CD4+ Invariant Natural Killer T Cells Protect from Acute Graft-Versus-Host Disease Lethality through a Dramatic Expansion of Donor-Derived CD4+FoxP3+ Regulatory T Cells

Invariant natural killer T (iNKT) cells are a rare but potent immunomodulatory subset of lymphocytes in humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18). It has been shown previously that host iNKT cells require interaction with CD4⁺FoxP3⁺ regulatory T cells to protect from acute GVHD after conditioning with total lymphoid irradiation and anti-thymocyte globulin (Pillai et al., Blood 2009). In this study, we investigated the role of highly purified adoptively transferred donor-derived CD4⁺ iNKT cells in the setting of myeloablative conditioning. Balb/c (H-2Kd) recipient mice were irradiated with 8 Gy and transplanted with T cell-depleted bone marrow together with 1x10⁶ CD4/CD8 T lymphocytes (Tcon) from C57Bl/6 (H-2Kb) donor mice. Mice co-injected with as low as 5x10⁴ highly purified (>99%) CD4⁺ iNKT cells showed a significant survival benefit compared to mice receiving Tcon alone (p=.002). Consistently, weight and GVHD score improved in mice that received CD4⁺ iNKT cells. Signal intensity derived from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with CD4⁺ iNKT cells demonstrating inhibition of proliferation of alloreactive Tcon through CD4⁺ iNKT cells (p<.0001). In vivo CFSE proliferation assay confirmed decreased Tcon proliferation in peripheral lymph nodes (p<.0001), mesenteric lymph nodes (p=.03) and spleen (p=.0005). In addition, Tcon from CD4⁺ iNKT cell treated mice showed a significantly lower expression of activation markers CD44 and CD25. Interestingly, animals treated with CD4⁺ iNKT cells showed a robust expansion of donor-derived Neurophilin-1^-/lowCD4⁺FoxP3⁺ T cells in peripheral lymph nodes, mesenteric lymph nodes, spleen, gut, liver and skin. Moreover, CD4⁺ iNKT cells derived from NKG2D^-/- animals were significantly less effective in preventing acute GVHD lethality (WT vs. NKG2D^-/- p=.003). Co-injection of CD4⁺ iNKT cells did not abrogate GVL reactions of Tcon towards BCL₁ cells measured by bioluminescence imaging (p=ns). With α-GalCer and IL-2 in vitro expanded CD4⁺ iNKT cells had the same protective effect from lethal acute GVHD compared to freshly isolated CD4⁺ iNKT cells (p=ns). We conclude that low numbers of highly purified CD4⁺ iNKT cells protect from lethal acute GVHD in mice through a dramatic expansion of donor-derived CD4⁺FoxP3⁺ regulatory T cells with retained GVL effect. Despite the fact that iNKT cells are a rare cell population, the feasibility of in vitro expansion with retained functionality of CD4⁺ iNKT cells provide the basis for clinical translation.

EM and RN contributed equally to this study.