Cell Subsets In Patients Research Articles

Single-cell transcriptomics reveals IRF7 regulation of the tumor microenvironment in isocitrate dehydrogenase wild-type glioma.

Mutations in isocitrate dehydrogenase (IDH) are important markers of glioma prognosis. However, few studies have examined the gene expression regulatory network (GRN) in IDH-mutant and wild-type gliomas. In this study, single-cell RNA sequencing and spatial transcriptome sequencing were used to analyze the GRN of cell subsets in patients with IDH-mutant and wild-type gliomas. Through gene transcriptional regulation analysis, we identified the M4 module, whose transcription factor activity is highly expressed in IDH wild-type gliomas compared to IDH-mutants. Enrichment analysis revealed that these genes were predominantly expressed in microglia and macrophages, with significant enrichment in interferon-related signaling pathways. Interferon regulatory factor 7 (IRF7), a transcription factor within this pathway, showed the highest percentage of enrichment and was primarily localized in the core region of wild-type IDH tumors. A machine-learning prognostic model identified novel subgroups within the wild-type IDH population. Additionally, IRF7 was shown to promote the proliferation and migration of T98G and U251 cells in vitro, and its knockdown affected glioma cell proliferation in vivo. This study systematically established the regulatory mechanism of IDH transcriptional activity in gliomas at the single-cell level and drew a corresponding cell map. The study presents a transcriptional regulatory activity map for IDH wild-type gliomas, involving single-cell RNA sequencing and spatial transcriptomics to identify gene regulatory networks, machine learning models for IDH subtyping, and experimental validation, highlighting the role of IRF7 in glioma progression.

Investigation of Transcription Factor and Cytokine Gene Expression Levels in Helper T Cell Subsets Among Turkish Patients Diagnosed with ICF2 (Novel ZBTB24 gene Variant) and ICF3 (CDCA7 Variant) Syndrome.

Immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF), is a rare disease with autosomal recessive inheritance. ICF syndrome. It has been reported that ICF syndrome is caused by mutations in the DNMT3B (ICF1), ZBTB24 (ICF2), CDCA7 (ICF3), and HELLS (ICF4) genes. As a result of literature research, there are no studies on transcription factor and cytokine expressions of helper T cell subsets in ICF syndrome. In the study; Th1 (TBET, STAT1, STAT4), Th2 (GATA3, STAT6), Th17 (RORgt, STAT3), Treg (FoxP3, STAT5) transcription factors and the major cytokines of these cells (Th1; IFNG, Th2; IL4, Th17; IL17A-21-22, Treg; IL10, TGFβ) expressions were aimed to be evaluated by qRT-PCR. Patients (ICF3: three patients; ICF2: two patients), six heterozygous individual and five healthy controls were included in the study. All patients had hypogammaglobulinemia. Except for the CD19 cells of P2 from patients diagnosed with ICF3, the CD3, CD4, CD8, and CD19 cells in the other ICF3 patients were normal. However, the rates of these cells were low in patients with ICF2 syndrome. Factors belonging to patients' Th1, Th17 and Treg cells were significantly lower than the control. Additionally, novel mutation was detected in ZBTB24 gene (c.1121-2A > T). Our study is the first molecular study on Th cell subsets in patients with ICF syndrome and a new mutation that causes ICF2 syndrome has been identified.

Increased Frequency of Circulating Activated FOXP3+ Regulatory T Cell Subset in Patients with Chronic Lymphocytic Leukemia Is Associated with the Estimate of the Size of the Tumor Mass, STAT5 Signaling and Disease Course during Follow-Up of Patients on Therapy.

