Frequency Of Cells Research Articles

Physical inactivity exacerbates pathologic inflammatory signalling at the single cell level in patients with systemic lupus

Physical activity is an adjunctive therapy that improves symptoms in people living with systemic lupus erythematosus (SLE), yet the mechanisms underlying this benefit remain unclear. We carried out a cohort study of 123 patients with SLE enrolled in the California Lupus Epidemiology Study (CLUES). The primary predictor variable was self-reported physical activity, which was measured using a previously validated instrument. We analyzed peripheral blood mononuclear cell (PBMC) single-cell RNA sequencing (scRNA-seq) data available from the cohort. From the scRNA-seq data, we compared immune cell frequencies, cell-specific gene expression, biological signalling pathways, and upstream cytokine activation states between physically active and inactive patients, adjusting for age, sex and race. We found that physical activity influenced immune cell frequencies, with sedentary patients most notably demonstrating greater CD4+ T cell lymphopenia (Padj=0.028). Differential gene expression analysis identified a transcriptional signature of physical inactivity across five cell types. In CD4+ and CD8+ T cells, this signature was characterized by 686 and 445 differentially expressed genes (Padj<0.1). Gene set enrichment analysis demonstrated enrichment of proinflammatory genes in the TNF-α signalling through NF-kB, interferon-γ (IFN-γ), IL2/STAT5, and IL6/JAK/STAT3 signalling pathways. Computational prediction of upstream cytokine activation states suggested CD4+ T cells from physically inactive patients exhibited increased activation of TNF-α, IFN-γ, IL1Β, and other proinflammatory cytokines. Network analysis demonstrated interconnectivity of genes driving the proinflammatory state of sedentary patients. Findings were consistent in sensitivity analyses adjusting for corticosteroid treatment and physical function. Taken together, our findings suggest a mechanistic explanation for the observed benefits of physical activity in patients with SLE. Specifically, we find that physical inactivity is associated with altered frequencies and transcriptional profiles of immune cell populations and may exacerbate pathologic inflammatory signalling via CD4+ and CD8+ T cells. This work was supported by the US National Institutes of Health (NIH) (R01 AR069616, K23HL138461-01A1, K23AT011768) the US CDC (U01DP0670), and the CZ Biohub.

Effects of dopamine receptor antagonists and radiation on mouse neural stem/progenitor cells

BackgroundDopamine receptor antagonists have recently been identified as potential anti-cancer agents in combination with radiation, and a first drug of this class is in clinical trials against pediatric glioma. Radiotherapy causes cognitive impairment primarily by eliminating neural stem/progenitor cells and subsequent loss of neurogenesis, along with inducing inflammation, vascular damage, and synaptic alterations. Here, we tested the combined effects of dopamine receptor antagonists and radiation on neural stem/progenitor cells. MethodsUsing transgenic mice that report the presence of neural stem/progenitor cells through Nestin promoter-driven expression of EGFP, the effects of dopamine receptor antagonists alone or in combination with radiation on neural stem/progenitor cells were assessed in sphere-formation assays, extreme limiting dilution assays, flow cytometry and real-time PCR in vitro and in vivo in both sexes. ResultsWe report that hydroxyzine and trifluoperazine exhibited sex-dependent effects on murine newborn neural stem/progenitor cells in vitro. In contrast, amisulpride, nemonapride, and quetiapine, when combined with radiation, significantly increased the number of neural stem/progenitor cells in both sexes. In vivo, trifluoperazine showed sex-dependent effects on adult neural stem/progenitor cells, while amisulpride demonstrated significant effects in both sexes. Further, amisulpride increased sphere forming capacity and stem cell frequency in both sexes when compared to controls. ConclusionWe conclude that a therapeutic window for dopamine receptor antagonists in combination with radiation potentially exists, making it a novel combination therapy against glioblastoma. Normal tissue toxicity following this treatment scheme likely differs depending on age and sex and should be taken into consideration when designing clinical trials.

Infants < 90 days of age with late-onset sepsis display disturbances of the microbiome-immunity interplay.

