bluuebunny/crossref_metadata_2025 huggingface dataset:
Itaconate deficiency results in prolonged bacterial burdens and enhanced T cell responses during Francisella tularensis infection
Authors: Leighton Ian , Roberts Lydia , Wehrly Tara , Bosio Catharine M
Published: February 2023

Shifts in cellular metabolism and associated metabolites regulate immune cell function. Recently, it has been shown that the metabolite itaconate plays a critical role in modulating inflammatory responses. Using animals that cannot synthesize itaconate due to an absence of aconitate decarboxylase 1 (AOCD KO), we previously established the lack of itaconate increased susceptibility to high dose challenge with the live vaccine strain (LVS) of Francisella tularensis in terms of both bacterial clearance and survival. However, ACOD KO mice that survived sublethal LVS infection had improved survival against a lethal challenge with virulent F. tularensis (Ftt). Since appropriate coordination of both macrophage and T cell function is essential for surviving Ftt, we next determined if the improved survival of ACOD KO mice was due to alterations in macrophage and/or T cell function. LVS immune ACOD KO mice had increased numbers of lung resident and splenic effector CD4+ and CD8+ T cells compared to WT mice following resolution of infection. Utilizing an in vitro macrophage-T cell co-culture system we did not observe any difference in the ability of WT and ACOD KO T cells to control LVS replication in macrophages when T cells were co-cultured with genetically matched macrophages. However, when macrophage sources were mixed, we observed that regardless of the source of LVS immune T cells, WT macrophages controlled bacterial replication significantly better than ACOD derived macrophages. Together, these findings suggest that the lack of itaconate results in increased and prolonged bacterial burdens in vivo, which leads to greater expansion of effector T cells that confer enhanced protection during Ftt challenge.
Chromatin accessibility differences of human monocyte identify age associated inflammatory heterogeneity.
Authors: Singh Amit K , Kaileh Mary , Mazan-Mamczarz Krystyna , Palagani Ajay , Roy Roshni , Sarantopoulou Dimitra , Ubaida-Mohien Ceereena , Moore Ann Zenobia , Qian Yong , Yin Jinhu , Bektas Arsun , Ding Jun , De Supriyo , Zukley Linda , McKelvey Julia , Weng Nan-Ping , Ferrucci Luigi , Sen Ranjan
Published: February 2023

Human aging is associated with low grade chronic inflammation termed, inflammaging, which is a significant risk factor for morbidity and mortality in elderly. The innate immune system is master regulator of this process via regulating expression of cytokines and other key mediators. We assayed chromatin landscape of the human monocytes during healthy aging with an aim to predict the inflammation associated contemporary and imminent states of monocytes. Our deep sequencing ATAC-seq data from healthy donors of the GESTALT cohort identified, chromatin landscape signatures defining heterogeneity of monocytes. Differentially open chromatin (DOC) clustered old individual monocytes in two distinct groups, while young donors demonstrated ethnicity-based differences. DOC’s heterogeneity of old donors was characterized by presence of intergenic enhancer-like elements dominated by the DNA binding motif of the transcription factor NF-κB. We identified the relationships of age-associated DOC with DNA methylation, gene expression and proteome of monocytes and serum. Finally, the intracellular cytokine staining analysis using multi-parameter flow cytometry demonstrated the DOC’s influence on the monocyte’s response to LPS stimulation. Our data revels the age associated heterogenous functional states of monocytes which can help us understanding decision making mechanisms during an immune response.
Non-Thermal Histotripsy Ablation of Osteosarcoma Causes a Local Acute Immune Response
Authors: Sereno Jacqueline , Hendricks Alissa Danielle , Allen Irving C , Vlaisavljevich Eli , Imran Khan Mohammad , Tuohy Joanne
Published: February 2023

While surgery, chemotherapy, and radiotherapy are available as treatments for osteosarcoma, there is only a 60% 5 year overall survival rate. To address this need, novel therapies are being developed and investigated. Histotripsy is a non-invasive, non-thermal focused-ultrasound ablation therapy that uses acoustic cavitation to mechanically lyse targeted cells. Recent evidence suggests that it is immunomodulatory, and can shift the tumor microenvironment to being more pro-inflammatory. However, given histotripsy’s mechanical mechanism, it is not established to be able to treat stronger tissues such as osteosarcomas at a dose that will allow for the preservation of immune stimulating molecules. In this study, mice with subcutaneous osteosarcoma DLM8 tumors were treated with histotripsy and acute changes to the local tumor immune response were analyzed 72 hours later. Using partial ablation to avoid off-target damage, a 40% average reduction in tumor size was achieved with targeted tissues being fully ablated. Through rtPCR, the levels of various cytokines associated with wound healing were significantly increased in the treated tumors following histotripsy treatment, with the greatest fold change observed in the concentrations for IL-6 and IL-13 over a given period of time. An increase in fold change for CD274, also known as PD-L1, was also observed, implicating the potential to pair histotripsy with checkpoint inhibitors for osteosarcoma therapy. The overall results of the study suggest that histotripsy is able to shift the tumor microenvironment following partial-ablation treatment. Thus, upon activation of the immune system, wound and tissue repair mechanisms are subsequently activated and responses are regulated.
Effect of the elderly human lung mucosa on Mycobacterium tuberculosis infection of the alveolar epithelium
Authors: Olmo-Fontanez Angelica M. , Scordo Julia M , Garcia-Vilanova Andreu , Maselli-Caceres Diego , Peters Jay , Clemens Daniel , Turner Joanne , Schlesinger Larry S. , Torrelles Jordi B.
Published: February 2023

