are linked to the TCR in the membranes of isolated T cells (3). These observations, taken together, have led to the suggestion

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1 Proc. Natl. Acad. Sci. USA Vol. 84, pp , August 1987 Immunology Coclustering of CD4 (L3T4) molecule with the T-cell receptor is induced by specific direct interaction of helper T cells and antigen-presenting cells (membrane dynamics/capping/double immunofluorescence) ABRAHAM KUPFER*, S. J. SINGER*, CHARLES A. JANEWAY, JR.t, AND SUSAN L. SWAIN* *Department of Biology, University of California at San Diego, La Jolla, CA 92093; and tthe Howard Hughes Medical Institute and Department of Pathology, Yale University School of Medicine, New Haven, CT Contributed by S. J. Singer, May 15, 1987 ABSTRACT Blocking studies with monoclonal antibodies have suggested that helper T cell recognition and triggering involve the CD4 (L3T4) accessory molecule as well as the T-cell receptor (TCR) that is linked to the T3 complex. We have investigated the surface distribution of L3T4 and TCR during the direct interaction of a cloned murine helper T-cell line with an antigen-presenting B-cell line. Using immunofluorescence microscopy, we show that in 1:1 cell'couples formed between the two cells, in which a specific interaction can be demonstrated, the L3T4 and the TCR become redistributed on the T-cell surface so that they are concentrated in the cell-cell contact region. This coclustering of L3T4 with TCR occurs only when the relevant antigen and appropriate major histocompatibility class II molecules are presented to the T cell, and it therefore requires the specific interaction of the TCR with it; complex ligand on the antigen-presenting cell. Effector T cells mediate two types of cellular immune responses: cytotoxicity, in which cytotoxic T lymphocytes (CTLs) cause the lysis of specific target cells; and help, in which helper T (Th) cells induce the proliferation and differentiation of specific antigen-presenting B cells (APCs). The specificity of these interactions is conferred at the molecular level by a clonotypic T-cell receptor (TCR) on the surface of the CTL or Th cell binding to a specific complex ligand on the surface of the syngeneic congener cell. This complex ligand consists of the specific antigen (Ag), or Ag fragment, together with a molecule of the major histocompatibility complex (MHC). For CTLs, the MHC molecule is generally class I, while for Th cells it is generally class II (Ia in the mouse). It is not clear, however, whether this discrimination between class I and class II MHC is a property of the TCR molecules themselves. The clonotypic Va and V# gene-encoded domains of the a and f3 polypeptides of the TCR can be identical for class I and class II recognition [although differences in the diversity-joining (D-J) regions may impart specificity] (1). The discrimination between class I and class II may therefore involve molecules other than, or in addition to, the TCR. Besides the clonotypic TCR, T cells express several monomorphic cell surface molecules that participate in the interactions with their congener cells. Among these accessory molecules, class II MHC-restricted T cells express CD4 (L3T4 in the mouse), whereas class I MHC-restricted T cells express CD8 (Lyt2 in the mouse) (for review, see ref. 2). Monoclonal antibodies to CD4 and CD8 block the responses of T cells recognizing class II and class I molecules, respectively. On the other hand, unlike the T3 complex, there is no evidence that these accessory molecules The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. are linked to the TCR in the membranes of isolated T cells (3). These observations, taken together, have led to the suggestion (2, 4) that CD4 and CD8 are at least in part responsible for the MHC class discrimination, perhaps by binding to monomorphic determinants