We hypothesized that previously healthy infants < 90 days of age with late-onset sepsis (LOS) have disturbances of the gut microbiome with yet undefined specific immunological patterns. We performed a prospective single-center convenience sample study between January 2019 and July 2021 in a case-control design. Routine diagnostics included conventional cultures (blood, cerebrospinal fluid, urine), PCRs and inflammatory markers in infants aged < 90 days with clinical LOS. We additionally analyzed blood lymphocyte subsets including CD4 + CD25 + forkhead box protein (FoxP3)+ Tregs and performed 16S rRNA sequencing of stool samples, both compared to age-matched healthy controls. Results were adjusted for potential confounders that may influence microbial composition. 51 infants with fever and clinical LOS were enrolled. Bacterial sepsis was diagnosed in n = 24 (47.1%) and viral infection in n = 13 (25.5%) infants, whereas in 14 (27.3%) infants the cause of fever remained undetermined. When compared to healthy controls, the gut microbiome of LOS infants at disease onset was characterized by a shift in community composition, specifically, decreased abundance of B. longum and an increase of Bacteroidia spp. Intriguingly, the abundance of B. longum negatively correlated with the frequency of blood CD4-positive cells in healthy controls but not in infants with LOS. At one year of age, we observed microbiome differences in infants with history of LOS when compared to healthy controls, such as an increased gut microbial diversity. Our data suggest potential signatures of the microbiome-immunity interplay in infants with LOS, which should be investigated further as possible targets for prevention.

CD40 agonist on patient-derived xenograft mice for the treatment of B-cell acute lymphoblastic leukemia.

Cluster of differentiation 40 (CD40) is expressed on B-cell acute lymphoblastic leukemia (B-ALL) cases. However, the effect of CD40 activation on B-ALL cells has never been tested in vivo. The aim of our preclinical study was to investigate the therapeutic potential of a CD40 agonist in the treatment of B-ALL using patient-derived xenograft (PDX) mouse models. Intravenous administration of the CD40 agonist significantly impeded B-ALL cell proliferation and growth in vivo, accompanied by rapid activation of the extracellular signal-regulated kinase (ERK) pathway, leading to the induction of apoptosis and disruption of cell cycle progression. Co-treatment with a specific inhibitor of ERK further demonstrated that CD40 stimulation induced the pro-apoptosis of B-ALL cells in an ERK-dependent manner. Proteomic analysis revealed alterations in key signaling pathways associated with B-ALL expansion and maintenance. Moreover, the CD40 agonist markedly reduced the frequency of leukemia-initiating cells and leukemia development in PDX mice. Our study showed that the CD40 agonist can be associated with chemotherapeutic agents such as vincristine and dexamethasone, and this combination showed improved effectiveness. Additionally, the CD40 agonist was more effective on pre-B-ALL (EGIL B-III) that expressed CD40, than on common B-ALL (EGIL B-II) that lacked CD40 expression. These findings suggest that CD40 agonists are promising immunotherapeutic candidates for pediatric B-ALL, warranting further clinical investigations to improve patient outcomes in CD40-expressing B-ALL.

Abstract A041: Detection of early-stage gastrointestinal cancers using micronuclei DNA from erythrocytes

Abstract Background: Gastrointestinal (GI) cancers account for over a third of cancer deaths. However, their early detection remains a challenge. Micronuclei (MN) are cytoplasm-resident subcellular structures, containing damaged chromosome segments that indicative of genomic instability. Increased MN frequency in hematopoietic cells is observed in patients with solid tumors. Our developed technique (WO2021/228246 A1) enables the purification and characterization of micronuclei DNA in erythrocytes from peripheral blood. By comparing MN-DNA from healthy donors (HDs) and GI cancer patients, we identified significant changes in read densities at specific genomic locations in patients, which were termed as tumor-associated MN-DNA (taMN- DNA) features. Here, we evaluated the potential of these features for early-stage GI cancer detection, including colorectal (CC), gastric (GC) and esophageal (EC) cancers. Methods: Peripheral blood (1-2 mL) was collected from healthy donors (HD) and cancer patients MN-DNA isolation and purification from erythrocytes. Participants were randomly divided into training, validation and independent test cohorts in a 7:2:1 ratio, maintaining similar cancer types, gender and age distribution. Distinct MN-DNA features between cancer patients and HDs were identified using sequencing data. Machine learning algorithms were applied using these features for cancer detection. Results: The study enrolled 1753 participants, including 987 HDs, 420 CC, 282 GC and 64 EC cases. Of these, over half were diagnosed with early-stage diseases; TNM stage 0-I accounted for 40.57%, stage II for 23.32%, stage III for 29.25% and stage IV for 6.87%. The pan-cancer model achieved 88.2% sensitivity with an overall specificity of 95.4%. Patients in individual caner types were detected from the tissue of origin classification with an overall accuracy of 91.9% based on pan-cancer at 95.4% specificity. Sensitivity was 90.8% for CC, 91.7% for GC and 100% for EC. For early- stage (stage 0-II) CC, GC and EC, the model demonstrated sensitivities of 97.4%, 94.7% and 100.00%, respectively, at 95.4% specificity. Conclusions: Unlike cell-free DNA, MN-DNA are chromosomal fragments in the cytoplasm. The abundance of erythrocytes in peripheral blood provides an easily accessible source for MN-DNA enrichment. This pilot study illustrates the potential of MN-DNA as a tool for early GI cancer detection, contributing to large-scale efforts towards developing an effective GI cancer screening test. Research sponsor: Timing Biotech. Citation Format: Haobo Sun, Xingyun Yao, Yuehua Han, Yurong Jiao, Xiangxing Kong, Chengcheng Liu, Qingqu Guo, Fei Meng, Honghao Liang, Hongbo Li, Xiuqin Fan, Jun Li, Xiaofei Gao. Detection of early-stage gastrointestinal cancers using micronuclei DNA from erythrocytes [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A041.

