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Objectives: Fatigue is a prominent feature of long COVID (LC) and may be related to several pathophysiologic mechanisms, including immune hyperstimulation. Aerobic endurance exercise training may be a useful therapy, with appropriate attention to preventing post-exertional malaise. Methods: Fourteen participants completed a pilot study of aerobic exercise training (twenty 1.5 h sessions of over 10 weeks). Cardiorespiratory fitness, 6 min walk distance, quality of life, symptoms, 7-day physical activity, immunophenotype, and inflammatory biomarkers were measured before and after exercise training. Results: The participant characteristics at baseline were as follows: 53.5 ± 11.6 yrs, 53% f, BMI 32.5 ± 8.4, 42% ex-smokers, 15.1 ± 8.8 months since initial COVID-19 infection, low normal pulmonary function testing, V. O2peak 19.3 ± 5.1 mL/kg/min, 87 ± 17% predicted. After exercise training, participants significantly increased their peak work rate (+16 ± 20 W, p = 0.010) and V. O2peak (+1.55 ± 2.4 mL/kg/min, p = 0.030). Patients reported improvements in fatigue severity (−11%), depression (−42%), anxiety (−29%), and dyspnea level (−46%). There were no changes in 6MW distance or physical activity. The circulating number of CD3+, CD4+, CD19+, CD14++CD16, and CD16++CD14+ monocytes and CD56+ cells (assessed with flow cytometry) increased with acute exercise (rest to peak) and was not diminished or augmented by exercise training. Plasma concentrations of TNF-α, IL-6, IL-8, IL-10, INF-γ, and INF-λ were normal at study entry and not affected by training. Conclusions: Aerobic endurance exercise training in individuals with LC delivered beneficial effects on cardiorespiratory fitness, quality of life, anxiety, depression, and fatigue without detrimental effects on immunologic function. [ABSTRACT FROM AUTHOR]

Introduction: This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Methods: Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP +CAF density, and immune response. Results: Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. Conclusions: These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development. [ABSTRACT FROM AUTHOR]

Background: Despite the improved survival observed in PD-1/PD-L1 blockade therapy, a substantial proportion of cancer patients, including those with non-small cell lung cancer (NSCLC), still lack a response. Methods: Transcriptomic profiling was conducted on a discovery cohort comprising 100 whole blood samples, as collected multiple times from 48 healthy controls (including 43 published data) and 31 NSCLC patients that under treatment with a combination of anti-PD-1 Tislelizumab and chemotherapy. Differentially expressed genes (DEGs), simulated immune cell subsets, and germline DNA mutational markers were identified from patients achieved a pathological complete response during the early treatment cycles. The predictive values of mutational markers were further validated in an independent immunotherapy cohort of 1661 subjects, and then confirmed in genetically matched lung cancer cell lines by a co-culturing model. Results: The gene expression of hundreds of DEGs (FDR p < 0.05, fold change < -2 or > 2) distinguished responders from healthy controls, indicating the potential to stratify patients utilizing early on-treatment features from blood. PD-1-mediated cell abundance changes in memory CD4 + and regulatory T cell subset were more significant or exclusively observed in responders. A panel of top-ranked genetic alterations showed significant associations with improved survival (p < 0.05) and heightened responsiveness to anti-PD-1 treatment in patient cohort and co-cultured cell lines. Conclusion: This study discovered and validated peripheral blood-based biomarkers with evident predictive efficacy for early therapy response and patient stratification before treatment for neoadjuvant PD-1 blockade in NSCLC patients. [ABSTRACT FROM AUTHOR]

Silicosis caused by engineered stone (ES-silicosis) is an emerging worldwide issue characterized by inflammation and fibrosis in the lungs. To our knowledge, only a few reports have investigated leukocyte/lymphocyte subsets in ES-silicosis patients. The present study was designed to explore the proportions of the main lymphocyte subsets in ES-silicosis patients stratified into two groups, one with simple silicosis (SS) and the other with a more advanced state of the disease, defined as progressive massive fibrosis (PMF). The proportions of B (memory and plasmablasts) cells, T (helper, cytotoxic, regulatory) cells, and natural killer (NK) (regulatory and cytotoxic) cells were investigated by multiparameter flow cytometry in 91 ES-silicosis patients (53 SS patients and 38 PMF patients) and 22 healthy controls (HC). Although the total number of leukocytes did not differ between the groups studied, lymphopenia was observed in patients compared to healthy controls. Compared with those in healthy controls, the proportions of memory B cells, naïve helper T cells, and the CD4+/CD8+ T cells' ratio in the peripheral blood of patients with silicosis were significantly decreased, while the percentages of plasma cells, memory helper T cells, and regulatory T cells were significantly increased. For the NK cell subsets, no significant differences were found between the groups studied. These results revealed altered cellular immune processes in the peripheral blood of patients with ES-silicosis and provided further insight into silicosis pathogenesis. [ABSTRACT FROM AUTHOR]

