Number Of Cell Subsets Research Articles

Autoimmune Regulator (AIRE) Deficiency Does Not Affect Atherosclerosis and CD4 T Cell Immune Tolerance to Apolipoprotein B.

Atherosclerosis is a chronic, lipid-driven disease of medium sized arteries which causes myocardial infarction and stroke. Recently, an adaptive immune response against the plaque-associated autoantigen Apolipoprotein B100 (ApoB), the structural protein component of low-density lipoprotein, has been implicated in atherogenesis. In healthy individuals, CD4+ T cells responding to ApoB mainly comprised regulatory T cells, which confer immune tolerance and atheroprotection. Mice and patients with atherosclerosis harbor increased numbers of proatherogenic ApoB-reactive T-helper cell subsets. Given the lack of therapies targeting proatherogenic immunity, clarification of the underlying mechanisms is of high clinical relevance. T cells develop in the thymus, where strong autoreactive T cells are eliminated in the process of negative selection. Herein, we investigated whether the transcription factor autoimmune regulator (AIRE), which controls expression of numerous tissue-restricted self-antigens in the thymus, is involved in mediating tolerance to ApoB and whether Aire deficiency might contribute to atherogenesis. Mice deficient for Aire were crossbred to apolipoprotein E-deficient mice to obtain atherosclerosis-prone Aire−/− Apoe−/− mice, which were fed a regular chow diet (CD) or western-type diet (WD). CD4+ T cells responding to the ApoB peptide p6 were analyzed by flow cytometry. We demonstrate that Aire deficiency influences neither generation nor activation of ApoB-reactive T cells and has only minor and overall inconsistent impacts on their phenotype. Furthermore, we show that atherosclerotic plaque size is not affected in Aire−/− Apoe−/− compared to Aire+/+ Apoe−/−, irrespective of diet and gender. In conclusion, our data suggests that AIRE is not involved in regulating thymic expression of ApoB or atherosclerosis. Alternative mechanisms how ApoB-reactive CD4 T cells are selected in the thymus will have to be investigated.

Severity of SARS-CoV-2 infection is linked to double-negative (CD27- IgD-) B cell subset numbers.

ObjectivesThe role of B cells in COVID‐19, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Here, we describe the novel landscape of circulating double-negative (DN) CD27− IgD− B cells in COVID‐19 patients, representing a group of atypical and neglected subpopulations of this cell lineage.MethodsUsing multiparametric flow cytometry, we determined DN B cell subset amounts from 91 COVID-19 patients, correlated those with cytokines, clinical and laboratory parameters, and segregated them by principal components analysis.ResultsWe detected significant increments in the DN2 and DN3 B cell subsets, while we found a relevant decrease in the DN1 B cell subpopulation, according to disease severity and patient outcomes. These DN cell numbers also appeared to correlate with pro- or anti-inflammatory signatures, respectively, and contributed to the segregation of the patients into disease severity groups.ConclusionThis study provides insights into DN B cell subsets’ potential role in immune responses against SARS‐CoV‐2, particularly linked to the severity of COVID‐19.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-021-01525-3.

Abstract 11303: Reduced Level of ErbB Receptors in Non-Myocyte Left Ventricular Cells in Patients with Diabetes Mellitus Undergoing Coronary Artery Bypass Grafting Surgery Contributes to Vascular Dysfunction

Introduction: ErbB signaling contributes to the regulation of vascular function through endothelial cell differentiation, proliferation, survival, and regulation of barrier integrity. Patients with diabetes (DM) are at an increased risk for endothelial dysfunction and worse outcomes after CABG. We investigated the level of ErbB expression on CD31 endothelial cells and CD105+ cells in DM and non-DM patients undergoing CABG. Methods: With IRB approval, left ventricular (LV) epicardial biopsies and blood samples from 25 patients (non-DM=9, DM=16), were obtained during surgery. Flow cytometry was used to determine the cell surface expression of ErbB receptors on cardiac endothelial and CD105+ cells isolated from the LV. Subpopulations of white blood cells were determined in blood samples. Plasma levels of IL-6, IL-8, and TNFα were measured. Results: Lower levels of ErbB1 and ErbB2 receptors expression on endothelial cells isolated from LV biopsies were found in patients with DM compared to non-DM control, and the expression of ErbB receptors negatively correlated with HgbA1c level. There were fewer CD105+ cells in DM patients (122 vs 153 cell/mg tissue, P=.021). Trends toward a negative association were found between the expression of ErbB2 in CD105+ cells and the number of neutrophils, as well as, HgbA1c level. No differences were found between DM and non-DM patients in the number of white blood cell subsets or plasma levels of IL-6, IL-8 and TNFα. Conclusions: We demonstrated that DM and high glucose blood levels are associated with reduced expression of ErbB1 and ErbB2 on endothelial cells and decreased number of CD105+ cells in the LV. Cardiac CD105+ are endothelial progenitor cells that differentiate in response to ErbB ligands. A decreased pool of CD105 endothelial progenitors in combination with down-regulation of ErbB1 and ErbB2 receptors in endothelial cells represent a novel mechanism for vascular dysfunction and adverse remodeling seen in diabetic patients.