Advanced chronic lymphocytic leukemia (CLL) is accompanied by increased circulating regulatory T cells (Tregs) and increased susceptibility to severe infections, which were also shown to entail a striking induction of FOXP3 expression in Tregs. As homeostasis of the most suppressive CD45RA-FOXP3high activated Treg (aTreg) subset differs, it is critical to analyse homeostatic signalling in Treg subsets. In this study, by using conventional and imaging flow cytometry, we monitored STAT5 signalling/phosphorylation (pSTAT5) and investigated Treg subsets in relation to the Binet stage, the total tumor mass score (TTM) and the disease course during a follow-up of 37 patients with CLL. The aTreg percentage was significantly increased among CD4+ T cells from patients with advanced disease and significantly correlated with the TTM. A subgroup of patients with higher aTreg percentages among CD4+FOXP3+ T cells at the start of therapy was characterised by more frequent episodes of severe infections during follow-up. The results suggesting that an aTreg fraction could represent a possible marker of a severe disease course with infectious complications. Augmented homeostatic STAT5 signalling could support aTreg expansion, as higher pSTAT5 levels were significantly correlated with an increased aTreg frequency among CD4+FOXP3+ T cells during the follow-up of patients on therapy, as well as following SARS-CoV-2 antigen-specific stimulation in vitro.

Impact of oxaliplatin and trastuzumab combination therapy on tumor markers and T lymphocyte subsets for advanced gastric cancer.

Advanced gastric cancer (AGC) remains a challenging malignancy with poor prognosis. The combination of oxaliplatin and trastuzumab has shown promising results in AGC treatment. This study aimed to investigate the effects of oxaliplatin and trastuzumab combination therapy on serum tumor markers and T lymphocyte subsets in patients with AGC and to explore their potential as predictive biomarkers for treatment response. To investigate the impact of oxaliplatin and trastuzumab combination therapy on serum markers and T cell subsets in patients with AGC. This prospective study enrolled 60 patients with AGC. All patients received oxaliplatin (130 mg/m2, every 3 weeks) and trastuzumab (8 mg/kg loading dose, followed by 6 mg/kg every 3 weeks) for six cycles. Serum carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), and cancer antigen 72-4 (CA72-4) were measured before and after treatment. T-lymphocyte subsets, including CD3+, CD4+, CD8+, and CD4+ /CD8+ ratios, were also evaluated. The clinical response was assessed using the Response Evaluation Criteria in Solid Tumors version 1.1. After six cycles of treatment, the CEA, CA19-9, and CA72-4 serum levels significantly decreased compared to baseline levels (P < 0.001). The percentages of CD3+ and CD4+ T lymphocytes increased significantly (P < 0.05), whereas the percentage of CD8+ T lymphocytes decreased (P < 0.05). The CD4+/CD8+ ratio also significantly increased after treatment (P < 0.05). Patients with a higher decrease in serum tumor markers (≥ 50% reduction) and a higher increase in CD4+/CD8+ ratio (≥ 1.5-fold) showed better clinical response rates (P < 0.05). Oxaliplatin and trastuzumab combination therapy effectively reduced serum tumor marker levels and modulated T lymphocyte subsets in patients with AGC. Combination therapy not only has a direct antitumor effect, but also enhances the immune response in patients with AGC. Serum tumor markers and T lymphocyte subsets may serve as potential predictive biomarkers for treatment response in patients with AGC receiving combination therapy.

Molecular investigations on T cell subsets in patients affected by Hypomorphic DCLRE1C Mutation

ABSTRACT Objective In this study, we explored the expression of transcription factors, cytokines, and co-stimulatory molecules within the helper T (Th) cell subsets (Th1, Th2, Th17 and Treg) of patients with hypomorphic DCLRE1C gene mutations. Methods The study comprised eight patients and five controls. Transcription factor and cytokine expressions of Th subsets and co-stimulatory molecules were investigated by qPCR and flow cytometric following T cell stimulation. The findings were compared between patients (non-HSCT) and with hematopoietic stem cell transplantation (HSCT). Results Flow cytometric analyses; while the Treg rate was significantly lower in non-HSCT than in controls (p = 0.010), the IFN-γ rate was significantly higher in patients than in the control and HSCT groups (p = 0.016, p = 0.022 respectively). Co-stimulatory molecule expressions were significantly lower in non-HSCT than in control (p < 0.001), and there was a significant improvement after HSCT. Post-stimulation qPCR analysis, significant changes were detected in non-HSCT/control, non-HSCT/HSCT and HSCT/control comparisons. Conclusions Our study is the first study to molecularly investigate Th cell subsets in hypomorphic DCLRE1C patients. It was determined that abnormalities in Th cell subsets still persisted despite HSCT. There are still many conditions to be explained in these patients, and we believe that our study may shed light on future studies.

