Frequency Of Cells Research Articles

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.

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

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. 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. 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. 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.

Brain temperature, brain metabolites, and immune system phenotypes in temporal lobe epilepsy.

Epileptogenesis is linked to neuroinflammation. We hypothesized that local heat production caused by neuroinflammation can be visualized non-invasively invivo via brain magnetic resonance spectroscopic imaging (MRSI) and MRSI-thermometry (MRSI-t) and that there is a relationship in patients with temporal lobe epilepsy (TLE) between MRSI-t and brain metabolites choline and myo-inositol and between neuroimaging and cellular and serum biomarkers of inflammation. Thirty-six (36) participants, 18 with temporal lobe epilepsy (13 females) and 18 age-matched healthy controls (nine females), were enrolled prospectively and underwent MRSI/MRSI-t; TLE participants also provided blood samples. Temperature was measured using creatine as a reference metabolite. Analysis of Functional NeuroImages 3dttest++ tool was used to analyze voxel-level group differences in temperature, choline, and myo-inositol. Associations with immune cell subsets, cytokines, and chemokines related to inflammation were quantified using correlation coefficients with significant relationships as noted. Patients with TLE showed elevated temperature, choline, and myo-inositol in the temporal lobes. Higher brain temperature was associated with higher levels of cytokines and chemokines, including GM-CSF, TNF, IL-1β, and IL - 12p70, and lower frequency of immune cells including CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, and classical monocytes. Higher choline was associated with higher levels of the cytokines including LT-α, IL-13, and IL-4, and higher myo-inositol was associated with a higher frequency of CD4+ T-cell and CD19+ B-cell subsets and higher levels of cytokines and chemokines including LT-α, IL-13, and CCL3. This study, for the first time, showed that in temporal lobes of patients with TLE temperature and metabolite changes correlate with cellular and serum biomarkers of inflammation. Our results provide support for further development of MRSI-t as a measure of neuroinflammation in epilepsy and potentially other neurological disorders and as an investigative and clinical tool. Neuroinflammation is associated with excessive heat production which can be visualized with magnetic resonance spectroscopic imaging and thermometry (MRSI-t). We prospectively investigated the relationship between MRSI-t and cellular and serum measures of peripheral inflammation in patients with temporal lobe epilepsy (TLE); we compared the results of MRSI-t in patients with TLE to healthy controls. We showed a relationship between the temperature elevations in TLE and elevations of various measures of peripheral inflammation. Our results support further development of MRSI-t as a measure of neuroinflammation in epilepsy and potentially other neurological disorders and as an investigative and clinical tool.

Loss of Y-chromosomal genes in macrophages induces paracrine phenotypic alterations in cardiac cells