As we age, there is an increased risk for developing tuberculosis (TB), a disease caused by the infectious agent Mycobacterium tuberculosis (M.tb). M.tb infection results in a bacterial deposition in the lung alveolus, where M.tb is bathed in alveolar lining fluid (ALF). We have published that age-associated changes in human ALF soluble components, such as increased levels of oxidized and dysfunctional innate proteins, accelerate M.tb growth within human alveolar macrophages. We are interested in studying the impact of human ALF on M.tb infection of non-professional phagocytes, such as alveolar epithelial cells (ATs). We hypothesized that M.tb exposure to elderly human ALF (E-ALF) drives increased M.tb replication and growth within ATs due to impairment of E-ALF innate soluble components. We observed that E-ALF exposed M.tb had significantly increased intracellular growth in ATs. Despite changes in intracellular bacterial growth, infected ATs with E-ALF-exposed M.tb did not show altered production of inflammatory mediators or cell activation. Interestingly, M.tb exposure to E-ALF did not alter intracellular trafficking, but it drove bacterial translocation to both endosomal and cytosolic compartments in ATs. Overall, rapid bacterial replication and increased growth within ATs was observed in M.tb exposed to E-ALF, and together with inadequate AT activation, could lead M.tb to potentially exploit the AT cytosol as a favorable intracellular niche for replication. These results emphasize the effects of elderly lung mucosa on M.tb infection of non-professional phagocytes (ATs), which may conceivably explain why the elderly population is more vulnerable to developing active TB disease.
SOCS1 mimetics attenuate autoimmune pathology in a murine model of lupus
Authors: Sharma Jatin , Collins Teresa , Lam Brandon , Cain Antia , Roach Tracoyia Roach , Cornaby Caleb , Polk Timothy B , Morel Laurence , Larkin Joseph
Published: February 2023

Autoimmune diseases are driven, in large part, by the aberrant activation of lymphocytes which mediate a dysregulated cytokine milieu and produce pathogenic, auto-reactive antibodies. Notably, many cytokines, including interferon gamma (IFN-γ), utilize the JAK/STAT pathway for signal propagation. Suppressor of Cytokine Signaling-1 (SOCS-1) is an inducible, intracellular protein known to regulate IFN-γ signaling through dampening of the JAK/STAT signaling cascade. We have previously shown that a peptide mimicking the kinase inhibitory region of SOCS1 (SOCS1-KIR) inhibited IFN-γ signaling and inflammation-driven disease progression. Using Fas deficient MRL/lpr mice, which spontaneously develop a lupus-like disease, we tested the hypothesis that SOCS1-KIR administration would alter lymphocyte phenotype and modulate lupus-associated pathologies. SOCS1-KIR administration reduced severity of skin lesions, autoantibody production, and modestly reduced kidney pathology. On a cellular level, peritoneal SOCS1-KIR administration enhanced Foxp3 expression in total splenic and follicular regulatory T cells, reduced the effector memory/naïve T lymphocyte ratio for both CD4+ and CD8+ cells, and reduced the frequency of germinal center B cells. Together, these data show that SOCS1-KIR treatment altered lymphocyte phenotype and suggest that therapeutic targeting of the SOCS1 pathway through peptide administration may have efficacy in mitigating autoimmune pathologies.
Countering Inflammatory Arthritis by 14-3-3zeta: A Novel Target
Authors: Chakravarti Ritu , Kim Joshua , Chun Krista , Durgala Johnathon , Joe Bina , Holoshitz Joseph , Chattopadhyay Saurabh
Published: February 2023

Inflammatory arthritis (IA) is a common inflammatory joint-destructive condition that affects millions of individuals worldwide. Increased immune dysfunction and bone loss have been observed both in IA affected humans and in animal models. Proinflammatory events responsible for RA pathogenesis have been studied in detail; however, the protective mechanism remains under-explored. Earlier, we reported that 14-3-3zeta (z) is a molecular determinant of skewing T cell polarization and interleukin-17A (IL-17A) signal transduction. Here we demonstrate that 14-3-3z is an endogenous suppressor of IA. Loss of 14-3-3z in a novel CRISPR/Cas9 edited knockout (KO) rat resulted in early-onset and severe arthritis in animal models of inflammatory arthritis. Arthritic 14-3-3z KO animals showed increased bone loss and immune cell infiltration in synovial joints. Rescue experiments to supplement the 14-3-3z by immunization during the pre-symptomatic phase resulted in significant arthritis suppression in both wild-type and 14-3-3z KO rats. Immunization generated a robust increase in 14-3-3z antibody and IL-17A, suppressed several cytokines, including IFN-g, TNF-a, and Cxcl-1, preserved tissue mineral bone density, and trabecular bone volume. Our findings, taken together with the prior report of the molecular function of 14-3-3z as an antigen, indicate that antigenic 14-3-3z is responsible for a previously unrecognized inflammatory arthritis-suppressive mechanism.
A metabolically engineered bacterial vaccine protects against arthritis in mice
Authors: de Figueiredo Paul , Guo Fengguang , Das Jugal , Ding Yufang , Jayaraman Arul , Alaniz Robert Christopher , Song Jianxun
Published: February 2023