A Single-Arm, Open-Label, Pilot Study of the JAK1 Selective Inhibitor Itacitinib for the Prophylaxis of Graft-Versus-Host Disease and Cytokine Release Syndrome in T-Cell Replete Haploidentical Peripheral Blood Hematopoietic Cell Transplantation

Introduction:Haploidentical peripheral blood allogeneic hematopoietic cell transplantation (PB haplo-HCT) can be complicated by graft-versus-host disease (GVHD) and cytokine release syndrome (CRS). Acute GVHD rates are higher with PB grafts compared with bone marrow, affecting 35-45% of patients and outcomes are poor in steroid refractory cases. Severe CRS occurs in 10-15% of patients receiving PB haplo-HCT and is associated with high non-relapse mortality and dismal one year overall survival between 25-30%. As interferon-γ and IL-6 are important mediators in both acute GVHD and CRS, we hypothesized that JAK1 inhibition with itacitinib could prevent these toxicities without impairing engraftment. Here we report the clinical outcomes from our pilot study of itacitinib with haplo-HCT (NCT03755414).Methods:Patients with AML, ALL, or NHL in remission undergoing PB haplo-HCT were treated with itacitinib 200 mg/day on days -3 through +100, followed by a taper. Myeloablative and reduced intensity conditioning were allowed. GVHD prophylaxis was tacrolimus, mycophenolate mofetil, and post-transplant cyclophosphamide. Primary outcomes were incidence of primary graft failure and incidence of grade III-IV acute GVHD. Secondary outcomes included incidence and severity of CRS (graded by Lee criteria). Peripheral blood and serum samples were banked prior to conditioning and on days -1, 1, 3, 7, 14, 28, 60, 100, end of treatment, and time of diagnosis of acute GVHD. Matched control samples were collected from patients undergoing haplo-HCT off clinical trials. Correlative studies include flow cytometry (FACS) with five 28-color panels for cellular subsets, mass cytometry (CyTOF) with 40-colors for intracellular signaling events, single cell RNA sequencing, and serum cytokine and chemokine measurements.Results:Twenty of a planned 20 patients completed enrollment and underwent haplo-HCT between 11/2019 and 3/2021. Median age at transplant was 49 (21-74). Diagnoses were AML (13), ALL (5) and NHL (2). Median follow up is 319 days, with 18/20 beyond 180 days. There were no cases of engraftment failure with short median times to neutrophil (14 days, range 12-20) and platelet (14 days, range 7-54) engraftment (historical 16 and 25 days, respectively). There were no cases of grade III-IV acute GVHD. The incidence of grade II acute GVHD on day 100 was 15%. Two patients developed grade I-II skin acute GVHD during itacitinib taper and responded to resumption of a higher dose. There were no cases of extensive chronic GVHD. There were no cases of severe CRS (historical rate 17%), with 90% of patients having grade 1 CRS and 10% having no CRS. Furthermore, no anti-IL6R or steroid therapy was used. Overall survival at day 180 was 90% (95% CI 75-100%) by Kaplan-Meier estimate. Incidence of relapse at 180 days was 5.5% (95% CI 0-15.6%). Refined GVHD and relapse-free survival at 180 days was 83% (95% CI 68-100%). All patients had full donor engraftment and >95% chimerism at day 100.FACS and CyTOF have been performed and analyzed for the day 28 time point from 14 patients and four controls. Flow cytometry revealed no difference in cell subset numbers between controls and patient samples. CyTOF revealed differences in intracellular signaling molecules between itacitinib and control patients - including higher Ki-67, pNF-κB, and Caspase3 in controls. Phospho-Stat1 and pStat3 were lower CD4 T subsets. FACS and CyTOF at remaining time points, single cell RNA sequencing, and serum cytokine and chemokine measurements are underway and will be presented at the ASH 2021 meeting.Conclusions:Itacitinib with PB haplo-HCT was safe with no engraftment failure and prompt engraftment. Rates of acute and chronic GVHD were low, without increased risk of relapse or transplant related mortality. Severe CRS was not seen in this trial, and no anti-IL6 or steroid therapy was used. Flow cytometry demonstrated comparable immune reconstitution in terms of cell lineage and number between treated patients and controls. Mass cytometry revealed lower Ki-67, pNF-κB, and caspase3 levels, among other markers, which suggest lower immune cell activity, proliferation, and apoptosis. An extension cohort of 20 additional patients is enrolling. Multi-platform correlative studies are underway, comparing samples from haplo-HCT patients treated with and without itacitinib. [Display omitted] DisclosuresUy: Astellas: Honoraria, Speakers Bureau; Novartis: Consultancy; Agios: Consultancy; Jazz: Consultancy; Genentech: Consultancy; AbbVie: Consultancy; GlaxoSmithKline: Consultancy; Macrogenics: Research Funding. Ghobadi: Atara: Consultancy; Amgen: Consultancy, Research Funding; Wugen: Consultancy; Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding. Jacoby: Abbvie: Research Funding; Jazz: Research Funding. Pusic: Syndax: Other: Advisory Board. Schroeder: Equillium Inc: Honoraria; Janssen: Honoraria; Sanofi Genzyme: Honoraria.

