Combined CCR5 and CXCR3 Blockade Attenuates Murine Agvhd through Alternating Donor-Derived T Cell Distribution and Function

Acute graft-versus-host disease (aGVHD) is one of the major complications and a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Chemokines and their cognate receptors play an important role in directing the migration of donor derived T cells to target organs. Chief among these chemokine receptors are CXCR3 and CCR5. Our previous study found that CCR5 blockade combined with cyclosporine A in a murine model could attenuate the severity of aGVHD in a less extent. This might be due to multiple expression of chemokine receptors on immune cell, only one kind receptor blockade might not completely block the migration of T cells to aGVHD target organs. In this study, we investigated whether combined CCR5 and CXCR3 blockade could further attenuate murine aGVHD, and also explored its immunologic mechanism.

BALB/c(H-2Kd)recipients received 8.0 Gy of total body irradiation (⁶⁰Coγsource) prior to transplantation followed by the infusion of bone marrow cells (BMCs) and splenic cells (SCs) from C57BL/6J(H-2Kb)or CCR5 knockout mice(H-2Kb)to establish an allogeneic bone marrow transplantation model. Body weight change, clinical scores of aGVHD, degree of histopathologic damage and survival rate were dynamically recorded to assess the efficacy of CCR5 and CXCR3 blockade in this transplantation model. Mechanically, donor derived lymphocytes were labeled with fluorescence dye to detect their distribution in vivo. Immunohistochemical analysis for CD4+ and CD8+ were performed on target organs of recipient mice at 7 days post-transplantation. Flow cytometry was used to calculate the absolute number of T cells in target organs, and to detect its proliferation, activation and differentiation in the spleen. ELISA was used to determine the concentration of T cells related cytokines.

The results showed that both CCR5 and CXCR3 are enhanced during the aGVHD process. Although transplantation of allogeneic BMCs and SCs induced severe clinical aGVHD with high aGVHD scores (4.67±0.33) and a lethality of 90%, combined CCR5 and CXCR3 blockade attenuated clinical signs of aGVHD, reduced aGVHD scores (1.25±0.25, P< 0.001), reduced aGVHD-induced death (a lethality of 40%, P< 0.05). Compared with aGVHD group, combined CCR5 and CXCR3 blockade reduced liver histologic score (3.3±0.3 versus 1.3±0.3, P< 0.05), lung histologic score (3.6±0.3 versus 1.3±0.3, P< 0.05) and intestine histologic score (3.7±0.3 versus 1.3±0.3, P< 0.05). On days 7 after transplantation, more fluorescent signal emanated from the spleen and from lymph nodes after the use of CCR5 and CXCR3 blockade. Also combined CCR5 and CXCR3 blockade reduced the infiltration of CD4+ and CD8+ T cells into the liver and intestine, and retained part of CD4+ and CD8+ T cells within SLOs. As for the effector function of donor T cells, the results showed that combined CCR5 and CXCR3 blockade dampened T-cell activation, suppressed the percentage of Th1 and Tc1, and induced Treg induction. Additionally, combined CCR5 and CXCR3 blockade altered the concentrations of IFN-γ and IL10. Furthermore, the transplantation of CCR5-/- splenic cells with CXCR3 blockade showed a stronger protection effect on aGVHD.

Combined CCR5 and CXCR3 blockade further attenuated aGVHD. aGVHD inhibition was associated with alternated donor T-cell distribution, limited donor T-cell activation and negatively impacted T-cell effector function in vivo. These data suggest that the combined use of CCR5 and CXCR3 blockade might be applicable for aGVHD prophylaxis in clinical settings.