Owing to their immunoprotective properties, natural killer (NK) cells are critical for the innate immune response to pathogens, as well as a new wave of cancer immunotherapy that harnesses natural cytotoxicity. We sought to study the genetic and epigenetic drivers behind human-specific NK cell receptors, so that we can better understand the underlying cellular function. Here, we present a transcriptomic, proteomic (CITE-seq), and chromatin (single nuclei ATAC-seq) profiling of human peripheral NK cell subsets, which was then compared with genomic databases. Through integrative multi-omics, we demonstrate that CD56(bright) versus CD56(dim) NK cell subsets have differential distal regulatory element (DRE) landscapes, with fewer accessible DREs in the CD56(dim) NK cells. We combine our epigenetic data, deposited Hi-C, and human genetic data to show mechanisms governing the NCAM1 (encoding CD56) and the killer cell immunoglobulin-like receptors (KIRs) loci. We identify an NCAM1 DRE that binds STAT3 in most NK cells, while identifying a genetic cohort that has motifs for binding repressive BLIMP1 at the DRE and resulting in less CD56 expression. Together, our findings reveal novel epigenetic and transcriptomic systems for the regulation of NK cell receptors driving NK cell cytotoxicity and diversity.

Abstract Aging is a major risk factor for severe Streptococcus pneumoniae ( Spn ) infection and pneumonia-associated – major adverse cardiac events (PA-MACE), yet underlying mechanisms remain unclear. Using young and aged murine models, we show that aging exacerbates bacterial burden, mortality, and cardiac dysfunction following Spn infection, associated with impaired macrophage bacterial killing. Single-cell RNA sequencing infected hearts revealed extensive age-dependent remodeling across immune and stromal compartments. Aged mice exhibited heightened pro-inflammatory myeloid responses, with increased neutrophil infiltration characterized by elevated S100A8/9 and LCN2 and reduced antimicrobial programs. Macrophages displayed defective efferocytosis, including disruption of the GAS6–AXL axis. Aging also drove expansion of an infection-responsive fibroblast population with inflammatory signatures and reduced extracellular matrix gene expression. These changes were linked to oxidative stress and impaired glucose oxidation. Notably, anti-inflammatory treatment rescued cardiac dysfunction, implicating excessive inflammation as a central driver of PA-MACE. Together, these findings define mechanisms linking aging to pneumococcal cardiac complications and identify potential therapeutic targets.

The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive "type III" responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1b. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12RB1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. The nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12RB1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFNg, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.

CD8+ virtual memory T (TVM) cells rapidly respond to infection via antigen-independent bystander effector functions. While it is recognized that TVM cells arise independently of foreign antigen encounter, the mechanisms governing their development are not fully understood. Here, we identify the Ikaros transcription factor Aiolos as a negative regulator of TVM cell programming. We observe enhanced frequencies and numbers of TVM in the spleen, liver, and blood of unchallenged Aiolos-deficient (Ikzf3-/-) mice and in the lungs 1-day post-infection with influenza A virus (IAV). Furthermore, Ikzf3-/- TVM cells produce elevated IFN-γ and granzyme B in response to cytokine stimulation. Importantly, Aiolos-deficient mice control IAV more rapidly and exhibit reduced morbidity, indicating enhanced TVM cell functionality. Mechanistically, Aiolos represses the expression of the transcription factor Eomes and the IL-15R subunit CD122, known positive regulators of TVM gene program. Collectively, these findings establish Aiolos as a molecular repressor of TVM programming and responses. The mechanism(s) controlling CD8+ virtual memory T cell (TVM) development are still under investigation. Here, using uninfected or IAV-challenged Ikzf3-deficient mice, the authors identify the transcription factor Aiolos as a negative regulator of TVM cell development by repressing Eomes and IL-15/STAT5 signaling.

