Third Party Invariant Natural Killer T Cells Protect from Lethal Graft-Versus-Host Disease through Donor CD4+CD25+FoxP3+ Regulatory T Cells

Invariant natural killer T (iNKT) cells are potent regulators of immune responses in humans (TCRa Va24-Ja18) and mice (TCRa Va14-Ja18). We showed previously that host and donor iNKT cells play a critical role for graft-versus-host disease (GVHD) prevention and tolerance induction following allogeneic hematopoietic cell transplantation (HCT). In this study, we explored the role of adoptively transferred third party CD4⁺ iNKT cells in a murine model of allogeneic HCT across major histocompatibility barriers. After 8 Gy total body irradiation BALB/c (H-2K^d) recipient mice were transplanted with T cell-depleted bone marrow together with 1x10⁶ CD4⁺/CD8⁺ T cells (Tcon) from C57BL/6 (H-2K^b) donor mice. Adoptive transfer of sorted (purity >95%) 5x10⁴ CD4⁺ iNKT cells from FVB/N (H2-K^q) third party mice significantly improved survival (p<0.001) while retaining Tcon mediated graft-versus-tumor (GVT) effects against BCL1 and A20 lymphoma cells (p=0.002). Notably, CD4⁺ iNKT cells from third party mice were as protective as CD4⁺ iNKT cells from donor mice (p=0.50). Consistently, weight and GVHD scores improved in mice that received a single injection of third party CD4⁺ iNKT cells as compared to animals that received Tcon alone. Bioluminescence from expanding luciferase expressing alloreactive Tcon was significantly decreased while showing a Th2-biased cytokine profile in animals treated with third party CD4⁺ iNKT cells (p=0.003). Interestingly, inhibition of Tcon proliferation was comparable to animals that received CD4⁺ iNKT cells from donor mice (p=0.90). Although we found that third party CD4⁺ iNKT cells were rejected by day +10 after allogeneic HCT, adoptive transfer resulted in a robust expansion of luciferase expressing donor Treg using C57BL/6 FoxP3^DTR/GFP/luc mice as a source of Tcon (p=0.006, Figure 1). Depletion of Treg from the graft by injection of diphtheria toxin abrogated both donor Treg expansion and protection from GVHD lethality through third party CD4⁺ iNKT cells. In conclusion, low numbers of purified and adoptively transferred third party CD4⁺ iNKT cells protect from GVHD lethality through activation and expansion of donor Treg with retained GVT effects. Despite the fact that iNKT cells are a rare cell population, the in vivo activity of small numbers of cells and feasibility of in vitro expansion provide the basis for clinical translation.

Figure 1. Representative bioluminescence images of BALB/c mice that received Tcon alone or Tcon together with adoptively transferred third party CD4⁺ iNKT cells. Tcon derived from FoxP3^DTR/GFP/luc C57BL/6 mice with the expression of the luciferase gene being under the control of the FoxP3 promoter.