The Journal of Immunology

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by suppressing excessive activation of effector T cells. Although several mechanisms of Treg-mediated suppression have been described, the molecular signals that contribute to this regulation remain incompletely understood. WNT signaling, best known for its roles in development and tissue homeostasis, has recently emerged as an important regulator of immune function, but its contribution to Treg-mediated immune suppression is largely unknown. Here, we show that Tregs preferentially express multiple canonical WNT ligands, including WNT2B, WNT3, WNT7B, and WNT10B, compared with conventional CD4+ T cells. These WNT proteins were detected intracellularly in Tregs, and WNT2B and WNT3 were actively secreted into culture supernatants. Conventional CD4+ T cells expressed Frizzled receptors capable of sensing these ligands. Pharmacological inhibition of canonical WNT signaling using the antagonist mDKK-1 enhanced CD4+ T cell activation and proliferation and increased pro-inflammatory cytokine expression, while anti-inflammatory IL-10 remained unchanged. Together, these findings identify Tregs as a source of canonical WNT ligands and suggest that Treg-derived WNT signaling contributes to the suppression of effector CD4+ T cell responses. This work reveals a previously underappreciated pathway through which Tregs regulate immune activity and identifies WNT signaling as a potential target for modulating inflammatory immune responses.

{gamma}{delta} T cells provide mucosal defense against infection while also contributing to tissue repair. However, data regarding the effect of the human lung environment on {gamma}{delta} T cell functionality remains limited. To address whether lung inflammation impacts {gamma}{delta} T cell functionality, we analyzed lung and matched hilar lymph node (LN) tissue from deceased donors and patients with interstitial lung disease (ILD). We performed high-parameter spectral flow cytometry to examine the expression pattern of phenotypic biomarkers and assess ex vivo function. We identified lung-specific enrichment of {gamma}{delta} T cells with an effector memory phenotype relative to matched regional LN. We then used an ex vivo stimulation approach to interrogate the capacity to protect against infection (granzyme B [GzmB], interferon-{gamma} [IFN{gamma}] and tumor necrosis factor [TNF]) and promote epithelial cell proliferation (amphiregulin [AREG]). We found that {gamma}{delta} T cells in lung and LN from deceased donors had similar functional properties. While {gamma}{delta} T cell populations from ILD lungs largely maintained cytokine production capacity, expression was diminished relative to LN counterparts. Importantly, lung {gamma}{delta} T cells maintained polyfunctional GzmB, IFN{gamma} and TNF expression across cohorts. Overall, we report human lung {gamma}{delta} T cells are regionally distinct with conserved functionality in a fibrotic environment.

Innate immunity and innate immune cell death provide a critical first line of defense against disease. However, excess cell death leads to pathological inflammation. ZBP1 is an innate immune sensor that is central to this balance between defense and inflammation as a driver of inflammatory lytic cell death, PANoptosis. Activation of ZBP1-dependent PANoptosis downstream of diverse triggers has roles in both host defense and disease pathology, making ZBP1 an attractive therapeutic target. Therefore, understanding the distinct roles of ZBP1 in different cell types, organ systems, and tissues is critical to identify therapeutic strategies. Although ZBP1 regulates PANoptosis in multiple cell types, there are limited tools to interrogate its function in a cell type-specific manner. Here, we report the generation of a Zbp1-floxed mouse line (Zbp1fl/fl) for investigation of ZBP1 in distinct cell populations. We crossed Zbp1fl/fl mice to LysMcre mice to selectively deplete Zbp1 from the myeloid compartment, which did not alter immune homeostasis. Bone marrow-derived macrophages (BMDMs) from Zbp1fl/fl mice had normal ZBP1 expression and PANoptosis activation, while those from Zbp1fl/flLysMcre mice exhibited markedly reduced ZBP1 expression and were biochemically and functionally protected from ZBP1-driven PANoptosis; these effects were validated using known triggers of the ZBP1-PANoptosome--IAV, nuclear export inhibition plus IFN, and ethanol. These findings demonstrate this new Zbp1fl/fl mouse as a versatile tool that can be utilized with a variety of Cre-drivers to study ZBP1 in a wide array of distinct cell types. Given the critical role of ZBP1 in disease, this tool will inform the development of therapeutic strategies.