Expression of GPR56 reflects a hypoactivated state of circulating B cells and is downregulated in B cell subsets in patients with early-stage lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer, and the early detection and diagnosis of this disease are crucial in reducing mortality rates. The timely diagnosis of LUAD is essential for controlling tumour development and enabling early surgical treatment. GPR56 is a vital G protein-coupled receptor and its role in T lymphocytes has received considerable attention. However, its function in B cells remains unclear. This study aimed to investigate the significance of GPR56 in LUAD. We found that GPR56 exhibited a significant increase in circulating plasmablasts and a decrease in new memory B cells. GPR56 expression in B cells was significantly reduced after LPS stimulation and the proportion of HLA-DR+ and CD40+ proportions were also decreased in GPR56+ B cells after stimulation. Additionally, GPR56 exhibited significant down-regulation in circulating B cell subsets of early-stage LUAD patients, and there were significant correlations between GPR56+ B cell subsets and tumour markers. In conclusion, GPR56 could reflect the hypoactivation state of B cells and the decreased proportion of GPR56+ B cell subset in LUAD patients can signify the active humoral immunity in vivo. The expression of GPR56 in B cells could potentially hold value in the early diagnosis of LUAD.

Single-cell transcriptomic analysis reveals a decrease in the frequency of macrophage-RGS1high subsets in patients with osteoarticular tuberculosis

BackgroundCell subsets differentially modulate host immune responses to Mycobacterium tuberculosis (MTB) infection. However, the nature and functions of these subsets against osteoarticular tuberculosis (OTB) are unclear. Here, we aimed to understand the phenotypes and functions of immune cell subsets in patients with OTB using single-cell RNA sequencing (scRNA-Seq).MethodsPathological and healthy adjacent tissues were isolated from patients with OTB and subjected to scRNA-Seq. Unsupervised clustering of cells was performed based on gene expression profiles, and uniform manifold approximation and projection was used for clustering visualization.ResultsThirteen cell subsets were identified in OTB tissues. scRNA-seq datasets of patients and healthy controls (HCs) showed that infection changed the frequency of immune cell subsets in OTB tissues. Myeloid cell examination revealed nine subsets. The frequency of macrophage-RGS1high subsets decreased in OTB tissues; this increased MTB susceptibility in an SLC7A11/ferroptosis-dependent manner. Immunohistochemistry assays and flow cytometry for patients with OTB and osteoarticular bacterial infection (OBI) and HCs verified that the frequency of macrophage-RGS1high subset decreased in OTB tissues and blood samples, thereby distinguishing patients with OTB from HCs and patients with OBI.ConclusionThe macrophage-RGS1high subset levels were decreased in patients with OTB, and would be up-regulated after effective treatment. Therefore, the clinical significance of this study is to discover that macrophage-RGS1high subset may serve as a potential biomarker for OTB diagnosis and treatment efficacy monitoring.