Abstract Background At the intersection of gender medicine and ageing, mosaic loss of the Y chromosome (mLoY) in hematopoietic cells has emerged as a novel cardiovascular risk factor. As the most prevalent chromosomal aberration in men, mLoY is age-associated and characterised by interindividual varying frequencies of LoY cells in blood. It is associated with an increased risk for cardiovascular disease and mortality. A murine model of mLoY demonstrates myocardial fibrosis and reduced lifespan. However, the interactions between LoY hematopoietic and cardiac cells remain elusive. It is uncertain, whether the loss of single genes or combinations of multiple genes drive the observed effects, impeding the development of targeted therapies. Purpose We aimed to examine the impact of individual Y-chromosomal genes in macrophages concerning their paracrine effects on cardiac cells types. Methods and results To identify target genes, we analysed two single-nuclei RNA sequencing datasets of patient-derived PBMCs and THP-1 monocytic cells for their expression of Y-chromosomal genes. We identified nine genes that exhibited robust and concordant expression in both datasets. Seven were protein coding (RPS4Y, DDX3Y, EIF1AY, ZFY, UTY, USP9Y and KDM5D) and two were non-coding (TTTY14 and LINC00278). Using siRNA pools, we achieved >50% reductions in mRNA content in THP-1 M0 macrophages for all genes (p<0.05) and collected conditioned media (CM). As previous data suggested increased myocardial fibrosis in mLoY, we assessed the impact of CM on primary human cardiac fibroblasts. Notably, treatment with CM from ZFY, TTTY14 or LINC00278 silenced macrophages induced collagen 1A1 protein expression by 1.44-, 1.30- and 1.86-fold, respectively (all p<0.05), suggesting that loss of these genes might contribute to myocardial fibrosis. To explore potential effects on cardiomyocytes, neonatal rat cardiomyocytes were exposed to CM. We observed reduced beating frequencies upon treatment with CM from ZFY, TTTY14 or UTY silenced macrophages (relative fold-changes 0.45, 0.40, 0.53, respectively; all p<0.05). While suggestive of cardiomyocyte stress, we found no changes in the expression of the stress markers Nppa or Nppb, possibly indicating direct effects on ion transporters. Given the strong association of cardiovascular disease with endothelial cell dysfunction, we investigated the effects of CM on human umbilical vein endothelial cells (HUVEC). CM from ZFY and KDM5D silenced macrophages induced ICAM protein expression in HUVEC by 1.9- and 1.6-fold, respectively (both p<0.05). Conclusion Loss of individual Y-chromosomal genes in monocytic cells elicits distinct paracrine effects on various cardiac cell types. Particularly, silencing of ZFY in macrophages demonstrated profound effects on all investigated cell types, warranting further investigation. Additionally, two long non-coding RNAs, previously unexplored in cardiovascular disease, influenced paracrine activity of macrophages.

Gingival mesenchymal stem cells derived from patients with rheumatoid arthritis treat experimental arthritis

AbstractA therapeutic strategy using mesenchymal stem cells (MSCs) has been accepted as a novel therapy for treating rheumatoid arthritis (RA). Human gingiva‐derived MSCs (GMSCs) are superior in regulating immune responses. Autologous MSCs are the optimal candidate to avoid the potential risks of allogenic MSCs. However, whether autologous GMSCs from RA patients are therapeutic remains unknown. In this study, we compared the therapeutic efficacy of GMSCs derived from patients with RA (RA‐GMSCs) and that from health donors (H‐GMSCs) in vivo and in vitro. Then, we utilized RNA‐sequencing, the molecular and cellular assays to determine the immunomodulatory molecules that contribute to the therapeutic effect of RA‐GMSCs on both collagen‐induced arthritis (CIA) and humanized synovitis models. We demonstrated that GMSCs derived from patients with RA (RA‐GMSCs) and health donors (H‐GMSCs) shared a similar expression of immunomodulatory molecules. Moreover, RA‐GMSCs were as effective as H‐GMSCs in suppressing T‐cell proliferation, proinflammatory cytokines secretion, as well as osteoclast differentiation in vitro. In addition, RA‐GMSCs had a robust therapeutic effect on the CIA model. Importantly, RA GMSCs can survive for at least 24 days in a CIA mouse model and can be distributed in the spleen, lymph nodes, and joints. Specifically, RA‐GMSCs decreased the frequency of Th1 and Th17 cells whereas enhanced Treg cells, reducing the joint histopathological scores of lymphocytes, osteoclasts, and cartilages. Moreover, RA‐GMSCs were also effective in suppressing inflamed synoviocyte proliferation, migration, and invasion in vitro, and cartilage invasion in a humanized synovitis model in vivo. Our study implies that manipulation of RA‐GMSCs is therapeutic in CIA mice and humanized synovitis models and may have therapeutic potential in RA patients using autologous GMSCs in the future.

Nuclear receptor corepressor 1 controls regulatory T cell subset differentiation and effector function.

