Immunol. Cell Biol (1990)68, 199-205

Mapping the dextran sulfate binding site on CD2 Hilary S. Warren* and Christopher R. Parish^ *Cancer Research Unit. Woden Valley Hospital Australian Capital Territory and ^Division of Cell Biology, John Curtin School of Medical Research. Australian National University. Canberra. Australia (Submitted 2 March 1990. Accepted for publication 16 May 1990.) Summary This study has analysed the binding of a series of anti-CD2 monoclonal anlibodies (MoAbs) to T cells in the presence of the suUated polysaccharide dextran sulfatc (2 3 sulfates/ monosaccharide, 500 kDa) (DXS) to define Ihe DXS binding site on CD2. The results show that DXS interacts primarily at theTl 11 epitope. Thus fiveanti-CD2 MoAbs which bound tothcTI 12 epitope were inhibited in their binding by DXS. In contrast, seven anti-CD2 MoAbs that totally inhibited sheep red blood cells (SRBC) rosetting (identifying the Til I epitope) were unaReeted in their binding to T cells in the presenee oI'DXS. Three MoAbs which partially inhibited SRBC rosetting and thereby definingonly part of theTl 1 [ epitope. were also inhibited in their binding by DXS. Consistent with the conelusion that (he DXS binding site on CD2 is associated with the Tl 12 epitope was the observation that interaetion of DXS with CD2 resulted in augmented binding of the four MoAbs defining theTl 13 epitope, possibly reflecting an increased expression of the TI li (activation. CD2R) epitope of CD2. Collectively, the data presented support the notion that a natural ligand for theTl 12 epitope of CD2 will be identified as a sulphated carbohydrate structure.

INTRODUCTION The structure of the CD2 (50 kDa, et7throcyte receptor) molecule and the gene eneoding CD2 are now well defined (I -4). CD2 is a transtnetnbrane glycoprotein (1.3) whose external domain consists of three functional epitopes identified by monoclonal antibodies (MoAbs). The Tl 11 epitope is involved in cell adhesion as exemplified by sheep erythrocyte (SRBC) binding. The Tl I2 and Tl I3 epitopes are involved in T cell activation with TI Ii epitope expression being augmented upon T cell activation and after treatment of resting T cells with anti-Tlh MoAb (5). It is assumed that MoAb interaction at the T111. T112 and T113 epitopes of CD2 and the consequent f cell activation process reflects an interaction of naturally occurring ligands involved in physiological T cell activation. Indeed there is compelling evidence that the LFA-3 molecule is a tigand fortheTIli epitope Address for correspondence: Dr H, S. Warren. Canecr Research Unit, Woden Valley Hospital. P.O. Box 11. Woden. ACT 2606. Australia. Ahhreviations used in ihis paper: BSA. bovine serum albumin; PBS. phosphate buffered saline; DXS. dextran sulfate; FMF. flow microfluorimetr>'; IL-2, interleukin-2; MoAb, monoclonal antibody; SRBC, sheep red blood cells; AET, 2-aminoethylisothiouronium bromide hydrobromide; Con-A. eoncanavalin-A.

of CD2 both for T cell adhesion (6-8) and T cell activation (8-10). The identity of the ligands for the T112 and T113 epitopes remains to be established. In previous studies (11) it was shown that CD2 interacts specifically with the sulfated polysaccharide dextran sulfate (2.3 sulfates/monosaccharide. 500 kDa) (DXS). providing evidence that a ligand for CD2 is a sulfated carbohydrate structure. In this study we have identified the DXS binding site on CD2 using a panel of 18 anti-CD2 MoAbs reacting with the different epitopes of CD2. The results show that DXS binds primarily to the Tl h epitope indicating that the natural ligand for the TII2 epitope will be identified as a sulphated carbohydrate structure. MATERIALS AND METHODS Cells Mononuclear cells (PBMC) were isolated from human peripheral blood by sedimentation over FieollPaque(Pharmaeia. Uppsala. Sweden). Selected resting Teells, defined as interleukin-2 (lL-2) non-responsive, were isolated from PBMC by proeedures previously described (12). In brief. PBMC were cultured at lO^/mL for 3 days with a lymphocyte conditioned medium (13) containing 5 unils/mL IL-2. Thereafter therestingeells were isolated by sedimentation at high Percoll (Pharmacia. Uppsala, Sweden) densities

