Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 2603-2607, April 1990 Immunology Role of interaction of CD2 molecules with function- associated antigen 3 in T-cell recognition of nominal antigen (cell adhesion/cytotoxic T cells/T-cell activation/T-cell receptor) SHIGEO KOYASU*t*, TREBOR LAWTON*, DAVID NovICK*, MICHAEL A. RECNY*§¶, ROBERT F. SILICIANO11, BARBARA P. WALLNER**, AND ELLIS L. REINHERZ*tt *Laboratory of Immunobiology, Dana-Farber Cancer Institute, and Departments of tPathology, ttMedicine, and 1Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; §Procept, Inc., Cambridge, MA 02139; 'Division of Molecular and Clinical Rheumatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205; and **Biogen Research Corporation, Cambridge, MA 02142 Communicated by Max D. Cooper, January 3, 1990 (receivedfor review December 1, 1989)

ABSTRACT The role of the interaction of CD2 molecules expressing human CD2 have suggested that CD2-LFA-3 with lymphocyte function-associated antigen 3 (LFA-3) in interaction is important for the T-cell recognition process (7, facilitating nominal antigen recognition by T was 8). However, virtually nothing is known about the mecha- studied by utilizing an HLA-DR4-restricted CD4+ cytotoxic nism by which CD2-LFA-3 interaction augments T-cell rec- human T-cell clone specific for human immunodeficiency virus ognition-in particular, whether the interaction influences envelope glycoprotein gpl20 as a responder and murine flbro- recognition of nominal antigen and whether this conjugate blasts transfected with human class II major histocompatibility pair interacts with TCR during the antigen recognition pro- complex (MHC) and/or human LFA-3 molecules as antigen- cess. To analyze the contribution of CD2-LFA-3 interaction presenting cells (APC). Although expression of the DR4 re- to T-cell recognition of nominal antigen, we have examined striction element in fibroblasts is sufficient for T-cell recogni- the nature of physiologic interactions between the CD4' tion of a gpl20 peptide asjudged by induction ofproliferation, HLA-DR4-restricted human cytotoxic T-cell clone Een- coexpression of human LFA-3 on DR4+ APC decreases the 217.5, which is specific for the H3DCG human immunode- molar requirement of nominal antigen by greater than one ficiency virus 1 envelope glycoprotein gp120 (9), and murine order ofmagnitude. Both LFA-3 and the relevant class H MHC fibroblasts into which human class II MHC restricting ele- molecules are necessary for antigen-independent conjugate ments and LFA-3 were selectively transfected. Such a sys- formation, but the binding is further enhanced by specific tem, in which only defined human molecules are expressed nominal antigen. CD2-LFA-3 interaction is independent of on antigen-presenting cells (APC), allows for a controlled T-cell receptor-MHC interaction and contributes directly to analysis of individual receptor-ligand pairs. the stabilized conjugate between the and LFA-3-bearing APC; soluble CD2 and monoclonal antibodies to LFA-3 and CD2 reduce T-cell-APC binding to the level mediated by MATERIALS AND METHODS nominal antigen and MHC. During conjugate formation, CD2 Materials. A peptide corresponding to residues 410-429 of but not CD3 molecules are reorganized into the cell-cell gp120 ("gpl20 peptide") was synthesized on an Applied interaction site in an antigen-independent manner. Thus, re- Biosystems 431A automated peptide synthesizer and purified organization and/or coassociation of CD2 with CD3 molecules by reverse-phase HPLC using a Waters Deltapak C18 column. is not essential for T-cell activation. The secreted membrane anchor-lacking forms of CD2 and CD4 molecules were prepared in a baculovirus system as The ability ofT lymphocytes to recognize specific antigens in described (10, 11). The following monoclonal antibodies the context of class I and class II major histocompatibility (mAbs) were used in this study: Leu4 (anti-CD3); l9Thy5D7 complex (MHC) molecules is determined by the unique (anti-CD4); 3T4-8B5 (anti-T111); 