Longitudinal Follow-up Reveals Peripheral Immunological Changes Upon Tick Bite in a-Gal-Sensitized Individuals.

α-Gal syndrome is characterized by specific IgE (sIgE) antibodies to the carbohydrate galactose-α-1,3-galactose (α-Gal) and delayed onset of allergic symptoms after ingestion of mammalian meat. While tick bites are assumed to mediate sensitization, the immune response to tick bites has not yet been investigated. To investigate the peripheral immune response to tick bites in humans over time. In a longitudinal cohort study, immunological reactions associated with tick bites (Ixodes species) were analyzed within 1 day (V1), 2 weeks (V2), 1 month (V3), and 3 months (V4) after the occurrence of a bite. sIgE, sIgG, and sIgG subclass levels, as well as 10 cytokines, were quantified. Deep immune phenotyping was performed using mass cytometry. A total of 4 controls and 10 patients were bitten by a tick and followed up over 3 months. None of the controls developed sIgE to α-Gal, and sIgE increased in all patients from V1 until V2/V3, as did IL-8 levels. We noted a significant increase in CD19+ B cells and B-cell subpopulations, as well as a decrease in γδ CD56+ T cells in patients between V0 and V1. At V1, frequencies of plasmacytoid dendritic cells (pDCs) and γδ CD56+ T cells were lower in patients than in controls. Our study provides evidence of significant changes in several immune cell populations in α-Gal sensitized patients, along with increased levels of IL-8 and sIgE. This is the first exploratory study to investigate longitudinal peripheral immune profiles in patients and controls bitten by ticks.

Abstract 4137935: Adipogenesis in Bone Marrow Niche under Cardiac Stress Worsens Cardiac Function

Background: We have recently reported that repetitive cardiac decompensation with multimorbidity often experienced by patients with heart failure (HF) is attributed to epigenetic modifications of hematopoietic stem cells (HSCs) in the bone marrow (BM). HF reprogrammed HSCs differentiation and altered tissue macrophage homeostasis. These findings demonstrate that the BM can carry an innate immune memory of cardiac stress that may exacerbate HF and predispose other organs to pathology. Aims: Because the stemness of HSCs is mainly regulated by mesenchymal stromal cells (MSCs) in the BM niche, we investigated phenotypic alterations of MSCs under cardiac stress. Methods&amp;Results: Transcriptome analysis of MSCs showed preferential differentiation toward adipocytes in murine pressure overload models. In vitro assays and histological BM sections also support this finding. Furthermore, single-cell RNA sequencing of MSCs demonstrated that the percentage of adipocyte-primed MSCs increased in proportion to the severity of cardiac dysfunction, and also correlated with the frequency of myeloid-lineage progenitor cells. To investigate the influence of adipo-lineage MSCs on HSCs, we conducted BM transplantation supplemented with MSCs from control or HF mice. Recipient mice transplanted with HF-MSCs showed significant increases in myeloid-biased multipotent progenitors in BM and myeloid cells in peripheral blood. Additionally, the number of proinflammatory cardiac macrophages was significantly increased in the HF-MSCs group, promoting cardiac fibrosis and dysfunction. Conclusions: Our results demonstrated that the BM niche could perceive cardiac stress in the form of adipocytic skewing of MSCs in the setting of HF, which changed the differentiation behavior in HSCs and ultimately led to further deterioration of cardiac function through the impaired differentiation of circulating monocytes into cardiac macrophages. Therefore, suppressing the adipocytic differentiation of MSCs could have a novel therapeutic potential to avoid repeated HF events.