Distinct profiles of immune cell populations underlie in-stent restenosis: a cluster analysis approach

Abstract Background In-stent restenosis (ISR) is a major challenge in patients with coronary artery disease due to its association with poor clinical outcomes, quality of life and costs. ISR etiopathogenesis remains unclear, but traditional risk factors cannot fully explain ISR burden. Inflammation-driven loss of endothelial homeostasis and neoatherosclerosis are thought to hallmark ISR. Recently, a number of immune cell subsets have been related to vascular repair failure and endothelial damage, such as angiogenic T-cells (Tang), endothelial progenitor cells (EPC), senescent T-cells (CD4+CD28null), monocyte subsets and low-density granulocytes (LDG). However, these subsets have not been studied in ISR and an integrative analysis is lacking. Purpose 1) to evaluate potential alterations in vascular repair and endothelial damage cellular mediators in ISR and 2) to identify profiles associated with clinical features. Methods Case-control study including 30 patients with ≥1 previous stent implantation (15 bare metal stents (BMS) and 15 drug-eluting stents (DE)) which suffered restenosis and 30 patients with ≥1 BMS without restenosis, both confirmed in a second angiogram performed by clinical symptoms >8 months after index procedure. Cellular mediators of vascular homeostasis were quantified by flow cytometry based on their surface markers in peripheral blood (EPC: CD34+VEGFR2+CD133+; EC: CD34-VEGFR+CD133-; Tang: CD3+CD31+CXCR4+; senescent T-cells: CD4+CD28null) or in peripheral blood mononuclear cells (monocyte subsets, ACE expression; total LDG: CD15+; and LDG subsets: CD15+CD14-CD16- and CD15+CD14lowCD16+). Results Patients with ISR exhibited decreased circulating Tang (p=0.005) and EPC (p<0.001), whereas CD4+CD28null and EC counts were higher (p<0.0001 and p=0.006) compared to ISR-free patients. No differences were observed in the frequency of monocyte subsets (all p>0.050), although ACE expression was found to be increased (non-classical p<0.001; and intermediate p<0.0001) in ISR. Moreover, no differences were noted in the total LDG population (p=0.092), but an increase in the CD14- compartment was observed in ISR (p=0.004). An unsupervised cluster analysis built with these subsets informed the presence of three profiles (Figure 1): group I (hallmarked by a profound impairment in vascular repair and augmented damage, suggestive of central haematopoiesis traits) exhibited an enhanced clinical risk profile compared to group II (hallmarked by a mid-altered vascular repair) and group III (hallmarked by CD16+ shifted LDG and ACE expression) (Figure 2). No differences were observed in stent types or traditional risk factors but hypertension. Conclusions Profound alterations in immune populations related to vascular repair and endothelial damage are found in ISR. Distinct cellular profiles can be distinguished within ISR, suggesting that different alterations may uncover different ISR clinical phenotypes, in terms of severity and extension. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIIIPrograma Intramural ISPA Figure 1Figure 2

Peripheral blood intermediate monocyte protease-activated receptor-2 expression increases during asthma exacerbations and after inhalation allergen challenge