The assembly of Tcrb and Tcra genes require double negative (DN) thymocytes to undergo multiple rounds of programmed DNA double-strand breaks (DSBs), followed by their efficient repair. However, mechanisms governing cell cycle checkpoints and specific survival pathways during the repair process remain unclear. Here, we report high-resolution scRNA-seq analyses of individually sorted mouse DN3 and DN4 thymocytes, which reveals a G2M cell cycle checkpoint, in addition to the known G1 checkpoint, during Tcrb and Tcra recombination. We also show that inactivation of GSK3β by phosphorylation on Ser389 is essential for DN3/DN4 thymocytes to survive while being stalled at the G1 and G2/M checkpoints. GSK3β promotes death by necroptosis, but not by apoptosis, of DN3/DN4 thymocytes during V(D)J recombination. Failure to inactivate GSK3β in DN3 thymocytes alters the Tcrb gene repertoire primarily through Trbv segment utilization. In addition, preferential recombination of proximal V segments in Tcra depends on GSK3β inactivation. Our study identifies a unique thymocyte survival pathway, enabling them to undergo cell cycle checkpoints for DNA repair during V(D)J recombination of Tcrb and Tcra genes. Thymocyte survival during cell cycle checkpoints for V(D)J recombination DNA repair determines TCRα/β repertoire.

Double-strand breaks represent the most dangerous form of DNA damage, and in resting cells, these breaks are sealed via the non-homologous end joining (NHEJ) factor Ligase IV (LIG4). Excessive NHEJ may be genotoxic, necessitating multiple mechanisms to control NHEJ activity. However, a clear mechanism of transcriptional control for them has not yet been identified. Here, we examine mechanisms governing Lig4 transcription in mammals, finding that most tissues maintain very low levels of LIG4 production. Select tissues upregulate LIG4, employing different strategies for genomic regulation. In developing lymphocytes, the Lig4 locus is devoid of long-range chromatin contacts; instead, its expression and role in immune development depend upon a promoter-proximal intronic regulatory element. Deletion of the Lig4 intronic regulatory element results in thymocyte-specific loss of Lig4 upregulation, defects in lymphocyte development and altered antigen receptor rearrangement. Our findings show the NHEJ gene, Lig4, is transcriptionally controlled to support stage-specific function concurrent with programmed DSBs. Moreover, we provide an example of how DNA cis-regulatory elements very close to a promoter can have substantial transcriptional effects.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor overall survival. Understanding how dysregulated immunity contributes to the development and progression of AML is an active area of investigation. Prior work has demonstrated functional defects in natural killer (NK) cells; however, the role of non-NK innate lymphoid cells (ILCs) in AML is incompletely understood. Conventional ILC3s are non-cytotoxic and regulate mucosal immunity through cytokine secretion. In this study, we discovered an expansion of ILC3s in both a murine model of AML and in AML patients. The transcription factor, aryl hydrocarbon receptor (AHR) is required for ILC3 development and function, and AML blasts have been shown to secrete AHR ligands. Modeling studies demonstrated ILC3 expansion was mediated by AHR activation in ILC precursors. ILC3s developed in leukemic settings had increased cytokine production, and co-culture of ILC3s significantly increased AML colony formation, which was mediated by ILC3-derived TNFα and GM-CSF. Furthermore, co-transfer of ILC3s with AML led to more rapid disease progression in vivo and human ILC3 frequency was associated with adverse risk stratification in AML patients. These data support a model in which AML promotes ILC3 expansion and function via an AHR-dependent mechanism to aid AML growth and survival.

Double-strand breaks represent the most dangerous form of DNA damage, and in resting cells, these breaks are sealed via the non-homologous end joining (NHEJ) factor Ligase IV (LIG4). Excessive NHEJ may be genotoxic, necessitating multiple mechanisms to control NHEJ activity. However, a clear mechanism of transcriptional control for them has not yet been identified. Here, we examine mechanisms governing Lig4 transcription in mammals, finding that most tissues maintain very low levels of LIG4 production. Select tissues upregulate LIG4, employing different strategies for genomic regulation. In developing lymphocytes, the Lig4 locus is devoid of long-range chromatin contacts; instead, its expression and role in immune development depend upon a promoter-proximal intronic regulatory element. Deletion of the Lig4 intronic regulatory element results in thymocyte-specific loss of Lig4 upregulation, defects in lymphocyte development, and altered antigen receptor rearrangement. Our findings show the NHEJ gene, Lig4, is transcriptionally controlled to support stage-specific function concurrent with programmed DSBs. Moreover, we provide an example of how DNA cis-regulatory elements very close to a promoter can have substantial transcriptional effects.

