Generation of adaptive regulatory T cells by alloantigen is required for some but not all transplant tolerance protocols

Because CD4CD25Foxp3 regulatory T cells (Tregs) are essential for the maintenance of self-tolerance, significant interest surrounds the developmental cues for thymic-derived natural Tregs (nTregs) and periphery-generated adaptive Tregs (aTregs). In the transplant setting, the allograft may play a role in the generation of alloantigen-specific Tregs, but this role remains undefined. We examined whether the immune response to a transplant allograft results in the peripheral generation of aTregs.

To identify generation of aTregs, purified graft-reactive CD4CD25 T cells were adoptively transferred to mice-bearing skin allograft. To demonstrate that aTregs are necessary for tolerance, DBA/2 skin was transplanted onto C57BL/6-RAG-1-deficient recipients adoptively transferred with purified sorted CD4CD25 T cells; half of the recipients undergo tolerance induction treatment.

By tracking adoptively transferred cells, we show that purified graft-reactive CD4CD25 T lymphocytes up-regulate Foxp3 in mice receiving skin allografts in the absence of any treatment. Interestingly, cotransfer of antigen-specific nTregs suppresses the up-regulation of Foxp3 by inhibiting the proliferation of allograft-responsive T cells. In vitro data are consistent with our in vivo data-Foxp3 cells are generated on antigen activation, and this generation is suppressed on coculture with antigen-specific nTregs. Finally, blocking aTreg generation in grafted, rapamycin-treated mice disrupts alloantigen-specific tolerance induction. In contrast, blocking aTreg generation in grafted mice treated with nondepleting anti-CD4 plus anti-CD40L antibodies does not disrupt graft tolerance.

We conclude that graft alloantigen stimulates the de novo generation of aTregs, and this generation may represent a necessary step in some but not all protocols of tolerance induction.