AbstractRegulatory T cells (Tregs) prevent autoimmunity through suppressive functions largely programmed by the transcription factor FOXP3. Healthy humans express approximately equivalent levels of two major alternatively spliced isoforms of FOXP3: a full-length version containing all coding exons (FOXP3-FL) and a version lacking exon 2 (FOXP3-{Delta}E2). However, sole FOXP3-{Delta}E2 expression causes lethal IPEX syndrome, and the FOXP3-{Delta}E2 isoform is elevated in several autoimmune diseases. These observations strongly suggest defects in suppression by FOXP3-{Delta}E2 Tregs which we investigated here using Foxp3-{Delta}E2 mice. In an influenza virus infection model, Foxp3-{Delta}E2 mice had an increased magnitude of the CD8+ T cell response during acute and memory formation phases of infection. Transcriptomic and chromatin accessibility analyses of homeostatic Foxp3-{Delta}E2 Tregs revealed impaired Treg programming, including reduced expression of inhibitory molecules such as Il2ra and chemokine receptors. Decreased cell surface CD25 expression on Foxp3-{Delta}E2 Tregs was associated with reduced IL-2 responsiveness in Foxp3-{Delta}E2 Tregs and, reciprocally, increased IL-2 responsiveness in CD8+ T cells from Foxp3-{Delta}E2 mice. Additionally, altered chemokine receptor expression resulted in diminished localization of Foxp3-{Delta}E2 Tregs to the T cell zone of the inflamed lymph node. Thus, Treg programming by the Foxp3-{Delta}E2 isoform impairs suppressive function, resulting in failure to restrain CD8+ T cells and aberrant immune responses. One Sentence SummaryFoxp3-{Delta}E2 expressing regulatory T cells have altered cellular programming which impairs their IL-2 sink function and co-localization with conventional T cells during priming, enhancing CD8+ T cell responses.

T cell development in the thymus requires tightly coordinated transcriptional programs that regulate lineage commitment, proliferation and differentiation. While key transcription factors controlling these processes have been extensively characterized, the contribution of the low expressed nuclear receptor Liver Receptor Homolog 1 (LRH-1, Nr5a2) in T cell development remains unexplored. Here, we investigated the role of LRH-1 in thymocyte maturation using an inducible ex vivo deletion system and in vivo Lck-Cre- and CD4-Cre-mediated LRH-1 knockout mouse models. We demonstrate that inducible LRH-1 deletion impairs early thymocyte development, identifying LRH-1 as a critical regulator of the double negative (DN)2/DN3 to DN4 transition. Early Lck-Cre-mediated deletion of LRH-1, but not CD4-Cre-mediated deletion at the double positive stage, resulted in markedly reduced thymic size and cellularity, indicating a stage-specific requirement for LRH-1 during thymopoiesis. Lck-Cre-mediated LRH-1 deletion led to a decreased frequency of mature CD4 T cells in peripheral lymphoid organs, while the remaining mature T cells were predominantly Cre reporter-negative and therefore escaped LRH-1 deletion. CD4 T cells that escaped Cre-mediated LRH-1 deletion exhibited impaired T cell activation marker expression and cytokine secretion. In vivo, these defects resulted in attenuated T cell effector function and compromised regulatory T cell-mediated protection in a T cell transfer model of colitis, indicating impaired effector and regulatory T cell function under (patho)physiological conditions. Collectively, our findings identify LRH-1 as a critical, previously unrecognized regulator of early thymocyte development, and establish its essential role in shaping functional peripheral CD4 T cell-mediated immune responses.