1OLD24CI (anti-T112); 9-49 clonotypic T-cell receptor (TCR)-CD3 complex which they (directed against the common determinant of human class II individually bear. The process of antigen recognition in- MHC molecule); 11.4.1 (anti-murine H-2Kk); TS2/9.1.1 (an- volves the physical interaction of a TCR with a nominal ti-human LFA-3); fluorescein isothiocyanate (FITC)- peptide antigen bound to a specific MHC molecule referred conjugated 1OLD24C1; and tetramethylrhodamine isothio- to as a restricting element. This process is not singularly cyanate (TRITC)-conjugated Leu4. dictated by the TCR complex itself, but rather is dependent Cells and Growth Conditions. The CD4' CD8- human on other structures, including CD4, CD8, CD2, and lympho- T-lymphocyte clone Een217.5 is a subclone ofEen217 that is cyte function-associated antigen 1 (LFA-1) (reviewed in refs. specific for gpl20 in a DR4-restricted manner (9). Nomen- 1-3). CD4 and CD8 structures bind to monomorphic regions clature and phenotype of the murine L cell fibroblast trans- of class II and class I MHC structures, respectively, thereby fectants used as APC in this study are shown in Table 1. The facilitating the interaction of the TCR with the MHC restrict- preparation of murine L cell fibroblasts transfected with ing element (reviewed in ref. 4). In addition to the MHC full-length cDNA encoding human class II DR a and / chains binding accessory structures, a set of adhesion structures was described previously (9, 12). Those cells were further facilitates non-antigen-specific interactions between T lym- transfected with Xmn I-linearized animal expression plasmid phocytes and their cognate partners. This set includes the BG8/LFA-3, which contains the full-length cDNA for the CD2/lymphocyte function-associated antigen 3 (LFA-3) re- transmembrane form ofLFA-3, and Sca-I-linearized plasmid ceptor-ligand pair (5, 6). Studies employing antibodies directed against CD2 or Abbreviations: APC, antigen-presenting cell(s); FITC, fluorescein LFA-3 as well as murine T-cell hybridoma transfectants isothiocyanate; LFA-1, lymphocyte function-associated antigen 1; LFA-3, lymphocyte function-associated antigen 3; mAb, monoclonal antibody; MHC, major histocompatibility complex; TCR, T-cell The publication costs of this article were defrayed in part by page charge receptor; TRITC, tetramethylrhodamine isothiocyanate. payment. This article must therefore be hereby marked "advertisement" *On leave from: Tokyo Metropolitan Institute of Medical Science, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Tokyo 113, Japan. 2603 Downloaded by guest on October 2, 2021 2604 Immunology: Koyasu et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 1. Phenotypes of L cell transfectants the human Een217.5 T-cell clone is cultured with the DR4+ Cell line Class II MHC LFA-3 LFA-3- murine L cell transfectant L89.2, it is stimulated to proliferate in expanding T-cell colonies and readily lyses L* - _ L89.2 only in the presence of specific peptide antigen derived L-HT16 - ++ from gpl20 of the human immunodeficiency virus H3DCG L17.8 DRw53t _ strain (Fig. 1). Using various L cell transfectants as APC, we L12.2 DR7 - examined the effect of CD2-LFA-3 interaction on the pro- L12.2.16 DR7 ++ liferative response of Een217.5 to gpl20 peptide at a range of L89.2 DR4 - concentrations. As shown in Fig. 2a, L89.2.1 could stimulate L89.2.11 DR4 proliferation of Een217.5 to the same degree as L89.2 but L89.2.1 DR4 ++ required <1/10th the molar concentration of peptide. *L cells were established from C3H/He mice (H-2k). L89.2.11, which expresses 1/5th the LFA-3 of L89.2.1, tExpression of class II MHC molecules among different positive stimulated Een217.5 at a lower concentration ofpeptide than transfectants was virtually identical except for L17.8, which ex- presses 1/5th the surface copy number. L89.2 but was less efficient than L89.2.1. As anticipated by tAll LFA-3 transfectants expressed virtually equivalent copy num- the MHC restriction of Een217.5, neither L cells nor L