Arthritis is an autoimmune disease that adversely affects the quality of life of patients. Treatment options for autoimmunity include the use of chemical and biologic agents that suppress inflammation. However, these can result in patients being vulnerable to opportunistic infections. Adoptive T regulatory (Treg) cell transfer can specifically block auto-inflammatory immune cells in an antigen-specific manner while maintaining an intact immune response to pathogens. However, isolating sufficient Treg cells for adoptive cell transfer therapy is challenging; therefore, improving Treg cell survival and function could have important impacts on adoptive Treg transfer therapy. To explore the use of bacterial vaccines as autoimmune therapies, we engineered an attenuated brucellosis vaccine (Brucella melitensis ΔvjbR Tna) to produce indole, a bacterial metabolite that induces Treg expansion. In the collagen induced arthritis (CIA) murine model, Treg transfer following BmΔvjbR Tna treatment decreased arthritis incidence and score. CD4+FoxP3+ T regulatory cells and CD8+ T cells in spleen and lymph nodes were increased in BmΔvjbR Tna treated CIA mice. BmΔvjbR Tna injection also promoted CD4+ cell proliferation, and increased IL-2 and IFN-g expression. The levels of some pro-inflammatory cytokines (IL-1β and IL-6) were also dramatically reduced in BmΔvjbR Tna infected bone marrow derived macrophages (BMDMs). Taken together, these findings indicate that the indole-producing BmΔvjbR Tna strain increases Treg cell populations and proliferation, suppresses proinflammatory cytokine secretion, and reduces autoimmune responses.
Preclinical development of universal SNAP-CAR T cell therapy
Authors: Ruffo Elisa , Kvorjak Michael , Adams Eric , Lohmueller Jason
Published: February 2023

Chimeric antigen receptors (CARs) are artificial T cell receptors that re-target patients’ T cells to specifically recognize and kill tumor cells. Despite remarkable success of anti-CD19 CAR therapy against refractory B cell leukemias, there are several limitations to CAR T cell therapies including toxicities and antigen-loss leading to relapse. To address these issues, we previously developed a “universal” CAR, SNAP-CAR, for which antigen-specificity is directed by co-administered tumor-targeted antibodies. Instead of directly recognizing a tumor antigen, the SNAP-CAR carries out an enzymatic reaction to fuse with antibodies conjugated to a benzylguanine (BG) tag. Activation and effector functions of SNAP CAR T cells can be re-targeted by antibody-BG conjugates to several antigens including: CD20, CD19, HER2, and EGFR. We are now developing SNAP-CAR T cells for potential clinical translation including optimization of the CAR expression construct and functional characterization in a human tumor xenograft mouse model. SNAP-CAR-T2A-LNGFR was cloned into a gamma-retroviral expression system, yielding a 10-fold greater expression level in primary human T cells compared to our previous lentiviral vector. SNAP-CAR T cells were potently activated and lysed tumor cells in a target antigen-specific manner in vitro. Challenging NSG mice with CD20+ Raji leukemia cells, significant tumor regression was observed with SNAP-CAR T cells + Rituximab-BG and anti-CD20-CAR positive control T cells but not SNAP-CAR T cells without antibody, as compared to untransduced T cells + Rituximab-BG. The SNAP-CAR provides a powerful system to program the antigen-targeting capability of human T cells with promise for cancer therapy.
Intracellular PD-L1 regulates DNA damage checkpoints and suppresses Chk1 and PARP inhibitor synthetic lethality
Authors: Kornepati Anand V , Deng Yilun , Dray Eloise , Murray Clare , Kari Suresh C , Osta Erica , Liu Zexuan , Boyd Jacob , Padron Alvaro , Reyes Ryan , Gupta Harshita B. , Clark Curtis A , Li Rong , Zhao Weixing , Vadlamudi Ratna , Sung Patrick , Curiel Tyler J.
Published: February 2023

Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.
BATF and IRF4 cooperate to counter exhaustion in tumour-infiltrating CAR T cells
Authors: Seo Hyungseok , Gonzales-Avalos Edahi , Zhang Wade , Lio Chan Wang “Jerry” , Rao Anjana , Hogan Patrick
Published: February 2023

Cooperative interactions among transcription factors are essential for gene transcription. We previously showed that NFAT and AP-1 (Fos-Jun) transcription factors cooperate to promote the effector functions of T cells, but that under conditions where it is unable to cooperate with AP-1, NFAT imposes a negative feedback programme of T cell hyporesponsiveness (“exhaustion”). Here we show that BATF and IRF4 cooperate to counter T cell exhaustion. Overexpression of Batf in CD8+ T cells expressing a chimeric antigen receptor (CAR) promoted the survival and expansion of tumour-infiltrating CAR T cells, increased their production of effector cytokines, decreased their expression of inhibitory receptors and the exhaustion-associated transcription factor TOX, and led to the generation of long-lived memory T cells that controlled tumour recurrence. These responses were dependent on the BATF-IRF interaction, since cells expressing a Batf mutant unable to interact with Irf4 did not survive in tumours and did not effectively delay tumour growth. We suggest that BATF overexpression is a therapeutically viable option for improving the anti-tumour responses of CAR TILs, by skewing their phenotypes and transcriptional profiles away from exhaustion and towards increased effector function.
Evaluating the Potential of Harnessing Anti-Leukemia T cells for Treating T-ALL
Authors: Triplett Todd A , Rios Joshua , Kottapalli Srividya , Weinberg Andrew D , Somma Alexander , Sandoval Trevor
Published: February 2023

