Lymphocyte Phenotype Research Articles

Immunophenotyping and Activation Status of Maternal Lymphocytes to Predict Spontaneous Preterm Birth in Women With Threatened Preterm Labor: A Prospective Observational Study.

Preterm birth (PTB) remains the leading cause of neonatal morbidity and mortality. Identifying women at high risk of spontaneous preterm labor (PTL) is challenging due to limited efficient diagnostic markers. Since human parturition involves inflammatory immune processes, we hypothesized that phenotyping of maternal peripheral lymphocytes might predict PTL. Therefore, we aimed to explore the relationship between maternal lymphocyte subpopulations and labor onset characterized by delivery within 7 days of admission in women hospitalized for PTL between 24 and 34 weeks of gestation. Lymphocyte subpopulations were obtained from peripheral blood samples and characterized by flow cytometry: activated and regulatory T cells, natural killer and B cells, and TH1/TH2/TH17 lymphocytes. Data analysis was conducted retrospectively based on the delivery within 7 days of admission. Among 167 women admitted for PTL, less than 10% delivered within 7 days post-admission. HLA-DR expression was significantly increased on CD4+CD8-, CD4-CD8+, and CD4+CD8+ lymphocytes in women who delivered within 7 days. Subset levels below 5% of CD4+CD8-HLA-DR+ lymphocytes and 20% of CD4+CD8+HLA-DR+ lymphocytes were associated with no probability of delivering within 7 days. Our study suggests that combining these two consecutive markers allowed us to identify 57% of women hospitalized for PTL with no probability of delivering within 7 days while retaining patients who delivered within 7 days. If prospectively validated, these markers may be able to identify patients at high risk of PTB and avoid a significant number of unnecessary admissions and healthcare costs. ANSM number: 2010-A00516-33; ClinicalTrials.gov identifier: NCT01340222.

Immune status in women after COVID-19 living in the subarctic region

Background. T cell immunity is known to play a central role in controlling SARSCoV- 2 (severe acute respiratory syndrome-related coronavirus 2) infection, so it is critical to understand its role in recovery from COVID-19 (coronavirus disease 2019), especially in the unfavorable conditions of the North.The aim. To assess the immune status of women after COVID-19 living in the subarctic region of the Russian Federation.Materials and methods. We examined a total of 50 women aged 36–46 years, including 38 vaccinated women 6 and 12 months after recovery from COVID-19 and 12 women (control group) who had not had COVID-19 and had not been vaccinated (Arkhangelsk region). We studied leukocyte count, leukogram and lymphocyte phenotypes (CD5+, CD8+, CD10+, CD16+, CD95+).Results. The contribution of monocytes to the formation of the adaptive immune response among female residents of the subarctic region decreases in those who have had mild COVID-19 and increases in those who have had moderate COVID-19. It was found that the imbalance in the formation of the immune response after COVID-19 is formed due to innate immunity (CD16+, neutrophils (r = 0.89; p < 0.001)), affects the adaptive immunity and depends on the severity of the disease and the time since recovery. The most significant contribution to the formation of the adaptive immune response was established due to cellmediated cytotoxicity (CD8+, CD16+) and the activity of apoptotic processes (CD95+) both after 6 and 12 months and does not depend on the severity of the COVID-19. The adaptive immune response formed 6 months after COVID-19 with a high level of cell-mediated cytotoxicity and activity of apoptotic processes prevailing over lymphoproliferation persists after 12 months, which indicates a tense state of immune homeostasis.Conclusion. In the examined women who had recovered from COVID-19, cellmediated cytotoxicity (CD8+, CD16+) is associated with the activation of the monocyte system, has a prolonged effect of up to 12 months and depends on the severity of the previous COVID-19 in 66.7–90 % of cases.

