Early Activation Marker Research Articles

The impact of adjuvanted and non-adjuvanted influenza vaccines on <i>in vitro</i> lymphocyte immunophenotype

Vaccination is the most effective method of influenza prophylaxis resulting in reduced frequency and severity of complications. Currently, for the prevention of influenza, inactivated split and subunit vaccines as the safest and promoting formation of protective level of strain-specific virus neutralizing antibodies are used. It is known that not all inactivated vaccines are effective enough for select human groups. While nowadays the level of public health is low, there is a need to improve the effectiveness of vaccines that should activate all chains of the immune system. In order to enhance intensity of influenza virus strain-specific antibody production, adjuvant vaccines exerting other mechanisms to activate humoral and cellular immunity compared to non-adjuvant vaccines have been used. The aim of the study was to examine lymphocyte immunophenotype in 27 healthy donors treated with polymer-subunit (immunoadjuvant) and non-adjuvanted split and subunit influenza vaccines.
 Materials and methods. Peripheral blood lymphocyte subpopulations were studied in vitro by flow cytometer FC-500 Cytomics (Beckman Coulter, USA) using FITC- and PE-labeled monoclonal antibodies (mAbs).
 Results. All examined influenza vaccines activate the effectors of cellular immunity, increasing the number of NK-cells (CD16/56), NKT-lymphocytes (CD3/CD16/56), B-lymphocytes (CD45/CD20), activated (CD3/HLA-DR) and cytotoxic (CD8/HLA-DR) T-lymphocytes, as well as cells bearing early activation marker (CD45/CD25). Among them the immunoadjuvant vaccine showed the greatest potential to induce cellular response eliciting regulatory mechanisms that prevent hyperactivation, stimulating growth of NK (CD16/56), NKT-cells (CD3/CD16/56), B-lymphocytes (CD45/CD20), activated (CD3/HLA-DR) and cytotoxic (CD8/HLA-DR) T-lymphocytes, T-regulatory cells (Tregs, CD4/CD25/Foxp3).
 Conclusion. Vaccination against influenza besides the formation of specific antibodies render a transient, immunomodulating effect that is more noticeable after immunoadjuvant vaccine. It can be assumed that vaccination of people with dysfunctions of the immune system an additional prophylactic effect will be observed.

P-352 Evaluation of CD25 and CD69 activation markers expression on peripheral blood cells subpopulations in endometriosis patients

Abstract Study question We aim to determine the percentages of CD25-positive and CD69-positive lymphocytes in peripheral blood of endometriosis (EMS) patients. Summary answer Higher percentages of lymphocytes expressing CD69 and CD25 markers in EMS and their correlation with the severity of the disease, indicate persistent activation of lymphocytes What is known already Exposure to antigens causes activation markers to appear on the surface of lymphocytes. Among them, we can distinguish early and late activation markers. The earliest one is CD69, which is expressed upon activation by the TCR receptor. CD69 plays a role in the proliferation and survival of activated T lymphocytes. CD25 is a moderate late activation marker, considered the most important marker of cellular response activation. The significance of CD69 and CD25 expression by T cells in endometriosis has not yet been determined. Increased CD69 expression on various lymphocyte subsets in peritoneal fluid in EMS has been reported. Study design, size, duration Within this prospective study between January 2016 and August 2018 immune diagnostics of the number of CD25-positive and CD69-positive lymphocytes in peripheral blood of EMS patients (firstly diagnosed during laparoscopy and confirmed histopatologically) and non-EMS controls were performed using flow cytometry analysis. Total number of 74 subjects were included in the study. All patients signed written informed consent before their enrollment in the study. Participants/materials, setting, methods We enrolled 54 subjects with previously untreated endometriosis and 20 healthy age-matched controls. Peripheral blood was collected from control patients to assess the immunophenotype of lymphocytes and measure expression of activation markers Cd25 and CD69 on several subtypes B and T lymphocytes. Diagnosis and assessment of the stage of the EMS was established during laparoscopy using rASRM score. Differences were considered statistically significant with a p < 0.05. Main results and the role of chance Significantly higher expression of the CD25 and CD69 antigen was found in both CD3+ T, CD4+ T and CD8+ T cells as well as CD19+ B cells. In each of the studied groups, a significance level of p < 0.001 was obtained. There was a weak positive correlation between the percentage of CD4+CD25+ T cells and the stage of endometriosis (R = 0.357; p = 0.008) and negative correlations between the percentage of endometriosis and the percentage of CD3+CD69+ T cells (R = −0.554; p < 0.001), T CD4+CD69 + (R = −0.554, p < 0.001) and T CD8+CD69 + (R = −0.553, p < 0.001). The expression of activation markers, CD25 and CD69 antigens in patients suffering from endometriosis with accompanying clinical symptoms was also evaluated. n the group of patients with endometriosis accompanied by infertility or pelvic pain, no statistical differences in the expression of CD25 and CD69 antigens were observed. The only clinical condition coexisting with statistically different expression of activation markers is adhesion disease accompanying endometriosis - a statistically significantly lower percentages of T CD3+CD69 + (p = 0.002), T CD4+CD69 + (p = 0.002) and T CD8+CD69 + (p = 0.002). Limitations, reasons for caution Small sample size is an important limitation of this study. In addition, evaluating immunological analysis only in serum samples does not let us drive any conclusions on the local changes of endometriosis lesions. Wider implications of the findings Clinical interpretation of changed expression of CD69 and CD25 in EMS could be useful in evaluating mechanisms of cellular activation, peripheral tolerance and immune imbalance involved in pathogenesis of endometriosis. Further studies to understand mechanisms underlying correlation between stage of the disease and expression of CD69 should be considered. Trial registration number not applicable

