Immunoregulatory role of CD8α in the veto effect: Transforming growth factor-b1 activation

Background. Allogeneic bone marrow cell (allo-BMC) infusion induces tolerance to incompatible renal allografts in rhesus macaques after depletion of peripheral T lymphocytes with cytolytic anti-T cell antibodies. The tolerogenic effect of allo-BMC, ascribed to a veto mechanism, associates with specific functional deletion of antidonor cytotoxic T lymphocyte precursor (CTLp), and is dependent on a CD8⁺ donor BMC subset. In previous studies, the CD8 molecule was implicated by loss of suppression after blocking interaction between CD8 on allo-BMC and major histocompatibility complex class Iα3 domain on CTLp. CD8 cross-linking on BMC induced secretion of active transforming growth factor-β1 (TGF-β1), suggesting a regulatory mechanism(s) operating via a CD8-mediated signaling pathway. Methods. CD8 on rhesus cells was cross-linked using IgG-conjugated beads, and TGF-β1 mRNA and protein were quantified. CD8⁺ cells were tested for veto activity by mixed lymphocyte reaction (MLR)-induced cell-mediated lymphocytotoxicity (CML) assay. Activated rhesus T cells exposed to TGF-β1 were examined for apoptosis by TdT-mediated end-labeling and annexin staining. Results. CD8 cross-linking induces accumulation of TGF-β1 mRNA and protein. Both CD3^- CD8⁺CD16⁺ and CD3⁺ CD8⁺CD16^- subsets of allo-BMC up-regulate TGF-β1 mRNA after CD8 cross-linking, and exhibit veto activity. The CD3^-CD8⁺CD16⁺ subset expresses more TGF-β1 mRNA and increased veto activity at low BMC/CTLp ratios. Exposure of activated T cells to TGF-β1 induces apoptosis. Conclusions. CD8⁺ allo-BMC are enriched for veto activity and activation via CD8 induces TGF-β1 mRNA and protein accumulation. These results agree with the hypothesis that paracrine TGF-β1 may be involved in peripheral deletion of alloreactive CTLp by CD8⁺ allo-BMC. We suggest that TGF-β1 overexpression by donor lymphohematopoietic cells may enhance tolerance induction.