Objectives: Myositis-specific antibodies (MSAs) and myositis-associated antibodies (MAAs) are associated with distinctive dermatomyositis (DM) clinical phenotypes. The aim of this study is to explicate the clinical and immunological features of MSAs-negative DM patients. Methods: A total of 515 individuals diagnosed with DM was screened from 2013 to 2022 and 220 DM patients were enrolled in this retrospective cohort. Clinical and laboratory data of these patients were analyzed. Results: MSAs-negative DM patients were categorized into two groups: MAAs-negative (MSAs (−)/MAAs (−)) group and MAAs-positive (MSAs (−)/MAAs (+)) group. The percentage of Raynaud's phenomenon (P=0.026) was higher in the MSAs (−)/MAAs (+) DM patients than the MSAs-positive DM patients and MSAs (−)/MAAs (−) DM patients. The proportion of rapidly progressive interstitial lung disease (RP-ILD) in the MSAs-negative DM patients was lower than that in the MSAs-positive group. The MSAs (−)/MAAs (+) group had a higher proportion of organizing pneumonia and usual interstitial pneumonia (P=0.011), and elevated eosinophils in their bronchoalveolar lavage fluid (P=0.008). Counts of lymphocytes (P=0.001) and CD16+CD56+ natural killer (NK) cells (P=0.012) were higher in the MSAs-negative group. Additionally, the percentage of CD4+TNFα+ (P=0.040), CD4+IFNγ+ (P=0.037), and CD4+IL-2+ (P=0.018) cells among total CD4+ T cells were higher in the MSA-negative DM patients compared with the MSAs-positive DM patients. Besides, MSAs-negative patients demonstrated a more favorable prognosis than MSAs-positive patients. Multivariable regression analysis identified advanced onset age, higher level of carcinoembryonic antigen (CEA), and RP-ILD as risk factors for mortality in DM patients. Conclusions: Compared with MSAs-positive group, MSAs-negative DM patients suffered less from organ involvement compared with MSAs-positive group and tend to have better prognosis. Key Points MSAs-negative DM patients exhibited distinct characteristics in comparison with MSAs-positive DM patients: • The MSAs (−)/MAAs (+) DM patients demonstrated a higher prevalence of organizing pneumonia (OP) and usual interstitial pneumonia (UIP), and elevated eosinophil counts in bronchoalveolar lavage fluid. • CEA levels were lower in MSAs-negative patients compared with MSAs-positive patients. • Elevated counts of lymphocytes and CD16+CD56+NK cells were identified in the MSAs-negative patients. Additionally, proportions of CD4+TNFα+, CD4+IFNγ+, and CD4+IL-2+cells among total CD4+T cells were higher in the MSAs-negative DM patients compared with DM MSAs-positive DM patients. • MSAs-negative DM patients had a more favorable prognosis than MSAs-positive DM patients. A multivariable regression analysis revealed the advanced onset age, high CEA levels, and RP-ILD were risk factors for mortality in DM patients. [ABSTRACT FROM AUTHOR]

This 27-color flow cytometry antibody panel allows broad immune-profiling of major leukocyte subsets in human whole blood (WB). It includes lineage markers to identify myeloid and lymphoid cell populations including granulocytes, monocytes, myeloid dendritic cells (mDCs), natural killer (NK) cells, NKT-like cells, B cells, conventional CD4 and CD8 T cells, γδ T cells, mucosa-associated invariant T (MAIT) cells and innate lymphoid cells (ILC). To further characterize each of these populations, markers defining stages of cell differentiation (CCR7, CD27, CD45RA, CD127, CD57), cytotoxic potential (perforin, granzyme B) and cell activation/proliferation (HLA-DR, CD38, Ki-67) were included. This panel was developed for quantifying absolute counts and phenotyping major leukocyte populations in cryopreserved, fixed WB collected from participants enrolled in large multi-site tuberculosis (TB) vaccine clinical trials. This antibody panel can be applied to profile major leukocyte subsets in other sample types such as fresh WB or peripheral blood mononuclear cells (PBMCs) with only minor additional optimization. [ABSTRACT FROM AUTHOR]

