Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8+ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8+ cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8+ T cells were adoptively transferred into PD-L1−/− recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1−/− mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8+ T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.

Dendritic cells (DCs) are antigen presenting cells with a dual role of priming T cells response and induction of peripheral tolerance. While CD11chigh DCs normally stimulate immunity (sDCs), CD11clowCD45rbhigh DCs were recently characterized as a regulatory subtype (rDC), which maintains low levels of costimulatory molecules after maturation. We found rDC levels were at least 2 times lower in bone marrow and spleens of CD48-/- C57BL/6 mice compared to their WT counterparts. Previously, we observed that aging CD48-/- mice developed spontaneous lupus-like autoimmune disease, indicated in elevated levels of autonuclear antibodies. We have shown that treatment of bone-marrow derived DCs with the neuropeptide, vasoactive intestinal peptide (VIP) and IL-10 strongly expanded the rDCs subtype, which produced high and low levels of IL-10 and IL-12, respectively. VIP-treated DCs of WT mice produced significantly higher amounts of IL-10, compared to CD48-/- DCs. We have also demonstrated that adoptive transfer of sDCs and rDCs subtypes together with OT-II-derived CD4+ T cells into WT and CD48-/- mice induced inhibition of OT-II-CD4+ T cell expansion only when WT-derived rDCs were transferred, as compared to expansion induced by their sDCs counterparts. These data suggest that dysfunctional and lower levels of rDCs of CD48-/- mice may be responsible for the development of spontaneous SLE in these mice when they are older.

Invariant NKT cells (iNKT cells) are a unique population of lymphocytes that have a biased TCR gene expression, are restriction by CD1d molecules, and rapidly produce a spectrum of cytokines following activation. Various studies have shown that positive costimulatory pathways can influence the activation of iNKT cells. However, little is understood about the regulation of NKT cells by negative costimulatory pathways. Here we show that PD-L1 and PD-1. but not PD-L2, are expressed on iNKT cells in spleen, liver and thymus. In vivo activation with α-GalCer lead to increased cytokine production and expansion of iNKT cells in the absence of the PD-L1 pathway. To study whether PD-L1 deficiency on NKT cells would affect antigen specific T cell responses we utilized CD8+ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 T cells after adoptive transfer into wild type recipients. However, this expansion was significantly augmented when OT-1 T cells were adoptively transferred into PD-L1-/- recipients. In a B16 melanoma metastasis model, there was an increased clearance of lung nodules when PD-L1-/- recipients were given dendritic cells loaded with α-GalCer and TRP-2 peptide. These data demonstrate that blocking PDL1:PD-1 interactions between NKT cells and cells of the adaptive immune system are important in mounting an effective anti-tumor immune response.