The recombination of Tcrb and Tcra genes requires several cycles of programmed DNA double-strand breaks (DSBs) in double-negative (DN) thymocytes, along with effective repair mechanisms. However, the regulatory processes governing cell cycle checkpoints and survival pathways during this repair process are still elusive. Here, we report high-resolution single-cell RNA sequencing (scRNA-seq) analyses of individually sorted DN3 and DN4 thymocytes. We show the presence of a G2/M cell cycle checkpoint as well as the known G1 checkpoint during Tcrb and Tcra recombination. We demonstrate that the inactivation of GSK3β through phosphorylation at Ser389 is crucial for the survival of DN3 and DN4 thymocytes while they are stalled at the G1 and G2/M checkpoints. GSK3β promotes cell death by necroptosis, rather than by apoptosis, during V(D)J recombination of DN3 and DN4 thymocytes. If GSK3β is not inactivated in DN3 thymocytes, it can alter the Tcrb gene repertoire primarily through changes in Trbv segment utilization. GSK3b alters the nucleotide composition of the N2 region of Tcrb by suppressing the expression of the Apobec3 deaminase during Tcrb V(D)J recombination. Additionally, the preferential recombination of proximal V segments in Tcra relies on the inactivation of GSK3β, and this change is induced by necroptosis. Our study identifies a unique survival pathway in thymocytes that helps them navigate cell cycle checkpoints for DNA repair during Tcr gene V(D)J recombination. NIH R01 AI051454 Hematopoiesis and Immune System Development (HEM)

Obesity is a major public health concern in the USA, affecting over 100 million individuals and negatively impacting multiple organ systems. However, its effects on immune responses remain underexplored. In this study, we investigated how obesity alters B cell function and demonstrated that high-fat diet (HFD)-induced obesity disrupts B cell subset ratios compared to lean controls. Interestingly, obese mice developed spontaneous germinal centers but paradoxically showed reduced efficiency in responding to NP-CGG immunization and LCMV infection, indicating altered B cell development and differentiation. Adoptive transfer experiments revealed that these obesity-induced B cell dysregulations are cell intrinsic, persisting even after the transfer of B cells from obese donors to lean hosts. Further cellular and metabolic analyses we performed, highlighted comprehensive remodeling in B cells, including altered surface marker expression, a metabolic shift toward fat utilization, and modified leptin receptor signaling sensitivity. Single-cell RNA sequencing uncovered unique transcriptional profiles in B cells from obese mice both at rest and during infection. Using B cell-specific leptin receptor-deficient mice, we confirmed the critical role B cell leptin signaling plays in mediating obesity-driven changes. Collectively, these findings provide novel mechanistic insights into the impact of obesity on humoral immune responses. Start up funds from the Ohio State University College of Medicine Seed grant from OSU Infectious Disease Institute NIH / NIGMS R35 GM155038 Immune Response Regulation: Cellular Mechanisms (IRC)

