VISTA (PD-1H) Is a Crucial Immune Regulator to Limit Pulmonary Fibrosis.

VISTA (V domain immunoglobulin suppressor of T cell activation, also called PD-1H [programmed death-1 homolog]), a novel immune regulator expressed on myeloid and T lymphocyte lineages, is upregulated in mouse and human idiopathic pulmonary fibrosis (IPF). However, the significance of VISTA and its therapeutic potential in regulating IPF has yet to be defined. To determine the role of VISTA and its therapeutic potential in IPF, the expression profile of VISTA was evaluated from human single-cell RNA sequencing data (IPF Cell Atlas). Inflammatory response and lung fibrosis were assessed in bleomycin-induced experimental pulmonary fibrosis models in VISTA-deficient mice compared with wild-type littermates. In addition, these outcomes were evaluated after VISTA agonistic antibody treatment in the wild-type pulmonary fibrosis mice. VISTA expression was increased in lung tissue-infiltrating monocytes of patients with IPF. VISTA was induced in the myeloid population, mainly circulating monocyte-derived macrophages, during bleomycin-induced pulmonary fibrosis. Genetic ablation of VISTA drastically promoted pulmonary fibrosis, and bleomycin-induced fibroblast activation was dependent on the interaction between VISTA-expressing myeloid cells and fibroblasts. Treatment with VISTA agonistic antibody reduced fibrotic phenotypes accompanied by the suppression of lung innate immune and fibrotic mediators. In conclusion, these results suggest that VISTA upregulation in pulmonary fibrosis may be a compensatory mechanism to limit inflammation and fibrosis, and stimulation of VISTA signaling using VISTA agonists effectively limits the fibrotic innate immune landscape and consequent tissue fibrosis. Further studies are warranted to test VISTA as a novel therapeutic target for the IPF treatment.

Aging gene signature of memory CD8+ T cells is associated with neurocognitive functioning in Alzheimer's disease.

BACKGROUND: Memory CD8+ T cells expand with age. We previously demonstrated an age-associated expansion of effector memory (EM) CD8+ T cells expressing low levels of IL-7 receptor alpha (IL-7Rαlow) and the presence of its gene signature (i.e., IL-7Rαlow aging genes) in peripheral blood of older adults without Alzheimer's disease (AD). Considering age as the strongest risk factor for AD and the recent finding of EM CD8+ T cell expansion, mostly IL-7Rαlow cells, in AD, we investigated whether subjects with AD have alterations in IL-7Rαlow aging gene signature, especially in relation to genes possibly associated with AD and disease severity. RESULTS: We identified a set of 29 candidate genes (i.e., putative AD genes) which could be differentially expressed in peripheral blood of patients with AD through the systematic search of publicly available datasets. Of the 29 putative AD genes, 9 genes (31%) were IL-7Rαlow aging genes (P < 0.001), suggesting the possible implication of IL-7Rαlow aging genes in AD. These findings were validated by RT-qPCR analysis of 40 genes, including 29 putative AD genes, additional 9 top IL-7R⍺low aging but not the putative AD genes, and 2 inflammatory control genes in peripheral blood of cognitively normal persons (CN, 38 subjects) and patients with AD (40 mild cognitive impairment and 43 dementia subjects). The RT-qPCR results showed 8 differentially expressed genes between AD and CN groups; five (62.5%) of which were top IL-7Rαlow aging genes (FGFBP2, GZMH, NUAK1, PRSS23, TGFBR3) not previously reported to be altered in AD. Unbiased clustering analysis revealed 3 clusters of dementia patients with distinct expression levels of the 40 analyzed genes, including IL-7Rαlow aging genes, which were associated with neurocognitive function as determined by MoCA, CDRsob and neuropsychological testing. CONCLUSIONS: We report differential expression of "normal" aging genes associated with IL-7Rαlow EM CD8+ T cells in peripheral blood of patients with AD, and the significance of such gene expression in clustering subjects with dementia due to AD into groups with different levels of cognitive functioning. These results provide a platform for studies investigating the possible implications of age-related immune changes, including those associated with CD8+ T cells, in AD.

Defining Clinical and Immunological Predictors of Poor Immune Responses to COVID-19 mRNA Vaccines in Patients with Primary Antibody Deficiency.