Epidermal growth factor receptor and programmed cell death-1 expression levels in peripheral T cell subsets of patients with non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths, in part due to its late diagnosis. Increased epidermal growth factor receptor (EGFR) expression in cancer cells is associated with a poor prognosis, and EGFR tyrosine kinase inhibitors are widely used in cancer treatment. This study aimed to clarify the relationship between EGFR expression on T cells and cancer prognosis in patients with non-small cell lung cancer (NSCLC). Forty patients with NSCLC and 40 healthy volunteers were included in this study. Peripheral CD4+T helper (Th1, Th2, Th9, Th17, Th1Th17, follicular and peripheral Th) and cytotoxic T lymphocyte (CD8+follicular and peripheral T) subsets were identified with flow cytometry according to their chemokine receptors. EGFR expression on T lymphocytes in relation to overall survival (OS) was investigated in patients with NSCLC. The patients [mean age (min-max) = 64.03 (45-83); 20 stage I-III and 20 stage IV] had increased EGFR expression on CD3+T, CD4+Th, Th1, Th2, and Th17 cells compared to the controls (p < 0.05). High EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells was associated with poor OS. Also, PD-1 expression on lymphocytes, CD3+T, and Th cells was increased in patients with NSCLC compared to controls. The high expression of EGFR and PD-1 on Th cells and the reduced percentage of lymphocytes and Th cells, especially in stage IV patients with NSCLC, revealed that increased EGFR activity may trigger apoptosis of Th cells and promote the development of metastases, while high EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells may be an independent poor prognostic marker in NSCLC.

CD69+ Vδ1γδ T cells are anti-tumor subpopulations in hepatocellular carcinoma

Background & aimsHepatocellular carcinoma (HCC), one of the malignancies with a wide expression of stress ligands recognized by Vδ1γδ T cells, has received much attention in adoptive immunotherapy of γδ T cells. In this study, we aimed to identify the potential anti-tumor Vδ1γδ T subpopulations in HCC. MethodsHealthy donors (HDs) and HCC patients were recruited from the Affiliated Cancer Hospital of Zhengzhou University. Blood and tumor tissue samples were obtained respectively. Bioinformatics methods were used to analyze total γδ T cells and subsets infiltration, overall survival of HCC patients with high and low infiltration level of Vδ1γδ T cells, and IFNG, granzyme A, granzyme B and perforin expression in TRDV1high/lowCD69high/low groups. CD69 expression and Vδ1γδT cells infiltration in HCC were detected by immunofluorescence. Phenotypic analysis of Vδ1γδ T cells in blood and tumor tissue samples were performed by flow cytometry. ResultsVδ1γδ T cells infiltrating in HCC were associated with better clinical outcome. Study in tumor micro-environment (TME) of HCC demonstrated that not total Vδ1γδ T but CD69+ Vδ1γδ subset infiltration was associated with smaller tumor volume. Moreover, HCC patients simultaneously with high TRDV1 and CD69 expression produced more effector molecules and had longer survival time. Since Vδ1γδ T cells in the tumor microenvironment were often difficult to access, we demonstrated that CD69+ Vδ1γδ T cells also existed in peripheral blood mononuclear cells (PBMC) of HCC and displayed enhanced cytotoxic potentials than HDs. Finally, we investigated the functions and found that CD69+ Vδ1γδ T cells exhibited stronger tumor reactivities when challenged by tumor cells. ConclusionsCD69+ Vδ1γδ T cells are functional Vδ1γδ T cell subsets in patients with HCC. Circulating CD69+ Vδ1γδ T cell is a promising candidate in immunotherapy of HCC.

Peripheral Blood Lymphocyte Subsets and Heterogeneity of B Cell Subsets in Patients of Idiopathic Inflammatory Myositis with Different Myositis-specific Autoantibodies.

Idiopathic inflammatory myopathies (IIM) are a group of myopathies that present with muscle weakness and multiple extra-muscular manifestations, in which lymphocytes play central roles in myositis pathogenesis. This study aimed to explore the clinical characteristics of lymphocyte subsets, especially B cell subsets, in patients with IIM. Our study included 176 patients with active IIM and 210 gender/age-matched healthy controls (HCs). Compared to HCs, patients have reduced counts of T cells, B cells, and natural killer cells. In addition, B cell subsets from 153 patients with IIM and 92 HCs were characterized. Patients had a lower percentage of memory B cells and translational memory B cells, while those patients were with an elevated percentage of CD19+ B cells, plasmablast and naïve B cells compared with HCs. Moreover, to further explore the heterogeneity of B cells in IIM, patients were categorized into three clusters based on clustering analysis. Cluster 1 was dominated by CD19+ B cells, Bregs and naïve B cells, cluster 3 was dominated by memory B cells and plasmablast, and cluster 2 had the highest proportion of translational memory B cells. Notably, patients in cluster 1 presented with higher CK levels, indicating muscle damage, whereas patients in cluster 3 showed a higher incidence of chest tightness. Our study indicated that lymphopenia is a common manifestation in patients with IIM. B cell subsets are abnormally expressed and showed high heterogeneity in patients with IIM. The patients with IIM were divided into three different clusters with different percentages of chest tightness and distinct CK levels.

