SummaryIn vitro studies have revealed that help for cytotoxic T lymphocyte (CTL) induction can be mediated through several pathways, including direct recognition of allogeneic class II antigens by CD4 § cells, direct recognition of altogeneic class I antigens by "CD4-independent" CD8 + cells, and "indirect" recognition of peptides of alloantigens presented in association with self class II molecules. Whereas good evidence for the two direct pathways is available in vivo, there is relatively little evidence to show that indirect recognition can initiate graft rejection. This study examined the role of indirect allorecognition during the generation of CTLs in mice as they rejected major histocompatibility complex (MHC) class II-deficient skin after depletion of CD8 + T cells in vivo. Recipients were depleted of CD8 + T cells by in vivo treatment with anti-CD8 monoclonal antibody and then grafted with allogeneic skin lacking MHC class II antigens. The mice rejected the skin grafts rapidly. Although flow cytometry showed marked depletion of CD8 + T cells in these mice, we found that (a) CD8 + CTLs were generated and sensitized to MHC class I antigens of the donor; (b) the generation of the CD8 + CTLs required the help in vivo of CD4 + cells, as well as priming with the allogeneic skin graft; and (c) the CD4 + T helper cells were sensitized indirectly to donor peptides presented in association with class II antigens on recipient antigen-presenting cells. These results provide evidence that indirect recognition can provide effective help for CTL induction during graft rejection, even when the cytotoxic T cells are sensitized by determinants expressed only on the donor graft. I~irect recognition describes the stimulation of recipient cells by allogeneic donor antigens presented in association with self MHC antigens on recipient APCs. Although indirect recognition represents the ordinary process by which T cells are sensitized during normal immune responses, its contribution to graft rejection is obscured by the powerful direct stimulation of T cells by donor APCs in allogeneic responses. Although it has been known for a long time that indirect recognition can occur during rejection of foreign tissue, and that it can play an effective role in allogeneic responses in vitro (1-3), it has not been clear whether this pathway alone can actually initiate graft rejection in vivo. This uncertainty has been especially important when the donor and recipient have been MHC mismatched, such that the determinants formed on recipient APCs would not be expressed on the cells of the donor graft.The recent availability of mice lacking MHC class II antigens has provided a new opportunity to examine the role of indirect recognition. Lacking class II antigens on their own APCs, grafts from these animals to normal recipients would be expected to stimulate CD4 + T cells only by the presentation of donor peptides in association with recipient class II molecules. However, because these grafts still express class I antigens, they can sensitize a...

To investigate the potential role for gene therapy in induction of tolerance to solid organ grafts, we used a murine model based on the introduction of an allogeneic MHC class I gene (H-2Kb) into hematopoietic cells of congenic animals differing only at the class I locus. The H-2Kb gene was placed into a retroviral vector under the control of regulatory sequences of the myeloproliferative sarcoma virus long terminal repeat. Transplantation of H-2Kb retrovirus-transduced autologous bone marrow into B10.AKM (Kk Ik Dq) recipients resulted in detectable levels of H-2Kb RNA and cell surface protein in lymphoid and myeloid lineages. H-2Kb expression was significant, although variable, in bone marrow Mac1+ cells, in splenic B cells, and in CD4+/CD8+ thymocytes 8 wk post-bone marrow transplantation. The recipients of the H-2Kb-transduced bone marrow showed specifically delayed rejection of B10.MBR (Kb Ik Dq) skin grafts disparate only for Kb. However, B10.MBR skin grafts were rapidly rejected when grafted simultaneously with skin grafts from B10 (Kb Ib Db) mice, a strain bearing additional third party alloantigens in association with Kb. Our experiments suggest that the hyporesponsive state induced by using the retrovirally mediated gene transfer model is characterized by the persistence of H-2Kb-specific cytolytic T cell precursors, which may be inactive because of deficient T cell help. Tolerance to Kb induced by this approach may therefore be restricted to T helper cell lineages.