Subpopulations Of Peripheral Blood Mononuclear Cells Research Articles

Toxicity effects of Cannabidiol (CBD) on immune cells

Background: Cannabis extract has a long history of being used in the treatment and prevention of several medical conditions. The utilization of cannabis extracts, whether for medical or localized purposes, is widely observed. In cannabis extract, cannabidiol (CBD) is one of the most important non-psychoactive compounds. Several studies have demonstrated that CBD has several benefits in the treatment of various medical conditions. Nevertheless, CBD has also been demonstrated to suppress both innate and adaptive immune responses. Despite CBD has claimed to have many benefits, the toxicity of CBD is often pointed out and discussed. Nonetheless, the data on the toxicity effects of CBD on immune cells are limited. Objectives: In this study, we aimed to investigate the toxicity effects of various concentrations of CBD on immune cells, including CD4 T cells, CD8 T cells, B cells, NK cells, and monocytes. Materials and methods: Various concentrations of peripheral blood mononuclear cells (PBMCs) were treated with various concentrations of CBD or relative concentrations of methanol as a diluent control for 12, 24, and 48 hrs. Cell morphology was observed using flow cytometry. The percentage of cell death in the treated cells was determined by cell viability assay. In addition, the toxic effects of CBD on PBMC sub-populations were determined by staining with fluorochromeconjugated zombie viability dye and fluorochrome-conjugated monoclonal antibodies specific to each cell sub-population. Then, the percentage of cell death in each sub-population was assessed using flow cytometry. Results: CBD at concentrations of 40 and 80 µM showed toxicity effects on PBMCs. At these concentrations, CBD induced both cell morphological changes and cell death. While 20 µM CBD induced different effects, ranging from none to mild and high toxicity. The toxicity of CBD at 20 µM concentration depends on the individual. In contrast, CBD at ten µM and below showed no toxicity to PBMCs. The observed toxic effects of CBD occurred in all sub-populations of PBMCs, including CD4 T cells, CD8 T cells, B cells, NK cells, and monocytes. Conclusion: CBD has toxicity effects on immune cells. These effects depend on CBD concentrations, PBMC concentrations, and the duration of CBD exposure. Our findings emphasize the importance of awareness for CBD users when consuming CBD.

Distinct Immune and Molecular Profiles of Hepatitis-Associated Aplastic Anemia

Background: Hepatitis-associated aplastic anemia (HAAA) is characterized by the development of hematopoietic failure within months following an acute episode of non-infectious hepatitis. Although disease presentation with pancytopenia and a hypocellular marrow in HAAA is similar to non-hepatitis immune AA (non-HAAA), the immune and molecular profile in HAAA has not been completely elucidated. Here, we aimed to assess HAAA patients for cytokines and presence of clonal events linked to AA, the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones and somatic mutations in peripheral blood mononuclear cells (PBMC) subpopulations, and compared to a non-HAAA cohort. Methods: A cohort of HAAA patients (n=30), age and sex-matched non-HAAA patients (n=24) and healthy controls (n=23) seen at the National Institutes of Health since 2003 were retrospectively screened for a panel of inflammatory cytokines and growth factors by Luminex, and somatic mutations in sorted CD3-, CD3+CD4+, and CD3+CD8+ PBMC fractions by error-corrected DNA sequencing. A customized panel with 42 myeloid-related and 49 autoimmunity-related genes at minimum limited of detection of 0.1% were used. All samples were before any immunosuppressive treatment (IST). Cytokine levels and frequencies of PNH clones and clonal hematopoiesis (CH) in different fractions were compared among groups. Results: HAAA patients were in average young (median age=23); 37% were at age < 18 (Table 1). Although disease severity and blood counts were similar to the non-HAAA cohort, immune and molecular markers of immune AA were distinct among cohorts. The frequency of PNH clones was significantly lower in HAAA compared to the non-HAAA cohort (15% v 58%, p=0.0008). The median size of PNH clones in HAAA and non-HAAA was 1.4% and 7.4%, respectively (p =0.03). The cytokine profiles performed in the sera of 22 HAAA before IST were marked by significantly higher levels of IL-1ra (p=0.0008), CXCL-10 (p=0.0038), and VEGF (p=0.0023) in comparison to 31 non-HAAA assessed patients; IL-6 (p=0.0002) and CD40L (p=0.0001) levels were significantly lower than in non-HAAA. As reported in immune AA, HAAA patients had higher levels of TPO (p=0.0001), G-CSF (p=0.0001), and EPO (p=0.0001) than controls; lower levels of CCL-5 (p=0.0001) and CCL-11 (p=0.0025) were seen in the HAAA. FLT3-L levels were markedly elevated in both AA cohorts when compared to HC. The clonal landscape was also strikingly different between HAAA and non-HAAA in all fractions evaluated (Figure 1). In HAAA, 3 out of 21 assessed patients (14%) had CH in the CD3- fraction at maximum variant allele frequency (VAF) of 1.8%; one patient had two RUNX1 mutations while a JAK2 and DNMT3A mutation were found in the other two patients. No mutations were found in CD4+ and CD8+ compartments in subjects with HAAA. In contrast, 13 of 24 non-HAAA patients (54%) had CH in the CD3- populations, which was dominated by PIGA, BCOR, and DNMT3A clones, a pattern consistent with the known clonal landscape of immune AA. The frequency of non-HAAA with CH in the CD4+ and CD8+ compartments was 7/24 (29%) and 8/24 (30%) patients, respectively; CH in these fractions was similar to those found in CD3- dominated by mutations in PIGA, BCOR, and DNMT3A at VAFs ranging from 0.2% to 15% (Figure 1). CH in lymphoid-related genes was only found in a single patient with a STAT3 p.S614Rmutation at VAF of 3% exclusive to the CD8+ fraction. Conclusion: HAAA has a distinct immunologic and molecular profile than non-HAAA patients. PNH clones were less frequent in HAAA and patterns of cytokines were significantly different than the non-HAAA cohort. CH in PIGA and BCOR, highly associated with AA, were not present in our small HAAA cohort. Our results indicate a distinct underlying pathophysiology that initiates the immune destruction resulting in marrow failure in HAAA patients. Confirming these findings in a larger cohort and correlating with clinical outcomes is ongoing.

Label-free microfluidic isolation of functional and viable lymphocytes from peripheral blood mononuclear cells.

The separation of peripheral blood mononuclear cells (PBMCs) into constituent blood cell types is a vital step to obtain immune cells for autologous cell therapies. The ability to separate PBMCs using label-free microfluidic techniques, based on differences in biomechanical properties, can have a number of benefits over other conventional techniques, including lower cost, ease of use, and avoidance of animal-derived labeling antibodies. Here, we report a microfluidic device that uses compressive diagonal ridges to separate PBMCs into highly pure samples of viable and functional lymphocytes. The technique utilizes the differences in the biophysical properties of PBMC sub-populations to direct the lymphocytes and monocytes into separate outlets. The biophysical properties of the monocytes and lymphocytes from healthy donors were first characterized using atomic force microscopy. Lymphocytes were found to be significantly stiffer than monocytes, with a mean cell stiffness of 1495 and 931 Pa, respectively. The differences in biophysical properties resulted in distinct trajectories through the microchannel terminating at different outlets, resulting in a lymphocyte sample with purity and viability both greater than 96% with no effect on the cells' ability to produce interferon gamma, a cytokine crucial for innate and adaptive immunity.

Peripheral blood mononuclear cells exhibit increased mitochondrial respiration after adjuvant chemo- and radiotherapy for early breast cancer.

Adjuvant chemo- and radiotherapy cause cellular damage to tumorous and healthy dividing cells. Chemotherapy has been shown to cause mitochondrial respiratory dysfunction in non-tumorous tissues, but the effects on human peripheral blood mononuclear cells (PBMCs) remain unknown. We aimed to investigate mitochondrial respiration of PBMCs before and after adjuvant chemo- and radiotherapy in postmenopausal patients with early breast cancer (EBC) and relate these to metabolic parameters of the patients. Twenty-three postmenopausal women diagnosed with EBC were examined before and shortly after chemotherapy with (n = 18) or without (n = 5) radiotherapy. Respiration (O2 flux per million PBMCs) was assessed by high-resolution respirometry of intact and permeabilized PBMCs. Clinical metabolic characteristics and mitochondrial DNA (mtDNA) content of PBMCs (mtDN relative to nuclear DNA) were furthermore assessed. Respiration of intact and permeabilized PBMCs from EBC patients significantly increased with adjuvant chemo- and radiotherapy (p = 6 × 10-5 and p = 1 × 10-7 , respectively). The oxygen flux attributed to specific mitochondrial complexes and respiratory states increased by 17-43% compared to before therapy initiation. Similarly, PBMC mtDNA content increased by 40% (p = 0.002). Leukocytes (p = 0.0001), hemoglobin (p = 0.0003), and HDL cholesterol (p = 0.003) concentrations decreased whereas triglyceride (p = 0.01) and LDL (p = 0.02) concentrations increased after treatment suggesting a worsened metabolic state. None of the metabolic parameters or the mtDNA content of PBMCs correlated significantly with PBMC respiration. This study shows that mitochondrial respiration and mtDNA content in circulating PBMCs increase after adjuvant chemo- and radiotherapy in postmenopausal patients with EBC. Besides the increased mtDNA content, a shift in PBMC subpopulation proportions towards cells relying on oxidative phosphorylation, who may be less sensitive to chemotherapy, might influence the increased mitochondrial respiration observed iafter chemotherapy.

Small RNA transcriptome analysis using parallel single-cell small RNA sequencing

miRNA and other forms of small RNAs are known to regulate many biological processes. Single-cell small RNA sequencing can be used to profile small RNAs of individual cells; however, limitations of efficiency and scale prevent its widespread application. Here, we developed parallel single-cell small RNA sequencing (PSCSR-seq), which can overcome the limitations of existing methods and enable high-throughput small RNA expression profiling of individual cells. Analysis of PSCSR-seq data indicated that diverse cell types could be identified based on patterns of miRNA expression, and showed that miRNA content in nuclei is informative (for example, cell type marker miRNAs can be detected in isolated nuclei). PSCSR-seq is very sensitive: analysis of only 732 peripheral blood mononuclear cells (PBMCs) detected 774 miRNAs, whereas bulk small RNA analysis would require input RNA from approximately 106 cells to detect as many miRNAs. We identified 42 miRNAs as markers for PBMC subpopulations. Moreover, we analyzed the miRNA profiles of 9,533 cells from lung cancer biopsies, and by dissecting cell subpopulations, we identified potentially diagnostic and therapeutic miRNAs for lung cancers. Our study demonstrates that PSCSR-seq is highly sensitive and reproducible, thus making it an advanced tool for miRNA analysis in cancer and life science research.

Abstract OT2-01-06: Phase II trial of palbociclib plus endocrine therapy followed by combination of pembrolizumab, palbociclib and endocrine therapy in patients with hormone receptor positive metastatic breast cancer

Abstract Background: The combination of CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is standard-of-care for patients with hormone receptor positive (HR+) HER2- metastatic breast cancer (MBC). Immune modulatory effects of CDK4/6i are well documented preclinically but poorly understood in the clinical setting. Our previous study combining letrozole, palbociclib and pembrolizumab in patients with HR+ MBC (NCT02778685) showed a promising complete response rate of 31%. Dynamic changes in peripheral blood mononuclear cell (PBMC) subpopulations indicated that palbociclib may increase CD8+ TEMRA (terminally differentiated effector memory cells) and CD4+ TEM (effector memory cells) and enable immune activation. The current cohort 3 was designed to study the immune modulatory effect of palbociclib as an immune-priming agent with a biomarker enriched design. Methods: Women with ECOG 0-1, HR+ HER2- MBC, RECIST 1.1 measurable disease, no prior therapy for MBC were enrolled. Patients with endocrine therapy, including aromatase inhibitor +/- ovarian suppression or fulvestrant, were eligible. A palbociclib + ET lead-in design was used, starting on day -28 followed by combination therapy with pembrolizumab added on C1D1. Peripheral blood and tumor biopsy at baseline and on-treatment were collected to allow in-depth analysis of biomarkers predicting response to the combination. The primary endpoint was to evaluate if the palbociclib potentiated immune responses as a “priming” agent through PBMC analysis and on treatment tumor biopsy. Secondary endpoints included other immune cell subsets and changes that follow the combination with pembrolizumab. With 25 patients, assuming a standard deviation of 0.51 in the relative change in classic monocytes in PBMCs, there is 90% power to detect a relative change of log(C1D1/baseline) of 34.5% with a type I error (two-sided) of 0.05. Results: Between August 2020 and April 2022, 16 patients were enrolled in cohort 3. Currently 11 patients have adverse event (AEs) and 16 patients have response data. Median age was 57 years (39-72). 8/11 (73%) were non-Hispanic white, 1/11 (9%) Hispanic, 1/11 (9%) Asian, and 1/11 (9%) African American. 87% patients had grade 3 AEs, and 30% had grade 4 AEs. Grade 3 AEs were 9/11 (82%) neutropenia, 5/11 (45%) leukopenia, 1/11 (9%) elevated LFTs, and 1/11 (9%) each lymphopenia, hot flashes, febrile neutropenia, and pneumonitis. Grade 4 AEs were 1/11 (9%) lymphopenia. 8/16 (50%) patients achieved a partial response (PR), 5/16 (31%) had stable disease (SD), and 1/16 (6%) had progression of disease (PD) by RECIST 1.1. Additionally, 2/16 (13%) patients were too early to determine best overall response. Response rate (CR+PR) was 50%. PBMCs and tumor microenvironment profiling are ongoing. Conclusion: The combination of palbociclib, pembrolizumab and ET is well tolerated, and a response rate of 50% was identified in HR+ MBC patients who received this combination as front-line therapy. Dynamic changes in peripheral blood mononuclear cells and tumor microenvironment profiling are ongoing. Citation Format: Yuan Yuan, Colt A. Egelston, Weihua Guo, Susan E. Yost, Paul H. Frankel, Christopher Ruel, Mireya Murga, Aileen Tang, Norma Martinez, Daniel Schmolze, Daphne Stewart, James Waisman, Kelly Yap, Joanne Mortimer, Niki Tank. Phase II trial of palbociclib plus endocrine therapy followed by combination of pembrolizumab, palbociclib and endocrine therapy in patients with hormone receptor positive metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-01-06.

Machine learning empowered multi-stress level electromechanical phenotyping for high-dimensional single cell analysis

Microfluidics provides a powerful platform for biological analysis by harnessing the ability to precisely manipulate fluids and microparticles with integrated microsensors. Here, we introduce an imaging and impedance cell analyzer (IM2Cell), which implements single cell level impedance analysis and hydrodynamic mechanical phenotyping simultaneously. For the first time, IM2Cell demonstrates the capability of multi-stress level mechanical phenotyping. Specifically, IM2Cell is capable of characterizing cell diameter, three deformability responses, and four electrical properties. It presents high-dimensional information to give insight into subcellular components such as cell membrane, cytoplasm, cytoskeleton, and nucleus. In this work, we first validate imaging and impedance-based cell analyses separately. Then, the two techniques are combined to obtain both imaging and impedance data analyzed by machine learning method, exhibiting an improved prediction accuracy from 83.1% to 95.4% between fixed and living MDA-MB-231 breast cancer cells. Next, IM2Cell demonstrates 91.2% classification accuracy in a mixture of unlabeled MCF-10A, MCF-7, and MDA-MB-231 cell lines. Finally, an application demonstrates the potential of IM2Cell for the deformability studies of peripheral blood mononuclear cells (PBMCs) subpopulations without cumbersome isolation or labeling steps.

Characterization of circulating immune cells and correlation with Tie2/Angiopoietins level in well differentiated neuroendocrine gastroenteropancreatic tumors: a cross-sectional analysis.

The immune environment represents a new, but little explored, tool for understanding neuroendocrine neoplasms (NENs) behavior. An immunosuppressed microenvironment is hypothesized to promote NENs progression. A missing profiling of circulating leukocyte and peripheral blood mononuclear cells (PBMCs) subpopulations would open new perspectives in the still limited diagnostic-therapeutic management of NENs. A cross-sectional case-control pilot study was performed recruiting 30 consecutive subjects: 15 patients naïve to treatment, with histologically proven gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) and 15 healthy controls, matched for age and sex. PBMCs subpopulations were studied by flow cytometry. Soluble Tie2 (sTie2), Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) were evaluated by ELISA. Immune cell profiling revealed a significant lower CD3-CD56+ natural killer (NK) cell count in NETs vs controls (p = 0.04). NK subset analysis showed a reduced relative count of CD56+CD16+ NK cells (p =0.002) in NETs vs controls. Patients with NET showed a higher percentage of CD14+CD16++ non-classical monocytes (p = 0.01), and a lower percentage of CD14+CD16+ intermediate monocytes (p = 0.04). A decrease in percentage (p = 0.004) of CD4+ T-helper lymphocytes was found in NET patients. Evaluation of cellular and serum angiopoietin pathway mediators revealed in NET patients a higher relative count of Tie2-expressing monocytes (TEMs) (p < 0.001), and high levels of Ang-1 (p = 0.003) and Ang-2 (p = 0.002). Patients with GEP-NET presented an immunosuppressed environment characterized by a low count of cytotoxic NK cells, a high count of anti-inflammatory non-classical monocytes, and a low count of T-helper lymphocytes. Higher levels of TEMs and angiopoietins suggest a crosstalk between innate immunity and angiogenic pathways in NETs.

CLL-050 Induction of Neutralizing Antibodies in Chronic Lymphocytic Leukemia Patients After SARS-CoV-2 mRNA Vaccination: A Monocentric Experience

Background: SARS-CoV-2 infection increases the clinical risk and mortality of patients with chronic lymphocytic leukemia (CLL). Covid-19 vaccinations have the potential to protect individuals from severe infection, but little is known about the antibody response profile in these patients after vaccination. We compared the anti-receptor binding domain (RBD) and neutralizing antibody responses of 27 patients with CLL to those of healthy donors after mRNA Covid-19 vaccination. Methods: Of the 27 patients enrolled in the study, 21 received the BNT162b2 mRNA vaccine and 6 received the Spikevax mRNA vaccine. Four patients were in clinical remission after treatment, 10 were treatment-naïve, and 13 were under treatment at the time of vaccination. Blood samples were taken before vaccination and after the second dose, with a median time between of 23 days. The levels of anti-RBD IgG, IgM, and IgA were evaluated by ELISA on recombinant RBD; the neutralizing antibody response was evaluated by spike protein inhibition assay (SPIA). We also performed a fluorescence-activated cell sorting study of peripheral blood mononuclear cell subpopulations, a disease burden assessment using blood indicators (lactate dehydrogenase, beta-2-microglobulin, lymphocytic count) and imaging, and an evaluation of IgG levels at the time of vaccination. Results: A comparison of anti-RBD antibody seroconversion (IgG, IgM, and IgA) and neutralizing antibodies present after vaccination between 27 patients with CLL and healthy control subjects indicated that CLL patients had a considerably lower response rate (50% vs. 100%) and lower antibody levels than the healthy controls. We found an inverse correlation between the spleen dimension and lymphonodal area. By stratifying patients according to the presence of neutralizing antibodies after vaccination, we found a larger prevalence of SPIA positives than SPIA negatives in treatment-naïve patients (60%); however, patients undergoing treatment had a much lower percentage of SPIA positives (23.1%). Conclusions: Only around half of vaccinated CLL patients acquire detectable anti-RBD and neutralizing antibodies, according to our findings. Furthermore, we discovered a substantial difference in the rates of detectable anti-SARS-CoV-2 antibodies between patients who were treatment-naïve/in clinical remission and those on CLL-directed treatment. The persistence and burden of disease represent a surrogate of vaccine failure, probably due to the persistence of immune dysfunction.

Presence of regulatory T-cells in endometrial cancer predicts poorer overall survival and promotes progression of tumor cells

PurposeEndometrial cancer (EC) is one of the most common gynaecologic malignancies. Tumor infiltrating regulatory T-cells (Treg) have been reported to have a prognostic impact in many malignancies. Immunotherapeutic strategies are gaining interest for advanced and recurrent EC cases, where treatment options are rare. Our study was aimed at determining the value of Treg in EC progression.MethodsEC specimens from 275 patients and 28 controls were screened immunohistochemically for the presence of Treg represented by FoxP3. Correlations with clinicopathological and survival parameters were performed. Functional assays were performed using EC cell lines Ishikawa + and RL95-2 after co-culturing with isolated CD4 + CD25 + CD127dim Treg. To assess the influence of EC on the composition of peripheral blood mononuclear cells (PBMC), flow cytometric analyses were performed.ResultsWe found that an increased infiltration of Treg was associated with high grades and a reduced overall survival. Treg were almost absent in endometrium tissues from healthy control patients. Co-culture of tumor cells with CD4 + CD25 + CD127dim Treg led to functional changes: enhanced invasion, migration and viability indicated that increased levels of Treg in the tumor microenvironment may promote tumor growth. Furthermore, we found that the presence of EC cells led to phenotypic changes in PBMC, showing significantly increased levels of CD25 and FoxP3.ConclusionOur results indicate that the presence of Treg in the EC tumor environment is associated with a poorer outcome. A remarkable impact of Treg on tumor cell behaviour and vice versa of tumor cells on PBMC subpopulations support this notion mechanistically. Our findings provide a basis for focusing on Treg as potential future therapeutic targets in EC.

EP08.01-040 Peripheral Blood Cells as Predictors for Efficacy of Immunotherapy in Patients with Advanced Non-small Cell Lung Cancer

In lung cancer immunotherapy, biomarkers to guide clinical decisions are limited to PD-L1. We explore whether one or several peripheral blood mononuclear cells (PBMC) subpopulations from treatment-naïve patients with advanced non-small-cell lung cancer are associated with the efficacy of anti-PD-1 immunotherapy.

Peripheral Blood Mononuclear Cells Predict Therapeutic Efficacy of Immunotherapy in NSCLC.

Simple SummaryBiomarkers to guide clinical decisions and efficacy are limited in advanced non-small cell lung cancer’s anti-PD-1 immune checkpoint inhibitors. We prospectively explored baseline peripheral blood mononuclear cells in order to asses’ immunotherapy predictors in this setting. We included 39 patients diagnosed with non-small cell lung cancer treated with immunotherapy in the study group and 40 patients with advanced malignancies treated with non-immunotherapy treatment, as control group. We detected that high baseline levels of circulating T cell subpopulations related to tissue lymphocyte recruitment are associated with poorer outcomes of immunotherapy-treated advanced non-small cell lung cancer patients, and these differences were specific to immunotherapy-treated patients.In lung cancer immunotherapy, biomarkers to guide clinical decisions are limited. We now explore whether the detailed immunophenotyping of circulating peripheral blood mononuclear cells (PBMCs) can predict the efficacy of anti-PD-1 immunotherapy in patients with advanced non-small-cell lung cancer (NSCLC). We determined 107 PBMCs subpopulations in a prospective cohort of NSCLC patients before starting single-agent anti-PD-1 immunotherapy (study group), analyzed by flow cytometry. As a control group, we studied patients with advanced malignancies before initiating non-immunotherapy treatment. The frequency of PBMCs was correlated with treatment outcome. Patients were categorized as having either high or low expression for each biomarker, defined as those above the 55th or below the 45th percentile of the overall marker expression within the cohort. In the study group, three subpopulations were associated with significant differences in outcome: high pretreatment levels of circulating CD4+CCR9+, CD4+CCR10+, or CD8+CXCR4+ T cells correlated with poorer overall survival (15.7 vs. 35.9 months, HR 0.16, p = 0.003; 22.0 vs. NR months, HR 0.10, p = 0.003, and 22.0 vs. NR months, HR 0.29, p = 0.02). These differences were specific to immunotherapy-treated patients. High baseline levels of circulating T cell subpopulations related to tissue lymphocyte recruitment are associated with poorer outcomes of immunotherapy-treated advanced NSCLC patients.

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients.

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients.

Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs.

Humans can be infected by zoonotic avian, pandemic and seasonal influenza A viruses (IAVs), which differ by receptor specificity and conformational stability of their envelope glycoprotein hemagglutinin (HA). It was shown that receptor specificity of the HA determines the tropism of IAVs to human airway epithelial cells, the primary target of IAVs in humans. Less is known about potential effects of the HA properties on viral attachment, infection and activation of human immune cells. To address this question, we studied the infection of total human peripheral blood mononuclear cells (PBMCs) and subpopulations of human PBMCs with well characterized recombinant IAVs differing by the HA and the neuraminidase (NA) but sharing all other viral proteins. Monocytes and all subpopulations of lymphocytes were significantly less susceptible to infection by IAVs with avian-like receptor specificity as compared to human-like IAVs, whereas plasmacytoid dendritic cells (pDCs) and myeloid dendritic cells were equally susceptible to IAVs with avian-like and human-like receptor specificity. This tropism correlated with the surface expression of 2-3-linked sialic acids (avian-type receptors) and 2-6-linked sialic acids (human-type receptors). Despite a reduced infectivity of avian-like IAVs for PBMCs, these viruses were not less efficient than human-like IAVs in terms of cell activation as judged by the induction of cellular mRNA of IFN-α, CCL5, RIG-I, and IL-6. Elevated levels of IFN-α mRNA were accompanied by elevated IFN-α protein secretion in primary human pDC. We found that high basal expression in monocytes of antiviral interferon-induced transmembrane protein 3 (IFITM3) limited viral infection in these cells. siRNA-mediated knockdown of IFITM3 in monocytes demonstrated that viral sensitivity to inhibition by IFITM3 correlated with the conformational stability of the HA. Our study provides new insights into the role of host- and strain-specific differences of HA in the interaction of IAVs with human immune cells and advances current understanding of the mechanisms of viral cell tropism, pathogenesis and markers of virulence.

Clinical significance of detection of mononuclear phagocyte subsets in blood and bronchoalveolar lavage fluid (BALF) in pulmonary sarcoidosis.

This study aimed to investigate the clinical significance of the detection of mononuclear phagocytes subsets in pulmonary sarcoidosis blood and bronchoalveolar lavage fluid (BALF). For this purpose, a total of 52 patients with pulmonary sarcoidosis were selected as the study group, 52 healthy people served as the "NC Group a" (peripheral blood mononuclear cell control group), 47 patients with chronic cough and no pulmonary sarcoidosis who underwent bronchoscopy were used as "control group b" (alveolar lavage fluid macrophage control group). Fasting peripheral blood and BALF were collected, and flow cytometry was used to detect monocytes and macrophage subpopulations. The monocytes and macrophage subpopulations of the study group were compared before and after treatment. The results showed that the proportion of CD14++CD16- subgroup of patients with pulmonary sarcoidosis was lower than that of healthy people (74.21±4.10% vs 84.32±4.76%); The proportion of CD14++CD16+ subgroups of patients with pulmonary sarcoidosis was higher than that of healthy people (7.42±4.08% vs 3.95±1.94%); The proportion of CD14+CD16++ subgroups of patients with pulmonary sarcoidosis was higher than that of healthy people in the control group, but the difference was not significant. After 2 months of treatment, the proportion of CD14++CD16- subgroups in peripheral blood mononuclear cells increased, and the proportion of CD14++CD16+ subgroups decreased. The proportion of M1 in patients with pulmonary sarcoidosis was lower than that in patients with non-pulmonary nodules (24.32±11.36% vs 47.03±13.86%); the proportion of M2 in patients with pulmonary sarcoidosis was higher than the proportion of M2 in patients with non-pulmonary nodules (75.40±10.23% vs 52.53±12.01%). After treatment, the proportion of M1 of BALF macrophages in patients with pulmonary sarcoidosis was increased (P<0.05), and the proportion of M2 was reduced (P<0.05). In general, detection of changes in peripheral blood mononuclear cell subpopulations and BALF macrophage subpopulations in patients with pulmonary sarcoidosis has certain clinical significance for the treatment.

SNHG15 is a negative regulator of inflammation by mediating TRAF2 ubiquitination in stroke-induced immunosuppression

BackgroundAbnormal expression of long noncoding RNAs (lncRNAs) has been reported in the acute stage of acute ischemic stroke (AIS). This study aimed to explore differential lncRNA expression in the subpopulations of peripheral blood mononuclear cells (PBMCs) from AIS patients and further evaluate its underlying mechanisms in stroke-induced immunosuppression.MethodsWe reanalyzed lncRNA microarray data and investigated abnormally expressed lncRNAs in the subpopulations of PBMCs by magnetic cell sorting and real-time quantitative PCR. The potential mechanism of small nucleolar RNA host gene 15 (SNHG15) was explored through in vitro and in vivo approaches.ResultsThe stroke-induced SNHG15 acted as a checkpoint to inhibit peripheral inflammatory responses. Functional studies showed that SNHG15 promoted M2 macrophage polarization. Mechanistically, SNHG15 expression was dysregulated through the Janus kinase (JAK)-signal transducer and activator of transcription 6 (STAT6) signaling pathway. SNHG15, localized in the cytoplasm, interfered with K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 2 and thereby repressed the activation of mitogen-activated protein kinase and nuclear factor kappa-B signaling pathways and prevented the production of proinflammatory cytokines. Administration of an adenovirus targeting SNHG15 improved stroke-induced immunosuppression in mice.ConclusionsThis study identified SNHG15 as a negative regulator of inflammation in stroke-induced immunosuppression, suggesting it as a novel biomarker and therapeutic target in stroke-associated infection.Trial registration ClinicalTrials.gov NCT04175691. Registered November 25, 2019, https://www.clinicaltrials.gov/ct2/show/NCT04175691.

Revisiting miR-155 in intervertebral disc degeneration: blood cell signature and local cell-free profiles

We unraveled the expression profiles of coding-noncoding RNAs in intervertebral disc degeneration (IDD). However, it remains elusive regarding miR-155 expression in peripheral blood mononuclear cells (PBMCs) and local extracellular space in IDD. The study aimed for investigating the miR-155 expression of PBMCs, extracellular miRNAs (ex-miRNAs) of human nucleus pulposus (NP) tissues, and morphological changes of cell death in the IDD process. Here, we harvested peripheral blood and NP samples from scoliosis and IDD patients as control and degenerative groups, respectively. Then standard Ficoll density-gradient centrifugation was used to isolate PBMCs. The two subpopulations of PBMCs were divided based on the cellular difference in adherence, i.e., monocytes/ macrophages and lymphocytes. Quantitative real-time PCR was used to evaluate miR-155 relative expression in PBMCs. ex-miRNAs were screened by comparison between GSE19943, GSE63492, and extracellular RNA (exRNA) atlas. The morphological changes of cell death subtypes in IDD were observed in transmission electron microscopy (TEM). Results indicate that the lower expression of miR-155 in PBMCs from IDD patients ascribed to alterations in monocytes/macrophages. Moreover, we identified 594 shared hsa-miRNAs as the intracellular miRNA expression profile and 258 miRNAs as the ex-miRNA expression profile in human NP tissue. miR-155 was amongst intracellular miRNAs in NP. TEM observation presented multiple endocytic secretory vesicles within human NP cells, implicating exosome transporting machinery. Typical necroptosis and pyroptosis were noted in human NP. Collectively, this study reveals miR-155 expression in PBMCs from IDD patients. Moreover, we propose ex-miRNA expression profile and necroptosis/pyroptosis in human NP tissue, shedding novel light on the etiology of IDD.