Inactivated Toxoplasma gondii nanovaccine boosts T-cell memory response in a seropositive yellow-footed rock wallaby (Petrogale xanthopus) – A case report from Copenhagen Zoo

Toxoplasma gondii is a ubiquitous parasite causing significant mortality in captive wildlife, especially marsupials. Historically, treatment has been unrewarding and no vaccine was available. An intranasal vaccine based on purified inactivated T. gondii was developed for toxoplasmosis prevention. A vaccination campaign started in early 2017 and was successful in preventing toxoplasma-related mortality in marsupials in many European and South American zoos. Amongst the vaccinated wallabies, about 30% were T. gondii seropositive before the vaccination, and no toxoplasma-related deaths were observed since the administration of the vaccine. The objective of this case study was to assess the potential effect of the vaccination on a seropositive wallaby. It is important to note that this vaccine doesn't induce any humoral response in sheep, and squirrel monkeys but induces a strong T-cell response. A T. gondii seropositive Yellow-footed rock wallaby (Petrogale xanthopus) from Copenhagen Zoo received two doses of the aforementioned intranasal vaccine. Blood samples were collected before each vaccination and used for peripheral blood mononuclear cell isolation. The impact of the vaccination on the lymphocyte phenotype was characterized by flow cytometry. Cell size, represented by forward scatter, and granularity, represented by side scatter parameters were analyzed. Two doses of the vaccine induced a respective 30.1 and 25.6% increase in cell size and granularity in lymphocytes stimulated with T. gondii antigens, as assessed by flow cytometry. These changes were likely correlated with T-cell activation, which indicates that two doses of the vaccine might have boosted the already-existing T-cell memory response against T. gondii in a seropositive animal. No morphological changes were observed in lymphocytes from an unvaccinated seronegative wallaby. This is the first documented case of boosting an already-existing cellular immune response against toxoplasmosis by the vaccine in a seropositive Yellow-footed rock wallaby.

HIV replication and tuberculosis risk among people living with HIV in Europe: A multicohort analysis, 1983-2015.

HIV replication leads to a change in lymphocyte phenotypes that impairs immune protection against opportunistic infections. We examined current HIV replication as an independent risk factor for tuberculosis (TB). We included people living with HIV from 25 European cohorts 1983-2015. Individuals <16 years or with previous TB were excluded. Person-time was calculated from enrolment (baseline) to the date of TB diagnosis or last follow-up information. We used adjusted Poisson regression and general additive regression models. We included 272,548 people with a median follow-up of 5.9 years (interquartile range [IQR] 2.3-10.9). At baseline, the median CD4 cell count was 355 cells/μL (IQR 193-540) and the median HIV-RNA level 22,000 copies/mL (IQR 1,300-103,000). During 1,923,441 person-years of follow-up, 5,956 (2.2%) people developed TB. Overall, TB incidence was 3.1 per 1,000 person-years (95% confidence interval [CI] 3.02-3.18) and was four times higher in patients with HIV-RNA levels of 10,000 compared with levels <400 copies/mL in any CD4 stratum. CD4 and HIV-RNA time-updated analyses showed that the association between HIV-RNA and TB incidence was independent of CD4. The TB incidence rate ratio for people born in TB-endemic countries compared with those born in Europe was 1.8 (95% CI 1.5-2.2). Our results indicate that ongoing HIV replication (suboptimal HIV control) is an important risk factor for TB, independent of CD4 count. Those at highest risk of TB are people from TB-endemic countries. Close monitoring and TB preventive therapy for people with suboptimal HIV control is important.

Lymphocytes Change Their Phenotype and Function in Systemic Lupus Erythematosus and Lupus Nephritis.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease, characterized by considerable changes in peripheral lymphocyte structure and function, that plays a critical role in commencing and reviving the inflammatory and immune signaling pathways. In healthy individuals, B lymphocytes have a major role in guiding and directing defense mechanisms against pathogens. Certain changes in B lymphocyte phenotype, including alterations in surface and endosomal receptors, occur in the presence of SLE and lead to dysregulation of peripheral B lymphocyte subpopulations. Functional changes are characterized by loss of self-tolerance, intra- and extrafollicular activation, and increased cytokine and autoantibody production. T lymphocytes seem to have a supporting, rather than a leading, role in the disease pathogenesis. Substantial aberrations in peripheral T lymphocyte subsets are evident, and include a reduction of cytotoxic, regulatory, and advanced differentiated subtypes, together with an increase of activated and autoreactive forms and abnormalities in follicular T cells. Up-regulated subpopulations, such as central and effector memory T cells, produce pre-inflammatory cytokines, activate B lymphocytes, and stimulate cell signaling pathways. This review explores the pivotal roles of B and T lymphocytes in the pathogenesis of SLE and Lupus Nephritis, emphasizing the multifaceted mechanisms and interactions and their phenotypic and functional dysregulations.

