Anti-CD4 Monoclonal Antibody Reduces the Dose of Cyclophosphamide Required to Induce Tolerance to H-2 Haplotype Identical Skin Allografts in Mice

Abstract

Cyclophosphamide (CP)-induced tolerance which consists of a single i.p. injection of 200 mg/kg CP 2 days after priming with 1 × 10 8 donor spleen cells (SC), leads to long-lasting donor-specific skin allograft tolerance in H-2 compatible, multiminor antigen incompatible, murine strain combinations. In this system, the optimal dose of CP has been suggested to be 200 mg/kg, however, such a dose of CP causes strong myelosuppression. In the present study, we therefore attempted to reduce the dose of CP by administering anti-CD4 monoclonal antibody (mAb) before donor cell priming in this toleranceinducing system. When C3H/He (C3H; H-2 k, Mls-1 b) mice were injected i.p. with 200 ltg anti-CD4 mAb on day -3, 1 × 10 8 AKR/J (AKR; H-2 k, Mls-1 a) SC plus 3 × 10 7 bone marrow cells (BMC) i.v. on day -2 and then 100 mg/kg CP i.p. on day 0, a long-lasting donor-specific skin allograft tolerance was established; furthermore, the decreases in the number of leukocytes and the concentration of hemoglobin (Hb) in the peripheral blood were all less in the C3H mice treated with this new combined protocol than in the C3H mice injected with 200 mg/kg CP following the previous protocol. In the periphery of these tolerant mice, the number of donor-reactive Vβ6 +CD4 + T cells decreased and mixed chimerism was observed on both days 14 and 80. On the other hand, in the mice injected with AKR SC, BMC plus 100 mg/kg CP without anti-CD4 mAb, the number of Vβ6 +CD4 + T cells decreased on day 14, and then recovered by day 80 when the mixed chimerism disappeared. These results therefore suggest that the combined use of anti-CD4 . mAb with CP can reduce the dose of CP without affecting the efficiency of inducing donor-specific tolerance, probably due to the enhancement of the destruction effect of donor-reactive T cells by CP.