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

Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses in both humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18). As the iNKT cell receptor and the glycolipid-presenting molecule CD1d interaction is highly conserved, we explored the role of adoptively transferred third party CD4⁺ iNKT cells in a murine model of allogeneic HCT. BALB/c (H-2K^d) recipient mice were irradiated with 8 Gy and 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 purified (>95%) 5x10⁴ CD4⁺ iNKT cells from FVB/N (H-2K^q) third party mice resulted in a significant survival benefit (p<0.001) while retaining Tcon mediated graft-versus-tumor (GVT) effects against A20 lymphoma cells (p=0.002). 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. Notably, CD4⁺ iNKT cells from third party mice were as protective as CD4⁺ iNKT cells from donor mice (p=0.50). Signal intensity deriving from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with third party CD4⁺ iNKT cells (p=0.003). Interestingly, inhibition of Tcon proliferation was similar to animals that received CD4⁺ iNKT cells from donor mice (p=0.90). In addition, adoptive transfer of third party CD4⁺ iNKT cells promoted a Th2-biased cytokine response of alloreactive donor T cells. Although we found that third party CD4⁺ iNKT cells were rejected by day +10 after allogeneic HCT, adoptive transfer of these cells resulted in a robust expansion of luciferase expressing donor CD4⁺FoxP3⁺ regulatory T cells (Treg) as measured by bioluminescence imaging (p=0.006). Using FoxP3^DTR C57BL/6 donor mice, depletion of Treg from the graft abrogated both donor Treg expansion and protection from GVHD lethality through third party CD4⁺ iNKT cells. We conclude that low numbers of highly purified and adoptively transferred third party CD4⁺ iNKT cells protect from lethal GVHD 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.