BackgroundMyeloid cells, especially dendritic cells and macrophages, play important roles in asthma pathophysiology. Monocytes (Mo) and macrophages express protease-activated receptor-2 (PAR-2), a proinflammatory serine protease receptor implicated in the pathophysiology of allergic airway inflammation. We have revealed that patients with severe asthma and those with a history of frequent asthma exacerbations exhibit increased PAR-2 expression on peripheral blood monocytes. ObjectiveTo determine PAR-2 expression on peripheral blood intermediate monocytes (IMMo) in subjects with increased airway inflammation, either as a result of an asthma exacerbation or after an inhalation allergen challenge. MethodsA total of 16 adults who presented to the emergency department with asthma exacerbations were recruited after giving an informed consent. After 2 weeks, 10 patients returned for follow-up. A total of 11 patients with mild asthma treated only with as-needed bronchodilators were recruited and underwent inhalation allergen challenge after providing an informed consent. Immune cell profiling was performed by whole blood flow cytometry in both groups of patients. ResultsPAR-2 expression in peripheral blood IMMo increased in patients with an asthma exacerbation compared with those with stable disease, but this expression decreased after treatment of the asthma exacerbation. Subjects with mild asthma had an increase in percentages of IMMo expressing PAR-2 after an allergen challenge. Patients who presented to the emergency department had lower dendritic cell and dendritic cell subset numbers in peripheral blood during exacerbation compared with after treatment. ConclusionIncreased PAR-2 expression on Mo during periods of increased airway inflammation may initiate a positive feedback loop leading to systemic inflammatory changes.

The Frequency of Natural Killer Cell Subsets in Patients with Acquired Immune Deficiency Syndrome with Deep Fungal Infections.

ObjectiveThis study aimed to investigate the expression of natural killer (NK) cell subsets in patients with acquired immune deficiency syndrome (AIDS) and deep fungal infections and the significance of such expression.MethodsA total of 829 patients with AIDS, who were treated in People’s Hospital of Deyang City our hospital between January 2011 and March 2019, were enrolled in the study. They were divided into two groups: those with human immunodeficiency virus (HIV) and invasive fungal infection (IFI) (HIV + IFI) (n = 390) and those with HIV and no IFI (HIV + non-IFI) (n = 439). Another 200 healthy volunteers were enrolled as the control group. The numbers of NK cell subsets in each group were compared.ResultsThe level of NK cells, number of NK cells in all lymphocytes, proportions of CD56bright, CD56dim, and CD56dim NK cells in NK cells, and the level of CD56–CD16+ NK cells were significantly lower in the HIV + IFI group than in the HIV + non-IFI group and control group (P < 0.05). Moreover, CD4+ T, CD4+/CD8+, and NK cells were negatively correlated with HIV-RNA expression (P < 0.05).ConclusionA combination of AIDS and deep fungal infection can change the immune status of a patient. This condition can be diagnosed early through the detection of NK cell expression.

Synergistic effects of AAGL and anti-PD-1 on hepatocellular carcinoma through lymphocyte recruitment to the liver.

Objective:Therapy for hepatocellular carcinoma (HCC) is a major challenge, and targeted therapies provide only a modest benefit in terms of overall survival. Treatment with antibodies to programmed cell death protein 1 (PD-1)/PD-L1 can restore the functions of tumor-infiltrating T cells in HCC and has shown clinical efficacy in 20% of patients with advanced HCC. Novel approaches are urgently needed to treat HCC and to augment the efficacy of immunotherapy.Methods:Tumor-bearing mice were treated with Agrocybe aegerita galectin (AAGL) alone or in combination with anti-PD-1, and the tumor sizes and lifespans of mice were determined. Transcriptome analysis, cytokine analysis, flow cytometry analysis of the number and proportion of immune cell subsets in the liver and spleen, and molecular and cellular analyses of tumors were used to define the underlying mechanisms.Results:AAGL significantly inhibited the growth of liver tumors in a dose-dependent manner. Furthermore, AAGL increased the expression of multiple cytokines and chemokines in tumor-bearing mouse livers; this effect was associated with the activation and migration of T cells and macrophages, in agreement with the in vitro results. Importantly, the aggregation of T cells and macrophages induced by AAGL in tumor-bearing mouse livers clearly enhanced the response to PD-1 blockade immunotherapy.Conclusions:The results showed that AAGL induced the activation and migration of lymphocytes to the liver, and that the combination of AAGL and anti-PD-1 may be a promising strategy for HCC treatment.

