The Absence of CC-Chemokine Receptor 8 on Donor Regulatory T Cells Impairs Their Ability to Prevent Lethal Acute Gvhd.

Abstract 3743 CC-Chemokine receptor 8 (CCR8) is a chemokine receptor expressed on the surfaces of activated effector T cells (T effs ) and resting regulatory T cells (T regs ). CCR8 has previously been shown to play a role in the trafficking of memory T cells into cutaneous and possibly pulmonary sites, and to contribute to the ability of T regs to properly interact with antigen presenting cells within secondary lymphoid tissue. Since the lungs and in particular the skin are frequent sites of tissue injury in those developing graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), we set out to study the contribution of CCR8 to GVHD pathogenesis using a haploidentical murine HSCT model. Given the emerging utilization of therapeutic donor T reg infusions as a means for preventing GVHD, we also explored the role that CCR8 plays in the protective immunosuppressive function of T regs in the HSCT setting. Methods: For our studies we used a haplotype-matched murine transplant model. C57BL/6 mice (B6; H-2 b ) functioned as donors, and B6 × DBA2 (B6D2; H-2 bxd ) mice served as recipients. For T eff studies, B6D2 mice were lethally irradiated to 950 rads on transplant day -1, and then administered 3×10 6 T cell depleted (TCD) B6 bone marrow (BM) cells +/− 4×10 6 CD25 − T effs isolated from the spleens of wild-type (WT) or CCR8 −/− B6 mice on day 0. For studies involving regulatory T cells, B6D2 animals were lethally irradiated on day -1 and administered WT B6 TCD BM cells +/− 1–1.25×10 6 CD4 + CD25 + T regs isolated from WT or CCR8 −/− B6 mice on day 0. 4×10 6 WT B6 T effs were then administered on transplant day +2. For some transplants, WT B6 and CCR8 −/− animals transgenic for enhanced green-fluorescent protein (eGFP + ) were used as T eff and/or T reg donors in order to study T cell trafficking following HSCT. Both stereo-fluorescence microscopy and anti-eGFP ELISA approaches were utilized for these in vivo trafficking experiments. The in vitro suppressive potential of WT and CCR8 −/− T regs was evaluated by examining their ability to inhibit T eff proliferation in a one way mixed lymphocyte reaction (MLR). Results: CCR8 was not required for the induction of lethal GVHD, with recipients of CCR8 −/− T effs demonstrating a 100% mortality rate by transplant day +32. However, CCR8 was important for T reg function. In vivo , CCR8 −/− T regs were significantly less effective at preventing GVHD lethality than WT T regs (see figure; P=0.013 for day +70 survival proportion comparison using Fisher9s exact test). When we evaluated donor T reg trafficking on transplant days 6–7 using eGFP + cells, no significant differences were noted between those animals receiving WT versus CCR8 −/− T regs . By transplant days 9–10, however, a generally higher eGFP signal was observed by stereo-fluorescence microscopy within the lymph nodes of those animals receiving WT eGFP + T regs compared to those given CCR8 −/− eGFP + T regs . In addition, significantly more eGFP was detected by ELISA in the spleens and colons of WT eGFP + T reg recipients (P=0.0015 and 0.021 respectively for mean total eGFP comparison between WT and CCR8 −/− T reg groups by student9s t test), and a strong trend for greater WT T reg accumulation was noted within host livers and lungs (P=0.059 and 0.066 respectively). Finally, CCR8 −/− T regs were relatively impaired in their ability to suppress T cell proliferation in vitro compared to WT cells, particularly at higher dilutions. Conclusions: CCR8 is not required for donor T effs to induce lethal GVHD in our HSCT model. However, CCR8 expression does appear to be important for the function of T regs in vitro and in vivo , particularly at limiting T reg dilutions. CCR8 is not required for the accumulation of donor T regs within any one recipient site. Rather, CCR8 appears to globally potentiate the accumulation of donor T regs within most recipient tissues, with the extent of this effect increasing over time. Future experiments will focus on elucidating a mechanism for this finding. Disclosures: No relevant conflicts of interest to declare.