecules. This phenomenon is associated with phosphorylation of serine residues and is likely to involve protein kinase C (Acres et al., 1986, 1987). The fact that p56~ck,which is associated with CD4 in resting cells, is no longer associated with CD4 after its internalization has suggested that sequestration of p56Ick may be required for T cell activation (Veillette et al., 1988; Hurley et al., 1989). Thus, the ability of CD4 to be internalized could be critical for its function as a coreceptor molecule. We observed that, similar to the human CD4, the substitution of the first, but not the third, of the three cytoplasmic serine residues of murine CD4 abolishes the ability of these molecules to be internalized. However, T cells expressing either of these mutant molecules secreted levels of IL-2 comparable to those produced by cells expressing the wild-type CD4. Thus, internalization of CD4 is not required for participation of the CD4-p56 Ick complex in T cell activation. This implies that the substrates of p56 ~ck are accessible prior to internalization of CD4 and that relocalization of p56 ~ck to a new cellular compartment is not required for antigen-mediated T cell activation. Minor Role of CD4 as an Adhesion Molecule in T Cell Activation Cell-cell adhesion interactions between T cells and their targets play a critical role in T cell activation (Springer, 1990). Many of these interactions are mediated by cell surface molecules of the integrin family, whose avidity for ligand can be influenced by occupancy of the TCR (Springer, 1990). Whether the CD4 and CD8 molecules also serve an adhesion function that is distinct from their roles as coreceptors in TCR-ligand interactions during T cell activation has remained controversial (Janeway, 1989). Both the CD4 and the CD8a molecules have the potential to promote adhesion interactions due to their intrinsic affinity for the MHC class II or I molecules, respectively (Doyle and Strominger, 1987; Norment et al., 1988). There is also strong evidence that CD8 and the TCR must interact with the same MHC molecule for activation of T cells (Potter et al., 1989; Salter et al., 1990). However, because CD4 and CD8 are intimately involved in TCR-MHC ligand interactions, it has been difficult to evaluate the respective contributions of their adhesion and signaling functions. Our results suggest that the adhesion function of CD4 plays only a minor role in T cell activation. Only a very small enhancement in IL-2 production was evident in cells expressing mutant CD4 molecules that could not bind p56jck. An adhesion-mediated function for CD8a was also not evident in these studies, despite the fact that the APCs also expressed class I molecules. These results are in contrast to previous studies with a CD8.dependent T cell hybridoma, in which the alloreactive response to a class I MHC ligand was only partially dependent on the cytoplasmic tail of CD8a (Zamoyska et al., 1989; Letourneur et al., 1990). Interestingly, we observed that the IL-2 response of 150 cells was less dependent on CD4 than that of 171 cells. In addition, tailless CD4 molecules had a greater effect on IL-2 production when expressed in 150 as compared to

171 transfectants. We believe that these two observations are related to the 5- to 10-fold higher level of MHC class il on the APCs recognized by 150 cells (data not shown). This hypothesis is also supported by the fact that 150 CD4-negative cells could not be stimulated with antigens when presented by FT7.2 APCs selected for lower class II MHC expression (our unpublished data). The correlation between the CD4 dependency of the IL-2 response and the MHC density may also explain some of the variability in the literature regarding the role of CD4 and CD8 in T cell activation. Taken together, our results demonstrate that the coreceptor function of CD4 is clearly dependent on the TCR-ligand interactions and that its putative adhesion function plays only a minor role in T cell activation. Possible Molecular Mechanisms for Participation of the CD4-p56 ~ck Complex in T Cell Activation and Ontogeny Although CD4 and the TCR complex are not covalently associated on the cell surface, several independent studies have shown that a subset of CD4 molecules exists in physical proximity of the TCR in resting or activated T cells (Kupfer et al., 1987; Saizawa et al., 1987; Anderson et al., 1988; Chuck et al., 1990). In addition, CD4 and CD8a are associated with p56~ck in resting cells (Rudd et al., 1988; Veillette et al., 1988). Our demonstration that CD4-p56/ok interactions are critical for T cell activation supports a model in which CD4 or CD8, already complexed with p56 ~ck, become physically associated with the TCR-CD3 complex upon recognition of the TCR ligand. This could occur through simultaneous interactions of the TCR and the appropriate CD4 or CD8 coreceptor with the same antigen-MHC ligand. As a result, p56 ~ck might be exposed to new substrates and/or regulatory proteins in the vicinity of the TCR-CD3 complex. Candidates for such proteins, include the r chain of the TCR complex p59 fyn, and the phosphotyrosine phosphatase CD45 (Veillette et al., 1989; Mustelin et al., 1989; Ostergaard et al., 1989). A critical issue in understanding the function of CD4 and CD8 molecules is to determine how they participate in thymic ontogeny, a complex process involving deletion of immature double-positive (CD4+8+) autoreactive thymocytes and positive selection of self-restricted single-positive (CD4+8 - and CD4-8 +) T cells. This process requires TCR-ligand interactions, whose outcome determines the developmental fate of individual T cells and the commitment to expression of either CD4 or CD8 molecules (Blackman et al., 1990). Direct involvement of CD4 and CD8 in thymic differentiation has been suggested by findings that anti-CD4 and anti-CD8 antibodies block development of T cells both in vivo and in thymic organ cultures (Ramsdell and Fowlkes, 1989; Zuniga-Pflucker et al., 1990). As our data suggest a minor role of the adhesion function of CD4 and CD8, it is likely that the required coreceptor function in thymic selection is a transmembrane signal mediated by p56Ick. It remains to be determined if the signals generated by the CD4-p56 Ick and CD8-p56 ~ckcomplexes are the same or different in developing thymocytes. Now that we have defined point mutations of CD4 that prevent the association with p56~ck,and hence the signaling function