Although cattle are the mammalian species with most global biomass associated with a huge impact on our planet, their immune system remains poorly understood. Notably, the bovine immune system has peculiarities such as an overrepresentation of γδ T cells that requires particular attention, specifically in an infectious context. In line of 3R principles, we developed an ex vivo platform to dissect host–pathogen interactions. The experimental design was based on two independent complementary readouts: firstly, a novel 12–14 color multiparameter flow cytometry assay measuring maturation (modulation of cell surface marker expression) and activation (intracellular cytokine detection) of monocytes, conventional and plasmacytoid dendritic cells, natural killer cells, γδ T cells, B and T cells; secondly, a multiplex immunoassay monitoring bovine chemokine and cytokine secretion levels. The experiments were conducted on fresh primary bovine blood cells exposed to Mycoplasmopsis bovis (M. bovis), a major bovine respiratory pathogen. Besides reaffirming the tight cooperation of the different primary blood cells, we also identified novel key players such as strong IFN-γ secreting NK cells, whose role was so far largely overlooked. Additionally, we compared the host–pathogen interactions at different temperatures, including commonly used 37 °C, ruminant body temperature (38–38.5 °C) and fever (≥ 39.5 °C). Strikingly, working under ruminant physiological temperature influenced the capacity of most immune cell subsets to respond to M. bovis compared to 37 °C. Under fever-like temperature conditions the immune response was impaired compared to physiological temperature. Our experimental approach, phenotypically delineating the bovine immune system provided a thorough vision of the immune response towards M. bovis and the influence of temperature towards that immune response. [ABSTRACT FROM AUTHOR]

Simple Summary: TLR-9, a vital component of our immune system, is found to be significantly involved in the progression of gastric cancer. This research also investigates its connection to EBV infection. By analyzing specific T and B immune cell groups that express TLR-9, along with the levels of soluble TLR-9 in the blood, the study compares these factors between individuals with gastric cancer and healthy volunteers. The aim is to understand how these immune markers relate to the severity of the cancer, its stage, type, and survival rates. Furthermore, the study delves into the presence of EBV genetic material and its potential impact on these immune responses. This research sheds light on the interplay between TLR-9, immune cell populations, and EBV in gastric cancer development. The relationship between Toll-like receptor 9 (TLR-9) signaling and its involvement with Epstein–Barr virus (EBV) in gastric cancer (GC) is complex and currently under study. This research intended to understand TLR-9's role in certain T and B lymphocytes and the serum levels of TLR-9 in GC patients versus healthy subjects. The team explored links between these immune markers and various GC traits, such as histological grade, tumor progression stages, cancer types, and survival rates. Additionally, the research sought to find if EBV genetic material influences these immune reactions. Using flow cytometry, TLR-9 levels in different immune cells were analyzed. At the same time, the amount of TLR-9 in the serum was determined. The results showed GC patients had varied TLR-9 levels compared to healthy subjects, with specific cells showing noticeable changes. When grouped by GC attributes, key relationships emerged between TLR-9 amounts, the histological grade, progression stages, and cancer types. A notable finding was the connection between TLR-9 levels and EBV genetic presence, suggesting possible interactions between TLR-9 responses and EBV-related GC processes. Survival data also hinted at TLR-9's potential as a predictor linked to clinical traits. Overall, this research emphasizes TLR-9's complex role in GC's immune responses, pinpointing its interactions with particular cells, clinical features, and EBV. The study unveils a complex web affecting GC and paves the way for new treatment avenues targeting TLR-9 pathways. [ABSTRACT FROM AUTHOR]

Many clinical features, besides cytogenetic and molecular abnormalities, can affect the prognosis of the patients with acute myeloid leukemia (AML). Within this context it remains unclear if and how platelet counts affect the outcome of AML patients. In the present study, we examined the platelet counts at diagnosis in 633 newly diagnosed adult patients with AML from January 2010 to April 2021, and divided the cases into the group with low level of platelet counts (≤30×109/L, n=316) and high level of platelet counts (>30×109/L, n=317) according to the median platelet counts. We then validated the prognostic significance and potential mechanism of platelet counts on the relevance of spectral features for diagnostic risk stratification, initial induction therapy response, treatment effect maintenance, long-term survival, leukemia stem cells (LSCs) proportion, immunomodulatory cytokines level and immune cell subsets proportion. The results suggested that AML patients with a high level of platelet counts at diagnosis were associated with a high-risk molecular cytogenetic stratification, low complete remission (CR) rate, poor leukemia free survival (LFS), high proportion of LSCs, high level of transforming growth factor-β (TGF-β) and interleukin-1β (IL-1β), high proportion of regulatory T cells (Tregs) and monocytic myeloid-derived suppressor cells (M-MDSCs). It was demonstrated that platelet might be an unfavorable prognostic biomarker and was associated with LSCs and immunomodulatory cytokines as well as immune cell subsets in AML. [ABSTRACT FROM AUTHOR]