IMMU-43. GUT MICROBIOTA SHAPE IMMUNE PATTERNS IN NAÏVE AND GLIOMA-BEARING HUMANIZED MICROBIOME MICE

Abstract Immunotherapy increases survival in mouse models of glioblastoma, but this is not observed in clinical trials. To address this issue, we employed a humanized microbiome (HuM) mouse model where fecal matter is transplanted from healthy human donors into gnotobiotic mice. Our previous research found that in the GL261 model of glioma, HuM mouse lines could be classified as either responders or nonresponders to anti-PD-1 therapy based solely on the gut microbiota unique to each HuM line. Here, we sought to determine the immune differences between responder and nonresponder HuM lines in both naïve (tumor-free) and glioma conditions. Single-cell RNA-sequencing (scRNA-seq) analysis of naïve colonic CD45+ cells showed that responder colons had higher frequencies of neutrophils and naïve B cells and lower frequencies of plasmablast and plasma cells. Notably, colonic neutrophils in naïve responders upregulated H2-Eb1 and Il1a compared to nonresponders. To validate the scRNA-seq data, flow cytometry was also performed on the naïve lymph nodes (LNs) and brains. The gut-draining LNs of responders had a higher frequency of CD45+ immune cells and CD4+CD8+ T cells and a lower frequency of Tregs than nonresponders. In naïve brains, responders had a higher frequency of CD45+ cells and macrophages than nonresponders and trended toward having fewer CD206+ macrophages. When mice were injected with GL261 tumor cells and treated with one dose of anti-PD-1, there was a trend for an increase in Nos2+ CD11b+ cells in both frequency and count compared to nonresponders, indicating an early and robust anti-tumor myeloid response in the responders. In summary, these results show how variations in the microbiota of HuM mice impact the immune environment across multiple organs in the baseline and tumor states, which potentially accounts for the positive response to anti-PD-1 therapy in the glioma model.

Perspectives on Ergodic Cancer Therapy Derived from Cloning Genome Chaos via In Vivo Rhabdomyosarcoma RA-2 Models: a Narrative Review.

This review explores articles concerning the experimental research cycle on genome instability in cell populations of highly malignant recurrent organotropic rhabdomyosarcoma RA- 2 in rats. Clonal analysis and cloning were pivotal components of this research, which relies on the frequency of cells with micronuclei and internuclear bridges to gauge the intensity of chromothripsis and break-fusion-bridge cycles. The efficacy of cloning, determined by these indicators, stemmed from the deliberate isolation of tumor stem cells, yielding clones in which chromothripsis activity and breakage-fusion-bridge cycles were sustained. Notably, it is plausible that the stem cells themselves, progenitors of these clones, harbor dicentric chromosomes and chromosomal fragments, contributing to the formation of "fatal micronuclei" in their karyotype. Cloning based on bridges and micronuclei has proven effective up to a certain threshold (15%-18%), reaffirming the predicted reproductive extinction of malignant cell populations under mutational pressure and genome chaos, as posited by the genetic theory of cell populations. Furthermore, this review highlights the potential of ergodic cancer therapy as a novel therapeutic strategy. Ergodic therapy offers promising prospects for late-stage and terminal malignant tumors, where conventional treatments may fall short due to advanced progression. Furthermore, by "enhancing chromothripsis" through the induction of additional micronuclei and bridges, ergodic cancer therapy seeks to increase genome chaos to a critical threshold, potentially halting malignant progression. This innovative approach presents opportunities to explore new drugs and targets for chromothripsis-based oncotherapy, addressing the pressing need for effective treatments in advanced stages of malignancy.