Cell 518

of CD4 in model T cells in vitro, we can progress to in vivo transgenic models to test the proposal that p56 ~ckdirectly regulates negative and positive selection in the thymus. The nature of the ligands that interact with the TCRs in immature thymocytes is poorly understood. It has been hypothesized that self-peptide analogs of superantigens such as MIs antigens play a role in the selection processes (Kappler et al., 1988). In this respect, it is interesting that, although the structural features of the TCR that determine its interaction with the s u p e r a n t i g e n - M H C complexes appear different from those that determine its interaction with p e p t i d e - M H C complexes (Marrack and Kappler, 1990), both stimuli show identical CD4 coreceptor requirements. This is unlikely to be the case in all instances of T cell-superantigen interaction, as murine and human CD8 ÷ cells have been reported to react with MIs1a and SEA, respectively (MacDonald et al., 1990; Fleischer and Schrezenmeier, 1988). The requirement for C D 4 - p 5 6 Ick c o m p l e x formation in SEA recognition, as demonstrated in this study, suggests that SEA and CD4 bind to nonoverlapping regions, distinct from the peptidebinding groove, of MHC class II molecules. It remains to be determined whether CD4 has a role in superantigen recognition in thymic ontogeny. The p56 Ick protein is thus far the only member of the s r c family of cytoplasmic protein tyrosine kinases that has been directly implicated in a cellular response. It is likely that a detailed analysis of the function of this kinase in systems such as those described in this report will shed much light on the general mechanism of action of this class of molecules. The demonstration that antigenspecific activation of T cells is d e p e n d e n t on the formation of CD4-p56/ck complexes thus provides an avenue for future studies.

Experimental Procedures Reagents, Antibodies, and Plasmids SEA was purchased from Toxin Tech. Inc. Phorbol myristate acetate (PMA), G418, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrezolium bromide (MTT), and polybrene were purchased from Sigma. MAbs GK1.5 (anti-murine CD4), 53-6.7 (anti-murina CD8a), OKT4 (anti-human CD4), and H57.597 (anti-routine TCR) were used as hybridoma supernatants or ascites. The amino-terminal p56~k antiserum is a polyclonal rabbit antiserum raised against a synthetic peptide corresponding to amino acids 39-64 of the murine p56ek protein and was a gift of Dr. Joseph Bolen. cDNA constructs of murina CD8a, CD4, and mutants thereof have been described previously (Turner et el., 1990), as has the human CD4 cDNA (Brodsky et el., 1990). cDNAs were transferred from the vector pSM into the retroviral expression vector pMV7 (Kirschmeier etal., 1988). pSMCD4 and pMV7 were first digested with Sail and Hindlll, respectively. Both DNAs were further treated with Klenow, digested with EcoRI, and ligated together. Plasmids encoding bm2T3.1 and BO4H9.1 a and I~chains are described elsewhere (Glaichenhaus et al., submitted). Cell Lines end Culture Cell lines 150 and 171 were generated by introducing the ~ and 13chain genes from two different lysozyme-specific T cell hybrids into the 58¢z-~- lymphoma (Letourneur and Malissen, 1989). The 150 cells are reactive with a 15 amino acid peptide (74-88) of hen lysozyme (NLCNIPCSALLSSDI) presented by the A brn12MHC molecule, while 171 cells are specific for the hen lysozyme (74-88) peptide analog (NLANIPASALLSSDI) associated with the Ab MHC molecule (Shastri, 1989). FT7.1 and FT7.2 cells lines were derived from L celt fibroblssts