cell bers of surface LFA-3 except for L89.2.11, which expresses 1/5th transfectants L-HT16, L17.8, L12.2, and L12.2.16 induced the surface copy number. Een217.5 to proliferate even in the presence of 1,uM specific peptide (data not shown). Although not shown, Een217.5 also pOPF, which carries the thymidine kinase gene (13). Trans- lysed L89.2.1 at a decreased peptide concentration relative to fected cells were selected in Dulbecco's modified Eagle's L89.2. medium (DMEM)/10% (vol/vol) fetal calf serum (FCS) con- The enhanced proliferation of Een217.5 in response to a taining 100 ,uM hypoxanthine/0.4 mM aminopterin/16 ,M suboptimal concentration of peptide presented by L89.2.1 thymidine (HAT) and were further analyzed for expression of versus L89.2 is clearly a consequence of CD2-LFA-3 inter- surface LFA-3 as well as class II molecules by indirect action. As shown in Fig. 2b, when anti-LFA-3 mAb was immunofluorescence. added to the culture stimulated by antigen and L89.2.1, the Proliferation Assays. Proliferation assays using fibroblast proliferative response was reduced to the same level as the cells as APC were performed as follows. Fibroblasts were response induced by L89.2. In contrast, the same antibody incubated with DMEM/10% FCS containing mitomycin C at did not affect the proliferative response to peptide plus L89.2. 10 ,g/ml and 1 mM thymidine for 4 hr. After extensive mAbs against the endogenous L cell H-2Kk murine class I washing, the cells were trypsinized and plated into 96-well MHC molecules did not show any inhibitory effect. These flat-bottom plates (2 x 104 per well). Een217.5 cells (5 x 104 results indicate that expression of the relevant class II MHC per well) were stimulated with those fibroblasts in the pres- molecules on APC is sufficient for T-cell proliferation but that ence ofgpl20 peptide at various concentrations for 24 hr. The coexpression ofLFA-3 reduces the concentration ofnominal T cells were then transferred into a second 96-well flat- antigen required for stimulation of the CD2+ T-cell clone. bottom plate seeded with mitomycin C-treated wild-type L These findings also support a role for CD4 in class II cells (2 x 104 per well) and cultured for a further 24 hr in the MHC-restricted antigen recognition involving both APC presence of [3H]thymidine at 1 ,Ci per well (1 Ci = 37 GBq) types, given that anti-CD4 mAb abrogates responses to 89.2 and recombinant interleukin 2 (Biogen) at 50 units/ml. The as well as 89.2.1. Conijugate Formation Between T Ceflsand APC. To elucidate incorporation of[3H]thymidine was measured by scintillation the importance of CD2-LFA-3 ligand pairs in facilitating the counting in triplicate samples. physical interaction between T cells and APC, we examined Cell to Cell Conijugate Formation. Fibroblasts were grown conjugate formation between T cells and APC (Fig. 3). L89.2 on glass coverslips (14 mm in diameter) and were pulsed with did not form conjugates with Een217.5 in the absence of the indicated concentration ofgpl20 peptide at 37°C for 1 hr. specific peptide (Fig. 3c) but clearly formed conjugates when After the fibroblasts had been washed, Een217.5 cells were pulsed with specific peptide (Fig. 3d). On the other hand, added and incubated at 37°C for another 1 hr. The wells were L89.2.1 formed conjugates with Een217.5 cells in the presence washed twice with DMEM/10o FCS and twice with Dul- or absence of peptide (Fig. 3 e and f), demonstrating the becco's phosphate-buffered saline (PBS). The remaining importance ofLFA-3 expression for antigen-independent con- cells were fixed with 3.7% (wt/vol) formaldehyde in PBS at jugate formation. However, L cells (Fig. 3a), L-HT16 (Fig. 37°C for 20 min and examined under a phase-contrast micro- 3g), or the gp120 peptide-pulsed L12.2 (Fig. 3b) did not bind scope. To quantify the binding, Een217.5 cells (0.5-1 x 107 per ml) were labeled with Na251CrO4 at 500S ,Ci/ml for 4 hr. After incubation of the labeled Een217.5 cells with fibro- blasts, the coverslips were washed as described above. The