Immunophenotyping of Baseline Bone Marrow Reveals a Specific Pattern of Immune Cells Associated with Greater Depth and Sustained Response in Newly Diagnosed Patients Randomized to Krd or Ktd Followed By Carfilzomib Maintenance or Control (AGMT MM 02 Study)

Immunophenotyping of Baseline Bone Marrow Reveals a Specific Pattern of Immune Cells Associated with Greater Depth and Sustained Response in Newly Diagnosed Patients Randomized to Krd or Ktd Followed By Carfilzomib Maintenance or Control (AGMT MM 02 Study)

Levels and Clinical Significance of Regulatory B Cells and T Cells in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy, whose immunological mechanisms are still partially uncovered. Regulatory B cells (Bregs) and CD4+ regulatory T cells (Tregs) are subgroups of immunoregulatory cells involved in modulating autoimmunity, inflammation, and transplantation reactions. Herein, by studying the number and function of Breg and Treg cell subsets in patients with AML, we explored their potential role in the pathogenesis of AML. Newly diagnosed AML patients, AML patients in complete remission, and healthy controls were enrolled. Flow cytometry was used to detect percentages of Bregs and Tregs. ELISA was conducted to detect IL-10 and TGF-β in plasma. The mRNA levels of IL-10 and Foxp3 were measured with RT-qPCR. The relationship of Bregs and Tregs with the clinicopathological parameters was analyzed. There was a significant reduction in the frequencies of Bregs and an increase of Tregs in newly diagnosed AML patients compared with healthy controls. Meanwhile, patients in complete remission exhibited levels of Bregs and Tregs comparable to healthy controls. Furthermore, compared with healthy controls and AML patients in complete remission, newly diagnosed AML patients had increased plasma IL-10 but reduced TGF-β. IL-10 and Foxp3 mRNA levels were upregulated in the newly diagnosed AML patients. However, there were no significant differences in IL-10 and Foxp3 mRNA levels between patients in complete remission and healthy controls. Bregs and Tregs have abnormal distribution in AML patients, suggesting that they might play an important role in regulating immune responses in AML.

CD16 expression on neutrophils predicts treatment efficacy of capecitabine in colorectal cancer patients

BackgroundEarly detection of capecitabine-resistance could largely increase overall survival of colorectal cancer (CRC) patients. Previous studies suggested examination of immune cells in peripheral blood would help to predict efficacy of chemotherapy.MethodsWe examined the immunological characteristics of peripheral blood in CRC patients with capecitabine treatment. We analyzed the relationships between the abnormal immune cell population in capecitabine-resistance patients and major clinical features. Furthermore, RNA sequencing, analyses of cell surface marker expression and the correlations with other major immune cell populations were performed using this population to explore the possible function of these cells.ResultsThe expression level of CD16 on neutrophils was down-regulated in capecitabine-resistant CRC patients. Patients with CD16low/−neutrophils after capecitabine therapy had adverse clinical features. What’s important, the change of CD16 expression level on neutrophils appeared much earlier than CT scan. RNA sequencing revealed that CD16low/−neutrophils in capecitabine-resistant patients had lower expression level of neutrophil-related genes, compared to CD16+neutrophils in capecitabine-sensitive patients, suggesting this CD16low/−population might be immature neutrophils. Furthermore, the expression level of CD16 on neutrophils in patients with capecitabine treatment was positively correlated with the number of anti-tumor immune cell subsets, such as CD8+T cell, CD4+T cell, NK cell and monocyte.ConclusionsOur findings indicated that CD16 expression on neutrophils in peripheral blood was a good prognostic marker for predicting efficacy of capecitabine in CRC patients.

The Increased Ratio of Blood CD56bright NK to CD56dim NK Is a Distinguishing Feature of Primary Sjögren's Syndrome.

Objective The aim of this study was to characterize the subsets of circulating CD56+ NK cells in pSS patients and their potential value in the diagnosis and/or prediction of prognosis in patients with pSS. Methods We included 52 pSS patients fulfilling the 2002 AECG criteria or 2012 ACR criteria and 20 age- and gender-matched healthy volunteers. The frequency and absolute number of NK cells and CD56 NK cell subsets in peripheral blood samples were detected by flow cytometry. Other laboratory parameters such as the IgG level and complement protein levels were extracted from the clinical system. Results Both the frequency and the absolute number of peripheral blood NK cells were reduced in pSS patients compared to healthy controls. The proportion of CD56bright NK cell subset was increased, and the proportion of CD56dim NK cell subset was decreased among NK cells, resulting in the ratio of CD56bright NK to CD56dim NK which was significantly elevated in pSS patients. ROC analysis indicated that the AUC of CD56bright NK/CD56dim NK ratio was 0.838, and the best diagnostic cut-off point was 0.0487 for pSS patients. Furthermore, this CD56bright NK/CD56dim NK ratio was positively correlated with the IgG level and negatively correlated with the complement protein C3 and C4 levels. More importantly, the CD56bright/CD56dim NK ratio was either slightly increased or not changed in other autoimmune diseases such as SLE and IgG4-related disease. Conclusion Our findings suggest that the ratio of blood CD56bright NK to CD56dim NK might have a diagnostic value relatively specific for pSS.

