Peripheral Blood Subpopulations Research Articles

Relationship of thymic area with clinical-epidemiological variables and values of T-lymphocyte subpopulations in peripheral blood of children with recurrent infections

BackgroundRecurrent infections in childhood are the main cause of remission to the immunology service. T lymphocytes generated in the thymus are essential for fighting infection, making the thymus area an important predictor of the immune system’s competence. This study aimed to identify the possible relationship of the thymic area with clinical-epidemiological variables and values of subpopulations of T lymphocytes in the peripheral blood of children with recurrent infections.MethodsWe conducted applied research using a transversal analytical design at the National Medical Genetics Center (Havana, Cuba), from January to August 2022. The study covered 73 children of which we analyzed clinical-epidemiological variables and the size of the thymus through ultrasound. Furthermore, we determined the relative and absolute values of the subpopulations of T cells using flow cytometry.ResultsOf the children studied, 65.8% had thymic hypoplasia. The children who breastfed for less than 6 months showed four times the risk of developing moderate-severe thymus hypoplasia (OR = 3.90, 95% CI: 1.21–12.61). A direct relationship was found between the area of the thymus and the child’s size (r = 0.238, p = 0.043) and weight (r = 0.233, p = 0.047). The relative values of CD3+ T lymphocytes decreased in the cases of mild hypoplasia (p = 0.018) and moderate-severe hypoplasia (p = 0.049). The thymus area was associated with the absolute cell count of CD8+ effector memory T cells (rs = −0.263, p = 0.024) and of the central memory T cells (r = −0.283, p = 0.015).ConclusionsBreastfeeding for less than 6 months, as well as the weight and size of the child, are related to their thymus area. The subpopulation values of T lymphocytes detected suggest that patients with thymic hypoplasia develop a contraction of CD3+ T cells, which can make them more vulnerable to infectious processes. This finding was combined with an expansion of the memory compartments of the subpopulations of CD8+ T cells, suggesting a greater susceptibility to intracellular viral and bacterial infections in these cases.

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Different Natural Killer Cell Subpopulations in Peripheral Blood during Pregnancy

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Different Natural Killer Cell Subpopulations in Peripheral Blood during Pregnancy

P127 Sphingosine-1-Phosphate Receptor Blockade with Etrasimod alters Lymphocyte Trafficking in Crohn's Disease: Insights from High-Dimensional Phenotypic Mapping

Abstract Background The blockade of lymphocyte trafficking from the lymph nodes (LNs) towards the intestinal mucosa is an effective approach for the treatment of ulcerative colitis (UC). Etrasimod is an oral, once-daily (QD), selective sphingosine 1-phosphate (S1P)1,4,5 receptor (S1PR) modulator, which is currently also under investigation for Crohn’s disease (CD). The therapeutic mechanism of action of S1PR modulators in the context of inflammatory bowel disease (IBD) is not completely understood. It is hypothesized that both B and T- cell populations are affected by this treatment and are retained in the peripheral LNs, reducing circulating lymphocytes and resulting in fewer immune cells available to traffic in the gastrointestinal tract. Etrasimod has been reported to induce a partial and selective reduction of lymphocyte subsets in the blood of healthy subjects, but the direct effect of S1PR modulators on LN leukocytes has not been evaluated. The aim of this study was to investigate changes in leukocyte subpopulations in peripheral LNs and peripheral blood (PB) in response to S1PR inhibition with etrasimod in patients with CD. Methods CD patients with moderate to severe disease activity, participating in the Phase 2, double-blinded, non-placebo-controlled portion of the CULTIVATE trial, were eligible for this sub-study. At baseline and after 14 weeks of etrasimod induction treatment (2 or 3 mg/day), 10cc of PB and 4 ultrasound guided biopsy samples of an inguinal LN were obtained. The isolated peripheral blood mononuclear cells (PBMCs) and LN leukocyte populations, were analyzed at single cell level via high-dimensional immunophenotyping through mass cytometry (CyTOF) at both timepoints. Results We observed that in the LNs, the numbers/percentages of naïve, central and effector memory T-helper cells, as well as, of CD8+ naïve T-cells were increased after treatment with etrasimod. There was a decrease in the proportion of these cell populations in peripheral blood. Circulating naïve and memory B-cells were reduced, while these subsets where also slightly reduced in the LN compartment. Innate immune cell populations were not significantly altered. Conclusion Etrasimod induction treatment resulted in an increase of both naïve and central memory T-lymphocyte subsets in inguinal LNs, with a corresponding decrease in the periphery. The effect on the frequency of B-cells was not consistent in the LNs and PB, while innate immunotypes and T-regs, were not significantly affected. Despite the limited sample size, these selective effects were consistent with previously published PB immunophenotyping studies with etrasimod. This study was conducted as a collaboration between Amsterdam UMC and Pfizer.