i) cells were then lysed by addition of0.5 ml of 1% Triton X-100 in water and radioactivities were determined with a y counter. Specific binding was determined after subtracting the radioactivity observed with coverslips containing L cells. c d Immunofluorescence Microscopy. Fibroblasts were grown on coverslips and cultured with Een217.5 cells as described above. After 3-60 min of incubation, cells were washed and fixed with 1% formaldehyde in PBS for 5 min at 37°C. Cells were then stained with FITC-conjugated anti-T112 at 1 ,g/ml and TRITC-conjugated Leu4 at 1 ,ug/ml. Specimens were FIG. 1. Antigen-specific induction of cytotoxic activity and pro- examined under an Axiophot fluorescence microscope with liferative response of Een217.5 cells. L89.2 fibroblasts cultured a Plan-Neofluor 100x objective (Zeiss). overnight in 24-well cluster dishes (105 per well) were incubated with (c, d) or without (a, b) 5 x 105 Een217.5 cells in DMEM/1O FCS RESULTS in the absence (a, c) or presence (b, d) of 1 ,uM gpl20 peptide. After CD2-LFA-3 Interaction Reduces the Concentration of Pep- 24 hr, cells were examined under a phase-contrast microscope tide Antigen Required to Induce T-Cell Proliferation. When without fixation. Downloaded by guest on October 2, 2021 Immunology: Koyasu et al. Proc. Natl. Acad. Sci. USA 87 (1990) 2605

a 1" 1No-Ag Ag v 6102

L '. ...:*.<,st' i. L12.2

4 ii t4 L89.2 L89.2

L892.I L89.2. 3,R4. LFA-3) C R4 LF *3

_ Antigen, juM b L-HT16 DR2[FA16 ± YA-3~~~~~~~~~~~~~~D7,LA3

+ c0LF-3

4- I~H2KK FIG. 3. LFA-3 and relevant class MHCe;molecule dependent conjugate formation betweenEen2*7.5 cells and L cell transfectants. 1892Z / L cell (a), L12.2 (b), L89.2 (c and d), L89.2.1 (e andf), L-HT16 (g), and L12.2.16 (h) were incubated with (b, d, f, h) or without (a, c, e, 0CD4 - g) 1 AtM gp120 peptide for 1 hr. After the fibroblasts had been washed, + c.F______3_ Een217.5 cells were added and incubated for another 1 hr. After 4J aH-2KK .______unbound cells were removed, bound cells were fixed and examined 2 6 S under a phase-contrast microscope. 3HjThymAdine x 10 incorporation, corn 3 The contribution of CD2-LFA-3 interaction in the en- hanced was an FIG. 2. Proliferative response of Een2l7.5 cells induced by binding of Een2l7.5 to L89.2.1 confirmed by specific antigen with murine L cell transfectants as APCs. (a) additional set of blocking studies. As shown in Fig. 4a, when Een2l7.5 cells (5 x 104 per well) were cultured in the presence of L89.2.1 was pretreated with anti-LFA-3 antibodies, the bind- various concentrations of gpl2O peptide for 24 hr with L89.2 (e), ing pattern of Een217.5 became essentially identical to that L89.2.11 (v), or L89.2.1 (0) cells as APC (2 x 104 per well). observed with L89.2. Consistent with this result, when the [3H]Thymidine incorporation is presented as the mean and SD of binding experiment was performed in the presence of soluble triplicate samples. (b) Een2l7.5 cells were stimulated in the presence T-cell surface the Een2w7.5 of various mAbs (a = anti). They were incubated with or without 1 glycoproteins, binding of to ,uM gp120 peptide (Ag) with L89.2 or L89.2.1 as shown. Staphylo- L89.2.1 was substantially inhibited by soluble CD2 but un- coccal A-purified anti-LFA-3 mAb (TS2/9) and anti-CD4 affected by soluble CD4 protein (Fig. 4b). Monomeric CD4 mAb (l9ThySD7) were used at 10 S.g/ml. Approximately equivalent was previously shown not to inhibit T-cell function because amounts of antibody from culture supernatant of the hybridoma of its low affinity for class MHC moleculessh (14). However, producing anti-H-2Kk mAb (11.4.1) were used (25%, vol/vol). to Een2l7.5 cells. Thus, the expression of LFA-3 alone was a p e wt _-- ai e t bid not sufficient to mediate stable cell adhesion. Furthermore, C I L12.2.16, which expresses both DR7 and LFA-3, did not form ok a n1- 3 .7 .Z I i~~ ~ ~ stable conjugates even in the presence of peptide (Fig. 3h). 