This newly established 24‐color (30‐marker) panel focuses on the characterization of the main human immune cell subtypes and was optimized for the analysis of human whole blood using a full spectrum flow cytometer. The panel covers all main leukocyte populations: neutrophils, eosinophils and basophils, monocytes (with additional subsets), dendritic cells, innate lymphoid cells and lymphocytes. As for lymphocytes, this panel includes CD4+ T helper, Treg cells, and CD8+ cytotoxic T cells. Further T cells subsets are included with special focus on invariant T cells: γδ T cells (including δ2TCR variant), invariant NKT cells and MAIT (mucosal‐associated invariant T cells) cells. Additionally, total B cells (including Bregs and plasmocytes), NK cells, and NKT cells are included. For the overall check of activation status of the analyzed immune cells we used HLA‐DR, CD38, CD57, CD69, PD‐1, and CD94. In addition, we used CD62L, CD45RA, CD27, and CD39 to describe the differentiation status of these cells. The panel was designed to maximize the information that can be obtained from surface markers in order to avoid the need for fixation and permeabilization steps. The presented multimarker panel offers the possibility to discover new immune cell subtypes which in patients and in cohort studies may lead to the identification of altered immune phenotypes and might give a link to immune system based or to certain other diseases. This panel was developed for a full spectrum flow cytometer equipped with a minimum of three lasers. We developed this panel using healthy human fresh blood, however it was also successfully used for staining of isolated human peripheral blood mononuclear cells (PBMC). [ABSTRACT FROM AUTHOR]

Introduction: Infections are a leading cause of morbidity and mortality in patients with multiple myeloma (MM). Methods: To examine the effects of modern second-generation novel agent therapy on immune cell subsets, in particular CD4+-T-cells, and infectious complications in patients with relapsed/refractory MM (RRMM), we conducted a prospective cohort study in 112 RRMM patients. Results: Substantially decreased CD4+-T-cells <200/µl before initiation of relapse therapy were detected in 27.7% of patients and were associated with a higher number of previous lines of therapy. Relapse therapy with carfilzomib or pomalidomide showed a significant further decrease of CD4+-T-cells. All novel agents led to a significant decrease of B-cell counts. Overall, infections were frequent with 21.3% of patients requiring antibacterial therapy within the first 3 months of relapse therapy, 5.6% requiring hospitalization. However, in the setting of standard antimicrobial prophylaxis in RRMM patients with very low CD4+-Tcells, no significant association of CD4+T-cell count and an increased risk of infection could be detected Discussion: Our findings imply that reduced CD4+-T-cell numbers and infections are common in patients with RRMM. We also demonstrate an association with the number of previous therapies and certain substances suggesting an increased need for personalized prophylaxis strategies for opportunistic infections in this patient cohort. [ABSTRACT FROM AUTHOR]

Maladaptive activation of the immune system plays a key role in the pathogenesis of chronic kidney disease (CKD). Our aim was to investigate differences in circulating immune cells between type 2 cardiorenal syndrome (CRS-2) patients and CKD patients without cardiovascular disease (CVD). CRS-2 patients were prospectively followed up, with the primary endpoint being all-cause and cardiovascular mortality. Method: A total of 39 stable males with CRS-2 and 24 male CKD patients matched for eGFR (CKD-EPI) were enrolled. A selected panel of immune cell subsets was measured by flow cytometry. Results: Compared to CKD patients, CRS-2 patients displayed higher levels of proinflammatory CD14++CD16+ monocytes (p = 0.04) and T regulatory cells (Tregs) (p = 0.03), lower lymphocytes (p = 0.04), and lower natural killer cells (p = 0.001). Decreased lymphocytes, T-lymphocytes, CD4+ T-cells, CD8+ T-cells, Tregs, and increased CD14++CD16+ monocytes were associated with mortality at a median follow-up of 30 months (p < 0.05 for all). In a multivariate model including all six immune cell subsets, only CD4+ T-lymphocytes remained independent predictors of mortality (OR 0.66; 95% CI 0.50–0.87; p = 0.004). Conclusion: Patients with CRS-2 exhibit alterations in immune cell profile compared to CKD patients of similar kidney function but without CVD. In the CRS-2 cohort, CD4+ T-lymphocytes independently predicted fatal cardiovascular events. [ABSTRACT FROM AUTHOR]

Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA- 4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti- PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti- PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection. [ABSTRACT FROM AUTHOR]

HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared with HIV-1B. The impact of HIV-1C Tat on the chemotaxis of the main lymphocyte subpopulations in the cerebrospinal fluid (CSF) and the peripheral blood (PB) is unclear. We hypothesized that there would be a lower frequency of specific lymphocyte subpopulations CD3+ or CD19+ in CSF in HIV-1C than in HIV-1B. The objectives were to detect the differences in the proportions of main lymphocyte subpopulations in CSF and PB, between people with HIV (PWH) and HIV-1-uninfected volunteers (PWoH) and in HIV-1B and HIV-1C. Lymphocyte immunophenotyping was studied in CSF and paired PB samples of PWH (n = 22) and PWoH (n = 14). Lymphocytes were analyzed within the CD45+ gated region. The proportions of CSF CD3+CD4+, CD3+CD8+, and CD3−CD19+ lymphocytes in CSF were comparable in HIV-1B and C. There was an increase in the proportion of CD3+CD8+ cells and a decrease in CD3+CD4+ T cells (ps = 0.016) in the CSF samples of the PWH compared with the PWoH group. In the PWH group, both CD3+CD4+ and CD3+CD8+ lymphocytes were significantly higher in the CSF than in the PB (p = 0.047 and 0.005). The proportion of CD3+CD4+ was lower and that of CD3+CD8+ was higher in the CSF samples of the aviremic group than that of HIV-negative control (p = 0.0008 and < 0.0001, respectively). HIV-1C Tat substitution (C30S) did not interfere with the CNS migration of the main lymphocyte subpopulations. This is the first study to evaluate these lymphocytes in CSF and PB of HIV-1C compared with HIV-1B. [ABSTRACT FROM AUTHOR]

Introduction: The immune system may involve and predict the different prognosis and therapy consequences. So, it's important to monitor and evaluate the immune status before and after treatments. Methods: Flow cytometry is the best technology to perform immune monitoring, because it can detect immune cells using small amount of sample in a short time. The whole blood is the ideal sample for immune status monitoring, since it includes almost all the immune cells and it's relatively easy to obtain and less invasive than bone marrow or lymph node. Results: Here we developed and validated a 10‐color panel with only four tubes containing 29 antibodies to monitor 90 immune cell subsets in 2 ml whole blood samples. The major immune cell populations detected by our panel included T cell subsets (CD3+total T, Th, Tc, Treg, CD8hi, CD8low, αβTCR, γδTCR, naïve, and memory T), T cell activation markers (CD25, CD69, and HLA‐DR) and one immune checkpoint PD1, B cell subsets (B1, switched memory, non‐switched, naïve B, and CD27‐IgD‐B cells), neutrophils, basophils, four monocytic cell subsets, dendritic cells (pDCs and mDCs), and four NK cell subsets. These panels of antibodies had been applied to monitor immune status (percentage and absolute number) in total 303 cases with various diseases, such as leukemia (AML, CML, MM, and ALL), lymphoma (B cells and NK/T cells), cancers (colon, lung, prostate, and breast), immune deficiencies, and autoimmune diseases. Conclusion: We provided proof of feasibility for clinical monitoring immune status and guiding immunotherapy by multicolor flow cytometry testing. [ABSTRACT FROM AUTHOR]

