The frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting renal allografts

Abstract

Abstract. We have estimated the frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting histoincompatible renal allografts. In a major plus minor antigen-incompatible DA-to-WF combination on day 4 post-transplantation, reverse protein A plaque assay demonstrated that in the graft the frequency of lymphoid cells secreting Ig was 1:850. A major locus-incompatible and minor locus-compatible, congeneic LBN-to-Lewis strain combination was then applied to estimate the specificity of the secreted antibody. The lymphoid inflammatory cells were fused with mouse myeloma cells, cultured under limiting dilution conditions, and assayed by ELISA to donor and irrelevant strain spleen cells. Among cells infiltrating the graft, the fusion frequency was 1:172 103 the frequency of Ig-producing hybrids 1:400 103 (i. e., this assay was approximately three log orders less sensitive than the reverse pA assay). The frequency of hybridomas secreting specific antibodies against donor MHC antigens was 1:720 103 (i. e., every second hybridoma deriving from inflammatory population produced specific Ig). In addition, there was at least one obviously polyspecific population of hybridomas, detectable only in the spleen and reactive with all rat strains tested with a frequency of 1:700 103. The inflammatory cells were also cultured directly under limiting dilution conditions, and the frequency of Ig-secreting cells was determined by ELISA. The frequency of inflammatory lymphocytes secreting detectable amounts of immunoglobulin in the supernatant was 1:14 103 in the graft (i. e., this assay was approximately one log order less sensitive than the reverse protein A plaque assay). Donor-strain reactive cells were not detected in the spleen or blood of nontransplanted Lewis (recipient) rats, nor were they detected in the blood of the transplant recipient. Our results show that B cells secreting antibodies against graft donor MHC antigens are present in the graft-infiltrating cells' inflammatory population. In the inflammatory population only approximately 1:850 of the inflammatory cells are engaged in Ig secretion, and at most 1:2 of these produce specific antibody to graft donor MHC antigens. This finding suggests that a nonspecific (polyclonal) Ig-producing population is also present in situ. These results are also concordant with the reported low frequency of specific T cells of both cytotoxic and helper type at the site of inflammation and emphasize the importance of nonspecific inflammatory (“delayed hypersensitivity”) mechanisms in acute allograft rejection.