Effect of circulating exosomes in patients with sepsis on T cell function

To investigate the effect of circulating exosomes (EXO) on T cell function in patients with sepsis. Plasma EXO were obtained by ultracentrifugation from 10 patients with sepsis admitted to the emergency intensive care unit of Guangdong Provincial People's Hospital Affiliated to Southern Medical University. Transmission electron microscopy observation, nanoparticle tracking analysis (NTA), and Western blotting were used to detect EXO markers to identify their characteristics. Furthermore, peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of 5 healthy volunteers, primary T cells were sorted by magnetic beads and expanded in vitro. After 24 hours of intervention with different doses (0, 1, 2.5, 5, 10 mg/L) of circulating EXO in patients with sepsis, T-cell activity was assessed using a cell counting kit-8 (CCK-8). The expression of T cell activation indicators CD69 and CD25 were observed using flow cytometry. Additional evaluations were performed on immunosuppressive indicators including the expression of programmed cell death 1 (PD-1) in CD4+ T cells and the proportion of regulatory T cell (Treg). The identification results confirmed that the successful isolation of EXO from the plasma of sepsis patients. The expression level of circulating EXO in sepsis patients was higher than that in healthy control group (mg/L: 48.78±5.14 vs. 22.18±2.25, P < 0.01). After 24 hours of intervention with 5 mg/L of plasma EXO from sepsis patients, T cells activity began to show suppression [(85.84±0.56)% vs. (100.00±0.00)%, P < 0.05]. As the dosage increased, after 24 hours of intervention with 10 mg/L of EXO, T cells activity was significantly suppressed [(72.44±2.36)% vs. (100.00±0.00)%, P < 0.01]. Compared with the healthy control group, after T cells intervention with plasma EXO from sepsis patients, the expression of early activation marker CD69 was significantly reduced [(52.87±1.29)% vs. (67.13±3.56)%, P < 0.05]. Meanwhile, there was an upregulation of PD-1 expression in T cells [(57.73±3.06)% vs. (32.07±0.22)%, P < 0.01] and an increase in the proportion of Treg [(54.67±1.19)% vs. (24.60±3.51)%, P < 0.01]. However, the expression of the late activation marker CD25 remained stable [(84.77±3.44)% vs. (85.93±2.32)%, P > 0.05]. Circulating EXO in sepsis patients induce T cell dysfunction, which may be a novel mechanism lead to immunosuppression in sepsis.

NANOSECOND PULSED ELECTRIC FIELD MODULATES IMMUNOPHENOTYPE OF LYMPHOCYTES

Introduction: Nanosecond pulsed electric fields (nsPEFs) have been shown to have anticancer effects, but little is known about the mechanisms modulating the immune landscape. According to the applied parameters (voltage, pulse duration, pulse delivery frequency, and a number of pulses), it is possible to achieve a specific effect modulating the immunophenotype of cancer cells. This study aims to identify changes in the expression profile of antigens defining lymphocyte maturity and function. Materials and Methods: We treated the Jurkat cell line with PEF. We tested different parameters of electroporation (2.5, 5, 7.5, 10, and 12.5 kV/cm, 100, 200 and 100 ns pulses, 25, 100, 250, and 500 pulses, 1, 10 and Hz). For the detection of cell permeabilization, Yo-Pro-1 and flow cytometry were employed. Cell viability was analyzed after 24h by MTT Assay. ATP levels in culture were measured using Cell-Titer-Glo® Assay after 24 hours. The expression of cell markers was determined using flow cytometry. Results: The obtained results confirmed that electroporation permeabilizes the cell membrane and modulates the immunophenotype of Jurkat cells. Moreover, we observed that the ATP release increases when higher electric fields are applied. Application of 10kHz-100Hz and 25 pulses of nsPEF induces an increase in the expression of CD154 costimulatory molecule, which regulates the immune response by priming T cells and activating immune cells. Simultaneously, we increase the expression of the early activation marker CD69, indicating the ability to activate lymphocytes. The application of the 10kHz parameter upregulated the expression of antigens involved in the recruitment of inflammatory cells (CD183), controlling homeostasis of peripheral T lymphocytes (CD127) and regulating platelet adhesion (CD61). Importantly, we thus noted there was no alteration in the expression of CD95, which can mediate the induction of apoptosis in cancer cells. On the contrary, significant CD7 subexpression was observed when 10Hz nsPEF was applied. In general, the cells have not undergone cell death with lower electric field parameters but have modified their immunophenotype. Based on our studies, we propose a mechanism in which the cells: (1) permeabilize the cell membrane, (2) increase the expression of lymphocyte-activation antigens, and (3) downregulate the expression of CD7. Conclusions: Concluding, we can state that nsPEF can be a promising approach in immunotherapy via enhancing the presentation of lymphocyte-activation antigens. Also, we proved that application of electric field treatment could modulate the expression profile of lymphocytes while avoiding cell death. Encore Abstract - previously submitted to regional or national meetings (up to < 1’000 attendees) The research was funded by: This research was founded by the Polish National Centre of Science of DAINA 2 (2020/38/L/NZ7/00342; PI: J. Kulbacka), and the Research Council of Lithuania grant (Nr. S-LL-21-4, PI: V. Novickij). Keywords: Diagnostic and Prognostic Biomarkers, Basic and Translational Science - Other No conflicts of interests pertinent to the abstract.