Type III interferons (IFNλ) are innate immune cytokines that limit viral replication and coordinate tissue repair through the induction of interferon stimulated genes (ISGs). This response must be tightly regulated to avoid excessive responses that result in the disruption of tissue barrier integrity or inefficient responses that allow for pathogen escape. Here we examine the contribution of Mitogen Activated Protein Kinase (MAPK) signaling on IFNλ-mediated antiviral activity. We find that extracellular-signal-regulated kinase 5 (ERK5), a poorly characterized member of the conventional MAPK family, potentiates the antiviral efficacy of IFNλ. Chemical inhibition and genetic targeting of ERK5 during IFNλ treatment of cells results in a decrease in ISG induction and impaired control of viral infections. This decrease in IFNλ antiviral efficacy in the absence of ERK5 kinase activity corresponded to lowered STAT1 phosphorylation, revealing a noncanonical role for ERK5 in STAT1 activation downstream of IFNλ. In contrast, type I IFN antiviral signaling is largely resistant to ERK5 modulation. Altogether, we identify ERK5 as a potentiator of STAT1 activation, ISG expression, and antiviral activity following type III IFN stimulation.SIGNIFICANCE Regulation of type III interferons (IFNλ) at mucosal barriers in response infection to mitigate viral replication and support barrier integrity. The specific mechanistic requirements for MAPK signaling to sustain IFNλ-mediated gene expression have remained elusive. Amongt the least characterized members of the MAPK family, the role of ERK5 in regulating host inflammatory responses has been hampered by off-target effects of kinase inhibitors. Here, we combine pharmacological and genetic approaches to specifically demonstrate that ERK5 promotes antiviral immunity in epithelial cells. Mechanistically, ERK5 enhances the activation of STAT1 in response to IFN stimulation to augment the transcription of IFN-stimulated genes. Our work demonstrates that therapeutic modulation of MAPK and IFN signaling pathway co-integration could distinguish between the protective and deleterious outcomes of IFN expression.One-sentence summary ERK5 potentiates IFN lambda responses.### Competing Interest StatementThe authors have declared no competing interest.

Abstract The prevalence of cardiac dysfunction and its vast implications for human health highlight the need to investigate the mechanistic processes allowing its pathological manifestations. Studies from our lab revealed Pseudomonas aeruginosa (P.a.) infection causes severe cardiac inflammation and dysfunction, despite absence of the bacteria in the heart. However, the underlying mechanism is unknown. Thus, we hypothesize cardiac fibroblast activation by PAMPs enhances cytokine and chemokine release, driving cardiac inflammation. To test our hypothesis in vitro, we used human monocyte derived macrophages (hMDMs) and cardiac fibroblasts (HCFs). We harvested conditioned media (CM) from P.a. infected hMDMs and exposed to HCFs. RNA was extracted at different time points, next generation mRNA sequencing was performed and supernatants were harvested to determine inflammatory cytokine. Our data revealed exposing CM to HCFs upregulates cytokine and chemokine gene expression. Also, we found CCL2, TNF-α and IL-1β levels are high in supernatants harvested from HCFs. To further investigate mechanisms controlling CCL2 expression in HCFs, we identified JNK was involved in induction of CCL2 in HCF. In conclusion, activation of HCFs releases chemokines (CCL2) which recruit leukocytes into heart tissue, causing severe cardiac inflammation. Ongoing and future studies are needed to dive further into mechanisms of this pathway, showing its potential impact in driving cardiac inflammation in vivo.

In contrast to the "helper" activities of most CD4+ T effector subsets, CD4+ cytotoxic T lymphocytes (CD4-CTLs) perform functions normally associated with CD8+ T and NK cells. Specifically, CD4-CTLs secrete cytotoxic molecules and directly target and kill compromised cells in an MHC class II-restricted fashion. The functions of these cells have been described in diverse immunological contexts, including their ability to provide protection during antiviral and antitumor responses, as well as being implicated in autoimmunity. Despite their significance to human health, the complete mechanisms that govern their programming remain unclear. In this article, we identify the Ikaros zinc finger transcription factor Eos (Ikzf4) as a positive regulator of CD4-CTL differentiation during murine immune responses against influenza virus infection. We find that the frequency of Eos+ cells is elevated in lung CD4-CTL populations and that the cytotoxic gene program is compromised in Eos-deficient CD4+ T cells. Consequently, we observe a reduced frequency and number of lung-residing, influenza virus-responsive CD4-CTLs in the absence of Eos. Mechanistically, we determine that this is due, at least in part, to reduced expression of IL-2 and IL-15 cytokine receptor subunits on the surface of Eos-deficient CD4+ T cells, both of which support the CD4-CTL program. Finally, we find that Aiolos, a related Ikaros family member and known CD4-CTL antagonist, represses Eos expression by antagonizing STAT5-dependent activation of the Ikzf4 promoter. Collectively, our findings reveal a mechanism wherein Eos and Aiolos act in opposition to regulate cytotoxic programming of CD4+ T cells.

