r 25-Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine. r 25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin. r 25OHD inhibits the potentiating effects of PKC-mediated TRPV1 activity. r 25OHD reduces T-cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity. r These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well-documented effects of vitamin D upon the nuclear vitamin D receptor. r The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8 þ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

Several unique waves of γδ T cells are generated solely in the fetal/neonatal thymus, whereas additional γδ T cell subsets are generated in adults. One intriguing feature of γδ T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus; however, it is less clear whether this paradigm holds true in adult animals. In this study, we investigated the relationship between maturation and thymic export of adult-derived γδ thymocytes in mice. In the Rag2pGFP model, immature (CD24+) γδ thymocytes expressed high levels of GFP whereas only a minority of mature (CD24−) γδ thymocytes were GFP+. Similarly, most peripheral GFP+ γδ T cells were immature. Analysis of γδ recent thymic emigrants (RTEs) indicated that most γδ T cell RTEs were CD24+ and GFP+, and adoptive transfer experiments demonstrated that immature γδ thymocytes can mature outside the thymus. Mature γδ T cells largely did not recirculate to the thymus from the periphery; rather, a population of mature γδ thymocytes that produced IFN-γ or IL-17 remained resident in the thymus for at least 60 d. These data support the existence of two populations of γδ T cell RTEs in adult mice: a majority subset that is immature and matures in the periphery after thymic emigration, and a minority subset that completes maturation within the thymus prior to emigration. Additionally, we identified a heterogeneous population of resident γδ thymocytes of unknown functional importance. Collectively, these data shed light on the generation of the γδ T cell compartment in adult mice.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted antigen (TRA) did not compromise tolerance. In this study, we determined that T cells that failed clonal deletion in this model were rendered functionally impaired in the thymus. PD-1 was induced in the thymus and established cell-intrinsic tolerance to TRA in CD8+ thymocytes independently of clonal deletion. PD-1 signaling in developing thymocytes was sufficient to induce tolerance but was dispensable for the initial maintenance of tolerance in the periphery. We showed that chronic exposure to high affinity antigen supported the long-term maintenance of tolerance in this model. Taken together, our study identifies a role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion and sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.Significance StatementThe establishment of T cell tolerance is critical to prevent autoimmune diseases. Apoptosis of highly self-reactive thymocytes is an important mechanism that enforces central tolerance. However, not all self-reactive thymocytes undergo apoptosis during development, and the fate of cells that evade this process is under-examined. Using bone marrow chimera, adoptive transfer, and thymic transplant experiments, we found acute PD-1 signaling is required to establish tolerance to tissue-restricted antigen (TRA) independently of clonal deletion. This tolerance is maintained by chronic exposure to tolerizing antigen but persists in the absence of PD-1 until late time points. Overall, this study identifies a role for PD-1 in establishing central tolerance, and it provides insight into the mechanism of PD-1 pathwaytargeting cancer immunotherapies.

The autoimmune disease of type 1 diabetes (T1D) results in the immune destruction of β-cells. Recent studies suggest supplementation of vitamin D along can significantly improve patients’ β-cell function and glycemic control possibly by dampening naïve T-cell activation. However, the underlying cellular mechanism for this effect has not been elucidated completely, especially as naïve T-cells possess absent or very low VDR expression. Therefore, the effects of Vitamin D on naïve T-cells may involve a VDR-independent pathway. Interestingly, TRPV1 channel activation is necessary for naïve T-cell activation. Our initial calcium imaging and electrophysiological data show that Vitamin D (25OHD) can partially activate TRPV1. 25OHD can inhibit capsaicin induced TRPV1 activity. We propose that vitamin D is a partial agonist of TRPV1, through direct binding to TRPV1 and modulating naïve T-cell activation. Furthermore, our flow cytometry studies confirm both 25OHD and 1,25OHD significantly reduce TNFα/INFγ and IL2/IL4 production of mouse CD4+ naïve T-cells after 24 hours activation. Our results support the concept that naïve T-cell activation can be dampened by vitamin D in a VDR-independent manner, via an as yet undescribed mechanism involving the modulation of TRPV1 activity. Moreover, in silico and point-muatgenic experiments indicate 25-OHD binds to the same region as known TRPV1 agonist and antagonists. These novel findings provide evidence of an additional pathway for the action of Vitamin D action and advance our knowledge of the underlying cellular mechanism by which vitamin D may beneficially regulate naive T-cell activation in autoimmune disease such as T1D. Disclosure W. Long: Research Support; Self; JDRF. M. Fatehi: None. S. Soni: None. R. Panigrahi: None. R.G. Kelly: None. K. Philippaert: None. A.J. Barr: None. M. Held: None. Y. Yu: None. S.A. Campbell: None. K. Ondrusova: None. T. Baldwin: None. J. Lemieux: None. P.E. Light: None. Funding JDRF