FOXP3+ regulatory T cells (Treg cells) are key for immune homeostasis. Here, we reveal that nuclear receptor corepressor 1 (NCOR1) controls naïve and effector Treg cell states. Upon NCOR1 deletion in T cells, effector Treg cell frequencies were elevated in mice and in in vitro-generated human Treg cells. NCOR1-deficient Treg cells failed to protect mice from severe weight loss and intestinal inflammation associated with CD4+ T cell transfer colitis, indicating impaired suppressive function. NCOR1 controls the transcriptional integrity of Treg cells, since effector gene signatures were already upregulated in naïve NCOR1-deficient Treg cells while effector NCOR1-deficient Treg cells failed to repress genes associated with naïve Treg cells. Moreover, genes related to cholesterol homeostasis including targets of liver X receptor (LXR) were dysregulated in NCOR1-deficient Treg cells. However, genetic ablation of LXRβ in T cells did not revert the effects of NCOR1 deficiency, indicating that NCOR1 controls naïve and effector Treg cell subset composition independent from its ability to repress LXRβ-induced gene expression. Thus, our study reveals that NCOR1 maintains naïve and effector Treg cell states via regulating their transcriptional integrity. We also reveal a critical role for this epigenetic regulator in supporting the suppressive functions of Treg cells in vivo.

IL-33 Induces a Switch in Intestinal Metabolites Revealing the Tryptophan Pathway as a Target for Inducing Allograft Survival

Background: IL-33, a pleiotropic cytokine, has been associated with a plethora of immune-related processes, both inflammatory and anti-inflammatory. T regulatory (Treg) cells, the main leukocyte population involved in immune tolerance, can be induced by the administration of IL-33, the local microbiota, and its metabolites. Here, we demonstrate that IL-33 drastically induces the production of intestinal metabolites involved on tryptophan (Trp) metabolism. Methods: naïve mice were treated with IL-33 for 4 days and leukocyte populations were analyzed by flow cytometry, and feces were processed for microbiota and intestinal metabolites studies. Using a murine skin transplantation model, the effect of Kynurenic acid (KA) on allograft survival was tested. Results: Under homeostatic conditions, animals treated with IL-33 showed an increment in Treg cell frequencies. Intestinal bacterial abundance analysis indicates that IL-33 provokes dysbiosis, demonstrated by a reduction in Enterobacteria and an increment in Lactobacillus genera. Furthermore, metabolomics analysis showed a dramatic IL-33 effect on the abundance of intestinal metabolites related to amino acid synthesis pathways, highlighting molecules linked to Trp metabolism, such as kynurenic acid (KA), 5-Hydroxyindoleacetic acid (5-HIAA), and 6-Hydroxynicotinic acid (6-HNA), which was supported by an enhanced expression of Ido and Kat mRNA in MLN cells, which are two enzymes involved on KA synthesis. Interestingly, animals receiving KA in drinking water and subjected to skin transplantation showed allograft acceptance, which is associated with an increment in Treg cell frequencies. Conclusions: Our study reveals a new property for IL-33 as a modulator of the intestinal microbiota and metabolites, especially those involved with Trp metabolism. In addition, we demonstrate that KA favors Tregs in vivo, positively affecting skin transplantation survival.

Bone marrow transplantation increases sulfatase activity in somatic tissues in a multiple sulfatase deficiency mouse model

BackgroundMultiple Sulfatase Deficiency (MSD) is an ultra-rare autosomal recessive disorder characterized by deficient enzymatic activity of all known sulfatases. MSD patients frequently carry two loss of function mutations in the SUMF1 gene, encoding a formylglycine-generating enzyme (FGE) that activates 17 different sulfatases. MSD patients show common features of other lysosomal diseases like mucopolysaccharidosis and metachromatic leukodystrophy, including neurologic impairments, developmental delay, and visceromegaly. There are currently no approved therapies for MSD patients. Hematopoietic stem cell transplant (HSCT) has been applied with success in the treatment of certain lysosomal diseases. In HSCT, donor-derived myeloid cells are a continuous source of active sulfatase enzymes that can be taken up by sulfatase-deficient host cells. Thus, HSCT could be a potential approach for the treatment of MSD.MethodsTo test this hypothesis, we used a clinically relevant mouse model for MSD, B6-Sumf1(S153P/S153P) mice, engrafted with bone marrow cells, Sumf1+/+, from B6-PtprcK302E mice (CD45.1 immunoreactive).ResultsAfter 10 months post-transplant, flow cytometric analysis shows an average of 90% of circulating leukocytes of donor origin (Sumf1(+/+)). Enzymatic activity for ARSA, ARSB, and SGSH is significantly increased in spleen of B6-Sumf1(S153P/S153P) recipient mice. In non-lymphoid organs, only liver and heart show a significant correction of sulfatase activity and GAG accumulation. Frequency of inflammatory cells and lysosomal pathology is significantly reduced in liver and heart, while no significant improvement is detected in brain.ConclusionsOur results indicate that HSCT could be a suitable approach to treat MSD-pathology affecting peripheral organs, however that benefit to CNS pathology might be limited.

