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

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 3×10⁶ T cell depleted (TCD) B6 bone marrow (BM) cells +/− 4×10⁶ 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 +/− 1–1.25×10⁶ CD4⁺CD25⁺ T_regs isolated from WT or CCR8^−/− B6 mice on day 0. 4×10⁶ 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).

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 Fisher's 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 student's 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.

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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_regsin 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.

No relevant conflicts of interest to declare.