0- ~~~ i I -",/ T- Collectively, the results imply that the expression of both T / 0' -T. ,, the 2-/2 relevant MHC restricting element and LFA-3 is required for _0E: ::~ '- t a _ . f~~~~~~~~~ the formation of stable antigen-independent conjugate forma- . 1 36 i _3 I _ tion in this system. Similar results were found with L89.2.11, f! ~ consistent with this view (data not shown). I ._1_I.... 1t " Io Next, we quantified the binding of 51Cr-labeled Een2l7.5 J `_31 0.1' j 336 I 3A 16, :11 0 3!6 3 16 cells to the various fibroblast populations. As shown in Fig. 4, Antigen. u M the binding ofEen2l7.5 to DR4+ L cell transfectants increased FIG. 4. LFA-3 and antigen dose-dependent binding of Een217.5 when APC were pulsed with increasing concentrations of cells to DR41 APCs. (a) L89.2 (O, *) and L89.2.1 (O, *) were antigen. Consistent with the earlier microscopic observations, incubated with various concentrations of gpl20 peptide in the conjugates between L89.2.1 and Een2l7.5 formed even in the absence (o, o) or presence (a, u) of anti-LFA-3 mAb TS2/9 at 10 absence of peptide. Fig. 4b shows that low or no significant ,ug/ml. After 1 hr of incubation, the fibroblasts were washed and 51Cr-labeled Een217.5 cells were added (105 cells with 10,000 cpm) binding of Een2l7.5 was detected above background with L and incubated for another 1 hr. Unbound cells were then removed by cells lacking DR4 such as L12.2, L12.2.16, or L-HT16 at any extensive washing and the bound radioactivities were determined in concentration of gpl2O peptide tested. These results indicate duplicate samples. (b) L-HT16 (v), L12.2 (o), L12.2.16 (.), L89.2 (o, that coexpression of LFA-3 and the specific MHC class II A, A), and L89.2.1 (e, i, *) were incubated with various concentra- restricting element on the APC is required for antigen- tions ofgpl20 peptide for 1 hr. After washing the cells, 51Cr-Een217.5 independent stable conjugate formation. Expression of an cells were added (105 cells with 3300 cpm) in the absence (o, c, v, irrelevant class II MHC molecule does not appear to contrib- o, *) orpresenceI" ofsoluble CD2 (e, A) or soluble CD4 (a, A) at 10 gM. After 1 hr of incubation, wells were extensively washed to remove ute to the conjugate formation. Note that the level of T-cell unbound cells and bound radioactivities were determined as de- binding to L89.2.1 was always higher than that with L89.2 at scribed above. The stippled areas indicate the background binding by any equivalent concentration of peptide (Fig. 4). untransfected L cells. Downloaded by guest on October 2, 2021 2606 Immunology: Koyasu et al. Proc. Natl. Acad. Sci. USA 87 (1990) Table 2. Effect of various mAbs on the binding of Een217.5 cells Phase to L cell transfectants Contrast FITC-CD2 TRITCIXCD3 Specific binding,* !r------F r -- gp120 cpm (% inhibition) mAb peptide L89.2 L89.2.1 None - 189 ± 94 840 ± 8 a Anti-CD4 - 170 ± 83 (3) 199 ± 9 (74) Anti-CD2 (T111) - 204 ± 87 (0) 177 ± 28 (79) L89.2 Anti-Ia (9-49) - 506 ± 28 (0) 1178 ± 291 (0) Ag None + 770 ± 105 1577 ± 8 Anti-CD4 + 176 ± 11 (77) 411 ± 75 (74) Anti-CD2 (T111) + 599 ± 8 (22) 679 ± 57 (57) b Anti-Ia (9-49) + 868 ± 148 (0) 1824 ± 51 (0) Purified mAb was used at 10 Ag/ml and gpl20 peptide was 1 ILM. *Specific binding was obtained by subtraction of cpm bound by L cells (374 cpm); 105 cells with 3300 cpm were added as an input. Results are mean ± SD for duplicate samples. c the binding of the T cells to L89.2 was affected by neither anti-LFA-3 antibodies nor soluble CD2 protein (Fig. 4). L89.2.1 mAbs against CD2 also inhibited the binding of Een217.5 to L89.2.1 but they did not inhibit the binding to L89.2 (Table No Ag 2). These results directly demonstrate that CD2-LFA-3 in- teraction makes a major contribution to the formation of d conjugates between T cells and APC. Furthermore, both the similarity in slope of binding curves of Een217.5 to L89.2.1 and L89.2 and the fact that the binding curve of Een217.5 to L89.2.1 becomes identical to the binding curve obtained with L89.2 in the presence of anti-LFA-3 mAb indicate that the contribution of antigen recognition by the TCR is indepen- e dent