*Purpose: Donor-specific tolerance offers many advantages over current lifelong non-specific immunosuppression strategies, but the mechanisms by which it may be durably maintained remain to be determined. Robust long-term graft-specific tolerance has been achieved in mouse models of heterotopic major mismatched heart transplantation through treatment with anti-CD154 combined with donor splenocyte transfusion (DST) at the time of transplantation. Using this model, we determined whether cell-intrinsic dysfunction in allospecific T cells persisting in tolerant heart recipients may contribute to robust transplant tolerance.

*Methods: C57BL/6 mice were given heterotopic Balb/c heart transplants. To establish tolerance, some recipients were treated with intravenous anti-CD154 and Balb/c splenocytes on the day of transplantation and intraperitoneal injections of anti-CD154 on days 7 and 14 relative to transplantation. We adoptively transferred 100,000 congenically-labeled allospecific CD4⁺ T cells of the TCR75 clone into heart recipients one day prior to transplantation to assess their function after rejection or establishment of tolerance. Greater than 35 days after transplantation, we analyzed the phenotype of these cells by flow cytometry or re-isolated them by fluorescence activated cell sorting to assess their function outside of the tolerant environment.

*Results: We show that alloreactive T cells are not completely deleted from tolerant heart recipients. Instead, they are maintained as a small population of antigen-experienced CD127^lo PD-1^hi cells, a phenotype which has been associated with exhausted T cells during chronic infection. Tolerant allospecific T cells expanded less upon re-challenge with donor antigen in secondary hosts, lacked the ability to produce IFN-gamma and TNF-alpha upon re-stimulation in vitro, were less potent providers of help to B cells when compared with T cells isolated from rejecting mice, and were less potent in rejecting skin allografts. Programming dysfunction required persistent stimulation of allospecific T cells, with the donor heart providing a reservoir of alloantigen. Indeed, removal of the allograft 1 week, but not 3 weeks, after transplantation prevented the establishment of T cell dysfunction.

*Conclusions: We have found that allospecific CD4⁺ T cells persisting in mice made tolerant to heart allografts using anti-CD154 and DST become intrinsically dysfunctional due to chronic stimulation with persisting alloantigen. Thus, achieving cell-intrinsic dysfunction in allospecific T cells may be desirable to maintain long-term transplantation tolerance.

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