T cell Acute Lymphoblastic Leukemia (T-ALL) is a devastating malignancy found primarily in pediatric populations. Although patient outcome has improved, standard of care for T-ALL has not progressed from intensive regimens of chemotherapy. Immunotherapy may offer an alternative approach for treating T-ALL with lower toxicity profiles. In the case of B-ALL, adoptive transfer of engineered cells (e.g. CAR cells) has had resounding success. Unfortunately this may not translate to T-ALL due to severe immunodeficiency from ablating the T cell compartment. These studies therefore sought to determine the therapeutic potential of harnessing anti-leukemia T cells that spontaneously arise in tumor-bearing hosts in response to T-ALL. As very little is known about the interplay between T-ALL and T cells, we initially examined changes to the T cell compartment in response to T-ALL during disease progression by transplanting primary leukemia cells into immune-competent, congenic mice. This model revealed an increase in effector memory CD8 T cells with increased levels of PD1 and OX40 that largely correlated with tumor burden. Importantly, co-targeting PD1 with OX40 resulted in drastic reduction in tumor burden while expanding actively proliferating CD8+GrzB+ T cells. To gain a better understanding of T cell interactions with T-ALL in situ, we have begun utilizing NanoString Technologies’ Digital Spatial Profiling platform. This platform has already shown distinct functional states of T cells between those interacting with leukemia compared with those at distal sights. Ongoing studies aim to identify therapeutic targets in multiple organs which can activate T cell populations of interest to maximize their anti-leukemia effector responses.
Diversity of draining lymph node-tumor overlapping T cell receptor repertoire reflects the anti-tumor effect of cancer immunotherapies
Authors: Aoki Hiroyasu , Ueha Satoshi , Tsunoda Mikiya , Ogiwara Haru , Shimizu Haruka , Shichino Shigeyuki , Matsushima Kouji
Published: February 2023

CD8+ tumor-infiltrating lymphocytes, the main effector cells of anti-tumor immune responses, are composed of various T cell clones. Numerous studies have demonstrated the presence of tumor-reactive T cell clones with high frequency in tumors. However, it remains unclear as to which of the two mechanisms, the expansion of limited tumor-specific clones or the induction of diverse tumor-reactive ones, is more beneficial for the action of immune checkpoint inhibitors (ICIs).

To address this question, we performed a T cell receptor repertoire analysis on B16F10 and on Lewis lung carcinoma tumor-bearing mice treated with immunotherapy including anti-PD-L1 monoclonal antibody (mAb) and anti-CD4 depletion mAb, which enhance the expansion of tumor-reactive CD8+ T cells. We focused on the T cell clones that overlapped between the tumor and draining lymph node (dLN) to analyze the T cell clones that were mobilized into the anti-tumor response after treatment.

In anti-PD-L1 and anti-CD4 treated mice, the total frequency of CD8+ dLN-tumor overlapping clones in the tumor was increased. Surprisingly, although the expansion of the top10 overlapping clones did not differ, ICI treatment increased the total frequency of the 11th or lower overlapping clones, indicating the broadening of the tumor-reactive T cell repertoire. Moreover, the administration of FTY720 to tumor-bearing mice, which inhibits egress of T cells from the LNs to circulation, reduced the total frequency of the 11th or lower overlapping clones in the tumor and reduced the long-term anti-tumor effect of anti-PD-L1 treatment.

These results suggest that mobilization of diverse tumor-reactive clones is essential for enhancing and persistent anti-tumor effect of ICIs.
Deep characterization of tumor infiltrating leukocytes using a combination of flow cytometry and single-cell multiomics
Authors: Shi Xiaoshan , Nakamoto Margaret , Middlebrook Aaron , Huang Wei , Lo Evelyn , Neuhoefer Patrick Thomas , Bornheimer Scott , Ghanekar Smita
Published: February 2023

Cancer immunotherapies have the potential to induce durable anti-tumor immune responses; however the efficacy of these treatments varies among patients and partially depends on the complex interplay between malignant cells and non-malignant cells within the tumor microenvironment. In the tumor microenvironment, T cells play a crucial role in eliminating tumor cells and orchestrating anti-tumor immune responses; in contrast immunosuppressive cell types can support tumor progression. Here we harnessed recent advances in single-cell multiomics that allow high-resolution functional characterization of intratumoral immune cells to improve our understanding of their dynamic relationships. Specifically, we utilized a One Solution Workflow to dissociate a solid tumor followed by in-depth dissection of the immune composition of the tumor microenvironment using both flow cytometry and the BD Rhapsody™ Single-Cell Analysis System. We characterized the cellular composition of tumor microenvironment using flow cytometry. Importantly, by examining the expression of mRNA and surface protein markers at single-cell resolution, we unraveled the complexities of the tumor microenvironment, elucidated cellular heterogeneity and identified cell-type specific gene signatures. We propose this One Solution Workflow can be adopted to understand the transcriptional and proteomic phenotypes of tumor infiltrating leukocytes for development of novel immune therapeutic strategies for cancer.