Validation of a hybrid approach to standardize immunophenotyping analysis in large population studies: The Health and Retirement Study

Traditional manual gating strategies are often time-intensive, place a high burden on the analyzer, and are susceptible to bias between analyzers. Several automated gating methods have shown to exceed performance of manual gating for a limited number of cell subsets. However, many of the automated algorithms still require significant manual interventions or have yet to demonstrate their utility in large datasets. Therefore, we developed an approach that utilizes a previously published automated algorithm (OpenCyto framework) with a manually created hierarchically cell gating template implemented, along with a custom developed visualization software (FlowAnnotator) to rapidly and efficiently analyze immunophenotyping data in large population studies. This approach allows pre-defining populations that can be analyzed solely by automated analysis and incorporating manual refinement for smaller downstream populations. We validated this method with traditional manual gating strategies for 24 subsets of T cells, B cells, NK cells, monocytes and dendritic cells in 931 participants from the Health and Retirement Study (HRS). Our results show a high degree of correlation (r ≥ 0.80) for 18 (78%) of the 24 cell subsets. For the remaining subsets, the correlation was low (<0.80) primarily because of the low numbers of events recorded in these subsets. The mean difference in the absolute counts between the hybrid method and manual gating strategy of these cell subsets showed results that were very similar to the traditional manual gating method. We describe a practical method for standardization of immunophenotyping methods in large scale population studies that provides a rapid, accurate and reproducible alternative to labor intensive manual gating strategies.

Short-Term Environmental Enrichment is a Stronger Modulator of Brain Glial Cells and Cervical Lymph Node T Cell Subtypes than Exercise or Combined Exercise and Enrichment

Physical exercise (PE) and environmental enrichment (EE) can modulate immunity. However, the differential effects of short-term PE, EE, and PE + EE on neuroimmune mechanisms during normal aging has not been elucidated. Hence, a cohort of 3-, 8-, and 13-month-old immunologically unchallenged C57BL/6 wild-type mice were randomly assigned to either Control, PE, EE, or PE + EE groups and provided with either no treatment, a running wheel, a variety of plastic and wooden objects alone or in combination with a running wheel for seven weeks, respectively. Immunohistochemistry and 8-color flow cytometry were used to determine the numbers of dentate gyrus glial cells, and the proportions of CD4+ and CD8+ T cell numbers and their subsets from cervical lymph nodes, respectively. An increase in the number of IBA1+ microglia in the dentate gyrus at 5 and 10 months was observed after EE, while PE and PE + EE increased it only at 10 months. No change in astroglia number in comparison to controls were observed in any of the treatment groups. Also, all treatments induced significant differences in the proportion of specific T cell subsets, i.e., CD4+ and CD8+ T naïve (TN), central memory (TCM), and effector memory (TEM) cells. Our results suggest that in the short-term, EE is a stronger modulator of microglial and peripheral T cell subset numbers than PE and PE + EE, and the combination of short-term PE and EE has no additive effects.

Dissecting the spatial heterogeneity of different immune cell subsets in non-small cell lung cancer

To date, most studies investigating the immune tumor microenvironment of non-small cell lung cancers (NSCLC) only consider a small number of immune cell subsets or do not reflect the distribution of these cells between different tumor compartments as they were performed on tissue microarrays (TMA). To address this, we analyzed the immune infiltrate in surgically resected NSCLCs, focusing on potential spatial heterogeneity.We evaluated 45 NSCLCs based on whole-slide sections using immunohistochemistry with eleven different antibodies (CD3, CD4, CD8, CD20, CD68, Gata3, FOXP3, T-bet, kappa, lambda, PD-L1).While most markers were relatively evenly distributed among different tumor compartments as well as within the same tumor compartment, some immune cell subsets showed a considerable variance. Notably, the immune infiltrate at the tumor invasion front was dominated by B cells. Concerning markers for T cell differentiation, FoxP3 (Th2) was predominantly expressed in stromal lymphocytes, while T-bet (Th1) was most commonly expressed in intraepithelial immune cells. Although most immune cell subtypes showed a heterogenous distribution within in the intraepithelial compartment, the results from a simulated TMA and core biopsy were mostly in line with the results from whole slide evaluation.Regarding disease specific survival, there were no clear correlations. Interestingly, patients with intraepithelial T-bet positive lymphocytes had a significantly better outcome (p = 0.039), however, this difference was not preserved in multivariate analysis.In conclusion, our study shows that the immune tumor microenvironment of NSCLCs is complex and partially heterogenous, especially concerning markers for T cell differentiation.