Regulatory T (Treg) cells are central mediators of immune tolerance and are generally considered to rely predominantly on mitochondrial metabolism rather than glucose-driven glycolysis. To define the role of glucose metabolism in Treg cells, we investigated the contribution of the hexose transporters GLUT1 and GLUT3. Genetic ablation of GLUT1 in T cells or selectively in Treg cells had minimal impact on Treg cell numbers, phenotype or immune homeostasis, indicating that GLUT1 is largely dispensable in this lineage. By contrast, deletion of GLUT3 in T cells resulted in a marked reduction in Treg cell numbers. However, it remained unclear whether this reduction reflected diminished IL-2 production by GLUT3-deficient conventional T cells or a cell-intrinsic requirement for GLUT3 in Treg cells. To investigate this, we generated mice with Treg cell-specific deletion of GLUT3. These animals developed severe systemic inflammation accompanied by lethal cellular and humoral autoimmunity. Mechanistically, GLUT3-deficient Treg cells exhibited reduced glycolytic activity and mitochondrial respiration, leading to impaired suppressive function and defective effector and follicular Treg cell differentiation. Collectively, our findings demonstrate a non-redundant requirement for GLUT3 in Treg cell metabolic fitness and immune regulation, refining the prevailing view that Treg cells operate largely independently of glucose metabolism. Our data further suggest that therapeutic strategies targeting glucose uptake and glycolysis in autoimmune and inflammatory diseases should account for potential adverse effects on Treg cell-mediated immune tolerance.

Mitochondrial dynamics are critical for T cell activation, differentiation, and survival. The inner mitochondrial membrane ATP-dependent metalloprotease YME1L1 regulates proteostasis and the processing of optic atrophy protein 1 (OPA1), thereby shaping mitochondrial cristae architecture and respiratory function in many cell types. Whether YME1L1 fulfils similar roles in lymphocytes remains unknown. Here, we examined YME1L1 function in T cells using conditional knockout mice lacking YME1L1 in lymphocytes (YME1L1{Delta}TB). YME1L1 expression increased upon T cell activation, yet its absence did not alter thymic development, peripheral T cell homeostasis, or the proportions of naive, memory, and regulatory subsets. T cell activation and proliferation in response to anti-CD3{varepsilon} stimulation were also unaffected. Mitochondrial parameters such as mass, membrane potential, and reactive oxygen species production, were largely preserved, with only modest, transient increases in oxidative stress detected in CD4 T cells lacking YME1L1. Electron microscopy revealed no major changes in mitochondrial size or roundness but showed increased cristae branching and reduced tortuosity, indicating subtle alterations in ultrastructure. Additionally, {gamma}{delta} T cells in YME1L1{Delta}TB mice exhibited a mild shift toward interferon-{gamma}-producing phenotypes at the expense of interleukin-17-producing subsets. Collectively, our data indicate that YME1L1, despite its requirement for OPA1 cleavage, is dispensable for T cell development and acute activation but may contribute to fine-tune mitochondrial architecture and {gamma}{delta} T cell effector programming. These findings highlight cell-type-specific redundancies in mitochondrial quality control and underscore the value of negative data in refining the understanding of mitochondrial regulation in immune cells.

Francisella tularensis is a Gram-negative bacterium that causes tularemia, a fatal zoonotic disease. F. tularensis has been used in the bioweapon programs of several countries. Its potential use as a bioterrorism agent led the CDC to classify F. tularensis as a Tier 1 Select Agent. The cytosolic sensor absent in melanoma 2 (Aim2) detects double-stranded DNA in the cytosol of infected cells and subsequently assembles a multiprotein complex known as the inflammasome. Inflammasome activation drives the secretion of IL-1{beta} and IL-18, key proinflammatory cytokines required for controlling F. tularensis infection. Prior studies have shown that F. tularensis actively suppresses Aim2 inflammasome activation; however, the underlying mechanism remains unknown. We hypothesized that F. tularensis suppresses Aim2-mediated responses by modulating the intracellular redox environment. We utilized an F. tularensis mutant lacking OxyR ({Delta}oxyR), a transcriptional regulator that controls the expression of major antioxidant enzymes. Our results show that macrophages infected with the {Delta}oxyR mutant exhibit significantly higher levels of Aim2-dependent Caspase-1 and IL-1{beta} than those infected with wild-type bacteria. The expression of interferon regulatory factor 1 and the guanylate-binding proteins GBP2 and GBP5, upstream signaling components of the Aim2 inflammasome, is markedly higher in {Delta}oxyR-infected macrophages than in controls. These changes were absent in {Delta}oxyR-infected NADPH oxidase-deficient macrophages, which are unable to generate reactive oxygen species. Collectively, these findings demonstrate that macrophage redox environment plays a key role in activating signaling components required for Aim2 inflammasome activation. This work advances our understanding of how F. tularensis-encoded factors subvert host innate immune defenses.