Background: Resolution of chronic hepatitis C virus (HCV) infection requires a complicated interaction between immune cell subsets. The effect of antiviral therapy on immune cell subsets remains to be defined. This study aimed to investigate the absolute count of certain immune cell subsets during therapy with pegylated interferon-α and ribavirin (PegIFN/RBV).Materials and Methods: Sixty HCV genotype 4-infected patients with compensated liver disease were treated with PegIFN/RBV therapy for 52 weeks. Efficacy was measured by studying the early virological response (EVR) at post-therapy week 12. Absolute counts of mature T cells, T helper cells, T cytotoxic cells, activated T cells, natural killer cells, natural killer/T (NKT) cells, B cells, and T regulatory cells (Treg), and the ratio of T helper to T cytotoxic cells were longitudinally analyzed by flow cytometry throughout the treatment and follow-up course.Results: Of the 60 genotype 4-infected subjects, 39 (65%) had EVR and 21 (35%) were non-EVR patients. In the first part of this study, there were significantly lower mean absolute count values of mature T, T cytotoxic, B, and NKT cells. Also, we detected statistically significantly lower mean values for the percentages of T cytotoxic, NKT, Treg, and activated T cells of HCV-infected patients at baseline values when compared with healthy subjects. After the initiation of PegIFN/RBV therapy, frequencies of T helper cells, activated T cells, Treg cells, B cells, and T helper:T cytotoxic ratio were found to be significantly lower in EVR patients than in non-EVR patients (p < 0.05). In contrast, frequencies of T cytotoxic and NKT cells were significantly increased in EVR patients when compared to non-EVR patients (p < 0.05).Conclusion: These results suggest a pattern of higher levels of T cytotoxic and NKT cells, and lower levels of T helper, activated T, Treg, and B cell populations in patients who respond favorably to PegIFN/RBV therapy. [ABSTRACT FROM AUTHOR]

This study investigates the prognostic value of immune cell subsets in assessing the risk of death in patients with sepsis. This retrospective study collected 169 patients from March 2020 to February 2021 at our hospital. Baseline data were collected from patients. The absolute values (Abs) and percentages (%) of immune cell subsets for lymphocytes, T cells, CD4+ cells, CD8+, B cells, NK cells, and NKT cells were measured using flow Cytometry. Among the included patients, 43 patients were in the nonsurvivor group and 126 patients were in the survivor group. The age of patients in the nonsurvivor survivor was higher than that of survivor group patients (P =.020). SOFA, APACHE II, C-reactive protein, and procalcitonin were higher in the nonsurvivor group than in the survivor group (all P values <.05). Multivariate regression analysis showed that lymphocytes (%) and SOFA were independent risk factors affecting patients' prognosis. Lymphocytes (%) have the highest area under the receiver operating characteristic (ROC) curve (0.812). The model area under the ROC curve for immune cell subsets was 0.800, with a sensitivity of 72.09%, and specificity of 79.27% (z = 7.796, P <.001). Analysis of patient prognosis by immune cell subsets diagnostic showed statistically significant differences in the grouping of cut-off values for all 5 indicators (all P <.05). The lymphocytes (%) and SOFA score are independent risk factors affecting the prognosis of patients. A moderate predictive power for mortality in sepsis patients by immune cell subsets model. [ABSTRACT FROM AUTHOR]

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD‐1, and PD‐1 receptor occupancy. Cell subsets include CD4+ and CD8+‐T‐cells, B‐cells, natural killer cells, classical‐, intermediate‐ and non‐classical monocytes, and myeloid‐ and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within‐ and between subject variations. The lower limit of quantification was 5.0% of PD‐1+ cells. Samples were stable for at least 153 days of storage at −80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD‐1+ immune cells, and number of PD‐1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+‐T‐cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Interleukin-33 (IL-33) is a IL-1 family member of cytokines exerting pleiotropic activities. In the steady-state, IL-33 is expressed in the nucleus of epithelial, endothelial, and fibroblast-like cells acting as a nuclear protein. In response to tissue damage, infections or necrosis IL-33 is released in the extracellular space, where it functions as an alarmin for the immune system. Its specific receptor ST2 is expressed by a variety of immune cell types, resulting in the stimulation of a wide range of immune reactions. Recent evidences suggest that different IL-33 isoforms exist, in virtue of proteolytic cleavage or alternative mRNA splicing, with potentially different biological activity and functions. Although initially studied in the context of allergy, infection, and inflammation, over the past decade IL-33 has gained much attention in cancer immunology. Increasing evidences indicate that IL-33 may have opposing functions, promoting, or dampening tumor immunity, depending on the tumor type, site of expression, and local concentration. In this review we will cover the biological functions of IL-33 on various immune cell subsets (e.g., T cells, NK, Treg cells, ILC2, eosinophils, neutrophils, basophils, mast cells, DCs, and macrophages) that affect anti-tumor immune responses in experimental and clinical cancers. We will also discuss the possible implications of diverse IL-33 mutations and isoforms in the anti-tumor activity of the cytokine and as possible clinical biomarkers. [ABSTRACT FROM AUTHOR]