Influence of combined functional resistance and endurance exercise over 12\u2009weeks on matrix metalloproteinase-2 serum concentration in persons with relapsing-remitting multiple sclerosis \u2013 a community-based randomized controlled trial

BackgroundThe relevance of regular moderate to intense exercise for ameliorating psychomotor symptoms in persons with multiple sclerosis (pwMS) is becoming increasingly evident. Over the last two decades, emerging evidence from clinical studies and animal models indicate immune regulatory mechanisms in both periphery and the central nervous system that may underlie these beneficial effects. The integrity of the blood-brain barrier as the main structural interface between periphery and brain seems to play an important role in MS. Reducing the secretion of proteolytic matrix metalloproteinases (MMP), i.e. MMP-2, as disruptors of blood-brain barrier integrity could have profound implications for MS.MethodsIn this two-armed randomized controlled trial 64 participants with relapsing-remitting MS (RRMS) (EDSS 0–4.0) will be allocated to either an intervention group or a passive wait list control group. The intervention group will perform 60 min of combined functional resistance and endurance exercises 3x per week over a period of 12 weeks in a community-based and publicly available setting. Changes in serum concentration of MMP-2 will be the primary outcome. Secondary outcomes are numbers of immune cell subsets, soluble (anti-) inflammatory factors, physical capacity, cognitive performance, physical activity behavior, gait performance, and patient-reported outcomes. All outcome measures will be assessed at baseline and after week 12 with an additional blood sampling before, during and immediately after a single training session in week 6.DiscussionTo our knowledge, this will be the first RCT to investigate both the acute and chronic effects of a community-based intense functional resistance and endurance exercise regimen in persons with RRMS. Combining analysis of biological and cognitive or psychological outcomes may provide a better understanding of the MS-specific symptomology.Trial registrationDRKS00017091; 05th of April, 2019; International Clinical Trials Registry Platform.

Algorithmic Clustering Of Single-Cell Cytometry Data-How Unsupervised Are These Analyses Really?

Algorithmic Clustering Of Single-Cell Cytometry Data-How Unsupervised Are These Analyses Really?

Immunomodulatory effect of mesenchymal stem cells in chemical-induced liver injury: a high-dimensional analysis

BackgroundThe efficacy of mesenchymal stem cell (MSC)-based therapy for acute liver injury (ALI) involves coordination with the hepatic immune system, a complex and coordinated network of immune-cell interactions. However, studies of the immunomodulatory effects of MSCs have focused on a limited number of cell subsets rather than a systematic assessment.MethodsCarbon tetrachloride (CCl4) was used to induce ALI in mice. To determine the efficacy of MSCs, ALI mice were injected with MSCs via the tail vein, and histopathological changes, survival rate, and the serum levels of liver enzymes were determined. To assess the immune response induced by MSCs, a mass cytometry panel of 43 metal isotope-tagged antibodies was used to characterize the hepatic immune compartment at days 1, 2, 3, and 7 after administration of MSCs or PBS.ResultsMSC treatment significantly alleviated CCl4-induced ALI and improved the survival rate. MSC treatment also modulated the hepatic immune system in terms of the distribution of immune-cell subsets and the phenotype of single cells. During the injured phase, MSCs inhibited a systemic response by reducing the numbers of Ly6ClowCD8+ TRM cells, conventional NK cells, and IgM+IgD+ B cells; suppressing the activation of Ly6ChiCD8+ TRM cells; downregulating MHC II and IgM expression in IgM+IgD+ B cells; and increasing the number of immunosuppressive monocyte-derived macrophages. During the recovery phase, MSCs promoted the retention of Ly6ClowCD8+ TRM cells and maintained the immunosuppressive activity of monocyte-derived macrophages. The response to MSC treatment differed between the injured and recovery phases, emphasizing the benefit of dynamic assessment of the immunomodulatory effects of MSCs.ConclusionsWe determined the immunomodulatory effects of MSC treatment on the subtype distribution and phenotypes of hepatic immune cells.

Bone marrow endothelial cell-derived interleukin-4 contributes to thrombocytopenia in acute myeloid leukemia.