Elevated circulating group-2 innate lymphoid cells expressing activation markers and correlated tryptase AB1 levels in active ascariasis

IntroductionAscaris lumbricoides infection is one of the most common soil-transmitted helminthiasis and IgE response to this helminth may increase the risk of asthma, bronchial hyperreactivity and atopy. There is not enough evidence showing the role of group-2 innate lymphoid cells (ILC2) in the pathogenesis of helminth infections in humans. Here, we aimed to investigate and characterize the influence of Ascaris lumbricoides infection on circulating ILCs in endemically exposed subjects.MethodsNon-infected (NI; n=16) and Ascaris-infected (AI; n=16) subjects from an endemic area were included. Two consecutive stool samples from each subject were examined by Kato-Katz to define parasite infection. Antibodies to the ABA-1 antigen of Ascaris and Ascaris extract were measured by ELISA. ILC subsets and their activation markers (CD25, CD69, thymic stromal lymphopoietin receptor (TSLPR) were evaluated in its PBMC by flow cytometry. Proximity extension assay (PEA) was performed to explore plasma proteins associated to infection.ResultsNo significant differences in the relative or absolute frequencies of total ILCs, ILC1, ILC2 and ILC3 cells were observed regarding the infection status. However, within AI group, IgE-sensitized subjects to ABA-1 had higher frequencies and counts of ILC2 (p<0.05). Frequencies of CD25+, CD69+ and TSLPR+ ILC2 were higher in AI compared to the NI (p<0.01). Additionally, egg burden was positively correlated with CD69+ ILC2 frequencies (r=0.67; p=0.005). Tryptase alpha/beta 1 (TPSAB1), GP6 and several plasma proteins associated with cell growth and granulocyte chemotaxis were highly expressed in the AI group (p<0.05). Interestingly, TPSAB1 levels were positively correlated with ILC2 expressing activation markers frequencies, egg burden and IgE levels against Ascaris.DiscussionAscaris infection is associated with increased expression of ILC2 activation markers and TPSAB1, which may contribute to the type-2 response.

Peripheral immune profiling of soft tissue sarcoma: perspectives for disease monitoring.

