Combination Anti-CD2 and Anti-CD3 Monoclonal Antibodies Induce Tolerance While Altering lnterleukin-2, lnterleukin-4, Tumor Necrosis Factor, and Transforming Growth Factor-β Production

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Abstract
Objective These studies were designed to elucidate the mechanism by which signals delivered by anti-CD2 monoclonal antibody (MoAb) interfere with activational signals delivered by anti-CD3 MoAb and induce long-term graft survival and tolerance. Summary Background Data Anti-CD2 or anti-CD3 MoAb can prolong allograft survival when administered alone. In combination, they synergistically prolong survival while reducing anti-CD3-associated cytokine toxicity. It was postulated that the mechanism of synergism and reduced cytokine toxicity was related to anti-CD2-induced alterations in anti-CD3-induced T-cell activation. Methods C57BL/6 (H-2b) mouse hearts were transplanted to CBA (H-2k) mice. The recipients received anti-CD2 and/or anti-CD3 MoAb intravenously only at the time of initial allografting. Serum from treated animals and culture supernatants from lymphocytes stimulated In vitro with anti-CD3 were examined for interleukin (IL)-2, −4, −6, and −10, tumor necrosis factor (TNF), and transforming growth factor-α (TGFα). RNA was isolated from lymphocytes from treated animals and examined for receptor and cytokine gene expression by northern hybridization or reverse transcribed and amplified by the polymerase chain reaction (PCR). Results Anti-CD2 and anti-CD3 MoAbs alone prolonged graft survival (22.0 ± 0.5 days and 28.0 ± 0.5 days, respectively; p < 0.02 and p < 0.01 vs. control, by Wilcoxon signed-rank test). Combined anti-CD2/anti-CD3 MoAbs synergistically prolonged survival indefinitely (> 150 days, p < 0.01) while decreasing cytokine toxicity. Second donor-specific allografts also showed long-term survival. The peak serum TNF concentration (2100 units/mL) was reduced 78% by anti-CD2 treatment (455 units/mL). Anti-CD2 inhibited anti-CD3-stimulated proliferation and in vitro production of IL-2 and IL-4, with no alteration of IL-6, IL-10, or TNF. Conversely, there was an increase in the immunosuppressive cytokine TGFα. PCR analysis showed that anti-CD2 reduced anti-CD3-stimulated IL-2 messenger RNA expression, and by northern analysis, anti-CD2 inhibited anti-CD3-stimulated increases in messenger RNA for the CD2 and CD3 receptors themselves. Conclusions The combination of anti-CD2 and anti-CD3 MoAbs induced a state of tolerance while decreasing anti-CD3-associated cytokine toxicity. The mechanism was related to anti-CD2-generated alterations in T-cell activation and gene expression.