Indicators of the immune status in patients with chronic wounds depending on the stage of the infectious process and the structure of the wound microbiota

The results of immunophenotyping of peripheral blood leukocytes were analyzed in 105 patients with chronic wounds ( the main group , wound duration more than 3 weeks ) at various stages of the infectious process : colonization (n=39), critical colonization (n=37) and infection (n=29). The comparison group consisted of 22 patients with acute wounds ( wound duration up to 4 days ), in which microorganisms were not detected in the wounds . In the main group , changes in immunological reactivity were revealed in the form of an increase in the relative content of T- lymphocytes and a decrease in NK cells , lower indicators of early activation markers on T- helpers and T- cytotoxic lymphocytes (CD4+/CD38+/CD3+, CD8+/CD38+/CD3+, CD3+/ CD71+) and HLADR on B- lymphocytes , a high level of expression of late markers of T- cell activation (CD3+HLADR+). In the comparison group , the levels of expression of integrins (CD11a, CD11b, CD11c) on neutrophils and lymphocytes , as well as the transferrin receptor (CD71) on neutrophils , monocytes and lymphocytes were higher than in the main group . An increase in the activity of neutrophils in the form of expression of CD11a molecules was revealed with the progression of the infectious process in a chronic wound , which may be an additional diagnostic sign of the transition from the stage of colonization to the stage of critical colonization and infection . The highest values of CD11a and CD11c on neutrophils were recorded in the presence of S. aureus monocultures in chronic wounds , the lowest values were recorded when P. aeruginosa and A. baumannii monocultures were isolated from wounds .

Antioxidation protein Nrf2 modulates glutamine metabolism to regulate activation-driven expansion of CD4 +T-cells

Abstract Nutrient metabolism governs the effector functions like activation, expansion, and subset differentiation of CD4 +T-cells, the orchestrators of adaptive immunity. The levels of reactive oxygen species (ROS), the metabolic byproducts of cellular metabolism are regulated by the antioxidation pathway, which is composed of Keap1 and Nrf2 proteins. We observed that, the expression of Nrf2 is temporally regulated post-activation in CD4 +T-cells (high on day 1 and low on days 2 and 3) indicating its role in CD4 +T-cell effector functions. It is yet unknown if and how the antioxidation pathway crosstalks with cellular metabolism to shape CD4 +T-cell effector responses. Here, using mice with T-cell specific deletion of Keap1 (Keap1-KO) or Nrf2 (Nrf2-KO) we deciphered the effects of high and low levels of Nrf2 (antioxidation) levels, respectively in CD4 +T-cells. Upon activation in vitro, Keap1-KO CD4 +T-cells with constitutively high levels of Nrf2 showed higher proliferation, increased expression of early activation markers and elevated TCR-mediated signaling compared to wildtype (WT) cells. In contrast, decreased proliferation and lower TCR-mediated signaling was noted in activated Nrf2-KO CD4 +T-cells. To dissect the underlying mechanisms, we performed metabolite analyses and activation in nutrient-limiting conditions. Interestingly, activated Keap1-KO CD4 +T-cells show upregulated glutamine metabolism as measured by metabolic intermediates and rely on glutamine for optimum activation and increased expansion. Together, our findings identify a mechanism by which antioxidation protein Nrf2 metabolically reprograms CD4 +T-cells and modulates their metabolic dependencies to support their activation and expansion.

Abstract 4159: The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy

Abstract Background: We previously found upregulation of the cyclooxygenase-2/prostaglandin E2 (COX-2/PGE2) pathway in the tumor microenvironment (TME) of cancer types that respond poorly to anti-PD-1 therapy, and showed that TME-resident cytokines such as IL-1B induce COX-2 expression in human tumor and myeloid cells. To investigate a potential role for COX-2/PGE2 in anti-PD-1 resistance, we studied PGE2-mediated effects on human myeloid and T cells and their dependence on prostaglandin (EP) receptor signaling. Methods: Monocytes (Monos) and T cells were enriched from normal donor peripheral blood by negative selection. PGE2 effects were tested on Monos activated with IFN-g (PD-L1 expression; FACS) or LPS (cytokine secretion; ELISA). PGE2 effects on monocytic dendritic cells (DCs) generated with GM-CSF+IL-4 and matured with CD40L were assessed by phenotyping for costimulatory molecules (CD80, CD86) and subpopulation markers (CD14, CD16, CD83). T cells activated with anti-CD3/CD28 +/- PGE2 for 2-6 days were assessed for expression of CD25, CD69, CD107a, CD137, OX40, GITR, LAG-3, PD-1, PD-1, PD-L1 and TIM3 (FACS); proliferation markers (Ki67, CFSE; FACS); cytokine secretion (IFN-g, IL-2, IL-10, TGF-b; ELISA); and metabolic function (Seahorse assay). In some experiments, cells were pre-incubated with inhibitors of EP2 (PF-04418948), EP4 (ONO-AE3-208), or the dual inhibitor TPST-1495 (Tempest Therapeutics). Results: Myeloid cell functions were suppressed by PGE2, which reduced IFNg-induced PD-L1 expression and modulated LPS-induced cytokine secretion. Furthermore, PGE2 inhibited DC maturation, evidenced by persistent CD14 and CD16 expression and a decreased CD83+ subset. In CD4 and CD8 T cells activated with anti-CD3/CD28, PGE2 reduced expression of CD25 and CD69 (early activation markers), CD107a (cytolytic potential), Ki67 (cell proliferation), co-stimulatory molecules (CD137, OX40, GITR) and checkpoint molecules (LAG-3, PD-1, PD-L1, TIM3) in a dose-dependent manner. PGE2 also reduced the CFSE-low (proliferating) population in activated CD4, CD8 and Treg cells. Secretion of proinflammatory cytokines IFN-g and IL-2, and inhibitory cytokines IL-10 and TGF-b from activated T cells was also suppressed by PGE2. These effects were specifically reversed by chemical inhibitors of EP2 and/or EP4. PGE2 exposure at the time of T cell activation was associated with depressed mitochondrial oxidation and cellular glycolysis, suggesting global effects on T cell metabolism. Conclusion: Understanding and preventing anti-PD-1 resistance is a critical goal in oncology. Our results suggest that the COX-2/PGE2 pathway exerts immunosuppressive effects on myeloid and T cells via the EP2 and EP4 receptors. COX-2 and PD-L1 are distinct immune checkpoints, providing a rationale to test anti-PD-1 in conjunction with COX-2 pathway inhibitors such as IL-1B antagonists and highly specific EP2 and EP4 inhibitors in patients with cancer. Citation Format: Shuming Chen, Seoho Lee, Tracee L. McMiller, Isaac Morales, Preethi Sankaran, Brian Francica, Robert D. Leone, Tom W. Dubensky, Suzanne L. Topalian. The COX-2/PGE2 pathway as a mediator of resistance to anti-PD-1 therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4159.

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools.

Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05-0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

Unraveling CD69 signaling pathways, ligands and laterally associated molecules.

CD69 is an early leukocyte activation marker involved in the regulation of the immune response. Initial in vitro studies evaluated its function using monoclonal antibodies until knock-out mice were developed. Subsequently, four ligands for CD69 have been identified, namely galectin-1, S100A8/S100A9 complex, myosin light chains 9 and 12, and oxidized low-density lipoproteins. In addition, several molecules are laterally associated with and regulated by CD69, including calreticulin and two transmembrane receptors, sphingosine-1-phosphate receptor (S1P1) and the heterodimeric amino acid transporter complex SLC7A5-SLC3A2 (LAT1-CD98). Recently, CD69 engagement has been shown to induce the expression of the immunoregulatory receptor programmed cell death-1 (PD-1) in T cells. The molecular signaling induced by CD69 has been explored in different scenarios and cell types. This review provides a perspective on the molecular pathways, ligands and cellular functions known to be regulated by CD69.

Comparative evaluation of effect of various means of basic and genetically engineered biological therapy on clinical course, rate of development of osteodestructive changes, quality of life and state of stress resistance in patients with rheumatoid arthritis (open observational study)

