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Long-term cardiac xenograft (pig to baboon) survival is correlated with presence of increased percentage of CD4+CD25HiFoxP3+ T regulatory cells

Avneesh Singh¹, Philip Corcoran¹, Marvin Thomas⁴, Robert Hoyt³, David Ayares², Keith Horvath¹, Muhammad Mohiuddin¹

¹Cardiothoracic Surgery Research Program, NHLBI / NIH, Bethesda, MD; ²Revivicor Inc, Blacksburg, VA; ³LAMS, NHLBI / NIH, Bethesda, MD, United States; ⁴DVR, ORS / NIH, Bethesda, MD; United States

The CD4+CD25HiFoxP3+ regulatory T (Treg) cells are suppressors of antigen activated immune reactivity. Due to their suppressive ability, these cells are considered a potential target in the development of transplant tolerance in vivo. In transplantation, several studies have compared the frequency of Treg cells (using different surface marker CD127 and Foxp3) and demonstrated that stable immunoppressed heart transplant patients have more Treg cells. In this study, we have analyzed the FoxP3-expressing CD4+CD25Hi Treg cells in the peripheral blood lymphocytes (PBL) of long term survived baboon recipients who have received a-GTKO.hCD46Tg pig cardiac xenografts.

Methods: Heterotopic cardiac xenotransplantation was performed from a-GTKO. hCD46Tg pig into baboons using ATG, anti CD20, Mycophenolate Mofitel (MMF), Cobra Venom Factor (CVF), and costimulation blockade (anti-CD154) immunosupression regimen. FACS analysis was done on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies to analyze the percentage of Treg cells in six baboons (n=6) that survived more than 2 months (ranging from 42 days to >180 days) after receiving cardiac xenografts.

Results: It was observed that the percentage of CD4+CD25Hi Treg cells (ranging from more than 40-70%) among CD4+ cells were increased in the PBL of recipients with long term functioning hearts as compared to naïve baboon (8-10%). To further confirm the phenotype of Treg cells, additional flow cytometry analysis were performed for FoxP3. Total percentage of CD4+CD25Hi FoxP3+ Treg cells within the CD4+CD25Hi cells was significantly increased (>80%) in long-term survivor as compared to naïve baboon. One of the baboons that underwent rejection and died after 75 days had lower percentage of CD4+CD25Hi Treg cells (< 35%).

Conclusion: Our results indicate that CD4+CD25Hi FoxP3 Treg cells may be involved in preventing or delaying the rejection process by controlling the activation/expansion of donor-reactive T cells thereby masking the anti-donor response leading to long term survival of cardiac xenograft.