JAK2 V617F Impairs Lymphoid Differentiation in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) arise from the clonal acquisition of a driver mutation in a hematopoietic stem cell (HSC), often decades before the emergence of clinical disease. A defining characteristic of an HSC is its potential to give rise to both myeloid and lymphoid hematopoietic lineages. Yet the most common MPN driver mutation, JAK2 V617F, has only rarely been observed in lymphocytes. We conducted this study to determine why the JAK2 V617F clone does not contribute to mature lymphocytes. We directly measured myeloid and lymphoid lineage contribution of normal and MPN HSCs in 133 patients with JAK2 V617F MPN. Peripheral blood subpopulations were fractionated via fluorescence-activated cell sorting (FACS) and JAK2 V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR. JAK2 V617F was enriched in neutrophils and maintained in erythroid progenitors (EPs) relative to HSCs. In contrast, JAK2 V617F alleles were largely absent in T and B cells relative to HSCs. Lymphopoiesis declines with age, potentially explaining the absence of JAK2 V617F in lymphocytes from older patients with MPN even if JAK2 V617F has no effect on lymphopoiesis. This “JAK2 V617F neutral” hypothesis predicts that patients who acquired the mutation at a younger age, and those who harbor the mutation for a longer duration, have a higher likelihood of accumulating JAK2mutated lymphocytes. However, we found that JAK2 V617F MAF in T and B cells across our cohort does not correlate with either patient age at diagnosis or duration of clinical disease. Thus, predictions of the “JAK2 V617F neutral” hypothesis are not supported by patient data. The alternative explanation is that JAK2 V617F impairs lymphocyte differentiation from mutated progenitors. We tested this “JAK2 V617F adverse” hypothesis by tracking JAK2 V617F during various stages of lymphopoiesis. To assess the relative bias of JAK2 V617F HSCs during the early stages of lymphoid and myeloid commitment, we measured JAK2 V617F MAF in common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs) and HSCs isolated by FACS from the peripheral blood of 79 patients with JAK2 V617F MPN. We found that while myeloid commitment from HSC to CMP was associated with an increase in mean JAK2 V617F MAF, there was no statistically significant change in mean MAF between CLPs and the parent HSCs across the cohort. Therefore, JAK2 V617F HSCs are not completely excluded from the earliest stages of lymphoid differentiation. To further define the extent to which JAK2 V617F affects lymphopoiesis, we used FACS to isolate CLPs and CMPs from 17 patients with JAK2 V617F polycythemia vera (PV) and differentiated each subpopulation in vitro into T cell progenitors (pro- and pre-T cells) and erythroblasts (EBs), respectively. We found that all CLP samples could be differentiated into T cell progenitors. However, we observed a marked depletion of JAK2 V617F alleles in progressively mature T cell progenitors (Figure 1). In contrast, JAK2 V617F alleles were further enriched in progressively mature erythroblasts. The in vitro differentiation potential of MPN progenitors thus supports the “JAK2V617F adverse” hypothesis. To determine the effect of JAK2 V617F on lymphopoiesis in vivo, we performed competitive transplantations of lethally irradiated CD45.1 mice with congenic CD45.2 donor whole bone marrow (WBM) cells harboring JAK2 V617F or wild type JAK2 (JAK2 WT). Measuring CD45.2 chimerism by FACS in lymphocytes and neutrophils from engrafted mice revealed that only CD45.2 cells harboring JAK2 V617F were preferentially depleted in lymphocytes (Figure 2). Therefore, JAK2 V617F HSCs displayed a competitive disadvantage relative to their JAK2 WT counterparts during lymphopoiesis in vivo. Myeloid proliferation in MPN driven by JAK2 V617F is well recognized but does not fully explain certain MPN complications such as increased risk of infections and second malignancies. Our findings demonstrate impaired lymphoid differentiation from JAK2 V617F stem and progenitor cells that may also contribute to clinical MPN phenotypes. Further study is ongoing to define actionable mechanisms through which JAK2 V617F interferes with T cell differentiation.

