CD8 EXPRESSION INCREASES SUPPRESSIVE ABILITY OF CD4+CD25+TREG AND IS A MARKER OF ANTIGEN-SPECIFICITY

Paul Wilcox ⁰; NirupamaD Verma ^0,0; CatherineM Robinson ^0,0; GiangT. Tran ^0,0; SuzanneJ. Hodgkinson ^0,0; BruceM. Hall ^0,0; Masaru Nomura ⁰; Nicole Carter ⁰; Rochelle Boyd ⁰

⁴Immune Tolerance Group, INGHAM Institute, Liverpool, Australia; ⁷Department of surgery , Keiwakai Ebetsu Hospital Nopporo Yoyogicho 81-7 , Ebetsu city Hokkaido 069-0817, Australia; ⁸Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia; ⁹Faculty of Veterinary Sciences, The University of Sydney, Sydney, Australia; ¹⁰South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales, Sydney, Australia

Aim: We aim to identify markers that can differentiate activated alloantigen-specific Treg from naïve CD4⁺CD8^-FOXP3⁺ tTreg.  We previously reported that tTreg cultured with alloantigen and IL-2 are induced to express IFN-gamma receptor (IFNGR) and IL-12 receptor β2 and we call these Ts1 cells. Ts1 cells have increased alloantigen-specific suppression of rejection and specific MLC responses.  Only a small portion of the tTreg cultured with IL-2 have specific receptor for alloantigen.   Here, we examined if the alloantigen-specific Treg were those induced to co-express CD8. 

Methods: Naïve CD4⁺CD8^-CD25⁺Treg from DA rats were cultured with PVG stimulator cells and IL-2 for 3-4d to induce Ts1 cells. 10-30% of cells were induced to co-express CD8. We compared the phenotypes and function of the CD4⁺CD8⁺CD25+Treg to the CD4⁺CD8^-CD25⁺T cells in vitro and in vivo.

Results: RT-PCR showed an increase in IFNGR and IL-12Rβ2 that characterizes Ts1 cells and was mainly in the CD8⁺ fraction as was IRF4, a transcription factor induced by TCR-activation.   In MLC, the proliferation to alloantigen and IL-2 measured by CSFE was higher in CD4⁺CD8⁺CD25⁺T cells compared to CD4⁺CD8^-CD25⁺T cells. 
In MLC, the CD8⁺ fraction suppressed response to PVG at 1:1012, and to third-party Lewis at 1:32. The CD4⁺CD8^-CD25⁺T cells had no enhanced suppression to PVG.  Unfractionated CD4⁺CD25⁺T cells suppressed at 1:32-1:64. In an adoptive transfer assay, IL-2 and alloantigen-activated Treg suppressed graft rejection mediated by naïve CD4⁺T cells at 1:10 and induce tolerance, whereas CD4⁺CD8^-CD25⁺FOXP3⁺T cells at 1:10 did not suppress rejection or induce tolerance.
DA rats rejecting PVG grafts or treated to induce tolerance were examined 10d post-transplant, 12-18% of CD4⁺CD25⁺T cells expressed CD8 vs <5% in naïve DA.  Whole CD25⁺T cells from both rejecting and tolerance-induced rats suppressed MLC to PVG at 1:256-1:512, and to Lewis at 1:16-1:32, while enriched CD4⁺CD8⁺CD25⁺Treg suppressed PVG at 1:1000 and did not suppress third-party.  The CD8^- fraction did not suppress. 

Conclusions: Our study indicated that CD8 expression on alloactivated CD4⁺CD8^-CD25⁺FOXP3⁺Treg is a marker of antigen-specificity.  The dual markers (CD4 and CD8) expressing Treg are potent and suppress responses to specific-alloantigen in vivo at <1:100 and in vitro at <1:1000.  Sorting CD8 expressing Treg may allow selective expansion of potent alloantigen-specific Treg for immune therapy and transplant tolerance induction.