Alterations in peripheral T- and B-cell subsets in patients with systemic sclerosis.

To determine the alteration of peripheral T and B cell subsets in patients with systemic sclerosis (SSc) and to evaluate their correlation with the progression of SSc. We recruited 47 SSc patients and 45 healthy controls (HCs) in this study. Demographic and clinical data were then collected. Flow cytometry was used to detect the proportions of 44 different T and B cell subsets in circulating blood. The proportion of total B cells (p = .043) decreased in SSc patients, together with similar frequencies of total T cells, CD4+ T cells, and CD8+ T cells in both groups. Several subsets of T and B cells differed significantly between these two groups. Follicular helper T cells-1 (Tfh1) (p < .001), helper T cells-1 (Th1) (p = .001), regulatory T cells (Treg) (p = .004), effector memory CD8+ T cells (p = .041), and cytotoxic T cells-17 (Tc17) (p = .01) were decreased in SSc patients. Follicular helper T cells-2 (Tfh2) (p = .001) and, helper T cells-2 (Th2) (p = .001) levels increased in the SSc group. Regulatory B cells (Breg) (p = .015) were lower in the SSc group, together with marginal zone (MZ) B cells (p < .001), memory B cells (p = .001), and non-switched B cells (p = .005). The modified Rodnan skin score (mRSS) correlated with helper T cells-17 (Th17) (r = -.410, p = .004), Tfh1 (r = -.321, p = .028), peripheral helper T cells (Tph) (r = -.364, p = .012) and plasma cells (r = -.312, p = .033). The alterations in T and B cells implied immune dysfunction, which may play an essential role in systemic sclerosis.

High frequency of circulating non-classical monocytes is associated with stable remission in relapsing-remitting multiple sclerosis

‘No evidence of disease activity (NEDA)’, judged by clinical and radiological findings, is a therapeutic goal in patients with multiple sclerosis (MS). It is, however, unclear if distinct biological mechanisms contribute to the maintenance of NEDA. To clarify the immunological background of long-term disease stability defined by NEDA, circulating immune cell subsets in patients with relapsing-remitting MS (RRMS) were analyzed using flow cytometry. Patients showing long-term NEDA (n = 31) had significantly higher frequencies of non-classical monocytes (NCMs) (6.1% vs 1.4%) and activated regulatory T cells (Tregs; 2.1% vs 1.6%) than those with evidence of disease activity (n = 8). The NCM frequency and NCMs to classical monocytes ratio (NCM/CM) positively correlated with activated Treg frequency and duration of NEDA. Co-culture assays demonstrated that NCMs could increase the frequency of activated Tregs and the expression of PD-L1, contributing to development of Tregs, was particularly high in NCMs from patients with NEDA. Collectively, NCMs contribute to stable remission in patients with RRMS, possibly by increasing activated Treg frequency. In addition, the NCM frequency and NCM/CM ratio had high predictive values for disease stability (AUC = 0.97 and 0.94, respectively), suggesting these markers are potential predictors of a long-term NEDA status in RRMS.