Features of Cell-Mediated Immunity in Children with Congenital Ichthyosis and Their Role in the Pathogenetic Management

Background. Congenital ichthyoses (CIs) are a heterogeneous clinical-etiological group of genodermatoses. Typical clinical symptoms of this disease, regardless of the form, are generalized erythroderma, peeling, itching, hyperkeratosis, severe structural and functional disorders of the epidermal barrier, other organs and systems. Patients have an extremely low quality of life due to changes in appearance, discomfort, constant disease symptoms. Thus far, there are no effective treatment methods for ichthyosis. That is why scientific search for new therapies is the topical issue in pediatrics and pediatric dermatology.Objective. The aim of the study is to examine the cell-mediated immunity state in patients with CI via assessment of the pattern of lymphocyte subpopulations in peripheral blood. The research was conducted to study the content of the main and small lymphocyte subpopulations in 86 patients with established diagnosis of CI aged from 1 month to 18 years. The diagnosis was made according to the clinical data and the results of molecular genetic testing. Comparative analysis of blood immunological indicators in children with CI and in patients with other immunemediated chronic dermatoses: atopic dermatitis (AD; n = 68) and psoriasis vulgaris (n = 55).Methods. The level of T lymphocytes, T helpers (Th), cytotoxic T lymphocytes (Tc), B lymphocytes, NK cells, Treg-cells (Treg), activated T helpers (Thact), Th17 lymphocytes in peripheral blood was evaluated via flow cytometry using monoclonal antibodies. Statistical analysis was performed via Statistica 10.0. Differences between the groups were assessed via Mann-Whitney non-parametric test, differences were considered significant at p < 0.05.Results. A significant increase of activated T-helpers level in peripheral blood was revealed in patients with CI and psoriasis compared to children with AD (p < 0.001), as well as an increased levels of B-lymphocytes and Treg in children with CI (p < 0.05).Conclusion. Children with CI have shown some features of cell-mediated immunity such as: pathological activation of Th lymphocytes, impaired terminal differentiation of naive CD4+ cells to Thact, Treg, Th17 lymphocytes and their proliferation. Comparative analysis of mentioned immunological indicators in children with CI, psoriasis and AD has shown comparable results of increased Thact lymphocytes levels in patients in CI and psoriasis groups. This results open up potential of using immunobiological drugs of psoriasis target therapy within the new management strategy for children with CI.

Prediction of outcome using CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocyte subpopulations in patients with complicated intra-abdominal infections.

There is still no study investigating the prognostic performance of CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocyte subpopulations in complicated intra-abdominal infections (cIAIs); therefore, we aimed to evaluate the association between monocyte subtypes and outcome in such patients. A single-center prospective study was conducted at a University Hospital Stara Zagora between November 2018 and August 2021. Preoperatively and on the 3rd postoperative day (POD), we measured the levels of CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes in peripheral blood using flow cytometry in 62 patients with cIAIs and 31 healthy controls. Nine of the 62 patients died during hospitalization. Survivors had higher pre-surgery percentages of CD14++CD16- classical monocytes and higher percentage of these cells predicted favorable outcome in ROC analysis (AUROC = 0.781, p = 0.008). The CD14++CD16+ intermediate monocyte percentages were higher in non-survivors both pre- and postoperatively but only the higher preoperative values predicted a lethal outcome (AUROC = 0.722, p = 0.035). For CD14+CD16++ non-classical monocytes, non-survivors had lower percentages on day 3 post-surgery and low percentage was predictive of lethal outcome (AUROC = 0.752, p = 0.046). Perioperative levels of monocyte subpopulations in peripheral blood show a great potential for prognostication of outcome in patients with cIAIs.

Effects of body mass index on the immune response within the first days after major stroke in humans

IntroductionImmunological alterations associated with increased susceptibility to infection are an essential aspect of stroke pathophysiology. Several immunological functions of adipose tissue are altered by obesity and are accompanied by chronic immune activation. The purpose of this study was to examine immune function (monocytes, granulocytes, cytokines) as a function of body mass index (BMI: 1st group: 25; 2nd group: 25 BMI 30; 3rd group: 30) and changes in body weight post stroke.MethodFat status was assessed using standardized weight measurements on days 1, 2, 3, 4, 5, and 7 after ischemic stroke in a cohort of 40 stroke patients and 16 control patients. Liver fat and visceral fat were assessed by MRI on day 1 or 2 [I] and on day 5 or 7 [II]. Leukocyte subpopulations in peripheral blood, cytokines, chemokines, and adipokine concentrations in sera were quantified. In a second cohort (stroke and control group, n = 17), multiple regression analysis was used to identify correlations between BMI and monocyte and granulocyte subpopulations.ResultsWeight and fat loss occurred from the day of admission to day 1 after stroke without further reduction in the postischemic course. No significant changes in liver or visceral fat were observed between MRI I and MRI II. BMI was inversely associated with IL-6 levels, while proinflammatory cytokines such as eotaxin, IFN-β, IFN -γ and TNF-α were upregulated when BMI increased. The numbers of anti-inflammatory CD14+CD16+ monocytes and CD16+CD62L− granulocytes were reduced in patients with higher BMI values, while that of proinflammatory CD16dimCD62L+ granulocytes was increased.ConclusionA small weight loss in stroke patients was detectable. The data demonstrate a positive correlation between BMI and a proinflammatory poststroke immune response. This provides a potential link to how obesity may affect the clinical outcome of stroke patients.

Immune cell subpopulations and serum neurofilament light chain are associated with increased risk of disease worsening in multiple sclerosis

Changes is lymphocyte subpopulations in peripheral blood have been proposed as biomarkers for evaluation of disease activity in multiple sclerosis (MS). Serum neurofilament light chain (sNfL) is a biomarker reflecting neuro-axonal injury in MS that could be used to monitor disease activity, response to drugs and to prognosticate disease course. Here we show a moderate correlation between sNfL and lymphocyte cell subpopulations, and our data furthermore suggest that sNfL and specific immune cell subpopulations together could predict future disease worsening in MS.

Influence of health resort treatment on the level of systemic inflammation in patients after new coronavirus infection

Background. Low-grade inflammation is currently one of the main risk factors for the development of adverse events in the cardiovascular system, including death from cardiovascular diseases or their complications that cause mortality in the postCOVID period.The aim of the study. To assess the impact of health resort treatment in the conditions of the Southern Coast of Crimea on clinical and functional parameters, as well as on the level of systemic inflammation in patients after a new coronavirus infection.Materials and methods. The study included 67 patients (54.9 ± 9.05 years) in the post-COVID period who underwent health resort treatment at the I.M. Sechenov Academic Research Institute of Physical Treatment Methods, Medical Climatology and Rehabilitation. Methods of health resort treatment included climatic treatment on the Southern Coast of Crimea, therapeutic breathing exercises, terrainkur, and various methods of respiratory therapy. In all patients, the study of the C-reactive protein (CRP) in peripheral blood upon admission and at discharge was carried out.Results. Despite a statistically significant improvement in most clinical and functional parameters, the level of CRP and the number of leukocytes and their subpopulations in peripheral blood in patients who underwent health resort treatment did not differ significantly (p > 0.05) from the baseline values obtained upon admission at the I.M. Sechenov Academic Research Institute of Physical Treatment Methods, Medical Climatology and Rehabilitation. The CRP index upon admission and at discharge corresponded to the lower limit of the interval specific for low-grade inflammation (from 3 to 10 mg/l).Conclusion. Our results indicate the lack of effectiveness of the presented plan of health resort treatment in the correction of low-intensity inflammation, as well as the necessity for deeper scientific research in the direction of studying the mechanisms of low-grade inflammation development and the methods of its management.

The link between lymphocyte subpopulations in peripheral blood and metabolic variables in patients with severe obesity.

Obesity, a public health problem, is a state of metainflammation that influences the development of chronic degenerative diseases, particularly in patients with severe obesity. The objective of this study was to evidence immunometabolic differences in patients with different degrees of obesity, including severe obesity, by determining correlations between lymphocyte subpopulations and metabolic, body composition, and clinical variables. Peripheral blood immune cells (CD4+, CD8+ memory and effector T lymphocytes) were analyzed, and measures of body composition, blood pressure, and biochemical composition (glucose, glycated hemoglobin (HbA1c), insulin, C-reactive protein (CRP), and the lipid profile) were carried out in patients with different degrees of obesity. The patients were classified according to total body fat (TBF) percentage as normal body fat, class 1 and 2 obesity, class 3 obesity, and class 4 obesity. The greater the TBF percentage, the more pronounced the differences in body composition (such as a decrease in the fat-free mass (FFM) that is defined as sarcopenic obesity) and the immunometabolic profile. There was an increase of CD3+ T lymphocytes (mainly CD4+, CD4+CD62-, and CD8+CD45RO+ T lymphocytes) and an increase in the TBF percentage (severity of obesity). The correlations between lymphocyte subpopulations and metabolic, body composition, and clinical variables demonstrated the existence of a chronic, low-intensity inflammatory process in obesity. Therefore, measuring the immunometabolic profile by means of lymphocyte subpopulations in patients with severe obesity could be useful to determine the severity of the disease and the increased risk of presenting obesity-associated chronic degenerative diseases.