Immune responses to coronavirus disease 2019 (COVID-19) mRNA vaccines in primary antibody deficiencies (PADs) are largely unknown. We investigated antibody and CD4+ T-cell responses specific for SARS-CoV-2 spike protein (S) before and after vaccination and associations between vaccine response and patients' clinical and immunological characteristics in PADs. The PAD cohort consisted of common variable immune deficiency (CVID) and other PADs, not meeting the criteria for CVID diagnosis (oPADs). Anti-S IgG, IgA, and IgG subclasses 1 and 3 increased after vaccination and correlated with neutralization activity in HCs and patients with oPADs. However, 42% of CVID patients developed such responses after the 2nd dose. A similar pattern was also observed with S-specific CD4+ T-cells as determined by OX40 and 4-1BB expression. Patients with poor anti-S IgG response had significantly lower levels of baseline IgG, IgA, CD19+ B-cells, switched memory B-cells, naïve CD8+ T-cells, and a higher frequency of EM CD8+ T-cells and autoimmunity compared to patients with adequate anti-S IgG responses. Patients with oPADs can develop humoral and cellular immune responses to vaccines similar to HCs. However, a subset of CVID patients exhibit impairment in developing such responses, which can be predicted by the baseline immune profile and history of autoimmunity.

Implication of IL-7 receptor alpha chain expression by CD8+ T cells and its signature in defining biomarkers in aging.

CD8+ T cells play an important role in host defense against infections and malignancies as well as contribute to the development of inflammatory disorders. Alterations in the frequency of naïve and memory CD8+ T cells are one of the most significant changes in the immune system with age. As the world population rapidly ages, a better understanding of aging immune function or immunosenescence could become a basis for discovering treatments of illnesses that commonly occur in older adults. In particular, biomarkers for immune aging could be utilized to identify individuals at high risk of developing age-associated conditions and help monitor the efficacy of therapeutic interventions targeting such conditions. This review details the possible role of CD8+ T cell subsets expressing different levels of the cytokine receptor IL-7 receptor alpha chain (IL-7Rα) and the gene signature associated with IL-7Rα as potential biomarkers for immune aging given the association of CD8+ T cells in host defense, inflammation, and immunosenescence.

IL-7 receptor alpha defines heterogeneity and signature of human effector memory CD8+ T cells in high dimensional analysis.

The IL-7 receptor alpha chain (IL-7Rα or CD127) can be differentially expressed in memory CD8+ T cells. Here we investigated whether IL-7Rα could serve as a key molecule in defining a comprehensive landscape of heterogeneity in human effector memory (EM) CD8+ T cells using high-dimensional Cytometry by Time-Of-Flight (CyTOF) and single-cell RNA-seq (scRNA-seq). IL-7Rα had diverse, but organized, expressional relationship in EM CD8+ T cells with molecules related to cell function and gene regulation, which rendered an immune landscape defining heterogeneous cell subsets. The differential expression of these molecules likely has biological implications as we found in vivo signatures of transcription factors and homeostasis cytokine receptors, including T-bet and IL-7Rα. Our findings indicate the existence of heterogeneity in human EM CD8+ T cells as defined by distinct but organized expression patterns of multiple molecules in relationship to IL-7Rα and its possible biological significance in modulating downstream events.

Dissecting alterations in human CD8+ T cells with aging by high-dimensional single cell mass cytometry.

We investigated the effect of aging on the multi-dimensional characteristics and heterogeneity of human peripheral CD8+ T cells defined by the expression of a set of molecules at the single cell level using the recently developed mass cytometry or Cytometry by Time-Of-Flight (CyTOF) and computational algorithms. CD8+ T cells of young and older adults had differential expression of molecules, especially those related to cell activation and migration, permitting the clustering of young and older adults through an unbiased approach. The changes in the expression of individual molecules were collectively reflected in the altered high-dimensional profiles of CD8+ T cells in older adults as visualized by the dimensionality reduction analysis tools principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE). A combination of PhenoGraph clustering and t-SNE analysis revealed heterogeneous subsets of CD8+ T cells that altered with aging. Furthermore, intermolecular quantitative relationships in CD8+ T cells appeared to change with age as determined by the computational algorithm conditional-Density Resampled Estimate of Mutual Information (DREMI). The results of our study showed that heterogeneity, multidimensional characteristics, and intermolecular quantitative relationships in human CD8+ T cells altered with age, distinctively clustering young and older adults through an unbiased approach.