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{P> I 0635 g/mL; osmolarily 304 mmol). Activated T cells were prepared by culliiring PBMC with ID |jg/mL concanavaiin-A (Con-A) (Sigma Chemical Co.. St Louis, MO, USA) for 4 days followed by growth in IL-2 for a further 2 days (13). Monoclonal antibodies MoAbs were provided through our participation in the 4th International Workshop on Human Leucocyte Differentiation Antigens. The MoAbs, their sources. and references where previously described, are listed in Table I. The MoAbs were diaiysed against PBS. sterile filtered, aliquoted and stored at —20°C. Purified anti-Tl I2 (lold24Cl) and anti-T! 13 (I mono2A6). used as standard reference co-stimulatory MoAb in the proliferation assays, were a generous gift from Dr E. Reinherz (Boston). immunofluorescence and flow cylometrv Procedures used were those described previously (II). In brief, T cells were prepared in PBS/OI%BSA for treatment with dextran suifate (2-3 sulfates/monosaccharidc 500 kDa (Pharmacia, Uppsala, Sweden) (DXS). DXS treatment was I h on ice. followed by incubation with predetermined concentrations of MoAb (30 min on ice) in the presence of DXS. Cells were washed and incubated (30 min on ice) with 1/10 dilution of lluoresceinated sbeep F(ab')2 anti-mouse IgG (Silenus-AMD, Dandcnong. Victoria. Australia). After washing, cells were fixed in 0 1 % paraformaldehyde in PBS and analysed usinga FACScan (BectonDickinson. Mountain View. CA, USA) flow cytometer. Results are expressed as per cent control McAb binding _ Peak FlUofMoAb binding with DXS-BackerQundFIU Peak FIU of MoAb binding wilhoul DXS-Background FIU where FIU designates fluorescence intensity units.

Rosette assays The T cell rosette assay used aminoethylisothiouronium bromide hydrobromide (AET)-treated SRBC (23) and rosettes were enumerated al 20 h. MoAb inhibition of T cell rosette formation was tested following incubation of T cells with optimum MoAb concentrations for 30 min on ice. Proliferation assays Culture media and conditions used were Ihose previously described (12), In brief. 5 X 10'' resting T cells were cultured in 0^2 mL volumes in round bottomed wells (Linbro Trays 76-013-05. Flow Laboratories, Sydney, Australia) with optimum concentrations of an IL-2 containing lymphocyte conditioned medium (I 3) and different dilutions of MoAb in the presence of" 0'05% SRBC, alone and with a suboptimum concentration of anti-Tl 1.1 (105 ng/mL purified MoAb lmono2A6) or a suboplimum concentration of antiT i l 2 (26 ng/mL purified MoAb lold24Cl). Proliferation was assessed by 5 h -'H-thymidinc incorporation on dav 3.

RESULTS Classification ofanti-CD2 MoAbs The results summarized in Table 2 show data on the ability of the anti-CD2 MoAbs to inhibit the binding of AET-treated SRBC to T cells. MoAbs which inhibit SRBC binding identify the Tl 11 epitope of CD2 (5). The results obtained are in agreement with those previously reported (Table 1). M-T20I was the only MoAb which differed in its ability to inhibit SRBC binding to resting and activated T cells. M-T20I only partially inhibited SRBC rosetting on activated T cellsbut totally inhibited rosetting with restingT

Table 1. Anti-CD2 MoAbs used in this studv. MoAb CLB-TlM/l RPA-2I0* 0-275* 39CI 5(CD2 9) T1I/3PT2H9 T11/3T48B5 M-T201* FllO-08* AICD2-1* CLB-TIl 2/1 6F1O3(CD2I)

X/3* anti-Til 2 GT-2 D-66 clone 1 VIT13 91

anti-Tl h

Source van Lier. Amsterdam Aversa, Stanford Bernard, Paris Mawas, Marseille Reinherz. Boston Reinherz. Boston Reiber. Munich Ptesner, Copenhagen Meuer, Heidelberg van Lier, Amsterdam Mawas, Marseille Pulford. Oxford Reinherz, Boston Bernard. Paris Bernard. Paris Knapp, Vienna Hansen, Seattle Reinherz, Boston

•Previously unclassified MoAbs.