Lymphocyte phenotyping in untreated children patients with chronic allergic asthma

This study aimed to isolate and phenotype lymphocytes in untreated children patients with chronic allergic asthma. To reach such aim the study involved (25) patients from children (17 male and 9 female) whom their ages where between (3-10) years, in addition to (15) apparently healthy children (9 male and 6 female) in the same ages involved as control group. The data demonstrated that there was a significant increase in the mean percentages of T-lymphocytes (CD3+ cells) in the peripheral blood of patients (66.75±0.29)**, in comparison with control group (43.58±0.19), a significant increase in the mean percentages of T-helper lymphocytes (CD4+ cells) in the peripheral blood of patients (51.14±0.55), in comparison with control group (39.17±0.23) and the mean percentages of B-lymphocytes (CD20+ cells) was also increased significantly in the peripheral blood of patients (29.63±0.20) when it compared with the mean percentages of the same cells in control group (18.60±0.80). Besides a significant decrease in the mean percentages of T-suppressor lymphocytes (CD8+ cells) in the peripheral blood of patients (11.31±0.05), in comparison with control group (16.42±0.15). Finally the results of this study showed a significant increase in the mean percentages of the ratio of (CD4+ cells/CD8+ cells) in the peripheral blood of patients (55.34±0.41), in comparison with control group (31.25±0.09).

Features of subset composition and functional activity of blood lymphocytes in tick-borne infections of different etiologies

Aim. To perform a comparative assessment of subset composition and functional activity of peripheral blood lymphocytes in patients with tick-borne encephalitis (TBE) and ixodid tick-borne borreliosis (ITBB) in the acute phase of the disease.Materials and methods. The study involved 22 patients with febrile and meningeal TBE, 15 patients with ITBB with and without erythema, and 11 healthy controls. Subset composition of blood lymphocytes was determined by flow cytometry. The blast transformation assay was applied to assess lymphocyte proliferation. Cytokine-producing activity of cells was studied in 24-hour incubated mononuclear cell cultures. Cytokine concentrations (interleukin (IL)-2, IL-4, IL-10, interferon (IFN)γ) were determined in the supernatants by the enzyme-linked immunosorbent assay (ELISA).Results. Patients with TBE demonstrated an increase in the proportion of helper – inducer T-cells, a pronounced decrease in the proportion and absolute count of cytotoxic T cells, and low T lymphocyte count compared to the control values. The study in ITBB patients revealed an increase in the helper – inducer T-cell count and the proportion of NK-cells, a decrease in the cytotoxic T cell count, and the T lymphocyte count comparable to normal values. The most significant decrease in the levels of phytohemagglutinin-induced lymphocyte proliferation was found in patients with TBE. Patients of both groups showed a decrease in IL-2 secretion in the mononuclear cell culture, a rise in IL-4 and IL-10 production, and IFNγ production levels comparable to control values.Conclusion. The study of TBE patients revealed relative lymphocytopenia with changes in the subset composition of lymphocytes characterized by an increase in the proportion of helper – inducer T-cells and a decrease in the absolute cytotoxic T lymphocyte count. Patients with ITBB demonstrated an increase in the proportion of NK-cells and a more pronounced imbalance in the T-helper / cytotoxic T lymphocyte ratio. Changes in the functional phenotype of lymphocytes, regardless of the etiology of tick-borne infection, were characterized by reduced proliferative reserve, low IL-2 secretion, increased IL-4 and IL-10 production, and depressed reactivity of lymphocytes with respect to IFNγ secretion.

Characterisation of the tumour microenvironment in primary and recurrent glioblastomas.