Studying the tumor microenvironment and surrounding lymph nodes is the main focus of current immunological research on soft tissue sarcomas (STS). However, due to the restricted opportunity to examine tumor samples, alternative approaches are required to evaluate immune responses in non-surgical patients. Therefore, the purpose of this study was to evaluate the peripheral immune profile of STS patients, characterize patients accordingly and explore the impact of peripheral immunotypes on patient survival. Blood samples were collected from 55 STS patients and age-matched healthy donors (HD) controls. Deep immunophenotyping and gene expression analysis of whole blood was analyzed using multiparametric flow cytometry and real-time RT-qPCR, respectively. Using xMAP technology, proteomic analysis was also carried out on plasma samples. Unsupervised clustering analysis was used to classify patients based on their immune profiles to further analyze the impact of peripheral immunotypes on patient survival. Significant differences were found between STS patients and HD controls. It was found a contraction of B cells and CD4 T cells compartment, along with decreased expression levels of ICOSLG and CD40LG; a major contribution of suppressor factors, as increased frequency of M-MDSC and memory Tregs, increased expression levels of ARG1, and increased plasma levels of IL-10, soluble VISTA and soluble TIMD-4; and a compromised cytotoxic potential associated with NK and CD8 T cells, namely decreased frequency of CD56dim NK cells, and decreased levels of PRF1, GZMB, and KLRK1. In addition, the patients were classified into three peripheral immunotype groups: "immune-high," "immune-intermediate," and "immune-low." Furthermore, it was found a correlation between these immunotypes and patient survival. Patients classified as "immune-high" exhibited higher levels of immune-related factors linked to cytotoxic/effector activity and longer survival times, whereas patients classified as "immune-low" displayed higher levels of immune factors associated with immunosuppression and shorter survival times. In conclusion, it can be suggested that STS patients have a compromised systemic immunity, and the correlation between immunotypes and survival emphasizes the importance of studying peripheral blood samples in STS. Assessing the peripheral immune response holds promise as a useful method for monitoring and forecasting outcomes in STS.

T cell immuno-phenotyping : a source of predictive biomarkers for autoimmune hepatitis relapse

Relapse after immunosuppression (IS) treatment withdrawal is frequent in patients with Autoimmune Hepatitis (AIH), and non-invasive biomarkers predictive of this risk are lacking. We assessed the frequency of circulating T cell subsets as potential biomarkers of disease activity and predictor of the risk of relapse after IS withdrawal. Serum levels of the cytokine B-cell Activating Factor (BAFF) were also investigated. Blood samples from 58 patients with active AIH, 56 AIH patients in remission, and 31 patients with NASH were analyzed. The frequency of activated CD4+ T peripheral helper (TPH) cells (CD4+CD45RA-CXCR5-PD1+CD38+) and of activated CD8+ T cells (CD8+CD45RA-PD1+CD38+) were assessed by flow cytometry. BAFF levels were determined by ELISA. Activated TPH and CD8+ T cell frequencies were significantly increased in patients with active AIH compared to remission AIH or NASH (TPH: 0.88% of total CD3+ vs. 0.42% and 0.39% respectively, p < 0.0001; CD8+ subset: 1.42% vs. 0.09% and 0.11% p < 0.0001). Among patients in remission undergoing treatment withdrawal (n = 18), those with increased frequencies of activated TPH (> 0.5% of total CD3+) and/or activated CD8+ T cells (> 0.18% total CD3+) had a higher risk of relapse (80% vs. 15% after 2 years, p = 0.0071). High BAFF serum concentration (> 213pg/ml) was also associated to a higher risk of relapse (57% vs. 11%, p = 0.0452). In conclusion, high frequency of activated TPH and of activated CD8+, as well as high levels of BAFF, before IS discontinuation, were significantly associated to a greater risk of relapse during the first two years. Thus, they represent promising biomarkers to provide personalized clinical follow-up for patients with AIH.

Regulator of G-Protein Signaling 2 Knockout in CD4+ T Cells Promotes Anti-Inflammatory T Cells, Enhancing Ovulation, and Oocyte Yield.

To determine the downstream effects on ovarian function and immune cell differentiation in the ovary and uterus using a model in which RGS2 was knocked out specifically in CD4+ T cells. Laboratory based experiments with female mice. Female congenic (fully backcrossed) and non-congenic (mixed strain) mice with CD4 T cell-specific RGS2 knockout. Four-week-old female CD4 RGS2 knockout (CD4 RGS2 KO ) mice and their littermate controls (CD4 RGS2 CTL ) were subjected to superovulation using pregnant mare serum gonadotropins. Oocyte numbers, lymphocyte populations in the ovary and uterus, and serum estradiol and progesterone concentrations. In non-congenic (mixed strain) mice, CD4 RGS2 knockout (KO) promoted higher oocyte ovulation and increased uterine total leukocyte numbers. Similarly, congenic (fully backcrossed strain) mice showed higher oocyte numbers and increased uterine total leukocytes in the CD4 RGS2 KO mice compared to CD4 RGS2 CTL mice. Pro-inflammatory CD4+ T helper (T H ) 1 and T H 17 cell frequencies in the ovary and uterus were unchanged, while Treg and T H 2 cell frequencies were elevated, along with increased concentrations of estradiol and progesterone in the serum of CD4 RGS2 KO mice. Our study highlights the important role of RGS2 in CD4+ T cells within the context of reproduction. The dysregulation of immune responses due to RGS2 knockout in CD4+ T cells appears to enhance oocyte production. Further research is warranted to elucidate the precise mechanisms by which RGS2 influences reproductive outcomes, including its impact on fecundability, endometrial receptivity, and successful implantation.