Transcriptomic and de novo proteomic analyses of organotypic entorhino-hippocampal tissue cultures reveal changes in metabolic and signaling regulators in TTX-induced synaptic plasticity

Understanding the mechanisms of synaptic plasticity is crucial for elucidating how the brain adapts to internal and external stimuli. A key objective of plasticity is maintaining physiological activity states during perturbations by adjusting synaptic transmission through negative feedback mechanisms. However, identifying and characterizing novel molecular targets orchestrating synaptic plasticity remains a significant challenge. This study investigated the effects of tetrodotoxin (TTX)-induced synaptic plasticity within organotypic entorhino-hippocampal tissue cultures, offering insights into the functional, transcriptomic, and proteomic changes associated with network inhibition via voltage-gated sodium channel blockade. Our experiments demonstrate that TTX treatment induces substantial functional plasticity of excitatory synapses, as evidenced by increased miniature excitatory postsynaptic current (mEPSC) amplitudes and frequencies in both dentate granule cells and CA1 pyramidal neurons. Correlating transcriptomic and proteomic data, we identified novel targets for future research into homeostatic plasticity, including cytoglobin, SLIT-ROBO Rho GTPase Activating Protein 3, Transferrin receptor, and 3-Hydroxy-3-Methylglutaryl-CoA Synthase 1. These data provide a valuable resource for future studies aiming to understand the orchestration of homeostatic plasticity by metabolic pathways in distinct cell types of the central nervous system.

NET-EN treatment leads to delayed HSV-2 infection, enhanced mucin and T cell functions in the female genital tract when compared to DMPA in a preclinical mouse model

Depot-medroxyprogesterone acetate (DMPA) and Norethisterone Enanthate (NET-EN) are progestin-only injectable contraceptives widely used by women in sub-Sharan Africa, where incidence of HIV-1 and HSV-2 infection remains high. Studies indicate that DMPA usage can increase the risk of HSV-2 infection, but limited data indicate no increased risk with use of NET-EN. We therefore investigated the effects of NET-EN and DMPA on susceptibility to vaginal HSV-2 infection in ovariectomized (OVX) mice and effects on immune responses, particularly in the vaginal tract (VT). OVX mice, when treated with NET-EN and infected intravaginally, had delayed genital pathology, decreased viral shedding, and extended survival compared to DMPA- or untreated OVX mice. CD4+ T cells isolated from VT showed no significant change in frequency with either contraceptive. However, DMPA significantly decreased the total number of VT CD4+ and CD8+ T cells and the number of IFN-γ producing CD4 and CD8 T cells and increased the percentage of CD4 and CD8 T cells producing TNF-α compared to untreated mice. In contrast, NET-EN significantly enhanced percentages of CD8+ T cells compared to DMPA treated mice, and frequencies of IFN-γ+ CD4 and CD8 T cells in the VT compared to untreated mice. Comparative analysis of splenic lymphocytes indicated that DMPA treatment resulted in reduction of CD4+ T cell frequency, but enhanced TNF-α+ CD4 T cells compared to untreated mice. NET-EN enhanced the frequency of CD8 T cells, as well as IFN-γ+ and TNF-α+ CD4, and IFN-γ+ CD8 T cells in the spleen compared to untreated mice. Importantly, we found DMPA treatment that significantly reduced mucin production, whereas NET-EN enhanced expression of cell-associated mucin in VT. High levels of mucin in NET-EN mice were associated with lower levels of HSV-2 virus detected in the vaginal tract. This study provides the first evidence that NET-EN treatment can delay HSV-2 infection compared to DMPA.

Immune responses in checkpoint myocarditis across heart, blood and tumour.

Immune checkpoint inhibitors are widely used anticancer therapies1 that can cause morbid and potentially fatal immune-related adverse events such as immune-related myocarditis (irMyocarditis)2-5. The pathogenesis of irMyocarditis and its relationship to antitumour immunity remain poorly understood. Here we sought to define immune responses in heart, tumour and blood in patients with irMyocarditis by leveraging single-cell RNA sequencing coupled with T cell receptor (TCR) sequencing, microscopy and proteomics analyses of samples from 28 patients with irMyocarditis and 41 unaffected individuals. Analyses of 84,576 cardiac cells by single-cell RNA sequencing combined with multiplexed microscopy demonstrated increased frequencies and co-localization of cytotoxic T cells, conventional dendritic cells and inflammatory fibroblasts in irMyocarditis heart tissue. Analyses of 366,066 blood cells revealed decreased frequencies of plasmacytoid dendritic cells, conventional dendritic cells and B lineage cells but an increased frequency of other mononuclear phagocytes in irMyocarditis. Fifty-two heart-expanded TCR clones from eight patients did not recognize the putative cardiac autoantigens α-myosin, troponin I or troponin T. Additionally, TCRs enriched in heart tissue were largely nonoverlapping with those enriched in paired tumour tissue. The presence of heart-expanded TCRs in a cycling blood CD8 T cell population was associated with fatal irMyocarditis case status. Collectively, these findings highlight crucial biology driving irMyocarditis and identify putative biomarkers.

Regional variations in hyperpolarized 129Xe lung MRI: Insights from CSI-CSSR and CSSR in healthy and irradiated rat models.

To compare pulmonary function metrics obtained with hyperpolarized xenon-129 (HXe) MRS, using chemical shift saturation recovery (CSSR) and CSI-CSSR, in healthy rats and a rat model of radiation-induced lung injury. HXe-MR data were acquired in two healthy rats and one rat with radiation-induced lung injury using whole-lung spectroscopy and CSI-CSSR techniques. The CSI-CSSR acquisitions were performed with both fixed TE and variable TE. Apparent alveolar septal wall thickness, gas transfer dynamics, and regional lung function were quantified and compared across acquisition methods. Spectral analysis included alignment of dissolved-phase frequency spectra using the membrane resonance as reference, segmentation of gas-phase (GP) frequency distribution, and characterization of gas uptake in the vasculature. Complex GP line shapes were observed in rat lungs, necessitating pixel-wise CSI analysis and membrane resonance alignment for improved quantification. Notable differences in alveolar septal wall thickness, dissolved-phase GP ratios, and GP and red blood cell frequencies were found between acquisition techniques and lung conditions. CSI-CSSR provided unique insights into regional lung function, including the identification of distinct GP frequency zones potentially corresponding to different airway structures, and the ability to map relative xenon gas transport. Metrics from fixed-TE and variable-TE acquisitions usually differed by less than 10%, but the latter yielded a 20% SNR gain. HXe-MRS and CSI-CSSR techniques provide similar but not universally interchangeable insights into lung function, particularly in the presence of pathology.

Identification of clonally expanded γδ T-cell populations during CAR-Tcell therapy.

Anti-CD19 Chimeric Antigen Receptor (CAR)-Tcell therapies have shown promise for treating B cell malignancies, but the clinical outcome is influenced by both the CAR-T product and the patient's immune system. The role of γδ Tcells in the context of CAR-T cell therapy remains poorly understood. This study investigates the transcriptional heterogeneity, clonal expansion and dynamics of γδ Tcells in patients undergoing anti-CD19 CAR-T cell therapy. Longitudinal single cell multi-omics analysis was performed on γδ Tcells from four patients receiving anti-CD19 CAR-T cell therapy. Single cell RNA-seq, antibody-based protein profiling (AbSeq) and full-length TCRγδ sequences revealed clonally expanded populations displaying plasticity in Tcell differentiation, and temporal dynamics of large clones, suggesting ongoing expansion and differentiation. Clonally expanded γδ Tcells had heterogeneous gene expression profiles, occupying seven transcriptionally distinct clusters. Analysis of chemokine markers indicated cluster-specific homing tendencies of circulating γδ Tcells to peripheral tissues. We found unexpectedly high frequencies of Vδ1 and Vδ3 cells in the blood with distinct gene and protein expression profiles. This analysis provides insights into the dynamic and heterogeneous nature of γδ Tcells following anti-CD19 CAR-T cell therapy, contributing valuable information for optimizing CAR-T cell therapies in B cell malignancies.

Antigen-specific T cell frequency and phenotype mirrors disease activity in DRB1*04:04+ rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is associated with high-risk HLA class II alleles known as the "RA shared epitope." Among prevalent shared epitope alleles, study of DRB1*04:04 has been limited. To define relevant epitopes, we identified citrullinated peptide sequences from synovial antigens that were predicted to bind to HLA-DRB1*04:04 and utilized a systematic approach to confirm their binding and assess their recognition by CD4 T cells. After confirming the immunogenicity of 13 peptides derived from aggrecan, cartilage intermediate layer protein (CILP), α-enolase, vimentin, and fibrinogen, we assessed their recognition by T cells from a synovial tissue sample, observing measurable responses to 8 of the 13 peptides. We then implemented a multicolor tetramer panel to evaluate the frequency and phenotype of antigen-specific CD4 T cells in individuals with anti-citrullinated protein antibody (ACPA)-positive RA and controls. In subjects with RA, CILP-specific T cell frequencies were significantly higher than those of other antigens. The surface phenotypes exhibited by antigen-specific T cells were heterogeneous, but Th1-like and Th2-like cells predominated. Stratifying based on disease status and activity, antigen-specific T cells were more frequent and most strongly polarized in RA subjects with high disease activity. In total, these findings identify novel citrullinated epitopes that can be used to interrogate antigen-specific CD4 T cells and show that antigen-specific T cell frequency is elevated in subjects with high disease activity.