Evidence for immune activation in pathogenesis of the HLA class II associated disease, podoconiosis

Available evidences suggest that podoconiosis is triggered by long term exposure of bare feet to volcanic red clay soil particles. Previous genome-wide studies in Ethiopia showed association between the HLA class II region and disease susceptibility. However, functional relationships between the soil trigger, immunogenetic risk factors and the immunological basis of the disease are uncharted. Therefore, we aimed to characterise the immune profile and gene expression of podoconiosis patients relative to endemic healthy controls. Peripheral blood immunophenotyping of T cells indicated podoconiosis patients had significantly higher CD4 and CD8 T cell surface HLA-DR expression compared to healthy controls while CD62L expression was significantly lower. The levels of the activation markers CD40 and CD86 were significantly higher on monocytes and dendritic cell subsets in patients compared to the controls. RNA sequencing gene expression data indicated higher transcript levels for activation, scavenger receptors, and apoptosis markers while levels were lower for histones, T cell receptors, variable, and constant immunoglobulin chain in podoconiosis patients compared to healthy controls. Our finding provides evidence that podoconiosis is associated with high levels of immune activation and inflammation with over-expression of genes within the pro-inflammatory axis. This offers further support to a working hypothesis of podoconiosis as soil particle-driven, HLA-associated disease of immunopathogenic aetiology.

Identification and validation of RNA-binding protein SLC3A2 regulates melanocyte ferroptosis in vitiligo by integrated analysis of single-cell and bulk RNA-sequencing

BackgroundThe pathogenesis of vitiligo remains unclear. The genes encoding vitiligo-related RNA-binding proteins (RBPs) and their underlying pathogenic mechanism have not been determined.ResultsSingle-cell transcriptome sequencing (scRNA-seq) data from the CNCB database was obtained to identify distinct cell types and subpopulations and the relative proportion changes in vitiligo and healthy samples. We identified 14 different cell types and 28 cell subpopulations. The proportion of each cell subpopulation significantly differed between the patients with vitiligo and healthy groups. Using RBP genes for unsupervised clustering, we obtained the specific RBP genes of different cell types in vitiligo and healthy groups. The RBP gene expression was highly heterogeneous; there were significant differences in some cell types, such as keratinocytes, Langerhans, and melanocytes, while there were no significant differences in other cells, such as T cells and fibroblasts, in the two groups. The melanocyte-specific RBP genes were enriched in the apoptosis and immune-related pathways in the patients with vitiligo. Combined with the bulk RNA-seq data of melanocytes, key RBP genes related to melanocytes were identified, including eight upregulated RBP genes (CDKN2A, HLA-A, RPL12, RPL29, RPL31, RPS19, RPS21, and RPS28) and one downregulated RBP gene (SLC3A2). Cell experiments were conducted to explore the role of the key RBP gene SLC3A2 in vitiligo. Cell experiments confirmed that melanocyte proliferation decreased, whereas apoptosis increased, after SLC3A2 knockdown. SLC3A2 knockdown in melanocytes also decreased the SOD activity and melanin content; increased the Fe2+, ROS, and MDA content; significantly increased the expression levels of TYR and COX2; and decreased the expression levels of glutathione and GPX4.ConclusionWe identified the RBP genes of different cell subsets in patients with vitiligo and confirmed that downregulating SLC3A2 can promote ferroptosis in melanocytes. These findings provide new insights into the pathogenesis of vitiligo.

The effects of low-dose IL-2 on Th17/Treg cell imbalance in primary biliary cholangitis mouse models