Glioblastoma patients have a dismal prognosis, due to inevitable tumour recurrence and respond poorly to immunotherapy. Tumour-associated microglia/macrophages (TAMs) dominate the glioblastoma tumour microenvironment and have been implicated in tumour progression and immune evasion. Early recurrent glioblastomas contain focal reactive regions with occasional fibrosis, chronic inflammation, TAMs and tumour cells. Surgical specimens from these tumours are rare and provide crucial insights into glioblastoma recurrence biology. This study aimed to characterise TAM- and lymphocyte phenotypes in primary vs early- and late-recurrent glioblastomas. Patient-matched primary and recurrent glioblastomas were compared between patients with early recurrences (n = 11, recurrence ≤6months) and late recurrences (n = 12, recurrence after 12-19 months). Double-immunofluorescence stains combining Iba1 with HLA-DR, CD14, CD68, CD74, CD86, CD163, CD204 and CD206 along with stains for CD20, CD3, CD8 and FOXP3 were quantified with software-based classifiers. Reactive regions in early recurrent tumours contained more TAMs (31.4% vs 21.7%, P = 0.01), which showed increased expression of CD86 (59.4% vs 38.4%, P = 0.04), CD204 (48.5% vs 28.4%, P = 0.03), CD206 (25.5% vs 14.4%, P = 0.04) and increased staining intensity for CD163 (86.4 vs 57.7 arbitrary units, P = 0.02), compared to late recurring tumours. Reactive regions contained more B-lymphocytes compared to patient-matched primary tumours (0.71% vs 0.40%, P = 0.04). Fractions of total, cytotoxic and regulatory T-lymphocytes did not differ. Early recurrent glioblastomas showed enrichment for TAMs, expressing both pro- and anti-inflammatory markers and B-lymphocytes. This may indicate a time-dependent response to immunotherapy explained by time-dependent alterations in the immune-microenvironment in recurrent glioblastomas.

Immune status of students with different levels of physical activity

The nature of changes in the immune system during physical work is a complex process involving many different mechanisms. Research in this direction is an urgent problem. The purpose of the research was to study the indicators of cellular and humoral immunity in students with different levels of physical activity. A total of 77 male students were examined, consisting of 3 groups: 1) with a low level of physical activity (n = 32) – students who were not involved in sports activities on a regular basis; 2) students with an average level of physical activity (n = 22) – beginner sambo wrestlers without sports categories; and 3) students with a high level of physical activity (n = 23) – highly qualified sambo wrestlers – first-class athletes, candidates for masters and masters of sports. In students of all groups, the quantitative content of various phenotypes of lymphocytes in the blood was studied by flow cytometry; serum immunoglobulin levels by laser nephelometry; phagocytic parameters by traditional methods; and oxygen-dependent metabolism of neutrophils by chemiluminescence. In students with a high level of physical activity, the content of T and B lymphocytes in the blood was significantly increased compared to similar indicators of the group with a low level of physical activity and the group of students who did not participate in sports. The concentration of class G immunoglobulin in the blood serum of students with a high level of physical activity significantly exceeded the corresponding values in students with an average level and students not engaged in sports. The highest concentration of class M immunoglobulin was observed in students with high levels of physical activity. Phagocytic activity and phagocytic count in individuals with high levels of physical activity and neutrophils were significantly higher than those of students with medium and low levels of physical activity. The rates of spontaneous and induced chemiluminescence in the groups with high and medium levels of physical activity were significantly higher than in students with low levels. The studies carried out indicate the positive effect of regular training physical activity on the factors of humoral and cellular links of immunity in students in the conditions of the educational environment of a higher educational institution.

Effects of parenteral nutrition supplemented with beta-hydroxy-beta-methylbutyrate on gut-associated lymphoid tissue and morphology in mice.

Parenteral nutrition (PN) without enteral nutrition (EN) leads to marked atrophy of gut-associated lymphoid tissue (GALT), causing mucosal defense failure in both the gut and the extraintestinal mucosal system. We evaluated the effects of beta-hydroxy-beta-methylbutyrate (HMB) on GALT and gut morphology in PN-fed mice. Experiment 1: male Institute of Cancer Research mice were assigned to the Chow (n = 12), Control (standard PN: n = 10), or H600 and H2000 (PN containing 600 mg/kg or H2000 mg/kg body weight of Ca-HMB: n = 12 and 10, respectively) groups. After 5 days of dietary manipulation, all mice were killed and the whole small intestine was harvested. GALT lymphocyte cell numbers and phenotypes of Peyer patch (PP), intraepithelial space, and lamina propria lymphocytes were evaluated. Experiment 2: 47 mice (Chow: n = 12; Control: n = 14; H600: n = 11; and H2000: n = 10) were fed for 5 days as in experiment 1. Proliferation and apoptosis of gut immune cells and mucosa, and protein expressions (mammalian target of rapamycin [mTOR], caspase-3, and Bcl2) were evaluated in the small intestine. Compared with the Controls, the Chow and HMB groups showed significantly higher PP cell numbers, prevented gut mucosal atrophy, inhibited apoptosis of gut cells, and increased their proliferation in association with increased mTOR activity and Bcl2 expression. HMB-supplemented PN is a potentially novel method of preserving GALT mass and gut morphology in the absence of EN.