SUBPOPULATIONS OF LYMPHOCYTES AND MONOCYTES IN PATIENTS` VENOUS BLOOD WITH ATRIAL FIBRILLATION OR ATRIAL FLUTTER ASSOCIATED WITH HYPERTENSION

Objective: The most common reason of arrhythmia onset nowadays is hypertensive desease. The aim of the study is to compare the subpopulations of lymphocytes (LCTs) and monocytes (MCTs) in blood of patients (pts) with paroxysmal and persistent forms of AF or atrial flutter (AFL) associated with arterial hypertension. Design and method: The study involved 103 patients with atrial fibrillation and flutter that occurred on the backdround of hypertension. Depending on the form of arrhythmia, patients were divided into three main groups: group I – with paroxysmal form of atrial fibrillation, group II – with a persistent form of atrial fibrillation, group III – with a persistent form of atrial flutter. The control groups included patients with hypertension, but without these arrhythmias and healthy individuals who entered groups IV and V, respectively. The lymphocytes and monocytes subpopulation in peripheral blood was assessed by flow cytometry. Results: Analyzing the lymphocyte subpopulations in peripheral blood of patients with atrial fibrillation and flutter (groups I, II and III), it was found that the number of cells with cytotoxic activity (NK and NKT) in both absolute count and percentage values was significantly higher than in healthy individuals. A statistically significant decrease of T-regulatory cells number was found in patients with arrhythmias compared to control groups (p < 0.05). In patients with AF and AFL associated with hypertension, compared to patients with hypertension without these rhythm disturbances or healthy individuals, there is an increased number of classical and intermediate monocytes subpopulations. Conclusions: 1. In patients with atrial fibrillation or atrial flutter associated with hypertension, compared to patients with hypertension without these rhythm disturbances or healthy people, there is an increased number of classical and intermediate monocytes fraction and a decreased content of T-regulatory cells in peripheral blood, an increased number of cells with cytotoxic activity (NK and NKT) compared to healthy individuals. These changes can be one of the factors for the activation of systemic and local inflammatory processes that can lead to fibrosis in the myocardium and the associated electrical and structural remodeling typical for AF and AFL.

Dynamics of T helper subpopulations in the critical period of severe injury in children

Severe mechanical injury is one of the main reasons behind children’s disability and mortality. Severe injury induces a complex host immune response to tissue injury, a parallel pro- and anti-inflammatory state, bearing an elevated risk for infectious complications (IC) and/or multiple organ failure (MOF). This study aimed to determine the informative immunological criteria of traumatic injury severity and prognosis outcome in children (severe injury group (SInj, ISS ≥ 16), n = 87; mild/moderate injury group (MInj, ISS < 16), n = 34) based on the assessment of absolute cell count (abs) and percentage of such T helper subpopulations as regulatory T lymphocytes – CD4+CD127lowCD25high(Treg), Th17 lymphocytes – CD4+CD161+ and CD4+CD127higtCD25high T cells(T127hi) in severe injury cases grouped by the outcome (favorable, n = 47; unfavorable, n = 40) and depending on IC (n = 16) and the development of MOF (n = 11) on the 1st, 3d , 5th, 7th, 14th day after injury. The control group was comprised of 80 apparently healthy children comparable in age and sex. An inverse relationship between severity of injury, degree of blood loss and outcome of injury was revealed with the abs of all Th populations, but for Th abs and Treg abs the most significant correlation was found (Spearman’s R ≤ -0,70, p < 0.00001). For SInj group, a pronounced decrease of Th abs, Treg abs, T127hi abs and Th17 abs, in the acute post-traumatic period with an increase to 14 days was revealed. The values of in the first day for indicators of patients with MInj group correspond to the values of control group and significantly differ from SInj group. There are different kinetics of percentage Th subpopulations in peripheral blood of children with severe injuries. The Th17%CD4+ and T127hi%CD4+ significant increase in 1st-3d and 3d -7th days after injury respectively in comparation with сontrol and MInj groups. There were no differences between groups in terms of Treg%CD4+. The lower-level Treg abs in trauma patients admitted to the ICU is significantly associated with develop the infectious complications and outcome of trauma. The Th17 abs is significantly reduced in 3-7th days after the injury in the SInj group with MOF. The results of the study indicate that in children levels of Treg, T127hi and Th17 is significantly associated with severity of injury and may be used to predict outcome of trauma and assess the risk of IC and MOF.