Sex-Based Selectivity of PPARγ Regulation in Th1, Th2, and Th17 Differentiation.

Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been recognized to regulate adaptive immunity through Th17 differentiation, Treg functions, and TFH responses. However, its role in adaptive immunity and autoimmune disease is still not clear, possibly due to sexual differences. Here, we investigated in vitro treatment study with the PPARγ agonist pioglitazone to compare Th1, Th2, and Th17 differentiation in male and female mouse splenic T cells. Pioglitazone treatment significantly inhibited various effector T cell differentiations including Th1, Th2, and Th17 cells from female naïve T cells, but it selectively reduced IL-17 production in male Th17 differentiation. Interestingly, pioglitazone and estradiol (E2) co-treatment of T cells in males inhibited differentiation of Th1, Th2, and Th17 cells, suggesting a mechanism for the greater sensitivity of PPARγ to ligand treatment in the regulation of effector T cell differentiation in females. Collectively, these results demonstrate that PPARγ selectively inhibits Th17 differentiation only in male T cells and modulates Th1, Th2, and Th17 differentiation in female T cells based on different level of estrogen exposure. Accordingly, PPARγ could be an important immune regulator of sexual differences in adaptive immunity.

Cell membrane penetrating function of the nuclear localization sequence in human cytokine IL-1α.

Cytokines are released from the cell, bind to their receptors, and affect cellular responses. The precursor form of interleukin 1 alpha (pIL-1α) has a nuclear localization sequence (NLS) that causes it to be localized to the nucleus and regulate specific gene expression. The amino acids of the NLS are basic amino acid-rich sequences, as is the cell penetrating peptide (CPP), which has been widely studied as a way to deliver macromolecules into cells. Here, we hypothesized that the NLS in pIL-1α (pIL-1αNLS) can penetrate the cell membrane and it could deliver macromolecules such as protein in vivo. We characterized cell membrane penetration ability of pIL-1αNLS or its tandem repeated form (2pIL-1αNLS) to enhance its intracellular delivery efficiency. 2pIL-1αNLS showed comparable protein delivery efficiency to TAT-CPP and it mediates endocytosis following heparan sulfate interaction. 2pIL-1αNLS conjugated enhanced green fluorescence protein was localized to the nucleus and the cytoplasm. Intra-peritoneal administration of 2pIL-1αNLS conjugated dTomato protein showed remarkable in vivo intracellular delivery efficiency in various tissues including spleen, liver, and intestine in mice. Moreover, cytotoxicity of 2pIL-1αNLS was not observed even at 100 µM. Our results demonstrate cell membrane-penetrating function of NLS in pIL-1α, which can be used as a safe therapeutic macromolecular delivery peptide.

IL-1 receptor 1 signaling shapes the development of viral antigen-specific CD4+ T cell responses following COVID-19 mRNA vaccination.

BACKGROUND: The innate immune cytokine interleukin (IL)-1 can affect T cell immunity, a critical factor in host defense. In a previous study, we identified a subset of human CD4+ T cells which express IL-1 receptor 1 (IL-1R1). However, the expression of such receptor by viral antigen-specific CD4+ T cells and its biological implication remain largely unexplored. This led us to investigate the implication of IL-1R1 in the development of viral antigen-specific CD4+ T cell responses in humans, including healthy individuals and patients with primary antibody deficiency (PAD), and animals. METHODS: We characterized CD4+ T cells specific for SARS-CoV-2 spike (S) protein, influenza virus, and cytomegalovirus utilizing multiplexed single cell RNA-seq, mass cytometry and flow cytometry followed by an animal study. FINDINGS: In healthy individuals, CD4+ T cells specific for viral antigens, including S protein, highly expressed IL-1R1. IL-1ß promoted interferon (IFN)-γ expression by S protein-stimulated CD4+ T cells, supporting the functional implication of IL-1R1. Following the 2nd dose of COVID-19 mRNA vaccines, S protein-specific CD4+ T cells with high levels of IL-1R1 increased, likely reflecting repetitive antigenic stimulation. The expression levels of IL-1R1 by such cells correlated with the development of serum anti-S protein IgG antibody. A similar finding of increased expression of IL-1R1 by S protein-specific CD4+ T cells was also observed in patients with PAD following COVID-19 mRNA vaccination although the expression levels of IL-1R1 by such cells did not correlate with the levels of serum anti-S protein IgG antibody. In mice immunized with COVID-19 mRNA vaccine, neutralizing IL-1R1 decreased IFN-γ expression by S protein-specific CD4+ T cells and the development of anti-S protein IgG antibody. INTERPRETATION: Our results demonstrate the significance of IL-1R1 expression in CD4+ T cells for the development of viral antigen-specific CD4+ T cell responses, contributing to humoral immunity. This provides an insight into the regulation of adaptive immune responses to viruses via the IL-1 and IL-1R1 interface. FUNDING: Moderna to HJP, National Institutes of Health (NIH) 1R01AG056728 and R01AG055362 to IK and KL2 TR001862 to JJS, Quest Diagnostics to IK and RB, and the Mathers Foundation to RB.