Objective. To study the effect of various basic and genetic engineering biological therapies (infliximab, rituximab, tocilizumab) used as first, second- and third-line drugs in combination with methotrexate on clinical course, quality of life (QOL), and the evolution of articular erosions and synovitis in patients with rheumatoid arthritis (RA) during 12 months of follow-up.Material and methods. This paper is an open observational study of the efficacy and safety of various basic and genetic engineering biological therapy agents (bDMARD) in patients with early active RA who are in the Mechnikov North-West State Medical University registry. Included are 151 patients with early RA (eRA), the average age is 58.2 ± 5.5 years, the disease duration is 6.5 ± 0.3 months, DAS 28-CRP – 4.48 ± 0.87. At the first stage, basic therapy was administered to patients with eRA by random sampling: sulfasalazine (CC) 2 g per day (group I – 55 patients), methotrexate (MT) – 20 mg per week (group II – 55 patients), or leflunomide (LF) 20 mg per day (group III – 41 patients). The follow-up was 12 months. At the second stage, 101 patients with persisting moderate (DAS 28: 3.2–5.1) and high levels of RA activity (DAS 28 &gt; 5.1) despite ongoing therapy with DMARDs were assigned MT at a dose of 25 mg per week (subgroup 1–25 patients), or MT – 20 mg per week in combination with infliximab (INF) – 3 mg/kg (subgroup of 2–25 patients), or – MT 20 mg per week and rituximab (RM) – two infusions of 1000 mg at the week 0 and 2 and then at the week 52nd and 54th with a duration of observation of 12 months. In the third stage, 20 patients (16 women and 4 men aged 28 to 67 years) with a high disease activity DAS 28-CRP despite previous therapy with MT and INF or RM were transferred to therapy with an IL6 – tocilizumab receptor blocker as a second-line or third-line drug. The duration of observation ranged from 6 months to five years.Results. By 12 months of treating DMARDs, clinical remission was achieved more often in the MT group than in the LF group (43.2 and 32.4 %) (p &lt; 0.05), and was accompanied by a decrease in the expression of markers of early activation of T-lymphocytes, IL-1β, IL-2 and IL-4. In the SS group, clinical remission was not observed, while there was a significant increase in the median of the total Sharpe score and erosion score relative to the baseline. The prescription of INF or PM as the first bDMARD after 12 months led to a significant increase in the physical and psychological components of the patients' QL and positive dynamics of the HAQ index compared to baseline values. The use of tocilizumab as a second-line and third-line drug in patients with active RA and the ineffectiveness of previous therapy for DMARD and bDMARD contributed to a significant decrease in DAS 28-CRP by as early as 24 weeks of follow-up. Cases of serious adverse reactions (AE) are not marked.Conclusions. There were no statistically significant differences in the dynamics of CRP, ESR, circulating immune complex and X-ray progression indicators between the groups of patients who received biological therapy of INF and RM, which confirms the possibility of ‘switching’ therapy from the first bDMARD to the drugs of subsequent lines with an increase in the clinical activity of RA. Tocilizumab can be a second- and third-line drug with an ‘escape’ effect from other bDMARDs.

Parameters of spontaneous and benzene-induced cell death of spermatozoa under in vitro conditions in male employees of an oil production enterprise with impaired fertility

Introduction. Every year there is a steady increase in the frequency of male infertility both in Russia and in other countries of the world with the concomitant low efficiency of recognizing the causes of its idiopathic forms. The explanation for the increase in infertility are: harmful production factors, including chemical ones, progressive deterioration of the quality of the urban environment, which has a significant impact on the reproductive health of the urbanized population, recurring economic crises that negatively affect the human psyche, mass consumption of substandard products and medicines. These factors, primarily chemical production, negatively affect the reproductive system, including the differentiation of male germ cells, as a cause of fertility disorders. The aim of the study to consider in vitro spontaneous and benzene-induced features of sperm apoptosis in male oil industry workers with impaired fertility. Materials and methods. We have conducted an experimental study of biological material (ejaculate) in 50 men with reproductive disorders (26-49 years old), employees of an oil production enterprise. To conduct the experiment in vitro, the researchers used seminal fluid samples. A benzene solution at a concentration of 0.001 micrograms/ml was used as an in vitro induction factor (selected experimentally). We divided the analyzed samples into 2 equal parts to study the spontaneous and benzene-induced expression of apoptosis markers (CD25+, CD95+, bax, p53, Annexin V-FITC and Propidium Iodide, Caspasa-3), which the researchers determined by flow cytometry. Results. There were no significant deviations of intracellular markers of sperm apoptosis (bax, p53, Annexin V-FITC+7AAD+, Annexin V-FITC+7AAD-, Caspasa-3), however, experts observed differences in the expression of CD95+ and CD25+ membrane proteins by 20% and 10% compared to the spontaneous level, which is associated with the acceleration of the events of programmed sperm death (p&lt;0.05). Conclusion. The results obtained in an in vitro experiment on modeling the apoptosis program by induction with hapten (benzene) showed that violations of sperm cell death are associated with overexpression of the key receptor for triggering apoptosis CD95, as well as a marker of early activation of CD25 cells, which reflects benzene modification of the cell death scenario along the path of its excessive stimulation.

Clinical and immunological features of the atopic dermatitis in children

Background: “atopic march” (AM) is the natural history of allergy mostly in children.&#x0D; The aim: to describe the production of biomarkers of allergic inflammation in dependence on the duration of AD.&#x0D; Patients and methods. 94 children suffered from atopic dermatitis (AD) have been included to the study. The severity of the disease has been estimated, serum level of total IgE, IL-4, interferon gamma and sIL-2R have been estimated.&#x0D; Results. Coexistence of AR and AD was diagnosed in 32/94 (34.0%) patients. Less severity of AD (SCORAD 32.3 ± 9.17 score vs 22.2 ± 2.29 score, p &lt; 0.05), but higher serum level of total IgE (Ме 123 [Q1-Q3 67–156] IU/ml vs Ме 53 [Q1-Q3 5–108] IU/ml, р &lt; 0.001), IL-4 (Ме 12.0 [Q1-Q3 8.7–16.1] pg/ml vs Me 6.0 [Q1-Q3 2.2–12.1] pg/ml, р = 0.0399) and sIL-2R (Ме 2.1 [Q1-Q3 0–20] IU/ml vs Me 0 [Q1-Q3 0–12.9] IU/ml, р = 0.3365) are detected in comorbid patients. Eposides of wheezing are associated with additional risk of AD (OR 2.3 [95% CI 1, 18–4,54]).&#x0D; Discussion. The development of AR in children with AD is considered as progression of natural history of allergy. In our study it was associated with higher level of IgE, IL-4 and sIL-2R. At the same time the comorbid patients had less severity of AD.&#x0D; Conclusion. The coexistence of AD and AR in 3-year-old children is associated with less severity of skin symptoms but higher frequency of sensitization to inhalant allergens than in AD. AM is accompanied by higher production of Th2-dependent parameters and markers of early activation of T-cells. The probability of the AM is higher in 2 times if there were wheezing episodes in the anamnesis.