Galectin-9 influences the Tim-3 molecule expression in natural killer different subpopulations

Galectin-9 is a b-galactoside binding lectin with expressed immunoregulatory activity. During pregnancy galectin-9 is produced by trophoblast cells and regulates the function of natural killer (NK) cells at the maternal-fetal interface via binding to Tim-3 (T-cell Ig and mucin domain-containing protein 3) molecules. Natural killer (NK) lymphocytes belong to the innate lymphoid cells, which have a cytotoxic effect on target cells and are capable of producing a large number of regulatory factors (cytokines, chemokines). Decidual NK have a tolerant phenotype and play a leading role in the regulation of invasive trophoblast growth and provide peripheral immune tolerance in the area of uteroplacental contact. Peripheral NK cells express Tim-3 molecules. Galectin-9 concentration is increased in peripheral blood during physiologic pregnancy. At pregnancy phenotype and functions of peripheral NK cells are changed to maintain the maternal–fetal immune tolerance. Peripheral NK cells migrate to the maternal-fetal interface and are transformed into a decidual NK-like phenotype cells. Galectin-9 concentration is decreased in women with a complicated pregnancy and miscarriage. However the galectin-9 effects on different NK cell subpopulations of peripheral blood are not investigated. Therefore, we studied the galectin-9 influence on phenotype transformation and Tim-3 expression of NK cells isolated from peripheral blood of healthy non-pregnant fertile women. CD56+NK cells were obtained by immunomagnetic separation and cultivated in vitro during 72 hours with cytokines (IL-2 and IL-15). Galectin-9 (5 ng/mL) and anti-Tim-3 (10 mg) antibodies were added to the NK cultures. Galectin-9 concentration is corresponded to its level during first trimester of physiologic pregnancy. The number of regulatory NK (CD16-CD56bright), cytotoxic NK (CD16+CD56dim/-) cells and Tim-3 expression on different NK subpopulations were assessed by flow cytometry. It was found that Tim-3 was expressed on all subpopulations of peripheral blood NK cells (CD16-CD56brightNK, CD16+CD56dimNK, CD16+CD56-NK). Incubation with galectin-9 increased the expression of Tim-3 on regulatory CD16-CD56brightNK cells and did not change on cytotoxic CD16+CD56dim/-NK cells. Galectin-9 reduced the percentage of cytotoxic CD16+CD56dimNK in culture, but did not influence the number of regulatory CD16-CD56bright NK and cytotoxic CD16+CD56-NK cells. Thus, galectin-9 regulates Tim-3 molecule expression and NK cell subpopulation distributions in vitro culture.

Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing.

Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirus-specific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor β-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed invivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific Tcells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific Tcells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter invivo and even persist in the absence of early viral reactivation.

Immune-Activated B Cells Are Dominant in Prostate Cancer.

B cells are multifaceted immune cells responding robustly during immune surveillance against tumor antigens by presentation to T cells and switched immunoglobulin production. However, B cells are unstudied in prostate cancer (PCa). We used flow cytometry to analyze B-cell subpopulations in peripheral blood and lymph nodes from intermediate-high risk PCa patients. B-cell subpopulations were related to clinicopathological factors. B-cell-receptor single-cell sequencing and VDJ analysis identified clonal B-cell expansion in blood and lymph nodes. Pathological staging was pT2 in 16%, pT3a in 48%, and pT3b in 36%. Lymph node metastases occurred in 5/25 patients (20%). Compared to healthy donors, the peripheral blood CD19+ B-cell compartment was significantly decreased in PCa patients and dominated by naïve B cells. The nodal B-cell compartment had significantly increased fractions of CD19+ B cells and switched memory B cells. Plasmablasts were observed in tumor-draining sentinel lymph nodes (SNs). VDJ analysis revealed clonal expansion in lymph nodes. Thus, activated B cells are increased in SNs from PCa patients. The increased fraction of switched memory cells and plasmablasts together with the presence of clonally expanded B cells indicate tumor-specific T-cell-dependent responses from B cells, supporting an important role for B cells in the protection against tumors.