Aging gene signature of IL-7 receptor alpha low effector memory CD8+ T cells is associated with neurocognitive functioning in Alzheimer's disease.

CD45RA+ effector memory (EM) CD8+ T cell expansion was reported in Alzheimer's disease (AD). Such cells are IL-7 receptor alpha (IL-7Rα)low EM CD8+ T cells, which expand with age and have a unique aging gene signature (i.e., IL-7Rαlow aging genes). Here we investigated whether IL-7Rαlow aging genes and previously reported AD and memory (ADM) genes overlapped with clinical significance in AD patients. RT-qPCR analysis of 40 genes, including 29 ADM, 9 top IL-7Ralow aging and 2 control genes, showed 8 differentially expressed genes between AD and cognitively normal groups; five (62.5%) of which were top IL-7Rαlow aging genes. Over-representation analysis revealed that these genes were highly present in molecular and biological pathways associated with AD. Distinct expression levels of these genes were associated with neuropsychological testing performance in 3 subgroups of dementia participants. Our findings support the possible implication of the IL-7Rαlow aging gene signature with AD.

T Cell Response to Influenza Vaccination Remains Intact in Adults with Congenital Heart Disease Who Underwent Early Thymectomy.

Introduction: T cells developed in the thymus play a key role in vaccine immunity. Thymectomy occurs during infant congenital heart surgery and results in an altered T cell distribution. We investigated if adults with congenital heart disease (ACHD) who underwent early thymectomy have a diminished response to influenza vaccination. Methods: Blood samples from ACHD with early thymectomy ≤ 1 year of age (ACHD-ET; n = 12), no thymectomy (ACHD-NT; n = 8), and healthy controls (HC; n = 14) were collected prior to and 4 weeks after influenza vaccination. Flow cytometric analysis of T cell subsets and vaccine-specific cytokine expressing CD4+ T cells as well as hemagglutination inhibition (HI) assays were completed. Results: The mean age of the cohort was 34 ± 10.6 years and similar in all groups. The mean frequencies of naïve CD4+ and CD8+ T cells were lower in ACHD-ET than in HC (32.7% vs. 46.5%, p = 0.027 and 37.2% vs. 57.4%, p = 0.032, respectively). There was a rise in the frequency of memory CD4+ and CD8+ T cells in the ACHD-ET group. The ACHD-NT had no statistical difference from either group. The frequencies of influenza-specific memory CD4+ T cells expressing IFN-γ and TNF-α were increased after vaccination across all groups (p < 0.05). Conclusions: ACHD-ET have fewer naïve T cells, suggesting immunosenescence. Despite this, they show an adequate T Cell response to vaccination in young adulthood. Our findings support routine vaccination is effective in this population, but research into older ACHD is necessary.

AHCC®, a Standardized Extract of Cultured Lentinula Edodes Mycelia, Promotes the Anti-Tumor Effect of Dual Immune Checkpoint Blockade Effect in Murine Colon Cancer.

Treatment strategies combining immune checkpoint blockade (ICB) with other agents have emerged as a promising approach in the treatment of cancers. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been reported to inhibit tumor growth and enhance immune cell function. Here we investigated whether AHCC® promotes the therapeutic effect of immunotherapy in cancers. A combination of oral AHCC® and dual immune checkpoint blockade (DICB), including PD-1/CTLA-4 blockade, had reduced tumor growth and increased granzyme B and Ki-67 expression by tumor-infiltrating CD8+ T cells in MC38 colon cancer bearing mice compared to a combination of water and DICB. In the same tumor bearing mice, AHCC® and DICB treatment also altered the composition of the gut microbiome with the increased abundance of the species of Ruminococcaceae family which is associated with increased therapeutic efficacy of immunotherapy. The anti-tumor effect of AHCC® and DICB was not found in MC38 tumor-bearing mice treated with antibiotics. These data suggest that AHCC® increases the anti-tumor effect of DICB by enhancing T cell function and affecting the gut microbiome.

