Absence of CCR4 Enhances the Function of Expanded Regulatory T-Cells.

Chemokine Receptor 4 (CCR4) has been shown to be important for the homing of effector T-lymphocytes (T_effs) to cutaneous and possibly pulmonary sites of inflammation. CCR4 is also expressed on the surface of regulatory T-lymphocytes (T_regs), and previous work has suggested a critical role for CCR4 for their in-vivo suppressive ability. Since the skin and lung are important sites of graft-versus-host disease (GVHD) morbidity, we set out to determine the contribution of CCR4 to both T_eff and T_reg function in a murine stem-cell transplant model.

Methods: C57BL/6 (B6) mice served as bone marrow (BM) donors, and B6xDBA/2 F1 (B6D2) mice functioned as recipients. T_effs and T_regs were obtained from wild-type (WT) or CCR4 knockout (CCR4^−/−) B6 mice. For T_eff studies, recipient animals were lethally irradiated and administered T-cell depleted (TCD) BM cells +/− splenic T_effs from WT or CCR4^−/− donors. For T_reg studies, mice received TCD BM + WT T_{effs +/−} naive or expanded T_regs from WT or CCR4^−/− donors.

Results: Animals receiving WT or CCR4^−/− T_effs all developed severe GVHD with an 88% mortality rate. Using flow cytometry, we demonstrated that WT and CCR4^−/− eGFP⁺ T_effs were able to accumulate in the skin and lungs of recipient animals at a similar frequency, suggesting a non-essential role for CCR4 in T_eff homing to these sites. Attention was next directed towards the influence of CCR4 on T_reg function in-vivo. Those animals receiving BM and T_effs without T_regs developed aggressive GVHD with 100% mortality. In contrast, when animals were administered BM and naïve T_regs from either WT or CCR4^−/− donors two days prior to receiving T_effs, all animals demonstrated 100% survival and only mild GVHD, suggesting a non-obligatory role for CCR4 for T_reg function in-vivo. Since CCR4 is upregulated on T_regs and T_effs after activation, we next examined whether its absence would exert a greater effect on the ability of expanded T_regs to protect against GVHD in-vivo compared to naïve cells. Animals were irradiated and received TCD BM plus T_{effs +/−} T_regs from WT or CCR4^−/− donors previously expanded in-vitro. Those animals receiving BM and T_effs without T_regs all developed severe GVHD with 100% mortality. Ex-vivo expanded WT T_regs, 80% of which expressed L-selectin at low levels, provided only marginal GVHD protection with 12.5% survival when given concurrently with T_effs at a 1:1 ratio. In contrast, expanded CCR4^−/− T_regs provided superior in-vivo GVHD protection, with 50% of mice surviving long term (see figure, p=.05 for WT versus CCR4^−/− T_reg groups). This enhanced protection in-vivo occurred despite an inferior ability of expanded CCR4^−/− T_regs to inhibit T-cell proliferation in an in-vitro mixed lymphocyte reaction compared to WT cells. Migration studies did not reveal a difference in the trafficking of expanded CCR4^−/− T_regs compared to WT cells except for a greater frequency of CCR4^−/− T_regs in the mesenteric lymph node on day +7.

Conclusions: Our data suggest that naïve CCR4^−/− T_regs are as efficient as WT cells in preventing GVHD, and that CCR4 is not required for the induction of GVHD by T_effs. Paradoxically we show that expanded T_regs function better to prevent GVHD in the absence of CCR4. Current work is underway to determine the mechanism for this finding.

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