Problem In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. Method of Study Peripheral blood mononuclear cells ( PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT- qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. Results In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8β. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. Conclusion Strong correlations between RT- qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophils are among the first defense against infection and closely involved in the initiation of inflammatory response. It is well recognized that this function of neutrophils was mainly mediated by phagocytosis, intracellular degradation, releasing of granules, and formation of neutrophil extracellular traps after sensing dangerous stress. However, accumulating data showed that neutrophils had a variety of important biological functions in both innate and adaptive immunities, far beyond cytotoxicity against pathogens. Neutrophils can differentially switch phenotypes and display distinct subpopulations under different microenvironments. Neutrophils can produce a large variety of cytokines and chemokines upon stimulation. Furthermore, neutrophils directly interact with dendritic cells (DCs), macrophages, natural killer cells, T cells, and B cells so as to either potentiate or down-modulate both innate and adaptive immunity. In the present review, we summarize the recent progress on the functional plasticity and the regulatory ability on immunity of neutrophils in physiological and pathological situations. [ABSTRACT FROM AUTHOR]

Immunosenescence results from a continuous deterioration of immune responses resulting in a decreased response to vaccines. A well-described age-related alteration of the immune system is the decrease of de novo generation of T and B cells. In addition, the accumulation of memory cells and loss of diversity in antigen specificities resulting from a lifetime of exposure to pathogens has also been described. However, the effect of aging on subsets of γδTCR+ T cells and Tregs has been poorly described, and the efficacy of the recall response to common persistent infections in the elderly remains obscure. Here, we investigated alterations in the subpopulations of the B and T cells among 24 healthy young (aged 19–30) and 26 healthy elderly (aged 53–67) individuals. The analysis was performed by flow cytometry using freshly collected peripheral blood. γδTCR+ T cells were overall decreased, while CD4+CD8− cells among γδTCR+ T cells were increased in the elderly. Helios+Foxp3+ and Helios−Foxp3+ Treg cells were unaffected with age. Recent thymic emigrants, based on CD31 expression, were decreased among the Helios+Foxp3+, but not the Helios−Foxp3+ cell populations. We observed a decrease in Adenovirus-specific CD4+ and CD8+ T cells and an increase in CMV-specific CD4+ T cells in the elderly. Similarly, INFγ+TNFα+ double-positive cells were decreased among activated T cells after Adenovirus stimulation but increased after CMV stimulation. The data presented here indicate that γδTCR+ T cells might stabilize B cells, and functional senescence might dominate at higher ages than those studied here. [ABSTRACT FROM AUTHOR]

Immunosenescence is a hallmark of the aging immune system, leading to increased susceptibility to infections in the aged population and decreased ability to eradicate infectious pathogens. These effects, in turn, result in an increased burden on the healthcare system due to elevated frequency and duration of hospital visits. Growing evidence suggests that cells of the innate immune system are central modulators for the initiation and maintenance of an adequate pathogen-specific response through the adaptive immune system. While there are many reports on age-dependent alterations and dysfunctions of the adaptive immune system, the underlying mechanisms and effects of natural aging on the composition of the innate immune system remain unknown. Here, we present the results obtained from the comprehensive immunophenotyping of innate leukocyte populations, examined for age-related alterations within different sub-populations assessed using multi-parametric flow cytometry. We compared peripheral blood mononuclear cells from 24 young (aged 19–30 years) and 26 elderly (aged 53–67 years) donors. For classical CD16+CD56dim NK cells, the fraction of CD62L+CD57+ was diminished in the elderly donors compared with young individuals, while the other investigated NK subsets remained unaffected by age. Both transitional monocytes and non-classical CD14+–CD16++ monocytes were increased in the elderly compared with the young. The populations of pDCs and mDC2 were decreased among the elderly. These data demonstrate that the dynamics of the mDC subsets might counteract decreased virus surveillance. Furthermore, these data show that the maturation of NK cells might gradually slow down. [ABSTRACT FROM AUTHOR]

Background: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays. Results: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles. Conclusions: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells' handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations. [ABSTRACT FROM AUTHOR]

Background: High quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays. Results: The mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles. Conclusions: Our findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells’ handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations. [ABSTRACT FROM AUTHOR]