Reference

Epitope type

14, 15, 16

Til]

17

Till T• i* l"l1

14, 18 14, 18

15, 16

17

5, 14 19,20 17, 19,21 22

14. 18 5. 14

Till

TII2 Tll2 TII2 TllVTlii TII3 TII3 Tlh Til,

201

DEXTRAN SULFATE BINDING SITE ON CD2 Table 2. Inhibition of SRBC rosetting by anti-CD2 MoAhs. Non-inhibitory MoAbs

Inhibitory MoAbs MoAb CLB-Tli-I/I RPA-2I0 O-275 39C1 5 TI1/3PT2H9

SRBC binding* (% of control) 0 0 0 0 0 0 0*; 58t 55

MoAb CLB-Tl 1-2/1 6F10-3 X/3 anti-Til 2 GT-2 D-66 clone 1 VIT13

SRBC binding (% of control) >95 >95 >95 >95 >95 >95t >95* >95t >95t

M-T201 91 FI10 08 79 AICD2I anti-Til 1 •Data were obtained usmg AET-treated SRBC and, unless indicated, with both resting T cells and activated T cells. The data are the average for two different donors. *Data obtained with resting T cells. *Data obtained with activated T cells.

cells. Two anti-CD2 MoAbs. FI 10.08 and A1CD2.1, appeared to bind to regions peripheral to the SRBC binding site since they partially inhibited SRBC binding (Table 2) and the size of the rosettes was smaller (data not shown). MoAbs identifying the TII2 and TII3 epilopes of CD2 were those which co-stimulated with suboptimal concentrations of anti-Tlli and anti-TIl2 respectively. The culture conditions included SRBC so that only low doses of the purified anti-Tl I3 or anti-TI h MoAb were required (24,25). Furthermore, IL-2 was added in these cultures so that T cell activation induced by the MoAbs only required that 1L-2R expression was induced and did not demand that the T cell activation signal was sufficient for IL-2 production as well (12). The amount of purified anti-TI \y or anti-Til 2 MoAb used was sufficient to induce only a weak proliferative response in the SRBC + IL-2 supplemented cultures (Table 3). The resting T cells did not proliferate in the presence of SRBC + IL-2, All anti-

CD2 MoAb were titrated to determine the optimum concentrations for use by FMF analysis. Some anti-CD2 MoAbs were mitogenic with SRBC + IL-2 as co-stimulants, and the dose responsecurve usually followed that for binding. When either anti-TI I.1 or anti-TI 12 MoAb was used as additional co-stimulant, the test antiCD2 MoAb was mitogenic at concentrations usually below that detected by FMF analysis. Data showing the mitogenic activity of MoAbs which did not bind to the T111 epitope of CD2 are presented in Table 3. The results obtained are in agreement with the previously reported TI I2 or TI I3 epitope designation of the MoAbs (Table 1). Some results obtained with the mitogenic assay used required comment. Some anti-CD2 MoAbs were mitogenic with SRBC + IL-2 without the need for antiTI I3 or anti-TI I2. Diluting those MoAbs to 1/64 of saturating concentrations revealed their co-stimulatory activity with either anti-TI I3 or atiti-Tlh.

Table 3. Mitogenic activity of anti-CD2 MoAbs. MoAb

TI I2 epitope MoAbs Mitogenic activity* (%max) SRBC SRBC + IL2+ low -t-IL2t dose anti-TI I3

TI I3 epitope MoAbs MoAb

Mitogenic activity* (%max) SRBC SRBC-I-IL2-1- low + IL25 dose anti-TI h

29 50 D-66 clone 1 95 >95 >95 >95 >95, 77*

MoAb CLB-Tll 2/1 6F103 X/3 anti-Til 2 GT-2

Binding %control 49 40 36 40 70

TII3 epitope MoAbs Binding %cDntroI MoAb D-66 clone 1 VIT 13 91 anti-Tll,

500 1200 134 145

42 36

•Results with activated T cells. 50 M-g/mL, and. with the exception of M-T201, identical results were obtained with resting and activated T cells. With M-T20! (on activated T cells), and with Fl 10.08 and AICD2I. where partial inhibition ofSRBCbindingtoT cells was observed (Table 2). binding to T cells was inhibited in the presence of DXS. Thus these three MoAbs which bind only in part totheTl 11 epitope, also bind near the DXS binding site of CD2. The five anti-CD2 MoAbs binding to t h e T l h epitope of CD2 were all inhibited in their binding to T ceils by DXS (Table 4, Fig. IB). In all cases inhibition of binding was never complete and for four of the MoAbs was in the range 3649% of control. MoAb GT-2 which interacted with the Tl l i a n d T l 13 epitopes of CD2 showed a weaker inhibition of binding (70% of control). The four anti-CD2 MoAbs binding to the T i l l epitope of CD2 showed augmented binding in the presence of DXS (Table 4). ThcTI It region MoAbs could be divided into two groups. ThcMoAbsinthefirst group, 9 1 andanti-Tl I3, were moderately expressed on resting T cells, and MoAb binding was augmented 1 •3-1-5-fold by DXS at both 5 and 50 ng/mL DXS {Fig. IC). The MoAbs in the second group. D-66 clonel and V1T13. reacted very weakly with resting T cells, and MoAb binding was augmented 5-12fold by DXS, and this was maximum at 5 |ig/mL DXS (Fig. 2D). The result obtained with GT-2. where DXS weakly inhibited MoAb binding, could reflect its binding to an exposed TI 13 epitope partly masking an inhibition of binding to the TI 12 epitope.