Germinal centers (GCs) are the site of antibody affinity maturation by the process of somatic hypermutation (SHM) and produce long-lived plasma cells (PCs). GC B cells circulate between two distinct zones: the light zone (LZ) and the dark zone (DZ). It has been demonstrated that the transcription factor NF{kappa}B cRel is required for B cell proliferation and GC B cell maintenance. cRel-deleted GC B cells show reduced SHM and affinity maturation. In contrast, transgenic overexpression of cRel does not affect SHM and results in little or no increase in affinity maturation. Therefore, the function of cRel in regulating SHM and GC B cell-derived PC generation remains unknown. To understand the function of cRel in GC B cell selection, we have used cRel fluorescence reporter mice, which provide insight into cRel function in GC B cells at the level of natural expression variation. We found that about 6-9% of LZ cells increased cRel expression compared to DZ cells, and high cRel-expressing LZ cells showed increased SHM and increased expression of IRF4 and cyclin D2, though cMyc expression remained similar. Combining single-cell RNA-Seq and flow cytometry, we revealed that high cRel-expressing LZ cells are enriched for precursor PCs (pre-PCs), not precursor memory B cells (pre-MBCs). Our findings provide insight into the physiologic function of cRel in regulating GC B cell output.

TNF is a potent proinflammatory cytokine that can induce cell death by activating the kinase RIPK1. The adaptor proteins TANK and AZI2 protect against cell death by recruiting TBK1 to the TNF receptor signaling complex, thereby inhibiting RIPK1. While deficiency of either adaptor alone is well tolerated, combined loss of TANK and AZI2 results in partial embryonic lethality and severe TNF- and RIPK1-driven autoinflammation. Here, we show that TANK/AZI2-deficient mice exhibit a striking expansion of regulatory T cells (Tregs), most of which display an effector phenotype with high expression of immunosuppressive genes. Although thymic Treg generation is modestly increased, Tregs arise predominantly in the periphery through a largely cell-intrinsic mechanism. The marked accumulation of effector Tregs suggested that the T-cell compartment may limit TNF-driven pathology in this model. Supporting this, T cell ablation in TANK/AZI2-deficient mice markedly exacerbates disease progression and enhances TNF-driven, RIPK1-mediated inflammation. Similarly, T cells protect against acute TNF-induced systemic inflammatory response syndrome by limiting RIPK1-mediated cell death. Together, our findings identify TANK and AZI2 as negative regulators of Treg formation, demonstrate that T cells restrain TNF-driven inflammation by limiting RIPK1-dependent cell death, and suggest that this protective effect is mediated primarily by Tregs.

Although the endolysosome system is central to intracellular recycling, signal transduction, and intercellular communication via exocytosis, its role in immunoregulation remains incompletely defined. We recently identified that CD4+ T cell-specific depletion of BLOC1S1, a component of multiprotein complexes regulating endolysosomal biology, predisposes toward type 2 (Th2) immunity. We therefore hypothesized that the study of BLOC1S1-deficient CD4+ T cells would expand our understanding of endolysosomal dynamics in Th2 function. Here, we demonstrate that CD4+ T cell BLOC1S1 deficiency resulted in aberrant lysosomal distribution, accumulation of endosomal vesicles, and increased exocytosis, which collectively correlated with enhanced Th2 immune responses. The phenotype was associated with upregulation of key components of the exocytosis machinery, including RAB11 and VAMP7. Functional inhibition of these vesicle trafficking proteins following siRNA knockdown of RAB11 and VAMP7 significantly attenuated Th2 cytokine secretion in BLOC1S1-deficient CD4+ T cells, highlighting their essential role in exosome-mediated cytokine export. Furthermore, exosomes derived from BLOC1S1-deficient CD4+ T cells promoted Th2 polarization in recipient cells, indicating a mechanism of intracellular amplification. Together, these findings identify BLOC1S1 as a critical regulator of lysosomal dynamics and exocytic vesicle fusion, thereby linking intracellular trafficking mechanisms to Th2 immune regulation.