The analysis of cerebrospinal fluid (CSF) with the assessment of CSF cell counts and proteins is an important method in the diagnostic workup of neurological diseases. As an addition to this standard approach, we here present data on the distribution of CSF immune cell subsets in common neurological diseases, and provide reference values along with cases of rare neurological diseases. CD4+ and CD8+ T cells, the CD4/CD8 ratio, B cells, plasmablasts, monocytes and NK cells in the CSF of 319 patients with inflammatory or non-inflammatory neurological diseases were analysed by seven-color flow cytometry. Diagnoses included headache, idiopathic intracranial hypertension, Guillain-Barré syndrome, multiple sclerosis, Lyme neuroborreliosis, bacterial and viral meningitis, human immunodeficiency virus (HIV) infection, stroke, and CNS malignancies, among others. T cells were the predominant population in the CSF with CD4+ T cells being more prevalent than CD8+ T cells. Mostly in HIV patients, and under other conditions of immunosuppression, CD4+ and CD8+ T cells were significantly altered and the CD4/CD8 ratio reduced. B cells and plasmablasts could hardly be detected in non-inflammatory diseases but were consistently elevated in inflammatory diseases. Monocytes were reduced in neuroinflammation and showed a negative correlation with B cells. NK cells were slightly elevated in neuroinflammation. Both monocytes and NK cells were slightly elevated in CNS malignancies. The analysis of immune cell subsets in the CSF adds valuable information to clinicians and is a promising tool for the differential diagnosis of neurological diseases. [ABSTRACT FROM AUTHOR]

Problem MUC16 ( CA125) released from ovarian tumors binds to NK cells and monocytes via the inhibitory receptor Siglec-9. Here, we investigate whether MUC16 also binds to circulating immune cells during pregnancy and in women with preeclampsia. Method of study MUC16 binding was monitored by flow cytometry and immunoprecipitation, and RT- PCR was used to monitor indigenous expression in immune cells. Serum CA125 levels were measured by a clinical assay. Results MUC16 was equally distributed on Siglec-9pos CD16pos/ CD56dim and CD16neg/ CD56br NK cells in the healthy pregnant and preeclampsia groups. While serum CA125 levels and number of NK and monocytes were similar, increased binding of MUC16 was observed on these immune cells in the preeclampsia cohort as compared to the healthy pregnant samples. Conclusion MUC16 binding to NK cells and monocytes likely contributes to tolerance of the fetal allograft from maternal responses and may also serve as a novel biomarker for preeclampsia. [ABSTRACT FROM AUTHOR]

Background: Cytokines play multiple roles during neuro-inflammatory processes and several cytokines have been studied in the context of specific diseases. This study provides a comprehensive picture of cerebrospinal fluid (CSF) changes during neuro-inflammation by analyzing multiple cytokines in combination with immune cell subsets and standard CSF parameters.Methods: Using multiplex assays, we simultaneously measured 36 cytokines (CCL1-3, CCL7, CCL8, CCL11, CCL13, CCL19, CCL20, CCL22-27, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, CXCL9, CXCL11-13, CXCL16, CX3CL1, IL2, IL4, IL6, IL10, IL16, GM-CSF, IFNγ, MIF, TNFα, and MIB1β) in the CSF and serum of 75 subjects. Diagnoses included clinically isolated syndrome and relapsing-remitting multiple sclerosis (MS, n = 18), secondary progressive MS (n = 8), neuro-syphilis (n = 6), Lyme neuro-borreliosis (n = 13), bacterial and viral meningitis (n = 20), and patients with non-inflammatory neurological diseases (NIND, n = 10). Cytokine concentrations were correlated with CSF standard parameters and CSF immune cell subsets (CD4 and CD8 T cells, B cells, plasmablasts, monocytes, and NK cells) quantified by flow cytometry.Results: We observed increased levels of multiple cytokines (26/36) in patients with neuro-inflammatory diseases when compared to NIND that consistently correlated with CSF cell count and QAlbumin. Most CSF cytokine concentrations correlated with each other, but correlations between CSF and serum values were scarce (3/36). Within the CSF compartment, CXCL13 showed a strong association with B cells when analyzing all patients, as well as patients with an intact blood-brain barrier (BBB). NK cells positively correlated with CSF concentrations of multiple cytokines (22/36) when analyzing all patients. These correlations were maintained when looking at patients with a disrupted BBB but not detectable in patients with an intact BBB.Conclusions: Under conditions of neuro-inflammation, multiple CSF cytokines are regulated in parallel and most likely produced locally. A combined increase of CSF CXCL13 levels and B cells occurs under conditions of an intact BBB. Under conditions of a disrupted BBB, CSF NK cells show significantly increased values and seem to have a major contribution to overall inflammatory processes, reflected by a strong correlation with multiple cytokines. Future studies are necessary to address the exact kinetics of these cytokines during neuro-inflammation and their relation to specific diseases phenotypes. [ABSTRACT FROM AUTHOR]