Eur. J. Immunol. zyxwvutsrq 1987.17: 1159-1165 Immunosuppression with CD4 monoclonal antibody pairs 1159 zy Shixin Qin’, Steve Cobbold, Helen Tighe, Richard Benjamin and Herman Waldmann Department of Pathology, University of Cambridge, Cambridge CD4 monoclonal antibody pairs for immunosuppression and tolerance induction* A pair of rat anti-mouse CD4 monoclonal antibodies (mAb) have been selected which bind to different epitopes of the molecule. Both the mAb are rat IgGZb and show clear synergistic activity in complement lysis zyxwvu in vitro. When injected together in vivo, they exhibit an improved immunosuppressive effect, compared to each antibody alone, on allogeneic graft rejection, humoral responses and on tolerance induction. Limiting dilution analysis indicates that the in vivo depletion of interleukin 2-producing cells is improved using both mAb by 2-%fold over that obtained with the individual anti- bodies. As little as 60 ng per mouse of the CD4 antibody pair was sufficient to allow the induction of tolerance to human y-globulin, even without elimination of the CD4’ cells. The results suggest that appropriate antibody pairs may be good candidates for effective immunosuppressive serotherapy in man. 1 Introduction The murine CD4 (L3T4, [l]), the homologue of human T4 and rat W3/25 [2-41, is a nonpolymorphic cell surface glycoprotein expressed on thymocytes and a major subpopulation of mature T cells which react with antigen presented on cells bearing major histocompatibility complex (MHC) class I1 gene prod- ucts [5]. The interaction has classically been studied in terms of “helper” function. As a result this population has come to.be called “helperhnducer” cells although they clearly exhibit other effector functions such as delayed-type hypersensitivity and cytotoxic activity 16-91. CD4 monoclonal antibodies (mAb) can inhibit in vitro T cell activation and proliferation to both antigens and mitogens [lo, 111. When administered in vivo the mAb are highly immunosuppressive [12-141. For these reasons CD4 mAb have been used in animal models for therapy of autoimmune disease [15, 161, prevention of graft rejection [12, 171 and tolerance induction [18-211. Reports thus far have largely associated immunosuppression with in vivo depletion of CD4 cells [12, 201. Cell surface glycoproteins of the size of CD4 (55 kDa) are likely to have multiple nonoverlapping epitopes. It is therefore possible to extend previous studies of CD4 mAb immunosup- pression by determining whether CD4 antibodies binding to different epitopes would be more potent than either mAb alone. There are three reasons for thinking that potency may be enhanced. First, at saturation each cell should bind double the number of CD4 antibodies. This might facilitate clearance of cells carrying less CD4 antigen. Second, there have been a number of reports of synergy for in vitro complement lysis by antisera [22] as well as mAb [23, 241 to distinct epitopes on a variety of cell surface molecules. Third, if CD4 molecules have an “adhesive” or “signalling” role then their blockade or redis- tribution may be mediated more efficiently by antibody pairs than by single mAb. [I 61851 zyxwvu * This work was supported by the Medical Research Council. Sponsored by the British Council. Correspondence: Shixin Qin, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 lQP, GB Abbreviations: Con A: Concanavalin A FITC: Fluorescein iso- thiocyanate HGG: Human y-globulin mAb Monoclonal antibody (ies) SRBC: Sheep red blood cells In the present study, a pair of CD4 mAb has been used for immunosuppression of mice. Both mAb were rat IgGZb, selected for the desirable potency in activating effector mechanisms of this isotype [12, 25, 261. The antibody pair is shown to exhibit clear synergistic lysis in vitro and demon- strates improved immunosuppressive and lytic potency in vivo. This study suggests that there may be benefits in selection of suitable CD4 antibody pairs as immunosuppressive agents. 2 Materials and methods 2.1 Animals CBA/Ca and (CBA X C57BL/10)F1mice were bred and kept in the conventional animal facility at the Department of Pathol- ogy, University of Cambridge, and were used at 6-8 weeks of age, 2.2 mAb and their biotinylation YTS 191.1 is a rat IgGZb CD4 mAb [12]. YTA3.1 was selected from a fusion of myeloma line Y31Ag 1.2.3 with rat spleen cells immunized against concanavalin A (Con A)-activated mouse splenocytes. The mAb used in the experiments were either spent culture media or partially purified by precipitating rat ascites fluid with 50% saturated ammonium sulfate. For biotinylation mAb were dialyzed against 0.1 M sodium hydrogen carbonate buffer (pH 9.3) and were then mixed in glass containers with NHS-LC-biotin (Pierce, Rockford, MD) which had been dissolved in dimethylformamide. The approxi- mate molar ratio of protein : biotin was 1 : 10 (120 pg NHS- LC-biotidl mg protein). After 3 h of incubation, antibodies were dialyzed against phosphate-buffered saline (PBS) and stored at 4°C with 0.05% azide. 2.3 Complement-mediated lysis Murine thymocytes, mesenteric lymph node cells and Con A- stimulated spleen cells (Con A blasts) were labeled with 3.3 yCi1ml ’lCr (Amersham Int., Bucks, GB) at 37°C for 1 h. Aliquots of 1 X 10’ cells were incubated with 50 pl mAb in U- bottom microtiter plates for 10 min at room temperature. Guinea pig serum was added as the complement source in a volume of 50 yl at a final concentration of 2%. The plates zy 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0014-2980/87/0808-1159$02.50/0