Patients with IgA Nephropathy Show Abnormal Frequencies of B cell Subsets and Unconventional T Cells and High Levels of Gd-IgA1 Coated Gut Bacteria

BackgroundMucosal inflammation is involved in the pathophysiology of Immunoglobulin-A nephropathy (IgAN), but peripheral immune phenotype analyses of IgAN patients often do not include unconventional T cells, the major subset in mucosal immunity. MethodsWe measured serum total IgA, gd-IgA1, secretory IgA, B cell activating factor (BAFF), and A proliferation-inducing ligand (APRIL) in 66 IgAN patients and 30 healthy controls (HC). We also quantified total IgA and gd-IgA1 in stool supernatant and coated on bacteria. In 35 patients and 14 controls, we performed extensive phenotyping (CyTOF) of circulating immune cells, including unconventional T cells [mucosal associated invariant T (MAIT) cells, γδ T, and NK T cells]. The results were validated using RNAseq data from a larger cohort of 179 patients with IgAN, 140 patients with minimal change disease (MCD), and 91 HC. ResultsIgAN patients had higher circulating levels of total IgA, gd-IgA1, and APRIL and higher IgA- and gd-IgA1-coated gut bacteria than controls, while serum levels of secretory IgA and BAFF did not differ between groups. IgAN patients showed more class switched memory and double negative B cells than controls. MAIT cells and γδ T cells were significantly lower, and CD4- CD8- NK T cells significantly higher in IgAN patients than in HC. We validated the significant decrease in MAIT cells in an independent cohort of patients with IgAN. ConclusionThe data indicate that patients with IgAN have increased circulating class switched memory and double negative B cells associated with abnormal T cell immunity, involving defects in unconventional T cell frequency. This may suggest putative alterations at mucosal sites due to cell migration leading to altered IgA production.

Buserelin Promotes the Differentiation and Function of Macrophage-Colony-Stimulating Factor-Producing T Helper Cells

ABSTRACT Background Buserelin has been used to treat central precocious puberty (CPP). However, it could potentially result in immune dysregulation to undermine patients’ health. Therefore, it is necessary to elucidate the effects of buserelin on immune cells. Here we explored buserelin-induced impacts on the differentiation and function of macrophage-colony-stimulating factor-producing T helper (ThGM) cells to uncover the immunoregulatory role of buserelin. Methods Rat CPP was induced by danazol injection followed by buserelin treatment. The frequencies of ThGM cells in the spleen and lymph nodes were evaluated by flow cytometry. ThGM cell generation and function were analyzed in cell culture assays. Cell signaling was measured by Immunoblotting. Results Buserelin increased the frequencies of splenic and lymph node ThGM cells. Buserelin promoted the in vitro differentiation and proliferation of ThGM cells. Buserelin-treated ThGM cells showed stronger supportive effects on other effector T helper cells. Buserelin induced the activation of the nuclear factor of activated T cells and extracellular signal-regulated kinase 1/2 in ThGM cells. Conclusion Buserelin enhances the differentiation and function of pro-inflammatory ThGM cells, thus increasing the risk of autoimmune or inflammatory disorders. Therefore, it is necessary to monitor ThGM cells in buserelin-treated children to prevent latent immune dysregulation.

Alterations in T cell immunity over 6–12 months post-COVID-19 infection in convalescent individuals: a screening study