Characterizing Human Peripheral Blood Lymphocyte Phenotypes and Their Correlations with Body Composition in Normal-Weight, Overweight, and Obese Healthy Young Adults.

Background and Objectives: Obesity-associated chronic low-grade inflammation supports various systemic alterations. In this descriptive study, 122 apparently healthy adults aged 20 to 35 years were voluntarily included and classified based on body mass index (BMI) as normal-weight (NW), overweight (OW), and obese (OB). This study aims to characterize peripheral blood (PB) lymphocyte (Ly) phenotypes and investigate their correlations with body composition indices (BCIs) in healthy young adults. Materials and Methods: The following BCIs were measured: waist circumference, hip circumference, height, waist-to-hip ratio, waist-to-height ratio, total body fat mass, visceral fat level, weight, and BMI. White blood cell count (WBC), Ly absolute count, serum TNF-α, and IFN-γ were quantified. Ly subpopulations were analyzed as follows: total TLy (TTLy-CD45+CD3+), early activated TLy (EATLy-CD45+3+69+), total NKLy (TNKLy-CD45+CD3-CD56+CD16+), NKdim (low expression of CD56+), NKbright (high expression of CD56+), BLy (CD45+CD3-CD19+), T helper Ly (ThLy-CD45+CD3+CD4+), and T cytotoxic Ly (TcLy-CD45+CD3+CD8+). Results: Higher BMI has significantly higher WBC and BLy (p < 0.0001; p = 0.0085). EATLy significantly decreased from NW to OB (3.10-NW, 1.10-OW, 0.85-OB, p < 0.0001). Only EATLy exhibited significant negative correlations with all the BCIs. A significantly higher TNF-α was observed in the OW and OB groups compared to the NW group. IFN-γ increased linearly but nonsignificantly with BMI. TTLy showed a nonsignificant positive correlation with both IFN-γ and TNF-α, while EATLy showed a negative correlation, significant only for IFN-γ. NKLy subpopulations exhibited a consistent negative correlation with TNF-α, significant only for NKdim (p = 0.0423), and a nonsignificant consistent positive correlation with IFN-γ. A nonsignificant negative correlation between age and both TNKLy (r = -0.0927) and NKdim (r = -0.0893) cells was found, while a positive correlation was found with NKbright (r = 0.0583). Conclusions: In conclusion, the baseline immunological profile of PB is influenced by excessive adipose tissue in healthy young adults.

High proportion of PD-1 and CD39 positive CD8+ tissue resident T lymphocytes correlates with better clinical outcome in resected human oesophageal adenocarcinoma

ObjectiveTo understand the CD8+ tumour infiltrating lymphocyte (TIL) compartment of oesophageal adenocarcinoma (OAC) with regards to markers of lymphocyte exhaustion, tissue residency and to identify possible reasons behind differential responses to therapy.DesignTumour samples from 44 patients undergoing curative resection for OAC were assessed by flow cytometry for presence of antigen-experienced TILs and markers of activation and exhaustion. Populations of PD-1 and CD39 positive OAC TILs were sorted, and bulk RNA sequencing undertaken using a modified SmartSeq2 protocol. Flow cytometric assessment of functionality was completed.ResultsA higher proportion of antigen experienced CD8+ OAC TILs was associated with improved survival following surgery; while, high double positivity (DP) for PD-1 and CD39 among these TILs also correlated significantly with outcome. These DP TILs possess a minority population which is positive for the markers of exhaustion TIM3 and LAG3. Transcriptomic assessment of the PD-1 and CD39 DP TILs demonstrated enrichment for a tissue resident memory T lymphocyte (TRM) phenotype associated with improved survival in other cancers, reinforced by positivity for the canonical TRM marker CD103 by flow cytometry. This population demonstrated maintained functional capacity both in their transcriptomic profile, and on flow cytometric assessment, as well as preserved proliferative capacity.ConclusionResected OAC are variably infiltrated by PD-1 and CD39 DP TILs, an abundance of which among lymphocytes is associated with improved survival. This DP population has an increased, but still modest, frequency of TIM3 and LAG3 positivity compared to DN, and is in keeping with a functionally competent TRM phenotype.