on class II and class I molecules, respectively. Such CD4-class II and CD8-class I binding could well make important contributions to the overall intercellular adhesivity, and antibodies to the accessory molecule might block by countering this effect. However, there are other possible explanations for antibody blocking. For instance, the accessory molecule might become associated with the TCR in some critical way (5) when the TCR is bound to its complex Ag/MHC ligand, and antibody might sterically inhibit this association. Another possibility is that the accessory molecules do not become bound to either MHC or TCR molecules but instead are involved in the transmission of a positive signal that is blocked by antibody or a negative signal that is induced by antibody (6, 7). Recently we have developed a model system to visualize the direct specific interaction ofth cells with APCs (8, 9). We have shown that specific Th-APC couples can be distinguished from nonspecific ones by intracellular reorganizations detected in the Th cells that occur only as a consequence of specific recognition. One such reorganization is the accumulation of the cytoskeletal protein talin within the Th cell under the plasma membrane, where it is in contact with the APC. This model system has now been used to examine the cell surface distributions of the TCR and L3T4 molecules during specific and nonspecific interactions of murine T-cell lines with B lymphoma cells. Using double immunofluorescence, we show that both TCR and L3T4 become clustered into the region of the Th cell membrane that is in contact with specific, but not nonspecific, APCs. On the other hand, the antibody-induced capping of TCR on the Th cell does not result in any detectable cocapping of L3T4, and vice versa. Possible interpretations of these findings are discussed. MATERIALS AND METHODS D10.G4.1, a cloned murine Th cell line, referred to here as D10, was used in all experiments in this paper. It is specific for the egg-white protein Ag conalbumin (Con) in conjunction with the class II MHC determinant I-Ak (10). As APCs, the B-hybridoma cell line LK35.2 (11), referred to here as LK, which expresses both lak and lad determinants, and the BCL1 in vivo B-cell tumor line (12), which expresses lad determinants, were used. Two rat monoclonal antibodies directed to Abbreviations: CTL, cytotoxic T lymphocyte; Th, helper T cell: TCR, T-cell receptor; APC, antigen-presenting B cell; Ag, antigen; MHC, major histocompatibility complex; Con, conalbumin; Ova, ovalbumin. 5888

2 Immunology: Kupfer et al. T-cell surface molecules were employed: KJ16, directed to the Vp8 family of the TCR (13), and GK1.5, directed to L3T4 (14). Mouse monoclonal antibody F23.1, directed to the V,8 family of the TCR (15), was used as well. Affinity-purified polyclonal rabbit antibodies to chicken gizzard talin were prepared as previously described (16). For the processing and presentation of the Ag, LK cells were pulsed overnight with either Con or the irrelevant egg-white Ag ovalbumin (Ova), each at 500 tkg/ml. In some experiments the LK cells were not pulsed with either protein. After washing away unbound antigen D10-LK cell conjugates were made by mixing equal numbers of LK cells and D10 cells. Aliquots from these cell mixtures, containing about 105 cells each, were plated on poly(l-lysine)-treated glass coverslips within 30 min after mixing and were fixed with 3% (wt/vol) formaldehyde 5 min later. The fixed cells were labeled with one of the rat monoclonal antibodies to a T-cell surface molecule. The membranes of the cells were then Proc. Natl. Acad. Sci. USA 84 (1987) 5889 rendered permeable by treatment with Triton X-100 and the cells were further labeled with affinity-purified rabbit antibodies specific for talin (9, 17). The secondary antibodies (Jackson ImmunoResearch, Avondale, PA) were affinitypurified