Flow Cytometric Identification and Enumeration of Monocyte Subsets in Bovine and Water Buffalo Peripheral Blood.

Monocytes are innate immune system key players with pivotal roles during infection and inflammation. They migrate into tissues and differentiate into myeloid effect cells (macrophages, dendritic cells) which orchestrate inflammatory processes and are interfaces between the innate and adaptive immune responses. Their clinical relevance to health and disease of cattle (Bos taurus) and water buffalo (Bubalus bubalis), two of the most important livestock species, has been highlighted in physiologic (pregnancy) and pathologic (mastitis, metritis, and viral infections) conditions. The existence of three different monocyte subsets in cattle was established by flow cytometry (FC), as follows: classical (cM; CD14++ CD16-/low ), intermediate (intM; CD14++/+ CD16+ ), and non-classical (ncM; CD14-/low CD16++ ) monocytes. FC applications for studying the immune system of cattle and water buffalo still have significant limitations. In this article, we describe some practical approaches to overcome these limitations and, in particular, allow the identification and enumeration of cM, intM, and ncM subpopulations in cattle and buffalo peripheral blood. Indeed, we propose the new procedure lyse/wash/no-centrifugation (L/W/NC) that can be combined with the FC absolute counting procedures and can overcome specific issues of the lyse/no-wash protocols (L/NW). Finally, for the first time, we demonstrated the existence of cM, intM, and ncM monocyte subsets also in the water buffalo, showing some interesting differences with cattle, such as the bubaline cM are mainly CD14+/++ /CD16+ . These subtle differences may influence inflammatory disease regulation in, for example, mastitis and metritis. The upregulation of CD16 expression on cM may reveal different monocyte priming, leading to different functional features of macrophages/dendritic cells in tissues after infection. © 2023 Wiley Periodicals LLC. Basic Protocol: Absolute count of cM, intM, and ncM without compensation Alternate Protocol: Absolute count of cM, intM, and ncM for single laser platform Support Protocol 1: In-house monoclonal antibody labeling using a Pacific Blue™ kit Support Protocol 2: In-house monoclonal antibody labeling using an Alexa Fluor® 647 kit Support Protocol 3: Titration of fluorochrome-conjugated antibodies.

Gas Chromatography-Mass Spectrometry Technology: Application in the Study of Inflammatory Mechanism in COVID-19 Patients.

SARS-CoV-2 infection in the human body induces a severe storm of inflammatory factors. However, its specific mechanism is still not clear. Gas chromatography–mass spectrometry (GC–MS) technology is expected to explain the possible mechanism of the disease by detecting differential metabolites. 15 COVID-19 patients and healthy controls were included in this study. Immune indicators such as hs CRP and cytokines were detected to reflect the level of inflammation in patients with COVID-19. The distribution of lymphocytes and subpopulations in peripheral whole blood were detected using flow cytometry to assess the immune function of COVID-19 patients. The expression of differential metabolites in serum was analyzed using GC–MS non-targeted metabolomics. The results showed that hs CRP, IL-5/6/8/10 and IFN-α in the serum of COVID-19 patients increased to varying degrees, and CD3/4/8+ T lymphocytes decreased. Additionally, 53 metabolites in the serum of COVID-19 patients were up regulated, 18 metabolites were down regulated, and 8 metabolites remained unchanged. Increased Cholesterol, Lactic Acid and 1-Monopalmitin may be the mechanism that causes excessive inflammation in COVID-19 patients. The increase of D-Allose may be involved in the process of lymphocyte decrease. In conclusion, the significance of our study is to reveal the possible mechanism of inflammatory response in patients with COVID-19 from the perspective of metabolomics. This provided a new idea for the treatment of COVID-19.

Peripheral blood subpopulations of Bregs producing IL-35 in women with endometriosis.

Interleukin 35 (IL-35) is involved in the pathogenesis of endometriosis by suppressing immunoreaction and promoting endometrial cell proliferation. It may also be an essential cytokine in forming the immunosuppressive functions of regulatory B lymphocytes (Bregs). The involvement of Bregs in the pathogenesis of endometriosis has not been previously investigated. In this study, we determined the frequencies of different Breg subpopulations, namely, B10, immature B-cells, and plasmablasts, and their abilities to produce IL-35 in women with endometriosis compared to healthy women. The frequencies of different subpopulations of Bregs producing IL-35 were measured in the peripheral blood of women with endometriosis (total pool), women with deep infiltration endometriosis (DIE), women with ovarian endometriosis, and healthy women as a control by flow cytometry. We observed a decrease in the percentage of B10 cells and plasmablasts in women with endometriosis and an increase in the percentage of these Breg populations producing IL-35 in the same experimental group. Interestingly, we also revealed that women with DIE had increased percentages of B10 cells and plasmablasts producing IL-35. Taken together, our findings are the first to reveal the frequencies of different subpopulations of Bregs producing IL-35 in women with endometriosis. The results suggest that IL-35 expression in B lymphocytes could be used as a peripheral marker of endometriosis; however, further studies are needed.