Alterations in high-dimensional T-cell profile and gene signature of immune aging in HIV-infected older adults without viremia.

Alterations in the components of the immune system occur with aging. The introduction of combination antiretroviral therapy (ART) has dramatically improved life expectancy in human immunodeficiency virus (HIV) infected individuals by suppressing viral replication and increasing CD4+ T-cell counts. Immunosenescence-like changes, including the expansion of memory CD8+ T cells with senescent features, are reported in young HIV-infected individuals who do not have clinically detectable viremia on ART. However, it is less known whether HIV infection affects the immunosenescent status in older HIV-infected individuals. Here, we addressed this question in older HIV-infected, HIV-uninfected, and frail individuals (all groups age ≥65 years) by examining a set of aging-associated genes in peripheral blood mononuclear cells (PBMCs) as well as by analyzing subsets of CD4+ and CD8+ T cells in depth using high-dimensional CyTOF analysis. Older HIV-infected individuals had increased expression of aging-associated genes such as CX3CR1 in PBMCs which are related to IL-7 receptor low effector memory (IL-7Rαlow EM) CD8+ T cells, a cell population known to expand with age. The subsets of IL-7Rαlow EM CD8+ T cells expressing senescent, cytotoxic, and inflammatory molecules, including CD57, perforin, and CX3CR1, as well as memory CD4+ T cells expressing CD161 and CXCR3, molecules associated with replication-competent HIV-1 harboring cells, were increased in older HIV-infected individuals. Overall, older HIV-infected individuals without detectable viremia on ART had augmented levels of age-associated immune alterations in PBMCs, suggesting that HIV infection has a persistent impact on senescence in older HIV-infected individuals despite the clinically controlled viremia.

Nucleotide-binding domain and leucine-rich-repeat-containing protein X1 deficiency induces nicotinamide adenine dinucleotide decline, mechanistic target of rapamycin activation, and cellular senescence and accelerates aging lung-like changes.

Mitochondrial dysfunction has long been implicated to have a causative role in organismal aging. A mitochondrial molecule, nucleotide-binding domain and leucine-rich-repeat-containing protein X1 (NLRX1), represents the only NLR family member that targets this cellular location, implying that NLRX1 probably establishes a fundamental link between mitochondrial functions and cellular physiology. However, the significance of NLRX1 function in cellular senescence, a key conceptual constituent in aging biology, is yet to be defined. Here, we demonstrate that molecular hallmarks involved in aging biology including NAD+ decline, and activation of mTOR, p53, and p16INK4A are significantly enhanced in NLRX1 deficiency in vitro. Mechanistic studies of replicative cellular senescence in the presence or absence of NLRX1 in vitro reveal that NLRX1-deficient fibroblasts fail to maintain optimal NAD+ /NADH ratio, which instigates the decline of SIRT1 and the activation of mTOR, p16INK4A , and p53, leading to the increase in senescence-associated beta-galactosidase (SA-ß-gal)-positive cells. Importantly, the enhanced cellular senescence response in NLRX1 deficiency is significantly attenuated by pharmacological inhibition of mTOR signaling in vitro. Finally, our in vivo murine studies reveal that NLRX1 decreases with age in murine lungs and NLRX1 deficiency in vivo accelerates pulmonary functional and structural changes that recapitulate the findings observed in human aging lungs. In conclusion, the current study provides evidence for NLRX1 as a crucial regulator of cellular senescence and in vivo lung aging.

Co-inhibitor expression on tumor infiltrating and splenic lymphocytes after dual checkpoint inhibition in a microsatellite stable model of colorectal cancer.