cotransfectsd with the (~and 13chain genes of the A b or Abin12class II MHC molecules, respectively (Ronchese et el., 1997). All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamina, 1 mM pyruvate, 50 I~M 2-mercaptoethanol, 100 Ulml penicillin G, and 100 I~g/mlstreptomycin with addition of 50 Ulml recombinant human IL-2 for CTLL cells. Preparation of Retrovlral Packaging Cell Lines and Ilmletlon of Infected T Cells cDNAs in the pMV7 vector were transfected into the packaging cell line PA317(Miller and Buttimors, 1986) using CaPO4 precipitation (Graham and van der Eb, 1973). After 48 hr, culture supernatants were harvested and used to infect ~2 cells in 100 mm dishes in the presence of polybrene (8 i~g/ml) for 2 hr. Cells were maintained in normal medium for a further 24 hr, and then G418 selection was applied (400 i.tg/ml) in order to establish stably transfected packaging cell lines. Such cell lines were prepared for all CD4 and CD8 constructs, and viral stocks isolated from them were used to infect 150 or 171 cells in the presence of polybrene (10 i~g/ml). In a typical experiment, 10 ml of cell supernatant was used to infect 5 x 10s T cells. After 48 hr, cells were collected by centrifugation and resuspended in fresh medium. After expansion in culture, 107 cells were analyzed by flow cytometry with appropriate primary antibodies as described below. Cells expressing CD4 or CD8ct (1%-5% of the overall population) were sorted and expanded for subsequent analysis. Each pool of cells was derived from at least 104 infected cells. Analysis of Cell Surface Expression Surface expression of murine CD4 was analyzed as follows: 106 cells were incubated with MAb GK1.5 at saturating concentration in 100 p.I of phosphate-buffered saline plus 2% fetal bovine serum for 20 min on ice. After washing, cells were stained under the same conditions with FITC-conjugated MAb (anti-murine IgG) and washed again. After resuspension in 1 ml of phosphate-buffered saline, 2% fetal bovine serum, and 0.2 I~g/ml propidium iodide (which stains nonviable cells and enables these cells to be gated out during analysis), cell surface fluorescence was analyzed on a FACS IV (Becton-Dickinson). The same procedure was used to analyze cell surface expression of human CD4 and murine CD8a and CD3 using MAbs OKT4, 53-6.7, and 500A2, respectively.

ImmunoprecipitsUon and Immunoblottlng Preparations of cell iysatas, immunoprecipitations, and immunoblots were performed as previously described (Turner et al., 1990). Briefly, T cells were washed twice with phosphate-buffered saline prior to lysis, and protein concentrations were determined by Bradford assay. For immunoprecipitationa, 1 mg protein aliquots of lysates were precleared with goat anti-rat IgG agarose (Sigma) when MAb GK1.5or 53-6.7 was used, or protein A-Sepharose CL-4B (Pharmacia) in the case of MAb OKT4. Incubations with antibodies and preparation of immune complexes for electrophorasis on 10o/o SDS-polyacrylamide gels were as described (Turner et al., 1990). Total endogenous or CD4(CDS)associated p56~ckwas detected on immunobiots by incubating 100 i~1 of T cell lysate overnight with the amino-terminal p56jck antiserum at 1:100 in TBST milk (150 mM MaCI, 10 mM Tris-,HCI [pH 8.0], 0.5% Twaen 20, 5% nonfat dry milk). After three washes in TBST milk and incubation with 12el-labeledprotein A (New England Nuclear) for 1 hr at the final concentration of 0.25 i~Ci/ml, blots were washed again in TBST and exposed to XAR film. T Cell Activation Antigen-spacific responses were assayed by incubating varying concentrations of antigens with 6 x 104 APCs (FT7.1 or FT7.2) and 1 x 10s responder T cells in a final volume of 200 p.I at 37°C. Overnight (20-24 hr) supernatants from duplicate cultures were collected and frozen at -20°C until assayed for IL-2 content. IL-2 was assayed by titrating supernatants in 2-fold dilutions with 1 x 104 CTLL indicator cells per well. After 24 hr, CTLL proliferation was assessed by pulsing with 20 i~1of MTT (at 5 mg/ml) for 4 hr and then solubilizing the cells with 150 Id of isopropanol in HCI 0.01 N. Plates were read on a Plate Reader EL309 (Bio-Tek Instruments) at ODsso nm. Total units of IL-2 per well were determined by comparisonto a recombinanthuman IL-2 standard.

CD4-p56/ck Interaction in T Cell Activation

519

Acknowledgments

We thank Elenie Callas for help with cytofluorometric analysis; Anthony Brown for advice on establishing packaging cell lines; Dr. Bernard Mallissen and Dr. Ronald Germain for the gifts of the 58a-~-, FT7.1, and FT7.2 cells; Dr. Joseph Bolen for generously providing us with the p56 ~k antiserum; and Drs. James Allison, Anthony DeFranco, Roger Perlmutter, and #.star Winoto for critical reading of the manuscript. N. G. and J. M. T. were supported by postdoctoral fellowships from the National Institutes of Health and the Science and Engineering Research Council, respectively. This work was supported by grants from the National Institutes of Health to N. S. and D. R. L. and by the Howard Hughes Medical Institute (D. R. L.). N. S. is a PEW Scholar in the Biochemical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC Section 1734 solely to indicate this fact. Received October 2, 1990; revised November 11, 1990. References

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