Activation of T-helper in vitro under the Influence of Yersinia pestis Antigens in People Vaccinated against the Plague

Relevance. The search for informative markers for assessing the immunological efficacy of a live plague vaccine remains an urgent scientific task. The goal of the studyAims was to make a comparative assessment of the level helper T-cells activation by CD69 and HLA-DR markers, in people, vaccinated against plague, in an in vitro test, using the disintegrated by ultrasonic Y. pestis cells, grown at temperature 28 °C as a specific stimulator.Materials &amp; Methods. A cytofluorimetric analysis of blood samples of 45 individuals vaccinated against the plague was carried out. The dependence of the result of cytological analysis of the two studied cell markers on the immunoregulatory index (IRI) of the vaccinated donor at the time of vaccination was revealed.Results. It was found that for newly vaccinated individuals, the T-helpers were more intensive and prolonged by the early activation marker, while for those revaccinated with the immunoregulatory index, at the time of vaccination more than 1.5, the more intense cellular response was by the late activation marker.Conclusions. Thus, the possibility of quantitative evaluation of the immunological efficacy of vaccination against plague, based on the identification of lymphocyte activation markers when stimulated with a specific antigen, is confirmed.

Effect of Mcl-1 Inhibitor, AMG-176, on T Cell Quality and Function

Introduction AMG-176 belongs to a class of small molecule inhibitors known as BH3 mimetics, which mimic the role of BH3-only proteins and inhibit pro-survival Bcl-2 family proteins. AMG-176 specifically inhibits the pro-survival Mcl-1 protein. Overexpression of anti-apoptotic protein Mcl-1 has been extensively reported in hematological malignancies including mantle cell lymphomas (MCL), diffuse large B cell lymphomas (DLBCL), and multiple myeloma (MM). Chromosomal aberration of Mcl1 was identified as one of the most important survival genes for MM, follicular lymphoma (FL), MCL, and DLBCL, and is associated with shorter patient survival. These hematological malignancies are targetable by FDA-approved chimeric antigen receptor (CAR) T cell therapies with varying efficacies as single agent regimens. The role of Mcl-1 in T cell biology could be further elucidated to improve therapeutic outcomes of malignancies characterized by Mcl-1 overexpression. Here, our team explores the effect of Mcl-1 inhibitor, AMG-176, on T cell quality and function. Methods Our lab obtained healthy human donor blood from consenting adults, then isolated pan T cells using microbead negative selection (Miltenyi Biotec). Following isolation, T cells were cryopreserved in cryopreservative medium consisting of 90% fetal bovine serum (Corning) and 10% dimethyl sulfoxide (Sigma-Aldrich). Upon thaw, T cells were cultured in complete RPMI supplemented with [10ng/mL] h-IL-7 (Peprotech) and [100IU/mL] h-IL-2, and activated for 24 hours using αCD3/αCD28 coated beads (Thermofisher). At 24 hours post-activation, cells were exposed to various concentrations of AMG-176 (MedChem Express). Following 72-hour exposure, T cells were harvested and assessed for ATP production, viability, proliferation, immunophenotype, and effector function. To determine ATP production, we used a commercially available luminescence-based assay (Promega) and obtained our readout using a BioTek Synergy HTX plate reader, T cell immunophenotype was identified following 72-hour exposure to AMG-176 at 0nM, 10nM, 100nM, 500nM, and 1uM concentrations. Samples from each cohort were stained with Live/dead yellow viability dye, CD3, CD4, CD8, CD69, CD25, and CD127, then acquired using a BD LSRII flow cytometer. Flow cytometry data was analyzed using FCS Express analytical software (De Novo Software). Intracellular cytokine staining was performed using markers for Granzyme B, Perforin, and IFN-γ. Results T cells exposed in vitro to 10nM and 100nM concentrations of AMG-176 exhibited greater ATP production (Fig 1.), viability, and proliferation (Fig. 2) than the unexposed control group. T cells exposed to 500nM or 1uM concentrations of AMG-176 exhibited unchanged or decreased ATP production, viability, and proliferation relative to the unexposed control group. T cells exposed to AMG-176 significantly increased CD8+ T cell frequency at 10nM and 100nM concentrations (Fig. 3), without affecting viability of CD3+ Pan T cells. Our flow data also indicates dose-dependent increase in expression of early activation marker CD69+. Conclusion Exposure of human T cells to Mcl-1 inhibitor, AMG-176, provides an increase in ATP production, viability, proliferation, and activation. These results shed light on the role of Mcl-1 in T cell biology. Furthermore, increases in human T cell viability, proliferation, and function as facilitated by AMG-176 could offer insight into the advancement of effector cell-based therapies, such as chimeric antigen receptor (CAR) therapy. We may employ AMG-176 alongside CAR T therapy in order to achieve more effective clinical outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Flow Cytometry Study of Cellular Immunity in Rhesus Monkeys after Experimental Infection with SARS CoV 2 Virus

Cellular immunity plays an important role in the pathogenesis and formation of protective immune defense against the SARS‑CoV‑2 virus.The aim of the work was to study the cellular immunity of rhesus monkeys applying flow cytometry after experimental infection with the SARS‑CoV‑2 virus.Materials and methods. Male rhesus monkeys were intranasally inoculated with the SARS‑CoV‑2 virus, Isolate B strain and hCoV-19/Russia/SP48-1226/2020 strain (abbreviated name U-2), at a dose of 5.0 lg PFU. Using flow cytometry, the levels of 21 populations/subpopulations of mononuclear cells in the peripheral blood of animals were determined before experimental infection with the pathogen and on day 14 after infection. SARS‑CoV‑2 coronavirus RNA was assessed using real-time polymerase chain reaction. Determination of the titer of virus-neutralizing antibodies to the SARS‑CoV‑2 virus in the blood sera of animals was conducted through neutralization test evaluating the ability to suppress negative colonies.Results and discussion. Infection with Isolate B strain culture has led to an increase in the relative content of total T-lymphocytes (p˂0.2), cytotoxic T-lymphocytes (p˂0.1), as well as monocytes expressing the early activation marker CD25 (p˂0.2). The decrease in levels has been observed for total B-lymphocytes (p˂0.2) and T-helper cells (p˂0.1). Infection with the U-2 strain culture revealed an increase in the relative content of monocytes expressing the early activation marker CD25 (p˂0.2). Thus, for the first time in the Russian Federation, flow cytometry was used to study the cellular immunity of rhesus monkeys before and after experimental infection with the SARS‑CoV‑2 virus. The obtained information can be used for studying the pathogenesis of SARS‑CoV‑2 infection, course, and outcome of the disease, and developing strategies for vaccination and treatment.

Studies of CD45+ and CD46+ expression on the peripheral blood lymphocyte subsets of the post-COVID patients

The SARS-CoV-2 virus can enter the cells using S1 viral spike (S) protein, not only by binding to ACE2, but also through other cellular receptors. These candidate receptors include CD46, which, like CD45, belongs to pan-leukocyte receptors and is expressed on all types of lymphocytes. In turn, SARS-CoV-2 infection is accompanied by damage to almost all compartments of the immune system, mainly T lymphocytes. The purpose of the study was to evaluate the expression levels of CD45+ and CD46+ in various subpopulations of lymphocytes in patients who had undergone SARS-CoV-2 infection.&#x0D; 72 patients who had undergone SARS-CoV-2 infection were examined. Using flow cytometry technique, we determined CD45+ and CD46+ (panleukocyte marker for lymphocyte gating), CD45+ and CD46+, CD3+ (T lymphocytes), CD45+ and CD46+, CD3+, CD4+ (helper inducers), CD45+ and CD46+, CD3+, CD8+ (cytotoxic T-lymphocytes), CD45+ and CD46+, CD3+, CD56+ (TNK cells) CD45+ and CD46+, CD3-, CD56+ (natural killers), CD45+ and CD46+, CD3-, CD19+ (B lymphocytes), CD45+ and CD46+, CD3+, CD4+, CD25+ (activated helpers, early activation of lymphocytes), CD45+ and CD46+, CD3+, HLA-DR (activated T lymphocytes late activation of lymphocytes). Our studies have shown that a decrease in CD46+ expression in T lymphocytes (CD3+) is accompanied by similar decrease of its expression in cytotoxic T lymphocytes (CD3+, CD8+), TNK (CD3+, CD56+), as well as in helpers T carrying markers of early activation (CD3+, CD4+, CD25+). At the same time, the most pronounced decrease was observed both among total T lymphocytes and cytotoxic T cells. In these patients, the expression level of CD46+ in B lymphocytes was slightly increased. Recent data suggest that there is no involvement of CD46 receptor on B lymphocytes. Our data suggest that the SARS-CoV-2 virus may affect the CD46 receptor. Such exposure may lead to promotion of the long-COVID (post-COVID) symptoms in such patients, thus requiring new approaches to correction of these disorders.

T cell kinetics reveal expansion of distinct lung T cell subsets in acute versus in resolved influenza virus infection.

Influenza virus infection is restricted to airway-associated tissues and elicits both cellular and humoral responses ultimately resulting in generation of memory cells able to initiate a rapid immune response against re-infections. Resident memory T cells confer protection at the site of infection where lung-resident memory T cells are important for protecting the host against homologous and heterologous influenza virus infections. Mapping kinetics of local and systemic T cell memory formation is needed to better understand the role different T cells have in viral control and protection. After infecting BALB/c mice with influenza virus strain A/Puerto Rico/8/1934 H1N1 the main proportion of activated T cells and B cells expressing the early activation marker CD69 was detected in lungs and lung-draining mediastinal lymph nodes. Increased frequencies of activated cells were also observed in the peripheral lymphoid organs spleen, inguinal lymph nodes and mesenteric lymph nodes. Likewise, antigen-specific T cells were most abundant in lungs and mediastinal lymph nodes but present in all organs studied. CD8+CD103-CD49a+ lung-resident T cells expanded simultaneously with timing of viral clearance whereas CD8+CD103+CD49a+ lung-resident T cells was the most abundant subset after resolution of infection and antigen-specific, lung-resident T cells were detected up to seven months after infection. In conclusion, the results in this detailed kinetic study demonstrate that influenza virus infection elicits adaptive immune responses mainly in respiratory tract-associated tissues and that distinct subsets of lung-resident T cells expand at different time points during infection. These findings contribute to the understanding of the adaptive immune response locally and systemically following influenza virus infection and call for further studies on the roles of the lung-resident T cell subsets.

BAP31 affects macrophage polarization through regulating helper T cells activation.

Previously, we reported that B cell receptor associated protein 31 (BAP31) is a positive regulator on T-cells activation. Helper T cells [cluster of differentiation 4+ (CD4+) T cells] can regulate macrophage activation in adaptive immune response against pathogens. In this study, we elucidate that M1 and M2 macrophages polarization is significantly suppressed in Lck Cre-BAP31flox/flox mice or the co-culture system of CD4+ T cells from Lck Cre-BAP31flox/flox mice and macrophages from WT mice. It means that BAP31 may affect the regulation of CD4+ T cells on macrophages. Further studies suggest that BAP31 deficiency significantly reduce the expressions of T helper 1 (Th1)/ Th2/ Th17/ Th9/ Th22/ Treg cells-related cytokines and transcription factors. The inhibition of macrophages activation caused by BAP31 knockdown is due to the reduction of IFN-γ and IL-4 secreted by Th1 and Th2 cells. BAP31 also affects the levels of early activation markers (CD69 and CD25) of CD4+ T cells. Moreover, BAP31 deficiency downregulates the expression of TCRαβ-CD3 complex, and the adaptor proteins p-Zap70, p-Lck, and p-Lat in TCR signaling pathway. These results demonstrate that BAP31 deficiency inhibits TCR/CD3-mediated activation in CD4+ T cells and adversely affects macrophages polarization. These findings establish a theoretical foundation for the study of BAP31 in immunotherapy.

C5a elevation in convalescents from severe COVID-19 is not associated with early complement activation markers C3bBbP or C4d.

Numerous publications have underlined the link between complement C5a and the clinical course of COVID-19. We previously reported that levels of C5a remain high in the group of severely ill patients up to 90 days after hospital discharge. We have now evaluated which complement pathway fuels the elevated levels of C5a during hospitalization and follow-up. The alternative pathway (AP) activation marker C3bBbP and the soluble fraction of C4d, a footprint of the classical/lectin (CP/LP) pathway, were assessed by immunoenzymatic assay in a total of 188 serial samples from 49 patients infected with SARS-CoV-2. Unlike C5a, neither C3bBbP nor C4d readouts rose proportionally to the severity of the disease. Detailed correlation analyses in hospitalization and follow-up samples collected from patients of different disease severity showed significant positive correlations of AP and CP/LP markers with C5a in certain groups, except for the follow-up samples of the patients who suffered from highly severe COVID-19 and presented the highest C5a readouts. In conclusion, there is not a clear link between persistently high levels of C5a after hospital discharge and markers of upstream complement activation, suggesting the existence of a non-canonical source of C5a in patients with a severe course of COVID-19.

PA-X protein assists H9N2 subtype avian influenza virus in escaping immune response of mucosal dendritic cells.

H9N2 subtype low pathogenicity avian influenza virus (AIV) poses a potential zoonotic risk. PA-X, a novel protein generated by PA gene ribosomal frameshift, is considered to be the virulence factor of H9N2 subtype AIVs. Our study found that rTX possessing PA-X protein enhanced the mammalian pathogenicity of H9N2 subtype AIVs compared with PA-X-deficient virus (rTX-FS). Furthermore, PA-X protein inhibited H9N2 subtype AIVs to infect dendritic cells (DCs), but not nonimmune cells (MDCK cells). Meanwhile, PA-X protein suppressed the phenotypic expression (CD80, CD86, CD40 and MHCII), early activation marker (CD69) and pro-inflammatory cytokines (IL-6 and TNF-α), whereas increased anti-inflammatory cytokine (IL-10) in DCs. After intranasally viral infection in mice, we found that PA-X protein of H9N2 subtype AIVs reduced CD11b+ and CD103+ subtype mucosal DCs recruitment to the nasal submucosa by inhibiting CCL20 expression. Moreover, PA-X protein abolished the migratory ability of CD11b+ and CD103+ DCs into draining cervical lymph nodes by down-regulating CCR7 expression. The rTX-infected DCs significantly impaired the allogeneic CD4+ T cell proliferation, suggesting PA-X protein suppressed the immune functions of DCs for hindering the downstream immune activation. These findings indicated that PA-X protein assisted H9N2 subtype AIVs in escaping immune response of mucosal DCs for enhancing the pathogenicity.