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Inhibition of TH2 responses by a secreted component of a common farm bacterium
Authors: Hollinger Maile Kananiokala , Camacho Daniel F , Hrusch Cara L , Barron Gabriel M , Velez Tania Estefania , Zamora-Pineda Jesus Bernabe , Knight Katherine , Sperling Anne I
Published: February 2023

The rise in incidence of asthma and allergic diseases over the past half century has led to the development of the hygiene hypothesis, which posits that lack of exposure to environmental microbes during childhood leads to inappropriate immune responses to innocuous antigens. Evidence for the hygiene hypothesis is particularly compelling in studies of rural farm children, where infants that live on farms had reduced prevalence of asthma at school age, as compared to their non-farming counterparts. In particular, the presence of exopolysaccharide (EPS) in farm dust independently predicted lower rates of asthma diagnosis. The purpose of this study is to investigate the protective effects of EPS from a common farm microorganism, the hay bacillus B. subtilis, in limiting TH2-type inflammation in a mouse model of asthma. Treatment of house dust mite (HDM)-sensitized mice with EPS was sufficient to block eosinophilia and TH2 accumulation in the lungs, without affecting accumulation of other TH subsets. These effects were dependent on TLR4, the putative receptor for EPS, as EPS no longer protected against eosinophilia and TH2 infiltration in Tlr4−\− mice. Additionally, lung-derived dendritic cells exposed to EPS and HDM in vivo were phenotypically distinct from dendritic cells recovered from mice treated with HDM alone. These data support a protective role for EPS in the inhibition of allergic asthma, potentially by affecting TLR4-dependent dendritic cell priming of TH2 responses.
Single-cell and spatial transcriptomics reveal NLRP3 inflammasome-mediated immunestromal interactions during vasculitis and cardiovascular inflammation.
Authors: Porritt Rebecca A , Zemmour David , Abe Masanori , Chen Shuang , Crother Timothy R , Shimada Kenichi , Arditi Moshe , Rivas Magali Noval
Published: February 2023

NLRP3 activation and IL-1β production are implicated in Kawasaki Disease (KD) pathogenesis, however a detailed description of molecular networks and cellular subsets involved is lacking. Here, in a murine model of KD vasculitis, we used single-cell RNA sequencing and spatial transcriptomics to characterize the cellular landscape of vascular tissues and observed infiltrations of innate and adaptive immune cells, associated with increased expression of Nlrp3 and Il1b. Monocytes, macrophages and dendritic cells were the main sources of IL-1β, whereas fibroblasts and vascular smooth muscle cells (VSMCs) expressed high levels of IL-1 receptor. Genetic inhibition of IL-1β signaling on VSMCs efficiently attenuated the development of cardiovascular lesions during murine KD. In addition, pharmacological inhibition of NLRP3 prevented the development of cardiovascular inflammation. Our results unravel the cellular diversity involved in IL-1β production and signaling in KD cardiovascular lesions and demonstrate that therapeutic strategies targeting NLRP3 might be beneficial for human KD.
Pericardial macrophages and their role in myocardial infarction
Authors: Hughes David M , Won Taejoon , Jurcova Ivana , Talor Monica V , Szarszoi Ondrej , Curnova Lenka , Striz Ilza , Hooper Jody E , Melenovsky Vojtech , Cihakova Daniela
Published: February 2023

Each year an estimated 790,000 people suffer a heart attack in the United States. With such a high incidence, it is important to understand the underlying cellular mechanisms at play during myocardial infarction. In this project we examined the role of pericardial macrophages in modulating the pathogenesis of myocardial infarction. We found that pericardial macrophages expressing the transcription factor GATA6 make up a majority of the cells in the healthy human’s pericardial fluid and have a regulatory function. However, patients with chronic ischemic cardiac disease had their GATA6+ macrophages replaced by inflammatory monocytes in the pericardial fluid. GATA6+ macrophages can be also isolated from murine pericardial fluid. We modeled myocardial infarction through permanent ligation of the left anterior descending artery in adult mice utilizing GATA6+ macrophage deficient LysCreGATA6fl/fl mice. LysCreGATA6fl/fl mice had worse fibrosis and more severe EKG abnormalities compared to control mice following myocardial infarction. Thrombi were also apparent in the hearts of LysCreGATA6fl/fl mice that were not present in control mice. In addition, mice lacking GATA6+ pericardial macrophages had a higher influx of inflammatory Ly6Chi monocytes to the myocardium during the early inflammatory phase of the disease, while pericardial macrophages did not traffic to the myocardium directly at any point. Therefore, we propose that pericardial macrophages inhibit late-stage cardiac remodeling following myocardial infarction by preventing the trafficking of inflammatory monocytes to the site of injury.
Tbet+ B cells facilitate iNKT cell expansion in murine liver and omentum during obesity
Authors: Enslow Benjamin , Vanz Carlo , Schute Travis , Parthasarathy Raksha , Dudley Elizabeth A. , Hägglöf Thomas , Leadbetter Elizabeth A.
Published: February 2023

Obesity is a burgeoning public health crisis, responsible for increasing chronic systemic inflammation which leads to metabolic disease in humans and mice. Understanding the contribution of immune inflammation to metabolic disease is critical for designing effective therapeutic interventions. Invariant Natural Killer T (iNKT) cells are enriched in the adipose tissue, liver, and omenta of both humans and mice, but their frequency is reduced in adipose during obesity. iNKT cells negatively regulate or positively activate other immune cells, including B cells, depending on the immunologic context. Adipose B regulatory cells protect against inflammation, but they are not the only subset of B cells in adipose tissue. Our preliminary studies find increased frequencies of inflammatory Tbet+ B cells in adipose tissue of obese humans and mice which correlates with weight gain in mice and increasing BMI in humans. We now extend those findings to consider iNKT cell interaction with B cells during obesity in the adipose, liver, and omental tissues. Flow cytometry confirmed iNKT cell-dependent expansion of Tbet+ CD11c+ B cells in adipose tissue of obese mice. Interestingly, similar expansion was not observed in liver or omental tissue from the same obese mice. Instead, the livers and omenta of obese mice demonstrated significant increases in iNKT cell frequency compared with lean mice. In contrast to the adipose tissue, the increase in liver and omental iNKT cells during obesity was missing in mice genetically deficient in Tbet+ B cells. Thus, we find that iNKT cell-dependent expansion of Tbet+ CD11c+ B cells within adipose tissue of obese mice is counter-balanced by a reciprocal Tbet+ B cell-dependent expansion of iNKT cells in liver and omentum.
Dectin-1 limits CNS autoimmunity through a non-canonical pathway
Authors: Deerhake M Elizabeth , Danzaki Keiko , Inoue Makoto , Cardakli Emre , Nonaka Toshiaki , Aggarwal Nupur , Barclay Will , Ji Ru-Rong , Shinohara Mari L
Published: February 2023