Checkpoint inhibitors have demonstrated clinical impact in colorectal cancer with deficient mismatch repair and high microsatellite instability. However, the majority of patients have disease with stable microsatellites that responds poorly to immunotherapies. Combinations of checkpoint inhibitors are under investigation as a way of increasing immunogenicity and promoting a robust anti-tumor immune response. The purpose of this study is to quantify the immune responses induced by mono and dual checkpoint inhibition in a mismatch repair proficient model of colorectal cancer (CRC). Tumor growth rates were monitored over time and compared between groups. We utilized fluorescence-activated cell sorting to analyze CD8+ and CD4+ T cells after treatment with either single PD-1 inhibition or dual PD-1 and CTLA-4 inhibition. Additionally, we sought to quantify the expression of co-inhibitory surface molecules PD-1, LAG3, and TIM3. Dual checkpoint inhibition was associated with a significantly slower growth rate as compared to either mono PD-1 inhibition or control (p < 0.05). Neither monotherapy nor dual checkpoint inhibition significantly affected the tumoral infiltration of lymphocytes. After treatment with dual inhibitors, infiltrating CD8+ T cells demonstrated significantly less expression of PD-1 (1700 vs. 2545 and 2462; p < 0.05) and LAG3 (446.2 vs. 694.4 and 707; p < 0.05) along with significantly more expression of TIM3 (12,611 vs. 2961 and 4259; p < 0.05) versus the control and anti-PD-1 groups. These results suggest that dual therapy with anti-CTLA-4 and anti-PD-1 antibodies significantly inhibits growth of microsatellite stable CRC by suppressing immunosuppressive checkpoints. Upregulation of TIM3 represents a potential escape mechanism and a target for future combination immunotherapies in CRC.

TLR4-mediated activation of mouse macrophages by Korean mistletoe lectin-C (KML-C).

Korean mistletoe lectin (KML-C) is an adjuvant that activates systemic and mucosal immune cells to release cytokines including TNF-alpha, which induces immunity against viruses and cancer cells. Although the immunomodulatory activity of KML-C has been well established, the underlying mechanism of action of KML-C has yet to be explored. When mouse peritoneal macrophages were treated with KML-C, both transcription and translation of TLR4 were upregulated. KML-C-induced TLR4 downstream events were similar to those activated by LPS: the upregulation of interleukin-1 receptor-associated kinase-1 (IRAK1); resulting in macrophage activation and TNF-alpha production. When TLR4 was blocked using a TLR4-specific neutralizing antibody, TNF-alpha production from the macrophages was significantly inhibited. Moreover, TLR4-deficient mouse macrophages treated with KML-C also secreted greatly reduced level of TNF-alpha secretion. Finally, TLR4 molecules were co-precipitated with KML-C, to which agarose beads were conjugated, indicating that those molecules are associated. These data indicate that KML-C activates mouse macrophages to secrete TNF-alpha by interacting with the TLR4 molecule and activating its signaling pathways.

The Effects of AHCC®, a Standardized Extract of Cultured Lentinura edodes Mycelia, on Natural Killer and T Cells in Health and Disease: Reviews on Human and Animal Studies.

Mushrooms have been used for various health conditions for many years by traditional medicines practiced in different regions of the world although the exact effects of mushroom extracts on the immune system are not fully understood. AHCC® is a standardized extract of cultured shiitake or Lentinula edodes mycelia (ECLM) which contains a mixture of nutrients including oligosaccharides, amino acids, and minerals obtained through liquid culture. AHCC® is reported to modulate the numbers and functions of immune cells including natural killer (NK) and T cells which play important roles in host defense, suggesting the possible implication of its supplementation in defending the host against infections and malignancies via modulating the immune system. Here, we review in vivo and in vitro effects of AHCC® on NK and T cells of humans and animals in health and disease, providing a platform for the better understanding of immune-mediated mechanisms and clinical implications of AHCC®.

Estrogen receptor α in T cells suppresses follicular helper T cell responses and prevents autoimmunity.