Immune phenotyping represents a pillar in diagnostics, characterization of new genetic defects, and understanding mechanisms of diseases. Cell population distribution often does not cover the intrinsic function changes that may contribute to disease. Outcome of signaling activation can be used as proxy for cell function. To overcome the limitation of sample availability and standardization of signaling assays, we developed a multiplex full spectrum cytometry phosphoflow assay allowing the study of 6 phospho-proteins representing BCR/TCR, MAPK, PI3K/Akt/mTOR and canonical NF-{kappa}B signaling pathways in 18 immune cell subpopulations. Maximal stimulation and temporal dynamics were studied in response to pan-stimuli, activating cells regardless of receptor, and targeted stimuli for T, B, and innate immune cells. We studied healthy individuals between 1-69 years and discovered subpopulations-specific responses. Furthermore, pediatric donors showed broad differences in B cell and T cell function compared to adults. Hence, we established a tool to assess multiple signaling pathways at once and provide age- and subpopulation-specific references for signaling outcome. SummaryMultiplex full spectrum flow cytometry-based phosphoflow assay across 18 immune cell subpopulations, 6 phospho-proteins in response to 6 stimuli at 4 time points in individuals aged 1-69 years, reveals distinct age- and subpopulation-associated signaling patterns in magnitude and dynamics of pathways activation.

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

T cells play an essential role in protecting tissues against pathogens and regulating tissue homeostasis. Previous studies highlight that T cells display tissue-specific phenotypic and functional properties, suggesting that T cells adapt to their local environment. Whether inflammation disrupts tissue-specific T cell adaptation remains poorly understood. To address this open question, we examined the T cell compartment - including conventional CD4 and CD8 T cells, regulatory T cells, {gamma}{delta} T cells, and MAIT cells - from healthy and inflamed human mucosal tissues. Using high-parameter spectral flow cytometry, we examined phenotype ex vivo and the functional capacity following stimulation, utilizing conventional gating and unsupervised clustering analysis approaches. Overall, we analyzed 65 tissue samples including mild, moderate, and severely inflamed oral gingiva, healthy and inflamed lung, along with healthy and inflamed tissue from the decidual-placental interface. Across these mucosal barrier tissues, we find that tissue location plays a dominant role in shaping the composition, phenotype, and functional capacity of the T cell compartment. Importantly, these tissue-specific adaptations were largely maintained during states of tissue inflammation. This included the ability to exert tissue repair functions, which was preserved across T cell subsets, even in severely inflamed tissues.

GPR65 has been shown to be a critical regulator of Th17 cell pathogenicity. Loss of GPR65 in mice results in a decrease in Th17 cells and reduced susceptibility to a mouse model of multiple sclerosis. The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of Th17 cells. In this study we performed RNA-seq to identify Th17 genes regulated by the CREB/CRTC2 pathway. Our RNA-seq analysis led us to uncover the first mechanism of regulation of the orphan receptor GPR65 by the CREB/CRTC2 pathway. We show that GPR65 is a target of the CREB/CRTC2 pathway through expression studies and chromatin immunoprecipitation. In addition, we show that targeting GPR65 with small molecules alters the expression of IL-17A. Understanding the regulation of GPR65 will be crucial in developing small molecules to treat patients with Th17 cell-mediated disorders.

Patients with a dominant mutation in the Rho GTPase RAC2, RAC2E62K, which hyperactivates the protein, suffer from a combined immunodeficiency characterized by recurrent bacterial and fungal infections and severe T cell lymphopenia. Patient neutrophils have elevated F-actin and superoxide production yet fail to control growth of S. aureus, and the mechanism underlying this killing defect is unknown. Here we report that hyperactive Rac2 primes neutrophils for primary granule degranulation, potentially depleting myeloperoxidase (MPO) needed for intraphagosomal microbial killing. Using a Rac2+/E62K mouse model, we show that mature bone marrow neutrophils have decreased side scatter, elevated surface CD63, and reduced intracellular MPO. Interestingly, bone marrow architecture and neutrophil development in the mice are normal. Rac2+/E62K neutrophils are hyperactivated, with increased CD11b expression, cell spreading, and bioparticle phagocytosis. In the spleen, Rac2+/E62K mice display extramedullary granulopoiesis and an accumulation of degranulating neutrophils. Splenic T cells, but not B cells, show elevated surface phosphatidylserine, an "eat me" signal that sensitizes them to phagocytic clearance and provides a candidate mechanism for the selective T cell lymphopenia. Together these findings suggest that hyperactive Rac2 compromises antimicrobial neutrophil function and drives selective T cell clearance in the spleen.