Innate immunity mediates both damage and repair of the central nervous system (CNS) in neurologic disorders. Although pathologic roles for innate immunity are well-described in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), protective aspects of the immune response are less understood. In this study, we sought to identify the function of Dectin-1, a C-type lectin receptor (CLR), in CNS autoimmunity. We found that Dectin-1 limited neuroinflammation in EAE while its canonical signaling mediator, Card9, promoted disease. Myeloid cells from the bone-marrow mediated the regulatory function of Dectin-1 in EAE and upregulated expression of Oncostatin M (Osm), a neuroprotective cytokine, through a non-canonical Card9-independent Dectin-1 signaling pathway. We found that the Osm receptor (OsmR) functioned specifically in astrocytes to reduce EAE severity. RNA-seq profiling of the Card9-independent Dectin-1 transcriptional program identified multiple targets with neuroprotective functions, including Osm, which were regulated by NFAT signaling. Our data describe a new mechanism of neuroprotective myeloid-astrocyte crosstalk regulated by a non-canonical Dectin-1 pathway.
Secreted IgM modulates the pool of IL-10+ B cells
Authors: McGettigan Shannon E , Diaz Lazaro Emilio Aira , Kumar Gaurav , Baumgarth Nicole , Debes Gudrun Fiona
Published: February 2023

IL-10+ regulatory B cells (Bregs) are increasingly recognized as critical for maintaining immune tolerance, and alterations in Breg numbers can have detrimental effects in inflammatory diseases, infection, and cancer. Bregs are found among most B cell subsets and act primarily by producing the immunosuppressive cytokine IL-10. Despite their importance, the signals that control Breg differentiation are not well defined. Here, we demonstrate that mice incapable of secreting IgM (sIgM−/−) have significantly increased IL-10+ Bregs in lymphoid organs compared to wildtype mice. These IL-10 producing Bregs are polyclonal and increased among all major B cell subsets: B-1, follicular B, marginal zone B, T2-marginal zone precursors, and transitional B cells. SIgM−/− B cells that develop in the presence of circulating IgM express IL-10 similar to that of WT B cells within the same mouse indicating that the expansions in IL-10+ B cell populations are not due to B cell-intrinsic effects based on the inability to secrete IgM. Mice that lack B cell-expressed high-affinity IgM receptor (FcμR) mirrored the IL-10 phenotype in their B-1 and MZ, but not follicular B cells, suggesting that secreted IgM can act as a negative regulator of IL-10 expression in B cell subsets via binding to surface-expressed FcμR, and potentially other IgM binding receptors. Our data indicate that IgM binding receptors control IL-10 competence in some B cell subsets thereby revealing potential novel avenues for altering Breg induction in disease settings. Funding: T32AI134646, R01AR067751, R01AI127389.
Macrophage Activation in the Lung during the Progression of Nitrogen Mustard Induced Injury is Associated with Altered miRNA Expression
Authors: Smith Ley Cody , Venosa Alessandro , Gow Andrew J , Zarbl Helmut , Laskin Jeffrey D , Laskin Debra L
Published: February 2023

Activated macrophages have been implicated in lung injury and fibrosis induced by the cytotoxic alkylating agent, nitrogen mustard (NM). Accumulating evidence suggests that phenotypic activation of macrophages during initiation, progression, and resolution of inflammatory responses is regulated by groups of miRNAs that act in concert to coordinate cellular activity. Herein, we determined if macrophage activation after NM injury was associated with altered miRNA expression. Male rats were exposed to NM (0.125 mg/kg, i.t.) and PCR array analysis of miRNAs involved in inflammation and fibrosis was performed in alveolar macrophages recovered from bronchoalveolar lavage. Results revealed unique and overlapping miRNA expression profiles in macrophages isolated 1, 3, 7, and 28 d post-NM. Seven miRNAs were upregulated in macrophages throughout the time-course, while three miRNAs were uniquely expressed 1 d post-NM, six at 3 d, six at 7 d, and nineteen at 28 d. Linkage with macrophage RNA-seq data showed differentially expressed miRNAs regulated mRNAs involved in STAT3, IL-6 and iNOS inflammatory signaling pathways at 1 d, and IL-4, PPAR and Sirtuin signaling at 28 d post-NM. Ingenuity Pathway Analysis predicted miR-21-5p, miR-29, miR-26a, miR-34a and let-7a as key regulators of macrophage mobilization, activation, and lipid homeostasis after NM. Collectively, these data show that NM promotes alterations in miRNA expression linked to lung macrophage responses during inflammatory injury and fibrosis. These findings provide clues on pathways regulating macrophage activation and may help identify novel therapeutic targets. This work was supported by NIH grants AR055073, ES004738, HL086621, ES005022, ES030984, and ES007148.
The impact of aging and senescence on CD4 T cell differentiation
Authors: Torrance Blake , Lorenzo Erica C , Keilich Spencer R , Al-Naggar Iman , Haynes Laura
Published: February 2023

