BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.