Direct evidence for clonal destruction of allo-reactive T cells in the mice treated with cyclophosphamide after allo-priming

It has previously been reported that a single i.p. injection of 200 mg/kg cyclophosphamide (CP) 2 days after priming with 10⁸ donor spleen cells (SC) leads to donor-specific skin allograft tolerance in H-2 compatible, multiminor antigen incompatible murine strain combinations. It is speculated that the i.v. injection of donor cells may result in synchronized proliferation of donor-reactive host T cells and subsequently administered CP may specifically destroy these proliferating T cells in the periphery. Although this unique action of CP is considered to be a principal mechanism in this method, direct evidence has not yet been obtained. In the present article, this in vivo destructive effect of CP is clearly demonstrated by assessing detailed kinetics of host-derived blastoid T cells and donor (Mls-1^a)reactive Vβ6⁺ T cells in the model system of C3H mice rendered tolerant to AKR. Frequencies of the blastoid cells and Vβ6⁺ cells, which increased as a result of AKR priming, decreased rapidly with the administration of CP. C3H mice, which received AKR SC alone, also exhibited partial deletion of Vβ6⁺ T cells, but both tempo and magnitude of decrease in the frequency of Vβ6⁺ cells were quite different from those of the C3H mice given AKR SC and CP, which showed more rapid and profound elimination of Vβ6⁺ T cells. In accordance with these kinetic studies, in vitro proliferative response to Mls-1^a antigens was greatly impaired in mice treated with SC and CP, whereas a low but appreciable response was detected in mice given SC alone. Furthermore, skin graft tolerance was not obtained in mice treated with SC alone, rather such mice rejected donor skin graft in an accelerated fashion, in contrast to the induction of profound skin ft tolerance in CP-treated mice. Thus, CP-induced clonal destruction of antigen-stimulated-and-then-proliferating T cells actually works in our tolerance-inducing method and is obviously distinct from the peripheral clonal deletion seen after Mls priming alone.