F(ab')2 fragments of rhodamine-labeled goat antirabbit IgG and biotin-labeled goat anti-rat IgG. These were first passed through columns of mouse IgG bound to Ultrogel AcA 22 (LKB) to eliminate binding to mouse IgG. The cells were further treated with fluorescein-streptavidin (Amersham International, Amersham, UK) to fluorescently mark the biotinylated antibodies. Microscopy and photography were carried out as previously described (16). For antibody-induced capping of L3T4, and the determination of whether TCR was cocapped with L3T4, D10 cells were incubated for 60 min at 37 C with the rat monoclonal antibody GK1.5, washed, and then treated with the biotinylated F(ab')2 fragments of goat anti-rat IgG for 30 min at 37 C. After washing, the cells were plated and fixed as described above. The fixed cells were then treated with the F23.1 mouse antibody to TCR, washed, and labeled with affinitypurified rhodamine-conjugated F(ab')2 fragments of goat anti-mouse IgG that had first been cross-absorbed on columns of rat IgG, as well as with fluorescein-streptavidin. For capping of the TCR and the determination of whether L3T4 was cocapped with the TCR this procedure was reversed, but biotinylated F(ab')2 fragments of goat anti-mouse IgG and rhodamine-conjugated F(ab')2 fragments of goat anti-rat IgG were used as the secondary antibody reagents. FIG. 1. Distribution of the TCR (A, D, G) and talin (B, E, H) in cell couples of D10 and LK that were pulsed overnight with the specific Ag Con (A-C) or the nonspecific Ag Ova (D-F), and a cell couple of D10 and BCL1 that was pulsed overnight with Con (G-J). Both here and in Fig. 2 single cell couples of the Th D10 and an APC are illustrated in rows of three panels. The right panel is the Nomarski image of the couple, the D10 always displayed to the left side of the couple; the middle panel shows the intracellular labeling for the cytoskeletal protein talin; and the left panel shows cell surface immunofluorescent labeling. Here, this labeling was with KJ16, which is specific for the TCR. (The bar in C represents 10,um.)

3 5890 Immunology: Kupfer et al. RESULTS Cell mixtures of D10 and Con-pulsed LK cells were immunolabeled with either of the rat monoclonal antibodies KJ16, specific for the TCR, or GK1.5, specific for L3T4. The same cell couples were also immunolabeled intracellularly with rabbit anti-talin antibodies. The labeling for talin was used as an independent measure to ascertain that, in the particular cell couple examined, the D10 cell had undergone the intracellular rearrangements that occur during specific, but not nonspecific, Th-APC interactions (8, 9). As expected, the immunolabeling for talin was highly concentrated at the cell contact region in the majority (90%) of the specific Proc. Natl. Acad. Sci. USA 84 (1987) D10-Con-pulsed LK cell couples (Figs. 1B and 2 B and E). Surface immunolabeling of the TCR with the KJ16 antibody showed a significant concentration of labeling at the contact regions in 70 ± 5% of the specific cell couples (Fig. 1A). With the GK1.5 antibody, a concentration of surface immunolabeling for L3T4 was detectable at the contact regions in 75 ± 5% of the specific cell couples, two examples of which are shown in Fig. 2 A and D. In order to determine whether the redistribution of these Th cell surface molecules into the cell contact site is Ag specific, LK cells that were pulsed with the irrelevant Ag Ova were mixed with D10 and immunolabeled as described above. Cell couples that formed in these mixtures were in general not FIG. 2. Distribution of L3T4 (A, D, G, J) and talin (B, E, H, K) in cell couples of D10 and LK that were pulsed overnight with the specific Ag Con (A-F) or the nonspecific Ag Ova (G-L). See legend of Fig. 1. Immunofluorescent labeling in A, D, G, and J was with GK1.5, which is specific for L3T4. (The bar in C represents 10,um.)

