Background and Objectives: Neuronal nitric oxide synthase (nNOS) overexpressed in melanoma plays a critical role in disease progression. Our previous studies demonstrated that nNOS inhibitors exhibited potent anti-melanoma activity and regulated PD-L1 expressions in the presence of interferon-gamma (IFN-γ). However, the role of nNOS in the melanoma immune response has not been well defined. Methods: Changes in gene expression profiles after nNOS inhibitor treatment were determined by transcriptomic analysis. A melanoma mouse model was used to determine the effects of nNOS inhibition on peripheral T cells and the in vivo anti-tumor activity of combining nNOS inhibitors with immune checkpoint blockade. Changes in human T cell activation through interleukin-2 (IL-2) production were investigated using an ex vivo co-culture system with human melanoma cells. Results: Cellular RNA analysis revealed significant changes in the genes involved in key signaling pathways after nNOS inhibitor HH044 treatment. Immunophenotyping of mouse peripheral blood mononuclear cells (PBMCs) after prolonged HH044 treatment showed marked increases in CD4+ and CD8+PD-1+ T cells. Ex vivo studies demonstrated that co-culturing human PBMCs with melanoma cells inhibited T cell activation, decreasing IL-2-secreting T cells both in the presence and absence of IFN-γ. PBMCs from a significant portion of donors (7/11, 64%), however, were reactivated by nNOS inhibitor pretreatment, displaying a significant increase in IL-2+ T cells. Distinctive T cell characteristics were noted at baseline among the responders with increased CD4+RORγt+ and reduced CD4 naïve T cells. In vivo mouse studies demonstrated that nNOS inhibitors, when combined with PD-1 blockade, significantly reduced tumor growth more effectively than monotherapy. Additionally, the median survival was extended from 43 days in the control mice to 176.5 days in mice co-treated with HH044 and anti-PD-1. Conclusions: Targeting nNOS is a promising approach to enhancing the anti-melanoma activity of immune checkpoint inhibitors, not only interfering with melanoma biological activities but also regulating the tumor microenvironment, which subsequently affects T cell activation and tumor immune response.

Epidemiological studies link exposure to viral infection during pregnancy, including influenza A virus (IAV) infection, with increased incidence of neurodevelopmental disorders (NDDs) in offspring. Models of maternal immune activation (MIA) using viral mimetics demonstrate that activation of maternal intestinal T helper 17 (TH17) cells, which produce effector cytokine interleukin (IL)-17, leads to aberrant fetal brain development, such as neocortical malformations. Fetal microglia and border-associated macrophages (BAMs) also serve as potential cellular mediators of MIA-induced cortical abnormalities. However, neither the inflammation-induced TH17 cell pathway nor fetal brain-resident macrophages have been thoroughly examined in models of live viral infection during pregnancy. Here, we inoculated pregnant mice with two infectious doses of IAV and evaluated peak innate and adaptive immune responses in the dam and fetus. While respiratory IAV infection led to dose-dependent maternal colonic shortening and microbial dysregulation, there was no elevation in intestinal TH17 cells nor IL-17. Systemically, IAV resulted in consistent dose- and time-dependent increases in IL-6 and IFN-γ. Fetal cortical abnormalities and global changes in fetal brain transcripts were observable in the high-but not the moderate-dose IAV group. Profiling of fetal microglia and BAMs revealed dose- and time-dependent differences in the numbers of meningeal but not choroid plexus BAMs, while microglial numbers and proliferative capacity of Iba1+ cells remained constant. Fetal brain-resident macrophages increased phagocytic CD68 expression, also in a dose- and time-dependent fashion. Taken together, our findings indicate that certain features of MIA are conserved between mimetic and live virus models, while others are not. Overall, we provide consistent evidence of an infection severity threshold for downstream maternal inflammation and fetal cortical abnormalities, which recapitulates a key feature of the epidemiological data and further underscores the importance of using live pathogens in NDD modeling to better evaluate the complete immune response and to improve translation to the clinic.
© 2024. The Author(s).

During thymic development, most γδ T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate γδ T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM/SAP signaling pathway regulates the development and function of multiple innate-like γδ T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent γδ TCR repertoire in mice. SAP deficiency resulted in both a significant loss of an immature Gzma+Blk+Etv5+Tox2+ γδT17 precursor population and a significant increase in Cd4+Cd8+Rorc+Ptcra+Rag1+ thymic γδ T cells. SAP-dependent diversion of embryonic day 17 thymic γδ T cell clonotypes into the αβ T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered γδ TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset. Together, the data support a model in which SAP-dependent γδ/αβ T cell lineage commitment regulates γδ T cell developmental programming and shapes the γδ TCR repertoire.
© 2024, Mistri et al.

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.
Published by Elsevier Inc.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.