Normal hematopoiesis can be disrupted by the leukemic bone marrow microenvironment, which leads to cytopenia-associated symptoms including anemia, hemorrhage and infection. Thrombocytopenia is a major and sometimes fatal complication in patients with acute leukemia. However, the mechanisms underlying defective thrombopoiesis in leukemia have not been fully elucidated. In the steady state, platelets are continuously produced by megakaryocytes. Using an MLL-AF9-induced acute myeloid leukemia mouse model, we demonstrated a preserved number and proportion of megakaryocyte-primed hematopoietic stem cell subsets, but weakened megakaryocytic differentiation via both canonical and non-canonical routes. This primarily accounted for the dramatic reduction of megakaryocytic progenitors observed in acute myeloid leukemia bone marrow and a severe disruption of the maturation of megakaryocytes. Additionally, we discovered overproduction of interleukin-4 from bone marrow endothelial cells in acute myeloid leukemia and observed inhibitory effects of interleukin-4 throughout the process of megakaryopoiesis in vivo. Furthermore, we observed that inhibition of interleukin-4 in combination with induction chemotherapy not only promoted recovery of platelet counts, but also prolonged the duration of remission in our acute myeloid leukemia mouse model. Our study elucidates a new link between interleukin-4 signaling and defective megakaryopoiesis in acute myeloid leukemia bone marrow, thereby offering a potential therapeutic target in acute myeloid leukemia.

Abstract P4-06-24: Microenvironment heterogeneity of triple-negative breast cancer reveals distinct immune escape mechanisms and potential driver events

Abstract Background The microenvironment phenotypes strongly affect the immunotherapeutic strategies for triple-negative breast cancer (TNBC). Although the multi-omics profile of TNBC has been comprehensively characterized, few studies have focused on the microenvironment phenotypes of TNBC. Methods With multi-omics data for the largest single-center TNBC cohort (n=386), we first established a TNBC-specific microenvironment cell signature. We further used single sample gene set enrichment analysis to calculate the relative number of microenvironment cell subsets in each sample. Then, we performed k-means clustering to classify the TNBC microenvironment phenotypes into heterogeneous clusters. Furthermore, we systematically analyzed the extrinsic and intrinsic immune escape mechanisms of different TNBC microenvironment clusters. In addition, we explored genomic alterations that might decrease immune infiltration in certain TNBC microenvironment clusters. Results We classified the TNBC microenvironment phenotypes into three heterogeneous clusters. Cluster 1 (type 1 “cold tumor”) had low microenvironment cells infiltration. Cluster 2 (type 2 “cold tumor”) was characterized by resting innate immune cells, fibroblasts and endothelial cells infiltration. Cluster 3 (“hot tumor”) was featured by adaptive immune cells infiltration. Analysis of immune escape mechanism revealed that an incapability to attract innate immune cells (resulting in failure of adaptive immunity) led to immune escape of cluster 1. The chemotaxis but inactivation of innate immunity (also leading to failure of adaptive immunity) and low tumor antigen burden resulted in immune escape of cluster 2. High expression of immune checkpoint molecules contributed to immune escape of cluster 3. In addition, we found that tumor infiltrating lymphocytes (TILs) were positively correlated with immune checkpoint molecules expression, while mutation load was negatively correlated with those indicators in triple-negative breast cancer. Analysis of enrichment pathways, mutations and somatic copy number variations between the “cold tumor” and “hot tumor” clusters revealed that amplification of MYC and activation of MYC-related pathways might decrease the immune infiltration of cluster 1. Mutations in PI3K-AKT pathway members and activation of fibroblasts-related pathways might decrease the immune infiltration of cluster 2. Conclusion Utilizing the largest single-center TNBC cohort with multi-omics data, our study first revealed the heterogeneity of the TNBC microenvironment, with translational significance both clinically and biologically. First, we identified a subtype of “hot tumor” in TNBC (cluster 3), for which immune checkpoint blockers (ICBs) might be effective. TILs and immune checkpoint molecules expression but not mutation load might predict the efficacy of ICBs. Second, we presumed some genomic alterations that might drive “cold tumor” formation in TNBC. Our study represents a step toward personalized immunotherapy for TNBC patients. Key Words triple-negative breast cancer, multi-omics, microenvironment heterogeneity, immune escape Citation Format: Xiao Y, Ma D, Zhao S, Jiang Y-Z, Shao Z-M. Microenvironment heterogeneity of triple-negative breast cancer reveals distinct immune escape mechanisms and potential driver events [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-24.