Background/aimsPrimary biliary cholangitis (PBC) is a chronic cholestatic liver disease. The imbalance of Th17/Treg cells has been reported in PBC patients. Low-dose IL-2 can alleviate disease severity through modulating CD4 + T cell subsets in patients with autoimmune diseases. Hence, the present study aimed to examine the effects and mechanism of low-dose IL-2 in PBC mouse models.MethodsPBC models were induced in female C57BL/6 mice by two immunizations with 2OA-BSA at two-week intervals, and poly I: C every three days. PBC mouse models were divided into the IL-2 treated and untreated groups and low-dose IL-2 was injected at three different time points. Th17 and Tregs were analyzed by flow cytometry, and the related cytokines were analyzed by ELISA. Liver histopathology was examined by H&E and immunohistochemical staining.ResultsTwelve weeks after modeling, the serum AMA was positive and the ALP was significantly increased in PBC mouse models (P<0.05). The pathology showed lymphocyte infiltration in the portal area, damage, and reactive proliferation of the small bile duct (P<0.05). The flow cytometric showed the imbalance of Th17/Treg cells in the liver of PBC mouse models, with decreased Treg cells, increased Th17 cells, and Th17/Treg ratio (P < 0.05). After the low-dose IL-2 intervention, biochemical index and liver pathologies showed improvement at 12 weeks. Besides, the imbalance of Th17 and Treg cells recovered. Public database mining showed that Th17 cell differentiation may contribute to poor response in PBC patients.ConclusionLow-dose IL-2 can significantly improve liver biochemistry and pathology by reversing the imbalance of Th17 and Treg cells, suggesting that it may be a potential therapeutic target for PBC.

Quantitative analysis of Tr1 lymphocytes in patients with type 2 diabetes mellitus

BackgroundType 2 diabetes mellitus (T2DM) is usually accompanied by a low-grade inflammatory phenomenon, which participates in the pathogenesis of different complications of this condition. The inflammatory response is under the regulation of different mechanisms, including T regulatory (Treg) lymphocytes. However, the possible role of type 1 T regulatory (Tr1) cells in T2DM has not been explored so far.AimTo carry out a quantitative analysis of Tr1 lymphocytes and other immune cell subsets in patients with T2DM and correlate these results with clinical findings and treatments.Materials and methodsSixty patients with T2DM and twenty-three healthy controls were included in the study. Biochemical and anthropometric variables were evaluated, and Tr1 lymphocytes (CD4+CD49+LAG-3+IL-10+) and other cell subsets (Th17, Th22 and Foxp3 + Treg cells) were analyzed in peripheral blood samples by multiparametric flow cytometry.ResultsSignificant increased levels of Tr1 cells were detected in patients with severe and mild disease, compared to healthy controls. In addition, CD4+IL-10+ lymphocytes were also increased in patients with T2DM. In contrast, similar levels of Foxp3+ Treg cells, Th17 and Th22 lymphocytes were observed in patients and controls. Likewise, no significant associations were detected between Tr1 cell levels and different clinical and laboratory parameters. However, those patients receiving glucagon-like peptide-1 receptor agonists (GLP-1-RA) showed similar levels of Tr1 cells than healthy controls, and significant lower numbers than untreated patients.ConclusionWe observed an increase in Tr1 and CD4+IL10+ lymphocyte levels in T2DM. Moreover, GLP1-RA treatment was significantly associated with normalization of the Tr1 levels. This highlights another potential immune dysfunction in patients with T2DM, which could participate in the pathogenesis of this condition.

Differential expression pattern of Bcl-2 family members in B and T cells in systemic lupus erythematosus and rheumatoid arthritis

ObjectiveThis study aimed to evaluate the expression level of anti-apoptotic Bcl-2 family proteins in B and T cells in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in relation to disease activity and the effect of various Bcl-2 family inhibitors (BH3 mimetics) as potential treatment.MethodsWe included 14 SLE patients, 12 RA patients, and 13 healthy controls to study anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 expression and cell survival in different B and T cell subsets using stimulation assays and intracellular flow cytometry. Effect of various BH3 mimetics was assessed by cell viability analyses.ResultsIn SLE, significant differences in Bcl-2 family members were confined to the B cell compartment with decreased induction of Bcl-XL (p ≤ 0.05) and Mcl-1 (p ≤ 0.001) upon CpG stimulation. In RA, we did not observe any differences in expression levels of Bcl-2 family proteins. Expression patterns did not correlate with disease activity apart from decreased induction of Mcl-1 in B cells in active SLE. After in vitro stimulation with CpG, plasmablasts were more viable after treatment with three different BH3 mimetics compared to naïve or memory B cells in control and patient cells. After activation, Mcl-1 inhibition was most effective in reducing plasmablast and T cell viability, however, less in patients than controls.ConclusionOur study provides evidence for the increased differential expression pattern of Bcl-2 family members in B and T cell subsets of patients with SLE compared to controls. Tested BH3 mimetics showed higher efficacy in controls compared to both autoimmune diseases, though nonsignificant due to low patient numbers.