Acute COVID-19 is a viral infection caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that results in dramatically decreased peripheral blood CD3+T cell count apparently due to alterations of thymic T cell maturation, that can persist long term afterwards. Therefore, we analyzed the levels of peripheral blood TRECs (T-cell receptor excision circles), and investigated the main alterations in peripheral blood T cell subsets in COVID-19 convalescents. We performed molecular quantification of TRECs with “TREC/KREC-AMP PS” kit and flow cytometric analysis of peripheral blood lymphocytes from three groups of patients. The first group contained 109 samples from COVID-19 convalescents (6–12 month post-acute COVID-19) with normal levels of TRECs (TRECn); the second was formed from COVID-19 convalescents (6–12 month post-acute COVID-19) with decreased levels of TRECs (TREClow, n = 29), and healthy control group (HC, n = 18). We noticed no significant differences between all three groups in CD3+T cell relative and absolute numbers. However, CD4+T cell frequencies were decreased in TREClow and TRECn groups compared to HC (40.8% (31.6; 50.1) and 46.4% (40.0; 53.0) vs 53.5% (47.36; 56.9), p 0.001 and р = 0.004, respectively). Furthermore, Th cell levels were decreased in TREClow patients vs HC and TRECn groups (701 cell/1 µL (478; 807) vs 1005 cell/1 µL (700; 1419), р = 0.020, and 876 cell/ 1 µL (661; 1046), р = 0.008, respectively). Finally, both groups of COVID-19 convalescents had increased frequencies of circulating CD8+T cells — 29.4% (20.7; 39.7) in TREClow group, 26.5% (21.1; 32.7) in TRECn group vs 21.3% (17.1; 26.0) in healthy controls (p = 0.024 and р = 0.026, respectively). In TRECn group, CD8+T cell count was elevated vs control range (508 cell/1 µL (372; 622) vs 356 cell/1 µL (247; 531), р = 0.044). Thus, COVID-19 convalescents (6–12 month post-acute COVID-19) showed an imbalance in CD4+and CD8+T cell level even at 6–12 months post-acute SARS-CoV-2 infection, and the observed changes in peripheral blood T cells could be closely related to the alterations in thymic T cell maturation and differentiation. Such a long-term decline in TREC levels in the circulation may have a profound impact on immune system functions and requires immunocorrection therapy.

Transiently boosting Vγ9+Vδ2+ γδ T cells early in Mtb coinfection of SIV-infected juvenile macaques does not improve Mtb host resistance.

Children living with HIV have a higher risk of developing tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Gamma delta (γδ) T cells in the context of HIV/Mtb coinfection have been understudied in children despite in vitro evidence suggesting γδ T cells assist with Mtb control. We investigated whether boosting a specific subset of γδ T cells, phosphoantigen-reactive Vγ9+Vδ2+ cells, could improve TB outcome using a nonhuman primate model of pediatric HIV/Mtb coinfection. Juvenile Mauritian cynomolgus macaques (MCM), equivalent to 4- to 8-year-old children, were infected intravenously (i.v.) with SIV. After 6 months, MCM were coinfected with a low dose of Mtb and then randomized to receive zoledronate (ZOL), a drug that increases phosphoantigen levels, (n = 5; i.v.) at 3 and 17 days after Mtb accompanied by recombinant human IL-2 (s.c.) for 5 days following each ZOL injection. A similarly coinfected MCM group (n = 5) was injected with saline as a control. Vγ9+Vδ2+ γδ T cell frequencies spiked in the blood, but not airways, of ZOL+IL-2-treated MCM following the first dose, however, were refractory to the second dose. At necropsy 8 weeks after Mtb, ZOL+IL-2 treatment did not reduce pathology or bacterial burden. γδ T cell subset frequencies in granulomas did not differ between treatment groups. These data show that transiently boosting peripheral γδ T cells with ZOL+IL-2 soon after Mtb coinfection of SIV-infected MCM did not improve Mtb host defense.

Metformin boosts doxorubicin efficacy and increases CD8 + T cell frequency in mouse breast cancer.

Breast cancer is a leading cause of cancer-related deaths among women worldwide. The presence and function of CD8 + T cells in the tumor microenvironment have been associated with better patient outcomes. Drug toxicity has posed a significant challenge to the clinical implementation of chemotherapy-based strategies in cancer treatment. In this study, we employed flow cytometry to investigate the potential synergistic immunomodulatory effects on CD8 + T cells in a mouse model of breast cancer by combining metformin, an anti-diabetic medication, with doxorubicin. Through flow cytometry analysis, we observed that the combination of the two drugs led to an increased percentage of CD8 + T cells compared to either drug alone. The modulation of HIF-1α and STAT3 gene expression in the combination group further confirmed the efficacy of this approach. Our findings suggest that combining metformin with doxorubicin can enhance the anticancer activity of doxorubicin and decrease its cytotoxicity in a 4T1 breast cancer mouse model.