An immune signature of postoperative cognitive decline: A prospective cohort study.

Postoperative cognitive decline (POCD) is the predominant complication affecting patients over 60 years old following major surgery, yet its prediction and prevention remain challenging. Understanding the biological processes underlying the pathogenesis of POCD is essential for identifying mechanistic biomarkers to advance diagnostics and therapeutics. This study aimed to provide a comprehensive analysis of immune cell trajectories differentiating patients with and without POCD and to derive a predictive score enabling the identification of high-risk patients during the preoperative period. Twenty-six patients aged 60 years old and older undergoing elective major orthopedic surgery were enrolled in a prospective longitudinal study, and the occurrence of POCD was assessed seven days after surgery. Serial samples collected before surgery, and one, seven, and 90 days after surgery were analyzed using a combined single-cell mass cytometry and plasma proteomic approach. Unsupervised clustering of the high-dimensional mass cytometry data was employed to characterize time-dependent trajectories of all major innate and adaptive immune cell frequencies and signaling responses. Sparse machine learning coupled with data-driven feature selection was applied to the pre-surgery immunological dataset to classify patients at risk for POCD. The analysis identified cell-type and signaling-specific immune trajectories differentiating patients with and without POCD. The most prominent trajectory features revealed early exacerbation of JAK/STAT and dampening of inhibitory κB and nuclear factor-κB immune signaling responses in patients with POCD. Further analyses integrating immunological and clinical data collected before surgery identified a preoperative predictive model comprising one plasma protein and ten immune cell features that classified patients at risk for POCD with excellent accuracy (AUC=0.80, P=2.21e-02 U-test). Immune system-wide monitoring of patients over 60 years old undergoing surgery unveiled a peripheral immune signature of POCD. A predictive model built on immunological data collected before surgery demonstrated greater accuracy in predicting POCD compared to known clinical preoperative risk factors, offering a concise list of biomarker candidates to personalize perioperative management.

Regulatory T cells modulate monocyte functions in immunocompetent antiretroviral therapy naive HIV-1 infected people

We previously demonstrated that the overall number of regulatory T (Treg) cells decrease proportionately with helper CD4+ T cells and their frequencies increase in antiretroviral therapy (ART)-naive human immunodeficiency virus type-1 (HIV-1) infected individuals. The question now is whether the discrepancies in Treg cell numbers and frequencies are synonymous to an impairment of their functions. To address this, we purified Treg cells and assessed their ability to modulate autologous monocytes functions. We observed that Treg cells were able to down modulate autologous monocytes activation as well as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production during stimulation with polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (poly-ICLC). This activity of Treg cells has been shown to be influenced by immunocompetence including but not limited to helper CD4+ T cell counts, in individuals with HIV-1 infection. Compared to immunosuppressed participants (CD4 < 500 cells/µL), immunocompetent participants (CD4 ≥ 500 cells/µL) showed significantly higher levels of transforming growth factor beta (TGF-β) and IL-10 (p < 0.001 and p < 0.05, respectively), key cytokines used by Treg cells to exert their immunosuppressive functions. Our findings suggest the contribution of both TGF-β and IL-10 in the suppressive activity of Treg cells.

The Power of Reagent Titration in Flow Cytometry.

Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration-response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution.