CD4+ Invariant Natural Killer T Cells Require NKG2D To Protect From Lethal Acute Graft-Versus-Host Disease

Invariant Natural Killer T (iNKT) cells are a rare cell population in humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18) that are characterized by a rapid release of immunoregulatory cytokines upon stimulation. The tolerogenic impact of host iNKT cells on graft-versus-host disease (GVHD) after conditioning with total lymphoid irradiation and anti-thymocyte globulin (TLI/ATG) has been shown previously (Lan et al., BBMT 2003). Moreover, we recently described a subset of CD49b+ NKT cells overlapping with iNKT cells and providing protection from acute GVHD mainly via an IL-4-dependent mechanism (Leveson-Gower et al., Blood 2011). Here, we investigated the role of highly purified adoptively transferred donor-derived CD4+ iNKT cells. Balb/c recipient mice were transplanted with T cell-depleted bone marrow together with 1x10⁶ CD4/CD8 T lymphocytes (Tcon) from C57Bl/6 donor mice after irradiation with 8 Gy. Mice co-injected with as low as 5x10⁴ freshly isolated and highly purified (>99%) CD4+ iNKT cells showed a significant survival benefit compared to mice receiving Tcon alone (p=.0015). 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=.0277) and spleen (p=.0005). CD4+ iNKT cells showed a Th2-biased cytokine profile with high levels of IL-4 and IL-13 in the presence of alloreactive Tcon challenged with irradiated allogeneic stimulators. Co-injection of CD4+ iNKT cells that were expanded 5-fold in vitro with α-GalCer and IL-2 had the same protective effect from lethal acute GVHD compared to freshly isolated CD4+ iNKT cells (p=.7987). Interestingly, CD4+ iNKT cells derived from NKG2D^-/- animals were significantly less effective in preventing acute GVHD lethality (WT vs. NKG2D^-/-p=.0027). In conclusion, low numbers of highly purified freshly isolated and cultured CD4+ iNKT cells protect from lethal acute GVHD in mice and require NKG2D. 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.