DISCUSSION The CD2 molecule has three functional epitopes identified by MoAbs. which are involved in cell

adhesion and T cell activation. The T l l i epitope binds the ligand LFA-3 (6-8) and was originally identified by the ability of MoAbs to inhibit SRBC rosetting (5). MoAbs identifying theTI I2 and Tl 13 epitopes are mitogenic for T cells and can be identified by their comitogenic activity with anti-Ttli or anti-Tlh. respectively {5,16.21,24.25). The TII3 epitope is described as an activation epitope because il becomes exposed (5) and/or augmented in expression (11,12) upon T cell activation. The majority of anti-CD2 MoAbs used in this study recognized cither the Tl 11. TI I2 or TI It epitope. Four MoAbs did not fit clearly into these epitope categories. MoAbs Fl 10 08 and AICD2-1 bound at least in part to the TI 11 epitope. as did M-T201 when tested on activated T cells, since they only partially inhibited SRBC binding. MoAbM-T201 totally inhibited SRBC binding to resting T cells suggesting that its inability to totally inhibit SRBC binding to activated T cells was due to some activationinduced conformational change in CD2 at the T i l l epitope. The MoAb GT-2 (19.20) preferentially recognizes the TII2 epitope but also recognizes the TI I3 epitope of CD2. It was previously shown that DXS interacted witb CD2 in an epitope specific manner (11). The results of the present study extend this observation and show that DXS interacts primarily with thcTl 12 epitope ofCD2 since DXS inhibited the binding of MoAbs recognizing the TII2 epitope. Three MoAbs which interacted partly with theTI 11 epitope were also inhibited in their binding by DXS demonstrating that DXS binds to a site on CD2 recognized by these MoAbs. This site is probably adjacent to the T i l l epitope since DXS inhibits SRBC rosetting (II). Inhibition of MoAb binding by DXS never exceeded 60%. suggesting that the DXS binding site on CD2 is not precisely defined by any ofthe MoAb studied. DXS is a large branch chain

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Structure and may cause inhibition of MoAb binding by steric hindrance. The interaction between DXS and CD2 leads to the augmented binding of MoAbs interacting with the Tl I3 epitope. We interpret this observation to indicate that the DXS interaction, like interaction of anti-Tl I2 MoAb with CD2 (5), leads to a conformational change in the molecule and the enhanced expression of the Tl I3 epitope. The alternative explanation that MoAbs to the T113 epitope are of weak affinity and that DXS may allow bivalent binding of MoAb to adjacent CD2 molecules seems unlikely since binding of MoAb D-66 clonel and VIT13 (an IgM antibody) was barely detectable on resting T cells and following DXS treatment MoAb binding increased 5-12-fold. In conclusion, the present studies provide evidence that DXS binds at a site on CD2 primarily identified by MoAbs to the Tl I2 epitope. This

interaction induces the expression ofthe Tl I3 {activation. CD2R) epitope of CD2. The data obtained are consistent with the notion that the Tl I2 ligand for CD2 is a sulfated carbohydrate structure. Acknowledgements We wish lo thank ihe organizers and participants of the 4th International Leucocyte Diflcrenliation Antigen Workshop for providing the anti-CD2 MoAbs used in these studies. We thank the ACT Red Cross Blood Transfusion Service and Ms J. Lenferink for preparation of buffy coats and lymphocyte isolation. Mr P. Baron {Haematology Department. Woden Valley Hospital) for assistance in FACSean operation. Ms Kaylyn Jorgensen-Fry and Ms Karen Jakobsen tor technical assistance, Dr Felicity Lynch for helpful eriticisms of the manuscript, and Dr R. G. Pembrey for his continuedsupport.Thiswork was supported in part by grants to H. S. Warren from the National Health and Medical Research Council of Australia.

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Mapping the dextran sulfate binding site on CD2.

This study has analysed the binding of a series of anti-CD2 monoclonal antibodies (MoAbs) to T cells in the presence of the sulfated polysaccharide de...
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