Revisiting miR-155 in intervertebral disc degeneration: blood cell signature and local cell-free profiles

We unraveled the expression profiles of coding-noncoding RNAs in intervertebral disc degeneration (IDD). However, it remains elusive regarding miR-155 expression in peripheral blood mononuclear cells (PBMCs) and local extracellular space in IDD. The study aimed for investigating the miR-155 expression of PBMCs, extracellular miRNAs (ex-miRNAs) of human nucleus pulposus (NP) tissues, and morphological changes of cell death in the IDD process. Here, we harvested peripheral blood and NP samples from scoliosis and IDD patients as control and degenerative groups, respectively. Then standard Ficoll density-gradient centrifugation was used to isolate PBMCs. The two subpopulations of PBMCs were divided based on the cellular difference in adherence, i.e., monocytes/ macrophages and lymphocytes. Quantitative real-time PCR was used to evaluate miR-155 relative expression in PBMCs. ex-miRNAs were screened by comparison between GSE19943, GSE63492, and extracellular RNA (exRNA) atlas. The morphological changes of cell death subtypes in IDD were observed in transmission electron microscopy (TEM). Results indicate that the lower expression of miR-155 in PBMCs from IDD patients ascribed to alterations in monocytes/macrophages. Moreover, we identified 594 shared hsa-miRNAs as the intracellular miRNA expression profile and 258 miRNAs as the ex-miRNA expression profile in human NP tissue. miR-155 was amongst intracellular miRNAs in NP. TEM observation presented multiple endocytic secretory vesicles within human NP cells, implicating exosome transporting machinery. Typical necroptosis and pyroptosis were noted in human NP. Collectively, this study reveals miR-155 expression in PBMCs from IDD patients. Moreover, we propose ex-miRNA expression profile and necroptosis/pyroptosis in human NP tissue, shedding novel light on the etiology of IDD.

Expression analysis of miRNAs from the DLK1-DIO3 locus in CD4+ and CD14+ cells in patients with relapsing-remitting multiple sclerosis

To analyze the expression of several previously unstudied miRNAs from the DLK1-DIO3 locus in peripheral blood mononuclear cells (PBMC) and in CD14+ and CD4+ cell subpopulations in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy individuals. MiRNA expression analysis was performed in PBMC, CD14+, and CD4+ cells of 14 patients who did not take immunomodulatory drugs, and 14 individuals without autoimmune and inflammatory diseases by reverse transcription followed by real-time PCR. Expression levels of 10 miRNAs selected based on different criteria were significantly higher in PBMC in men with RRMS compared to healthy men; in women, their expression did not change in a similar comparison. No mass unidirectional changes in the expression of these miRNAs in RRMS men were observed in CD14+ and CD4+ cells. High consistency in the expression of miRNA pairs was also not found. The analysis of PBMC showed the involvement of 10 more miRNAs encoded by genes from the DLK1-DIO3 locus in the development of RRMS. The main observed effect of the increase in the expression levels of these miRNAs in men with RRMS compared with healthy men is not achieved due to the contribution of CD14+ and CD4+ cells. This opens up possibilities for studying the involvement of miRNAs in other PBMC subpopulations in the pathological processes in RRMS.

Potential PD-L1 expressing cytotoxic T-cell immunopathology in Alzheimer disease.

Growing evidence suggested that adaptive immunity involved Alzheimer's disease (AD). Peripheral blood mononuclear cell (PBMC) subpopulations and expression of immune checkpoints such as programmed death-ligand 1(PD-L1) and programmed cell death protein-1 (PD-1) could play roles in Alzheimer disease. The interaction of PD-L1/PD-1 lead PD-1 expressing T cells to attenuate T cell-activating signals. Recent studies had demonstrated the blockade of PD-L1/PD-1 axis modified brain pathology and restored cognitive performance in tauopathy murine model. However similar human experiment suggested controversial results in AD patients. To observe the changes of PBMC subpopulations and PD-L1/PD-1 expression in AD and healthy controls, we utilized flow cytometry, and applied t-distributed stochastic neighbor embedding (t-SNE) algorithm as a new approach to analyze results. AD patients (n=16) with different disease severity (CDR= 1 - 2) and healthy controls (n=16) were enrolled. Expression levels of PD-L1/PD-1 in AD patients and healthy controls were derived by flow cytometric analysis parameters. Moreover, a dimensionality reduction algorithm for visualized t-SNE, was implemented to analyze the PD-L1/PD-1 expressing cell clusters between AD patients and healthy controls. We found no significant PD-1 expression intensity difference between cytotoxic T lymphocytes (CTL) of AD patients and healthy controls. In AD patients, subpopulation of CTL expressing PD-L1 was elevated by 85.53%. Further, PD-L1 expressing CTL clusters between AD patients and healthy controls were compared. We observed that densities of PD-L1high CTL clusters are significantly higher than the healthy controls. Upregulation of PD-L1 and PD-L1 expressing CTL were increased in peripheral blood of AD patients, linking to newly found strong correlation between the level of PD-L1 positive CTL and markers which indicated negative immune climate. Abnormal adaptive immune response of CTL might associate with concurrent viral infection, intracellular bacterial infection, and underlying cancer in AD patients. Our results suggested a novel strategy to prevent AD progression with regulation of PD-L1 expressing CTL, and notation of underlying intracellular infection and malignancy for which could relate to dementia progression.