CD4⁺CD25⁺ T Cells Can Inhibit CD8 T Cell Mediated GVHD: Requirement for In Vivo Recognition of Allogeneic Host MHC Class II Antigens.

CD4 regulatory T (Treg) cells have shown promise in the transplantation mileu including the ability to inhibit the development of graft vs host disease (GVHD) following allogeneic hematopoietic stem cell transplants (HCT). The antigen specificity of the Treg population(s) involved is not yet clear nor is the role of their activation following transplant. We are interested in determining the requirement for recognition of host MHC antigens following infusion of CD4⁺CD25⁺ T cells in an experimental model of GVHD. To clearly distinguish the requirements of regulatory vs GVH reactive cells, a model of CD8 T cell mediated GVHD was developed using highly purified BALB/c (H2^d) donor CD8⁺ T cells (Miltenyi column, 95-98%). CD8 T cells were transplanted together with T cell depleted (TCD) BALB/c BMC into 12.0 GY (6.0 Gy split dose) TBI conditioned C57BL/6 (B6, H2^b) recipients. To support development of GVHD by these cells, resistance was inhibited by treatment of recipients with anti-NK1.1mab (PK136) at Days -1, 0 and +7. BALB/c CD8⁺ T cells at doses of 5.0x10⁶ but not 2.5x10⁶ induced weight loss and some lethality in B6 recipients. 5x10⁶ CD8⁺ T cells were then transplanted into B6-MHC class II^{−/ −} recipients. GVHD symptoms including weight loss and lethality were readily apparent in these mice post-transplant. Interestingly, GVHD was consistently more severe with respect to the induction of weight loss and lethality in MHC Class II^{−/ −} vs B6-wt recipients. Highly enriched BALB/c CD4⁺CD25⁺ T cells (> 95%) were produced from spleen and lymph node cells following negative (B-cells, CD8 and NK) and positive (CD25) selection using Miltenyi magnetic bead columns. Co-transplant of 1x10⁶ CD4⁺CD25⁺ T cells together with BALB/c CD8⁺ T cells into B6 recipients inhibited GVHD as assessed by the absence of weight loss and lethality compared to B6 recipients of CD8⁺ T cells alone. In contrast, BALB/c CD4⁺CD25⁺ T cells failed to protect B6-MHC class II^{−/ −} recipients from severe CD8⁺ T cell mediated GVHD. These findings demonstrate that donor CD4⁺ T regulatory cells can suppress GVHD inducing CD8⁺ T cells after the former recognize host class II alloantigen following transplant. We hypothesize that activated CD4⁺CD25⁺ T regulatory cells inhibit GVH reactive T cells at the host APC interface. Future studies in this model can be designed to examine ex-vivo activated and expanded CD4⁺CD25⁺ T regulatory populations. Transplant of such cells will enable us to address questions regarding the importance of in vivo recognition of host class II in the regulation of GVHD by these cells.