4 Immunology: Kupfer et A morphologically distinguishable from the specific couples by Nomarski optics (8, 9). The majority (80 ± 5%) of these cell couples showed no detectable increase in immunolabeling at the cell-cell contact sites of D10 cells for talin (Figs. 1E and 2 H and K), the TCR (Fig. 1D), or L3T4 (Fig. 2 G and J). The Ia specificity of the specific cell-surface redistributions was examined by mixing Con-pulsed BCL1 cells (which express lad but lack Iak, to which the D10 cell is specific) with the D10 cells and carrying out the immunolabeling experiments on these nonspecific cell couples. The majority of these couples (80 ± 5%) displayed a uniformly dispersed immunolabeling for talin (Fig. 1H), the TCR (Fig. 1G), and L3T4 (not shown, but as in Fig. 2 G and J). Antibody-induced capping experiments were carried out on the D10 cells. The capping of L3T4 (Fig. 3A) did not result in any detectable redistribution of the TCR on the same cell (Fig. 3B), and conversely, the capping of the TCR with the F23.1 antibody (Fig. 3D) did not induce any detectable redistribution of L3T4 (Fig. 3C). DISCUSSION The experiments presented here indicate that the specific binding of a Th cell with an APC results in the redistribution of the TCR and of L3T4 into the region of the T-cell membrane that is in contact with the APC. In the absence of specific recognition, the cell couples that form do not exhibit a redistribution of either surface molecule (Figs. 1 and 2). Furthermore, anti-v138 antibody-induced capping of the TCR on D10 cells does not result in any detectable redistribution of L3T4, and vice versa (Fig. 3), demonstrating that L3T4 is not linked to the TCR in the membranes of isolated Th cells. Therefore the coclustering of L3T4 with TCR that is observed in the specific Th-APC couples is induced only by the cell-cell interaction. Other evidence (3) is consistent with the physical independence of L3T4 and TCR in Th cell membranes. While there are several alternative explanations for these findings (see below), these observations strengthen the case that L3T4 is somehow physically involved in the specific interaction of Th cells with APCs. In order to interpret these results, we must realize that a cell-cell interaction is not simply a matter of receptor-ligand Proc. Natl. Acad. Sci. USA 84 (1987) 5891 binding but is a dynamic process that involves the diffusibility of interacting proteins in the cell membranes. Consider the simple case of two cells P and Q, where P has on its surface receptor molecules A with binding affinity for ligand molecules a on Q. The interaction of P and Q is mediated at the molecular level by the binding of A to a. One of us predicted (18) that stable contacts can form between the two cells in which A and a molecules become collected in their respective fluid membranes into the regions of P-Q contact. Thus, if A and a molecules are present at appropriate concentrations, and A-a bonds are slow to dissociate relative to the diffusion times of A and a in their respective fluid membranes, then upon collision of P and Q cells the initial formation of a small number of A-a bonds could 4rapidly lead to an extended region of stable P-Q contact, as the result of the diffusion of many A and a molecules in their respective membranes into the growing region of contact, and the formation of an increasing number of A-a bonds. A and a molecules would thereby both become clustered into the contact region. This phenomenon, which might be termed "mutual capping" (because two molecules are involved) has been confirmed experimentally (19, 20) and analyzed theoretically (21). If one considers first our results with the TCR, which was found to be concentrated in the contact regions formed by the Th cell with the specific APC (Fig. 1A), but not with the nonspecific APC (Fig. 1 D and G), one could propose that the binding of the TCR to its specific complex ligand, Con/lak, produced a mutual capping of that receptor-ligand pair into the specific cell-cell contact regions. With the nonspecific Ag Ova presented on the same APC, or the specific Ag Con presented with the incorrect Ia, the cell-cell contacts that form do not show a clustering of the TCR, because the TCR does not bind to a nonspecific ligand on the APC. Such clustering of the TCR, and not simply its binding to the Ag/Ia ligand, may be required for the transmission of the signal to activate the Th cell. This suggestion is consistent with evidence (22) that a monoclonal antibody to the TCR could activate D10 cells, but the univalent Fab' fragment of that antibody could not. If one considers next the results with L3T4, the situation is interestingly different. It has been suggested (reviewed in ref. 2) that L3T4 molecules on Th cells bind to monomorphic FIG. 3. Double indirect immunofluorescent labeling of D10 cells after antibody-induced capping of L3T4 (A, B) or of the TCR (C, D). The capped cells (A, D) were simultaneously labeled for the other surface marker, the TCR in B and L3T4 in C. The capping of either surface molecule (arrows) did not cause a detectable redistribution of the other. (The bar in D represents 10,um.)

