CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Microbial Metabolites in the Maturation and Activation of Dendritic Cells and Their Relevance for Respiratory Immunity.

The respiratory tract is a gateway for viruses and bacteria from the external environment to invade the human body. Critical to the protection against these invaders are dendritic cells (DCs) - a group of highly specialized myeloid cells that monitors the lung microenvironment and relays contextual and antigenic information to T cells. Following the recognition of danger signals and/or pathogen molecular associated patterns in the lungs, DCs undergo activation. This process arms DCs with the unique ability to induce the proliferation and differentiation of T cells responding to matching antigen in complex with MHC molecules. Depending on how DCs interact with T cells, the ensuing T cell response can be tolerogenic or immunogenic and as such, the susceptibility and severity of respiratory infections is influenced by the signals DCs receive, integrate, and then convey to T cells. It is becoming increasingly clear that these facets of DC biology are heavily influenced by the cellular components and metabolites produced by the lung and gut microbiota. In this review, we discuss the roles of different DC subsets in respiratory infections and outline how microbial metabolites impact the development, propensity for activation and subsequent activation of DCs. In particular, we highlight these concepts in the context of respiratory immunity.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

Ptpn2 and KLRG1 regulate the generation and function of tissue-resident memory CD8+ T cells in skin.

Tissue-resident memory T cells (TRM cells) are key elements of tissue immunity. Here, we investigated the role of the regulator of T cell receptor and cytokine signaling, Ptpn2, in the formation and function of TRM cells in skin. Ptpn2-deficient CD8+ T cells displayed a marked defect in generating CD69+ CD103+ TRM cells in response to herpes simplex virus type 1 (HSV-1) skin infection. This was accompanied by a reduction in the proportion of KLRG1- memory precursor cells and a transcriptional bias toward terminal differentiation. Of note, forced expression of KLRG1 was sufficient to impede TRM cell formation. Normalizing memory precursor frequencies by transferring equal numbers of KLRG1- cells restored TRM generation, demonstrating that Ptpn2 impacted skin seeding with precursors rather than downstream TRM cell differentiation. Importantly, Ptpn2-deficient TRM cells augmented skin autoimmunity but also afforded superior protection from HSV-1 infection. Our results emphasize that KLRG1 repression is required for optimal TRM cell formation in skin and reveal an important role of Ptpn2 in regulating TRM cell functionality.

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

Mechanosensing by Peyer's patch stroma regulates lymphocyte migration and mucosal antibody responses.

Fibroblastic reticular cells (FRCs) and their specialized collagen fibers termed 'conduits' form fundamental structural units supporting lymphoid tissues. In lymph nodes, conduits are known to transport interstitial fluid and small molecules from afferent lymphatics into the nodal parenchyma. However, the immunological contributions of conduit function have remained elusive. Here, we report that intestinal Peyer's patches (PPs) contain a specialized conduit system that directs the flow of water absorbed across the intestinal epithelium. Notably, PP FRCs responded to conduit fluid flow via the mechanosensitive ion channel Piezo1. Disruption of fluid flow or genetic deficiency of Piezo1 on CCL19-expressing stroma led to profound structural alterations in perivascular FRCs and associated high endothelial venules. This in turn impaired lymphocyte entry into PPs and initiation of mucosal antibody responses. These results identify a critical role for conduit-mediated fluid flow in the maintenance of PP homeostasis and mucosal immunity.

Microbiota-Derived Short-Chain Fatty Acids Promote the Memory Potential of Antigen-Activated CD8+ T Cells.

Interactions with the microbiota influence many aspects of immunity, including immune cell development, differentiation, and function. Here, we examined the impact of the microbiota on CD8+ T cell memory. Antigen-activated CD8+ T cells transferred into germ-free mice failed to transition into long-lived memory cells and had transcriptional impairments in core genes associated with oxidative metabolism. The microbiota-derived short-chain fatty acid (SCFA) butyrate promoted cellular metabolism, enhanced memory potential of activated CD8+ T cells, and SCFAs were required for optimal recall responses upon antigen re-encounter. Mechanistic experiments revealed that butyrate uncoupled the tricarboxylic acid cycle from glycolytic input in CD8+ T cells, which allowed preferential fueling of oxidative phosphorylation through sustained glutamine utilization and fatty acid catabolism. Our findings reveal a role for the microbiota in promoting CD8+ T cell long-term survival as memory cells and suggest that microbial metabolites guide the metabolic rewiring of activated CD8+ T cells to enable this transition.

Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection.

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN-I) during viral infections, in response to triggering of endosomal Toll-like receptors (TLRs) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN-I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN-I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN-I downstream of the TLR9-to-MyD88-to-IRF7 signaling pathway without requiring IFN-I positive feedback, high IRF7 expression, or AP3-driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN-I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection.

Effective Priming of Herpes Simplex Virus-Specific CD8+ T Cells In Vivo Does Not Require Infected Dendritic Cells.

Resolution of virus infections depends on the priming of virus-specific CD8+ T cells by dendritic cells (DC). While this process requires major histocompatibility complex (MHC) class I-restricted antigen presentation by DC, the relative contribution to CD8+ T cell priming by infected DC is less clear. We have addressed this question in the context of a peripheral infection with herpes simplex virus 1 (HSV). Assessing the endogenous, polyclonal HSV-specific CD8+ T cell response, we found that effective in vivo T cell priming depended on the presence of DC subsets specialized in cross-presentation, while Langerhans cells and plasmacytoid DC were dispensable. Utilizing a novel mouse model that allows for the in vivo elimination of infected DC, we also demonstrated in vivo that this requirement for cross-presenting DC was not related to their infection but instead reflected their capacity to cross-present HSV-derived antigen. Taking the results together, this study shows that infected DC are not required for effective CD8+ T cell priming during a peripheral virus infection.IMPORTANCE The ability of some DC to present viral antigen to CD8+ T cells without being infected is thought to enable the host to induce killer T cells even when viruses evade or kill infected DC. However, direct experimental in vivo proof for this notion has remained elusive. The work described in this study characterizes the role that different DC play in the induction of virus-specific killer T cell responses and, critically, introduces a novel mouse model that allows for the selective elimination of infected DC in vivo Our finding that HSV-specific CD8+ T cells can be fully primed in the absence of DC infection shows that cross-presentation by DC is indeed sufficient for effective CD8+ T cell priming during a peripheral virus infection.