Estrogen receptor alpha (ERα) is a sex hormone nuclear receptor that regulates various physiological events, including the immune response. Although there have been some recent studies on ERα regarding subsets of T cells, such as Th1, Th2, Th17, and Treg cells, its role in follicular helper T (TFH) cells has not yet been elucidated. To determine whether ERα controls TFH response and antibody production, we generated T cell-specific ERα knockout (KO) mice by utilizing the CD4-Cre/ERα flox system (CD4-ERα KO) and then analyzed their phenotype. At approximately 1 year of age, CD4-ERα KO mice spontaneously showed mild autoimmunity with increased autoantibody production and CD4+CD44+CXCR5+Bcl-6+ TFH cells in the mesenteric lymph nodes and spleen. We next immunized 6-8-week-old CD4-ERα KO mice with sheep red blood cells (SRBCs), which resulted in an increased proportion of TFH cells and germinal center (GC) responses. In addition, 17ß-estradiol (E2) treatment decreased TFH responses in wild-type mice and suppressed the mRNA expression of Bcl-6 and IL-21. Finally, we confirmed that the production of high-affinity antigen-specific antibodies and isotype class switching induced by NP-conjugated ovalbumin immunization were elevated in CD4-ERα KO mice under sufficient estrogen conditions. These results collectively demonstrate that the female sex hormone receptor ERα inhibits the TFH cell response and GC reaction to control autoantibody production, which was related to estrogen signaling and autoimmunity.

Transcriptomic analysis of human IL-7 receptor alpha low and high effector memory CD8+ T cells reveals an age-associated signature linked to influenza vaccine response in older adults.

Here, we investigated the relationship of the age-associated expansion of IL-7 receptor alpha low (IL-7Rαlow ) effector memory (EM) CD8+ T cells with the global transcriptomic profile of peripheral blood cells in humans. We found 231 aging signature genes of IL-7Rαlow EM CD8+ T cells that corresponded to 15% of the age-associated genes (231/1,497) reported by a meta-analysis study on human peripheral whole blood from approximately 15,000 individuals, having high correlation with chronological age. These aging signature genes were the target genes of several transcription factors including MYC, SATB1, and BATF, which also belonged to the 231 genes, supporting the upstream regulatory role of these transcription factors in altering the gene expression profile of peripheral blood cells with aging. We validated the differential expression of these transcription factors between IL-7Rαlow and high EM CD8+ T cells as well as in peripheral blood mononuclear cells (PBMCs) of young and older adults. Finally, we found a significant association with influenza vaccine responses in older adults, suggesting the possible biological significance of the aging signature genes of IL-7Rαlow EM CD8+ T cells. The results of our study support the relationship of the expansion of IL-7Rαlow EM CD8+ T cells with the age-associated changes in the gene expression profile of peripheral blood cells and its possible biological implications.

Macrophage Migration Inhibitory Factor Regulates U1 Small Nuclear RNP Immune Complex-Mediated Activation of the NLRP3 Inflammasome.

OBJECTIVE: High-expression alleles of macrophage migration inhibitory factor (MIF) are linked genetically to the severity of systemic lupus erythematosus (SLE). The U1 small nuclear RNP (snRNP) immune complex containing U1 snRNP and anti-U1 snRNP antibodies, which are found in patients with SLE, activates the NLRP3 inflammasome, comprising NLRP3, ASC, and procaspase 1, in human monocytes, leading to the production of interleukin-1ß (IL-1ß). This study was undertaken to investigate the role of the snRNP immune complex in up-regulating the expression of MIF and its interface with the NLRP3 inflammasome. METHODS: MIF, IL-1ß, NLRP3, caspase 1, ASC, and MIF receptors were analyzed by enzyme-linked immunosorbent assay, Western blotting, quantitative polymerase chain reaction, and cytometry by time-of-flight mass spectrometry (CytoF) in human monocytes incubated with or without the snRNP immune complex. MIF pathway responses were probed with the novel small molecule antagonist MIF098. RESULTS: The snRNP immune complex induced the production of MIF and IL-1ß from human monocytes. High-dimensional, single-cell CytoF analysis established that MIF regulates activation of the NLRP3 inflammasome, including findings of a quantitative relationship between MIF and its receptors and IL-1ß levels in the monocytes. MIF098, which blocks MIF binding to its cognate receptor, suppressed the production of IL-1ß, the up-regulation of NLRP3, which is a rate-limiting step in NLRP3 inflammasome activation, and the activation of caspase 1 in snRNP immune complex-stimulated human monocytes. CONCLUSION: The U1 snRNP immune complex is a specific stimulus of MIF production in human monocytes, with MIF having an upstream role in defining the inflammatory characteristics of activated monocytes by regulating NLRP3 inflammasome activation and downstream IL-1ß production. These findings provide mechanistic insight and a therapeutic rationale for targeting MIF in subgroups of lupus patients, such as those classified as high genotypic MIF expressers or those with anti-snRNP antibodies.