With aging, the immune response and ability to return to homeostasis declines and the presence of senescent cells increases. In this study, we examined how aging and senescence impact Th subset differentiation during the response to influenza infection. In the lungs of aged mice during infection, there are significantly greater percentages of influenza-specific CD4 T cells expressing the transcription factor FoxP3, indicative of the regulatory CD4 T cell (Treg) subset, when compared to young. Higher levels of the cytokine TGFb, which can drive FoxP3 expression, were also found in the bronchoalveolar lavage (BAL) in these aged mice. Blocking TGFb with a neutralizing antibody reduced FoxP3 expression in lung CD4 T cells. Furthermore, treating aged mice with senolytic drugs prior to infection could reduce the percentage of FoxP3-expressing CD4 T cells as well as TGFb levels. Using an adoptive transfer model in which young CD4 T cells were transferred into aged hosts, we found that prior treatment of the aged hosts with senolytic drugs could reduce FoxP3 expression in the young donor cells, indicating that the senescent environment plays a role in aberrant Th subset differentiation. Finally, treatment with senolytic drugs induced differentiation of aged CD4 T cells to a healing Type 2 phenotype, which promotes a return to homeostasis, much like younger mice. These results suggest that senescent cells, via production of cytokines such as TGFb, have a significant impact on CD4 T cell differentiation, which could alter the outcome of an immune response and slow the return to homeostasis.
Subset distinct richness reduction and clonal expansion of human CD4+ and CD8+ αβ TCR repertoires with aging
Authors: achour achouak , Sun Xiaoping , Nguyen Thomas , Ko Annette , Cifello Jeffrey , Ling Chen , Sharma Jay , Hiroi Toyok , Zhang Yongqing , Chee Chia , Wood William , Wu Wells , Zukley Linda , Phue Je-Nie , Becker Kevin G. , Shen Rongfong , Ferrucci Luigi , Weng Nan-ping
Published: February 2023

A competent T-cell receptor (TCR) repertoire contains TCRs recognizing both novel and experienced antigens. It is widely believed that the human TCR repertoire declines with age, but its precise change in size and clonal distribution with age has not been fully determined. Here, we investigated how the size and content of TCR repertoire changes with age by analyzing the TCR repertoire of human CD4+ and CD8+ T cells and their naïve and memory subsets from 30 healthy adults aged from 25 to 85 at first visit and an average of 9-year follow-up as second visit by RNAseq. First, we calculated abTCR repertoire changes with age and found a profound reduction of the TCRa and TCRb richness in naïve CD8+ T cells and an increase of clonal expansion especially in memory CD8 T cells compared to CD4+ T cells. We also study the degree of overlap of TCRa/b repertoire between naïve and memory T cells in each subject at the same visit and found little overlap of TCRa/b unique sequences between naïve and memory CD4 T cells and ~40% of the total TCRs in CD8 T cells. Moreover, we showed elevated overlap of TCR sequences between two visits in both CD4 and CD8 T cells with memory CD8 T cells as the highest. Finally, we determined the degree of TCR clonotypes shares among different subjects via the analysis of the sharing of TCR sequences in the 30 healthy adults. As results, we observed increased sharing of identical TCR between CD4 and CD8 T cells suggesting that the public clonotypes may share with the common pathogens shared in the population, particularly in the old population. Our findings showed more profound reduction in the CD8+TCR repertoire with age compared to CD4+ TCR repertoire. All together provide evidence of ab TCR sequence-based age-associated changes of T cells and their subsets.
TCF-1 deficiency produces prion-like expansion of noncanonical regulatory T cells
Authors: Harris Rebecca , Mammadli Mahinbanu , Karimi Mobin A
Published: February 2023

Regulatory T cells (Tregs) are suppressive T cells which help to control and dampen immune responses. Tregs naturally suppress potentially autoreactive T cells, and are used in a variety of T cell-mediated therapies. While these cells are very useful for controlling improper or overactive immune responses in disease, they are found in low frequencies in the body, and their exact mechanisms of action are still unknown. Tregs typically express CD4, CD25, and FOXP3, with FOXP3 being considered a defining lineage transcription factor. However, in some contexts, noncanonical Tregs that are CD4+ FOXP3+ CD25− have been described, and have been shown to be suppressive in some cases. It is still unclear which transcription factors play a role in regulating these noncanonical Tregs.

We utilized a mouse strain with T-cell specific deletion of a critical transcription factor, T Cell Factor-1 (TCF-1) to determine the role of TCF-1 in controlling Tregs. We report that loss of TCF-1 specifically in T cells of mice results in cell-intrinsic expansion of CD25− Tregs. We also show that TCF-1-deficient T cells have a prion-like ability to influence Treg fate of nearby WT T cells. These CD25− Tregs are found in multiple peripheral tissues, express other functional Treg markers (CTLA-4 and IL-10), and are not the result of aberrant FOXP3 expression. Therefore, TCF-1 acts as a regulatory transcription factor for these CD25− Tregs. Modulation of TCF-1 may increase yield of noncanonical Tregs, providing benefit for T cell-mediated therapies and further study of noncanonical Tregs.
IRF4 regulates the proliferative capacity of activated B cells during B cell differentiation
Authors: Patterson Dillon , Scharer Christopher , Kania Anna , Mi Tian , Hicks Sakeenah L , Boss Jeremy M
Published: February 2023