Systemic lupus erythematosus (SLE) is associated with infection susceptibility and altered innate immune function. Monocyte metabolism is linked to appropriate cytokine release and bacterial containment. We investigated cytokine production and metabolic programming in the monocyte population from SLE patients and healthy controls following lipopolysaccharide (LPS) stimulation. SLE monocytes displayed increased IL-10, TNF, and IL-8 production, with impaired IL-1{beta} induction. Metabolic profiling revealed altered substrate use, with increased glucose dependence and reduced fatty acid and amino acid oxidation after LPS stimulation. SLE patients exhibited reduced numbers of classical monocytes, expansion of intermediate monocytes, and dysregulated subset-specific metabolic reprogramming in response to LPS. This descriptive study provides a cornerstone for (i) understanding infection susceptibility in SLE, (ii) subset-resolved immunometabolic profiling as a tool in autoimmunity, and (iii) developing future metabolic-targeted therapeutic strategies HighlightsO_LIDescriptive mapping shows SLE monocytes are proinflammatory with glucose dependence after LPS C_LIO_LIClassical and intermediate SLE subsets show divergent baseline metabolic preferences versus healthy C_LIO_LISLE subsets display aberrant LPS responses, i.e.. increased glucose and reduced fatty acid oxidation C_LIO_LIThis study provides a cornerstone for subset-resolved immunometabolism in infection susceptibility. C_LI

PRV-101 is a multivalent formalin-inactivated Coxsackievirus B (CVB) vaccine developed to prevent CVB infections, which are associated with increased risk of islet autoimmunity. While PRV-101 induces robust neutralizing antibody responses, its T-cell immunogenicity is unknown. We analyzed peripheral blood mononuclear cells from 25 healthy adults receiving three high or low PRV-101 doses or placebo in a Phase I randomized, placebo-controlled trial. CVB-reactive CD8 T-cell responses were assessed using HLA Class I multimers, and CD4 and T follicular helper (Tfh) responses were measured by activation-induced marker assays following stimulation with a CVB peptide library. PRV-101 elicited minimal CVB-reactive CD8 T-cell responses but robust CD4 and Tfh responses, peaking at week 12 and persisting through week 32. Responses were observed in both seronegative and seropositive individuals, consistent with effective immune priming and boosting. Tfh frequencies correlated with neutralizing antibody titers. Female participants exhibited higher peak Tfh responses than males. We conclude that PRV-101 elicits a CVB-protective immune profile, dominated by Tfh responses supporting durable humoral immunity and devoid of potentially diabetogenic cytotoxic T-cell responses. This profile invites further investigations in vaccine trials for type 1 diabetes prevention.

Viral pathogens cause neurologic sequelae during acute and post-acute phases of infection. CD8+ T cells are hypothesized to contribute to these effects, but the mechanisms through which they act are poorly understood. We posited that viral infections and/or antiviral immune responses induce DNA damage, which may underlie neuronal dysfunction. Using a model of neurotropic flavivirus infection, we found that genes associated with interstrand crosslinking (ICL) DNA damage were upregulated post-infection, temporally congruent with T cell infiltration. Using an in vitro co-culture system, our results demonstrate that CD8+ T cells induced ICL-like damage in primary neurons, independent of antigen-specific interactions or direct contact. Human transcriptomic data also showed overexpression of genes associated with ICL damage in the brains of people with Parkinsons disease, Alzheimers disease, and multiple sclerosis, which are neurologic diseases characterized by neuroinflammation. Together, these data indicate that CD8+ T cells cause genotoxic DNA damage in neurons, which may underlie the neurologic dysfunction seen in neurodegenerative conditions. SummaryResults indicate that CD8+ T cells induce interstrand crosslinking-like DNA damage in neurons independent of antigen-specificity in a mouse model of viral infection, in vitro primary cell culture system, and human neurologic diseases. These findings provide insight on the mechanistic connection between neuroinflammation and neurologic dysfunction.