of that of the interaction between CD2 and LFA-3. The effects ofother mAbs on Een217.5 conjugate formation L89.2 and L89.2.1 are shown in Table 2. Anti-CD4 mAb IL89.2.1 strongly inhibited the binding of Een217.5 cells to both L89.2 Ag and L89.2.1. Although not shown, anti-murine class I (H-2Kk) had no effect on the binding of Een217.5 to either APC type. f Surprisingly, the anti-human class II mAb, 949, which rec- ognizes a common determinant of human class II molecules, did not inhibit conjugate formation (Table 2), although it significantly inhibited T-cell cytotoxic activity (data not shown). FIG. 5. Reorganization of CD2 but not CD3 by conjugate forma- CD2 Mocules Lalize to the Interface at the Site of tion between Een217.5 cells and murine APC that expresses human LFA-3. L89.2 (a, b) and L89.2.1 (c-f) cells on coverslips were Conjugate Formation. Given the importance of CD2 in facil- incubated with (a, b, e, f) or without (c, d) 1 /uM gp120 peptide (Ag). itating T-cell antigen recognition events, we examined by After 1 hr of incubation, cells were washed and then incubated with immunofluorescence microscopy the distribution ofCD2 and Een217.5 cells for another 1 hr. Conjugates were stained with TCR-CD3 on Een217.5 cells during interaction with APC. As FITC-conjugated anti-T112 and TRITC-conjugated anti-Leu4 for 1 shown in Fig. 5 a and b, conjugates between Een217.5 and hr. (Bar = 50 /tm.) L89.2 in the presence of peptide uniformly displayed CD2 and CD3 molecules around the T-cell surface. On the other human LFA-3 molecules by the APC augments this process hand, the CD2 molecule is localized to the site of conjugate by reducing the concentration of specific nominal peptide formation in conjugates formed between Een217.5 and antigen required for effective T-cell recognition. For exam- L89.2.1. This is the case regardless of whether the specific ple, the concentration of the peptide required to stimulate peptide antigen is present or absent (Fig. 5 c-f). Importantly, 50% maximal proliferation (50%1 effective dose, ED50) of CD3 molecules did not colocalize with CD2 at the site of Een217.5 with L89.2 (DR4' LFA-3-) was 1 (.M, whereas the conjugate formation (Fig. 5 c-f). These findings show that ED50 values with L89.2.11 (DR4+ LFA-3 OW) and L89.2.1 reorganization of CD3 on T cells does not occur if the (DR4+ LFA-3hiPO) were 0.3 ,uM and 0.05 ,uM, respectively. expression ofhuman ligands on the cognate APC is restricted These results indicate that increasing the expression of the to DR and LFA-3. Nevertheless, these molecules are suffi- LFA-3 molecule on APC diminishes the ED50 for T-cell cient to stimulate the T-cell clone in the absence of any function. The reduction of ED50 by CD2-LFA-3 interaction relocalization of the TCR. observed herein is important in recognition of nominal anti- gen in vivo, particularly when the affinity of the TCR for DISCUSSION antigen-MHC is low. Alternatively, when the affinity ofTCR The present experiments provide insight into the interaction and antigen-MHC is high, the contribution of cell adhesion between T lymphocytes and their cognate partners. On the molecules may be relatively less. basis of analysis of cell-cell conjugate formation as well as The first step in the T-cell recognition process involves a T-cell cytotoxicity and proliferation, it is clear that expres- physical interaction between the T cell and the APC (1-6). sion ofhuman class II MHC molecules on the surface ofAPC The subsequent crosslinking of TCRs by an array of ligands is sufficient to trigger the T-cell recognition process upon results in T-cell activation. Under physiologic circum- addition ofnominal antigen. However, surface expression of stances, the APC fulfills this requirement because it ex- Downloaded by guest on October 2, 2021 Immunology: Koyasu et A Proc. Natl. Acad. Sci. USA 87 (1990) 2607 presses multiple peptide-bound MHC molecules on its. sur- associated on the T-cell surface as detected by coprecipita- face. It is likely that stable conjugate formation between