Cutaneous leishmaniasis caused by Leishmania parasites exhibits a wide range of clinical manifestations. Although parasites influence disease severity, cytolytic CD8+ T cell responses mediate disease. Although these responses originate in the lymph node, we found that expression of the cytolytic effector molecule granzyme B was restricted to lesional CD8+ T cells in Leishmania-infected mice, suggesting that local cues within inflamed skin induced cytolytic function. Expression of Blimp-1 (Prdm1), a transcription factor necessary for cytolytic CD8+ T cell differentiation, was driven by hypoxia within the inflamed skin. Hypoxia was further enhanced by the recruitment of neutrophils that consumed oxygen to produce ROS and ultimately increased the hypoxic state and granzyme B expression in CD8+ T cells. Importantly, lesions from patients with cutaneous leishmaniasis exhibited hypoxia transcription signatures that correlated with the presence of neutrophils. Thus, targeting hypoxia-driven signals that support local differentiation of cytolytic CD8+ T cells may improve the prognosis for patients with cutaneous leishmaniasis, as well as for other inflammatory skin diseases in which cytolytic CD8+ T cells contribute to pathogenesis.

Abstract Endometrial cancer (EC) responds variably to immune checkpoint blockade, warranting study of the tumor microenvironment (TME). Natural killer (NK) cells are cytotoxic innate lymphocytes that can directly kill tumor cells, yet their diversity, locations, and functions in the EC TME are unknown. CITE-seq analysis of fresh EC tumor samples revealed multiple NK cell populations in the TME, with distinct patterns of surface activating and inhibitory receptor expression and less cytolytic granule expression by tumor-derived conventional NK (cNK) and tissue-resident NK (trNK) cells compared to autologous blood cNK. Ex vivo flow cytometry revealed significantly lower expression of cytolytic granules by TME-derived NK cell subsets (granzyme B MFI: blood cNK 1.5e5 +/- 5.4e4, tumor cNK 7.9e4 +/- 2.9e4 p<0.05, tumor trNK 3.7e4 +/- 1.4e4 p<0.05, n=10; perforin MFI: blood cNK 1.3e4 +/- 3.1e3, tumor cNK 1.0e4 +/- 3.2e3 p=0.19, tumor trNK 3.5e3 +/-1.3e3 p<0.01, n=8). To identify NK cell subsets within the TME, we performed immunohistochemistry using CD3, CD56, granzyme B, and CD103 to demarcate cNK and trNK, respectively. We observed granzyme B+ cells primarily in the stroma (78.6 +/- 3.7% Stroma, 21.4 +/- 3.7% Glands, n=10, p<0.05) and CD103+ cells primarily among tumor cells (15.6 +/- 4.5% Stroma, 84.3 +/- 0.64% Glands, n=10, p=0.07). These data suggest that both cNK and trNK cell cytotoxicity is suppressed in the EC TME and ongoing work seeks to elucidate the mechanisms.

Epstein-Barr virus (EBV) is deemed a necessary, yet insufficient factor in the development of multiple sclerosis (MS). In this study, myelin basic protein-specific transgenic T cell receptor mice were infected with murid gammaherpesvirus 68 virus (MHV68), an EBV-like virus that infects mice, resulting in the onset neurological deficits at a significantly higher frequency than influenza or mock-infected mice. MHV68 infected mice exhibited signs including optic neuritis and ataxia which are frequently observed in MS patients but not in experimental autoimmune encephalomyelitis mice. MHV68-infected mice exhibited increased focal immune cell infiltration in the central nervous system. Single cell RNA sequencing identified the emergence of a population of B cells that express genes associated with antigen presentation and costimulation, indicating that gammaherpesvirus infection drives a distinct, pro-inflammatory transcriptional program in B cells that may promote autoreactive T cell responses in MS.