5 5892 Immunology: Kupfer et al. determinants of Ia molecules on APCs. The clustering of L3T4 (Fig. 2 A and D) into the contact regions of specific Th-APC couples might therefore appear to be due to a mutual capping of the putative receptor-ligand pair. However, if this were true, L3T4 should also have become clustered into the contact regions of the nonspecific couples. In particular, in couples formed by the Th cell with the B-lymphoma cell presenting the nonspecific Ag Ova (Fig. 2 G-L), the same L3T4Ia pair was presumably present in precisely the same amounts, yet no mutual capping of L3T4 resulted. The observed clustering of L3T4 in the specific Th-APC interaction is evidently dependent upon the specific binding of the TCR to its Ag/Ia complex ligand on the APC. There are a variety of possible explanations of this dependency. One possibility is as follows. For the L3T4Ia receptor-ligand pair the membrane concentrations and bond dissociation rates might not allow this pair to achieve mutual capping on its own. The mutual capping of the specific TCR-Ag/Ia pair, however, could serve to concentrate and immobilize the Ag/Ia complex in the APC membrane into the cell-cell contact region. The immobilization of Ia in the contact region could now allow the formation of L3T4*Ia bonds in the contact region, and the consequent recruitment of L3T4 into the contact region to form those bonds. A second possibility, a variant of the first, is that the mutual capping of the TCRAg/Ia pair may itself require the synergy of the L3T4-Ia interaction in order for it to occur (mutual cocapping). In both of these cases, the L3T4Ia interaction would contribute directly and critically to the adhesion of the Th cells and APCs. They are attractive possibilities because either one would provide a rationale for the association of CD4 with the restrictive recognition of class II MHC. A third possibility is that the specific TCRAg/Ia binding induces some protein conformational changes or biochemical changes in one or more of the Th or APC cell surface molecules. For example, the TCR itself may undergo a conformational change upon binding its specific ligand. This may result in an interaction of, for example, L3T4 with the ligand-bound TCR (or TCR-T3 complex) within the Th cell membrane, a possibility that has been suggested (23) to explain the apparent comodulation of L3T4 with the TCR that is induced by certain anti-tcr antibodies (but not by anti- V,08). The clustering of TCR into the cell-cell contact region that was produced by the mutual capping of the TCR-Ag/Ia pair would therefore result in a concentration of L3T4 into the contact region. If such were the case, binding of L3T4 to Ia on the APC might, or might not, be involved. In the former case, the clustered L3T4 in the contact region would contribute directly to cell-cell adhesion; in the latter case it would not. The L3T4 clustering might nevertheless be necessary for the processing of signals between the two cells. A direct interaction of CD8 with the TCR has also been suggested to explain synergistic effects of antibodies to CD8 in CTL activation (24). The inhibition of helper activity by antibodies to L3T4 could be rationalized for any of these possibilities. Inhibition might be ascribed, for example, to prevention by the antibody of the normal collection of L3T4 into the Th-APC contact region, with either reduction of cell-cell adhesion or blockage of the transmission of a critical signal as a consequence. Proc. Natl. Acad. Sci. USA 84 (1987) In addition to these possible explanations of our results, a considerable number of variants and combinations of the two could be entertained. The information available at present is insufficient to decide amongst them. While our results, therefore, do not define the mechanism by which L3T4 participates in Th-APC interactions, they strongly suggest that those mechanisms are critically dependent upon the interaction of the TCR with the specific Ag/Ia on the APC. We thank Mrs. Margie Adams, Mrs. Hannah Kupfer, Ms. Michele English, and Ms. Linda Walker for technical assistance. This work was supported in part by National Institutes of Health Grants AI (to A.K.), AI (to S.J.S.), AI and CA (to C.A.J.), and AI and AI (to S.L.S.). S.J.S. is an American Cancer Society Research Professor. 1. Rupp, F., Brecher, J., Giedlin, M. A., Mosmann, T., Zinkernagel, R. M., Hengartner, H. & Joho, R. H. (1987) Proc. Natl. Acad. Sci. USA 84, Swain, S. L. (1983) Immunol. Rev. 74, Oettgen, H. 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