a tion of CD2 with CD3 in digitonin lysates (17). If CD2 and T-cell clone and APC as effected by CD2-LFA-3 interaction CD3 are physically associated on the T-cell surface, CD3 mediates the reduction of ED50. Antigen-independent conju- molecules should localize with CD2 molecules, particularly gate formation increases the area and/or duration of stable in view of the fact that Een217.5 cells express CD2 at higher contact between the T cell and the APC, thereby increasing levels than CD3 (unpublished). the potential interaction between a TCR and an antigen- It has been reported that CD3 as well as CD4 molecules MHC complex. In the absence of the CD2-LFA-3 interac- reorganize into the interface of murine T cell/murine tion, conjugate formation is presumably achieved by direct conjugates in the presence of specific antigen (18). In such interaction of the TCR with the antigen-MHC complex and homogeneic conjugates, other cell surface molecule(s), in- CD4 with class II MHC molecules (either ligated to or cluding LFA-1, which binds intercellular adhesion molecule independent of the TCR) but requiring larger numbers of 1 (ICAM-1), could be facilitating the reorganization of TCR. antigen-MHC complexes on the APC to attain a T-cell Though reorganization of CD3 is not essential for the induc- activation threshold. tion ofT-cell function as shown herein, such an effect should Earlier studies with a monomeric extracellular CD2 segment further promote multipoint attachment between T cell and demonstrated that it interacted with surface-bound LFA-3 APC and the consequent recognition process. With this in molecules with an affinity that was in the micromolar range mind, we note that autologous human monocytes can stim- (10, 15). However, as reported herein, the reorganization of ulate Een217.5 cells equivalently to L89.2.1 at 1/10th the the CD2 molecule into the site ofconjugate formation, which concentration of peptide (ref. 9; unpublished results). This is a consequence ofa series of successive low-affinity binding likely reflects the contribution of other cell adhesion mole- steps, permits multimeric interaction at the cell-cell junction cules, including LFA-1/ICAM-1, and possibly the coordi- that then enhances the avidity of the T cell for its cognate nated movement of CD3, CD4, and CD2. partner. Whether this movement of CD2 into the site of The effectiveness of multiple adhesion mechanisms prob- conjugation is solely a result of a gradient formation of CD2 ably also accounts for the ability of the TCR to be triggered complexing with LFA-3 or results from redistribution by an by APC pulsed with nominal antigen at concentrations sev- active mechanism is yet to be determined. Nevertheless, eral orders ofmagnitude below the Kd ofthe nominal antigen- because CD2 reorganization occurs in the absence ofnominal MHC interaction (19, 20). CD2 is expressed on virtually all antigen, TCR triggering is not required to initiate this process. , including CD4+ CD8+ double-positive cortical In contrast, interaction of CD2 with LFA-3 is required, since thymocytes, which are subject to the thymic selection pro- even in the presence of TCR triggering, conjugation between cess. It is therefore likely that CD2-LFA-3 interaction will aT cell and LFA-3- APC does not initiate CD2 reorganization. modulate the process of selection by affecting the overall The present analysis has also uncovered certain features avidity ofthe for the cell or APC. about class II MHC function. First, it is evident that the TCR thymic epithelial can interact with class II MHC molecules in the absence of 1. Meuer, S. C., Acuto, O., Hercend, T., Schlossman, S. F. & Rein- antigen. This is the case for the TCR of Een217.5, since both herz, E. L. (1984) Annu. Rev. Immunol. 2, 23-50. LFA-3 and the specific DR4 restriction element must be 2. Marrack, P. & Kappler, J. (1986) Adv. Immunol. 38, 1-30. expressed on the APC for it to bind stably to the T-cell clone. 3. Davis, M. M. & Bjorkman, P. J. (1988) Nature (London) 334, Een217.5 does not bind, therefore, to the DR71 LFA-3+ cell 395-401. 