Interaction between Host Natural Killer T Cells and Donor CD4⁺CD25⁺ T_reg Cells Protects Against GVHD after TLI/ATS Host Conditioning and Bone Marrow Transplantation.

The murine non-myeloablative regimen of total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS) was recently translated into a transplantation strategy for human hematolymphoid malignancies, preventing recipients from acute graft-versus-host disease (GVHD) without tumor relapses (Lowsky et al, NEJM, 2005). To investigate GVHD protection, we transplanted 50 x 10⁶ bone marrow cells and 60 x 10⁶ splenocytes (BMT) from wild-type, CD4^−/−, IL-4^−/, or IL-10^−/− C57BL/6 (H-2^b) donor mice into wild-type or natural killer (NK) T cell deficient Jα18^−/− BALB/c (H-2^d) hosts following non-myeloablative host conditioning with TLI/ATS. Control wild-type BALB/c hosts received a conventional regimen, either 800cGy (lethal) or 450 cGy (sublethal) total body irradiation (TBI) and ATS with BMT from wild-type C57BL/6 donors. TLI/ATS-conditioned wild-type hosts given BMT from wild-type donors survived 100 days without evidence of GVHD. TBI/ATS-conditioned wild-type hosts given BMT from wild-type donors and TLI/ATS conditioned wild-type hosts given BMT from CD4^−/−, IL-4^−/−, or IL-10^−/− donors succumbed to GVHD, with marked donor CD8⁺ T cell accumulation demonstrated in liver, mesenteric lymph nodes (MLN), and colon at day 6. Given the dependence of GVHD protection on donor CD4⁺ cells and the donor cytokines IL-4 and IL-10, we investigated the role of donor CD4⁺CD25⁺Foxp3⁺regulatory T cells (CD4⁺T_regs) in GVHD protection after TLI/ATS and BMT. Wild-type TLI/ATS conditioned hosts given BMT from wild-type donors demonstrated a significant (p< 0.01) ten-fold increase in both the percentage and absolute number of donor CD4⁺CD25⁺Foxp3⁺T_regs present in the host spleen at day 6. These donor CD4⁺CD25⁺Foxp3⁺cells demonstrated typical IL-4 and IL-10 dominant cytokine secretion profiles on PMA/ionomycin stimulation and were functional both in vitro in donor-host MLR suppression and in vivo in secondary GVHD suppression assays. Using congenic markers (CD45.1/CD45.2), we demonstrate that these donor CD4⁺ T_regs are derived from splenic (peripheral) T cells within the donor cell inoculum. When wild-type donor bone marrow and spleen cells were rigorously CD25-depleted using magnetic bead selection prior to BMT into wild-type TLI/ATS treated hosts, donor CD4⁺CD25⁺Foxp3⁺ T_regs were not present in the host spleen at day 6, and all hosts demonstrated markedly increased donor CD8⁺ T cell accumulation in the liver, MLN, and colon at day 6 accompanied by GVHD after BMT. TLI/ATS-conditioned Jα18^−/− hosts given BMT from wild-type donors demonstrated low numbers of donor CD4⁺CD25⁺Foxp3⁺ T_regs in host spleen and GVHD similar to TBI/ATS controls. When wild-type host NK T cells were adoptively transferred before BMT into TLI/ATS-conditioned Jα18^−/− hosts, the use of non-CD25-depleted donor cells resulted in increased numbers of donor CD4⁺ T_regs and markedly reduced donor CD8⁺ T cell accumulation in the host liver, MLN, and colon at day 6, whereas CD25 depletion of donor cells resulted in loss of donor CD4⁺CD25⁺Foxp3⁺ T_regs in the host spleen and significantly increased donor CD8⁺ T cell accumulation. Our previous studies demonstrated that host invariant NK T cells are critical for GVHD protection after TLI/ATS host conditioning. The present studies show that host regulatory NK T cells can facilitate the expansion of donor CD4⁺CD25⁺Foxp3⁺ T_regs, and that this expansion plays a pivotal role in protecting TLI/ATS treated hosts from lethal GVHD after allogeneic BMT.