Regulatory T cell profiles in patients with N-methyl-ᴅ-aspartate receptor-antibody encephalitis.

Purpose Regulatory T cells (Tregs) have been implicated in the pathogenesis of several autoimmune disorders and used in adoptive cell transfer therapies. Neither have been explored in patients with autoimmune encephalitis where treated patient outcomes remain suboptimal with frequent relapses. Here, to identify new treatment strategies for autoimmune encephalitis, we sought to evaluate the proportion of circulating Tregs and Treg subpopulations in peripheral blood of patients with N-methyl-ᴅ-aspartate receptor-antibody encephalitis (NMDAR-Ab-E) and compared this with healthy controls. We compared the phenotype of peripheral blood Tregs in four adult NMDAR-Ab-E patients and four age- and sex-matched healthy controls using an 11-color flow cytometry assay panel for characterization of Tregs (CD4+ CD25+ FoxP3+) cells into naïve (chemokine receptor [CCR] 7+ CD45RA+), central memory (CCR7+ CD45RA-), and effector memory (CCR7- CD45RA-) cells. We also examined and compared the expression of the CCR6 by circulating Tregs and the respective Treg subpopulations between the study groups. The proportion of circulating Tregs was similar between patients with NMDAR-Ab-E and healthy controls but the proportion of naïve Tregs was lower in NMDAR-Ab-E patients (p = 0.0026). Additionally, the frequency of circulating effector memory Tregs was higher, and the proportion of circulating effector memory Tregs expressing CCR6 was lower, in NMDAR-Ab-E patients compared with healthy controls (p = 0.0026). Altered Treg homeostasis may be a feature of patients with NMDAR-Ab-E. Future studies with larger samples are warranted to validate these findings.

Половые различия в субпопуляционном составе лимфоцитов периферической крови при язвенном колите отражают специфические особенности иммунопатогенеза воспалительных заболеваний кишечника у мужчин и женщин

Objective. To identify the features of the composition of peripheral blood lymphocyte subsets in women with ulcerative colitis (UC). Patients and methods. The study included 32 women: 21 patients with UC and 11 healthy volunteers (control group) without inflammatory bowel disease (IBD). The major subsets of T and B lymphocytes were detected by multicolor flow cytometry. Results. The analysis of peripheral blood CD3+ T cell differentiation in women with UC in comparison with healthy controls showed that the Tcyt group had a significant decrease in naive Tcyt cells with an increase in the level of Tcyt cell subsets with pronounced effector properties (EM Tcyt and TEMRA Tcyt). In the subsets of central and effector memory Th cells in women with UC compared to healthy controls, Th17.1-CM cells were significantly different among central memory (CM) cells, and Th17-EM and Th17.1-EM cells were significantly different among effector memory (EM) cells. No significant differences were found in any of the Th17 cell subsets, but there was a tendency towards decreased levels of Th17.1 cells. The analysis of the subsets of T follicular helper (Tfh) cells revealed significant changes only in the level of Tfh17/Tfh22-EM cells (Tfh17-EM cell subset). No significant differences in the B cells were revealed, except for plasmocytes with IgD–CD27++ phenotype, which were increased in UC patients. Conclusion. Changes in peripheral blood T- and B-lymphocytes in women with UC can reflect not only activation of the peripheral immune system, but also induction of immunoglobulin production by B cells activated by T follicular helper cells. For the first time, specific differences between women and men in the total pool of Th17/Th22 cells, “double-positive” Th17, Tfh2 and Tfh17/Tfh22 cells were identified. In contrast to men, no manifestations of a Th2-specific response at the cellular level were observed in women with UC. The revealed sex-related differences in the composition of lymphocyte subpopulations in peripheral blood in UC reflect specific features of the IBD immunopathogenesis in men and women and can be considered in the management of patients with IBD. Key words: inflammatory bowel disease, ulcerative colitis, T helper cell subsets, B lymphocytes, sex-related differences