Identification of cell-intrinsic effects of mutated circulating cells due to clonal hematopoiesis in patients with heart failure

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and is associated with worse prognosis in patients with heart failure with reduced ejection fraction (HFrEF). Circulating immune cells derived from patients with HFrEF harboring CHIP mutations show enhanced proinflammatory signatures. However, it is unclear, whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 patients with HFrEF. Using an improved protocol to detect mutant cells on a single cell level (MutDetect-Seq), as illustrated in figure 1A, we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells). DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. Specifically, DNMT3A mutant monocytes exhibit altered gene expression profiles associated with inflammation and phagolysosome function (figure 1B). In addition, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards effector cells and away from a naïve T cell state. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes. Conclusion This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HFrEF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte as well as the skewing of mutated T cells towards effector cells provide novel insights into how CHIP might promote worse prognosis in heart failure patients.MutDetect-seq

Active tuberculosis patients have high systemic IgG levels and B-cell fingerprinting, characterized by a reduced capacity to produce IFN-γ or IL-10 as a response to M.tb antigens.

Tuberculosis (TB) is a bacterial infection caused by Mycobacterium tuberculosis (M.tb). B cells are the central mediator of the humoral response; they are responsible for producing antibodies in addition to mediating other functions. The role of the cellular response during the TB spectrum by B cells is still controversial. In this study, we evaluated the distribution of the circulating B cell subsets in patients with active and latent TB (ATB and LTB, respectively) and how they respond to stimuli of protein or lipid from M.tb. Here, we show that ATB patients show an immune fingerprinting. However, patients with drug-sensitive- (DS-TB) or drug-resistant- (DR-TB) TB have altered frequencies of circulating B cells. DS-TB and DR-TB display a unique profile characterized by high systemic levels of IFN-γ, IL-10, IgG, IgG/IgM ratio, and total B cells. Moreover, B cells from DR-TB are less efficient in producing IL-10, and both DS-TB and DR-TB produce less IFN-γ in response to M.tb antigens. These results provide new insights into the population dynamics of the cellular immune response by B cells against M.tb and suggest a fingerprinting to characterize the B-cell response on DR-TB.

The landscape of innate and adaptive immune cell subsets in patients with adult-onset Still's disease.

Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder. The understanding of the changes in adaptive immune cells and the crosstalk between innate and adaptive immune systems in AOSD is limited. This study aimed to examine the peripheral immune cell composition and inflammatory protein levels in AOSD patients. Twenty-nine active AOSD patients were enrolled. Flow cytometry was used to analyze the cell populations in peripheral blood. Antibody chips were utilized to detect the protein expression profile in serum. In active AOSD patients, there was an increase in the percentage of classical and non-classical monocytes among peripheral blood mononuclear cells. The proportion of natural killer (NK) cells decreased, with an increase in CD56dim NK1 cells and a decrease in CD56bright NK2 cells compared with healthy controls (HC). The percentage of naïve central memory T cells was decreased, while the percentage of effector and effector memory T cells was increased among adaptive lymphocytes. The proportion of naïve B and memory B cells was decreased, while plasma cells were increased in AOSD patients, indicating activation of the adaptive immune system. Additionally, the serum levels of 40 proteins were elevated in AOSD patients, primarily involved in cytokine-cytokine receptor interaction, inflammatory response, and regulation of MAPK cascade. Our findings showed the activation of the innate and adaptive immune system in AOSD. The protein-protein interaction analysis suggested potential communication between innate and adaptive cell subsets. These findings provide new insights into the pathogenesis of the disease and the development of targeted therapies.