4. Parnes, J. R. (1989) Adv. Immunol. 44, 265-311. line L12.2.16. Given the sizable dimensions of MHC mole- 5. Shaw, S., Luce, G. E. G., Quinones, R., Gress, R. E., Springer, cules relative to the nominal peptide antigens which they T. A. & Sanders, M. E. (1986) Nature (London) 323, 262-264. bind, one might anticipate that the side chains ofpolymorphic 6. Spits, H., van Schooten, W., Keizer, H., van Seventer, G., van de amino acids contribute to TCR interaction during the T-cell Rijn, M., Terhorst, C. & de Vries, J. E. (1986) Science 232, recognition process (3). Second, the results imply that CD4- 403-405. monomorphic MHC class II interaction is not, in itself, 7. Bierer, B. E., Peterson, A., Gorga, J. C., Herrmann, S. H. & Burakoff, S. J. (1988) J. Exp. Med. 168, 1145-1156. sufficient to mediate adhesion in this system, and they are 8. Moingeon, P., Chang, H.-C., Wallner, B. P., Stebbins, C., Frey, consistent with the general perception that CD4-Ia interac- A. Z. & Reinherz, E. L. (1989) Nature (London) 339, 312-314. tions are ofvery low affinity (11). Third, our findings strongly 9. Siliciano, R. F., Lawton, T., Knall, C., Karr, R. W., Berman, P., suggest that the polymorphic Ia determinant that interacts Gregory, T. & Reinherz, E. L. (1988) Cell 54, 561-575. with the TCR is physically separable from CD4-binding Ia 10. Sayre, P. H., Hussey, R. E., Chang, H.-C., Ciardelli, T. L. & Reinherz, E. L. (1989) J. Exp. Med. 169, 995-1009. residues because the anti-human class II mAb 9-49, which 11. Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, N. R., reacts with a monomorphic epitope on DR molecules, did not Chang, H.-C., Siliciano, R. F., Dorfman, T., Walker, B., Sodroski, inhibit antigen-MHC- and TCR-dependent conjugate forma- J. & Reinherz, E. L. (1988) Nature (London) 331, 78-81. tion. In contrast, previous studies have shown that such 12. Klohe, E. P., Watts, R., Bahl, M., Alber, C., Yu, W.-Y., Anderson, mAbs inhibit antigen- and TCR-independent conjugate for- R., Silver, J., Gregersen, P. K. & Karr, R. W. (1988) J. Immunol. mation resulting from CD4-monomeric class II MHC inter- 141, 2158-2164. 13. Wallner, B. P., Frey, A. Z., Tizard, R., Mattaliano, R. J., Hession, actions (14, 16). Given that these Ia-specific mAbs inhibit C., Sanders, M. E., Dustin, M. L. & Springer, T. A. (1987)J. Exp. T-cell function, the monomorphic determinants which they Med. 166, 923-932. recognize on class II must be important for triggering events, 14. Clayton, L. K., Sieh, M., Pious, D. A. & Reinherz, E. L. (1989) including those resulting from CD4-Ia interaction. Interest- Nature (London) 339, 548-551. ingly, anti-CD4 mAb inhibits both conjugate formation and 15. Richardson, N. E., Chang, H.-C., Brown, N. R., Hussey, R. E., T-cell triggering, indicating that the blocking effects of anti- Sayre, P. H. & Reinherz, E. L. (1988) Proc. Nati. Acad. Sci. USA 85, 5176-5180. CD4 and anti-class II mAbs are not equivalent. Whether 16. Doyle, C. & Strominger, J. L. (1987) Nature (London) 330, 256-259. anti-CD4 efficiently disrupts class II MHC-CD4 interaction, 17. Brown, M. H., Cantrell, D. A., Brattsand, G., Crumpton, M. J. & sterically blocks TCR class II-MHC interaction, or initiates Gullberg, M. (1989) Nature (London) 339, 551-553. negative signaling events remains to be determined. 18. Kupfer, A., Singer, S. J., Janeway, C. A., Jr., & Swain, S. L. Though CD2 molecules were reorganized into the site of (1987) Proc. Natl. Acad. Sci. USA 84, 5888-5892. 19. Buus, S., Sette, A., Colon, S. M., Jenis, D. M. & Grey, H. M. conjugate formation, the TCR of Een217.5 remained evenly (1986) Cell 47, 1071-1077. distributed on its cell surface as judged by anti-CD3 mAb 20. Allen, P. M., Matsueda, G. R., Evans, R. J., Dunbar, J. B., Jr., staining (Fig. 5). Our results are clearly inconsistent with a Marshall, G. R. & Unanue, E. R. (1987) Nature (London) 327, report suggesting that CD2 and CD3 molecules are physically 713-715. Downloaded by guest on October 2, 2021