Cell division is essential for B cell differentiation to a plasma cell (PC). While the cell division coupled changes in the expression of transcription factors that coordinate the PC program have been described, it is unclear how these factors coordinate the proliferative program. To address this, we used an adoptive transfer system and monitored the cell division/differentiation kinetics in response to LPS, NP-ficoll, and X31 influenza. In this system, wild-type (WT) B cells undergo at least 8 cell divisions before differentiating into PC. In contrast, Interferon Regulatory Factor 4-null (IRF4−/−) B cells began dividing normally but stalled during the proliferative response. To assess the scope of IRF4-dependent reprogramming, WT and IRF4−/− B cells in divisions 0, 1, and 3–6 were sorted for ATAC- and RNA-seq. RNA-seq data revealed hundreds of differentially expressed genes (DEG) when IRF4 was deleted that encompassed a number of gene sets critical for B cell reprogramming, including glycolysis, OXPHOS, and mTORC1 signaling. Further, MYC target genes became progressively dysregulated across divisions. Indeed, IRF4−/− cells failed to fully upregulate MYC compared to WT cells and consequently, we observed aberrant cell cycle distribution, cell growth defects, and fewer actively proliferating cells. Overexpressing MYC in IRF4−/− B cells rescued cell growth and significantly improved proliferation. ATAC-seq data also exposed hundreds of differentially accessible regions, the majority of which contained a known IRF4 binding motif and mapped to a corresponding DEG. Together, these data create a road map defining the role of IRF4 throughout differentiation, revealing a critical function in coordinating the proliferative response.
H3K27me3 demethylases restrict the magnitude of PC formation
Authors: Kania Anna , Price Madeline , Scharer Christopher , Boss Jeremy M
Published: February 2023

Robust differentiation of naïve B cell into antibody secreting plasma cells (PC) in response to foreign antigens is a cornerstone of the humoral immune response. PC formation is highly regulated and requires substantial epigenetic remodeling, including redistribution of histone modification H3 lysine 27 trimethylation (H3K27me3), which is associated with a repressive chromatin state and gene silencing. However, little is known about the role of the two demethylases, UTX and JMDJ3, that remove H3K27me3 in B cells. To examine whether these enzymes module PC formation, we generated mice with B cell specific deletion of UTX and JMDJ3 (dKO; Utxfl/flJmjd3fl/fl Cd19Cre/+). Stimulation of B cells with T cell independent antigens, LPS or NP-Ficoll, led to a significant increase in PC in dKO mice compared to CreCtrl mice (Cd19Cre/+). This phenotype was attributed to enhanced proliferation and differentiation of marginal zone (MZ), but not follicular B cells (FoB). dKO PC upregulated genes associated with oxidative phosphorylation metabolism and exhibited higher spare respiratory capacity. Furthermore, deletion of UTX and JMJD3 led to significant redistribution of accessible chromatin and H3K27me3. Regions with reduced chromatin accessibility in dKO PC were enriched for transcription factor motifs of known PC repressors suggesting a potential mode of regulation. Additionally, we demonstrated that UTX is the primary H3K27me3 demethylase regulating B cell differentiation and evaluated whether its demethylase activity is required to augment PC formation. Taken together, H3K27me3 demethylases are critical epigenetic regulators necessary restrain B cell differentiation.
Expression of the purinergic receptor P2RX7 marks early effector CD8+ T cells with increased potential for both long-lived circulating and resident memory cell generation
Authors: Silva Henrique Borges da , Vardam-Kaur Trupti , Carvalho Igor Santiago , Peng Changwei , Wanhainen Kelsey M , Jameson Stephen C.
Published: February 2023

Memory CD8+ T cells protect against secondary viral infections. They develop and maintain exclusively in circulation (e.g. central memory - Tcm) or are excluded from re-circulation (resident memory - Trm). Circulating memory CD8+ T cells arises from memory precursors (MPs) which express memory, pro-survival, and pluripotency molecules. Trm generation, in contrast, is still not fully understood. Previous reports suggest that Trm and Tcm have a common precursor present at the early effector phase; yet, the nature of this precursor is elusive. We have found that the extracellular ATP receptor P2RX7 promotes both Tcm and Trm generation. Moreover, P2RX7 is quickly upregulated in some (but not all) early effectors and early P2RX7 ablation hinders both Tcm and Trm generation. Thus, we postulated that P2RX7 in early effectors may mark a Tcm/Trm common precursor. Comparison between high (P2RX7hi) and low (P2RX7lo) P2RX7-expressing CD8+ T cells shows a correlation between memory markers and P2RX7, with CD62L+ Tcm precursors exclusively found in P2RX7hi cells. RNA-seq of P2RX7hi and P2RX7lo early effectors show survival, memory and pluripotency genes and pathways are upregulated in P2RX7hi cells. Conversely, many terminal effector and apoptosis genes are upregulated in P2RX7lo cells. P2RX7lo early effectors and MPs have increased apoptosis. Upon transfer into infection-matched mice, P2RX7hi MPs preferentially survive and form Tcm compared to P2RX7lo MPs. This is even more apparent when P2RX7hi and P2RX7lo early effectors are compared: approximately 100x more P2RX7hi cells form memory when compared to P2RX7lo cells, both Tcm and Trm. Our data suggest that P2RX7 in early effector CD8+ T cells promotes the generation of a common Tcm/Trm precursor.