SARS-CoV-2 spike aggravates lupus nephritis and lung fibrosis in systemic lupus erythematosus

ObjectiveCOVID-19 induces the development of autoimmune diseases, including SLE, which are characterised by inflammation, autoantibodies and thrombosis. However, the effects of COVID-19 on SLE remain unclear.MethodsWe investigated the effects of...

Aging-related biomarkers in testicular cancer survivors after different oncologic treatments.

Testicular cancer survivors (TCS) exposed to chemotherapy have an increased expression of CDKN2A/p16INK4a and a lymphocyte phenotype associated with immunosenescence. We seek to define whether the immunosenescent phenotype is associated with chemotherapy. Case-control study of TCS, disease-free ≥3 months and stratified by primary treatment modality into orchiectomy only, chemotherapy, or bone marrow transplant (BMT). Each group was compared with age-matched healthy controls (HC). We measured the relative proportions of lymphocyte subpopulations using flow cytometry, levels of C-reactive protein, and relative expression of CDKN2A/p16INK4a quantified by qPCR. We included 65 patients; 19 were treated with orchiectomy only, 35 received different doses of chemotherapy, and 11 underwent BMT. The chemotherapy and BMT groups had decreased naïve CD4 cells compared to HC. The chemotherapy group showed increased central and effector memory CD4 cells, as well as effector and terminally differentiated CD8 cells, compared to HC. Chemotherapy (chemotherapy 1.84 vs. HC 0.92; p < 0.01) and BMT (BMT 6.96 vs. HC 1.25; p < 0.005) groups had higher expression of CDKN2A/p16INK4a compared to HC. The orchiectomy group showed no significant difference with HC (orchiectomy 1.73 vs. HC 1.01; p = 0.17). CRP levels were higher in all groups when compared with HC; in the orchiectomy group, they were only marginally increased (chemotherapy 0.22 vs. HC 0.06; p < 0.01; BMT 0.26 vs. HC 0.06; p < 0.01; orchiectomy 0.09 vs. HC 0.07; p < 0.01). Among TCS, only patients exposed to cytotoxic agents developed an immunosenescent phenotype. This finding supports the attribution of this alteration to the cytotoxic treatment.

Stromal CD4 (+) T Cell Subsets Mediate Antitumor Cytotoxic Immune Responses in Human Colorectal Carcinoma.

The local immune response in colorectal cancer is closely related to prognosis and therapeutic efficacy. In this study, histological analyses were performed to determine the phenotype of tumor-infiltrating lymphocytes (TILs) and their infiltration in the stromal and intratumoral regions, aiming to elucidate their interactions and prognostic effects. Multiplex fluorescent labeling was performed using surgically resected colorectal cancer specimens to investigate the infiltration of CD45RO (+) TILs, which exhibit cytotoxicity, and subsets of CD4 (+) TILs, identified by their characteristic transcription factor expression. The degree of CD45RO (+) TIL infiltration in the entire observation field or stromal area was not associated with prognosis. However, a high degree of infiltration in the tumor nest (intratumoral area) was significantly associated with a favorable prognosis. CD4 (+) TILs and their subsets were not associated with prognosis. However, stratified analyses revealed that a high degree of infiltration of stromal CD4 (+) TILs and the subsets T helper (Th)1, Th2, Th17, and regulatory T cells is necessary for the association between high intratumoral CD45RO (+) TIL infiltration and favorable prognosis. A sufficient degree of infiltration of stromal CD4 (+) TIL subsets is required for intratumoral CD45RO (+) TILs to exert toxicity against cancer cells. This highlights the significance of stromal immune reactions in achieving effective cytotoxic immune responses in the intratumoral area and demonstrates the critical role of the spatial distribution pattern of TILs in exerting their functions.

Polygenic polymorphism is associated with NKG2A repertoire and influences lymphocyte phenotype and function

AbstractCD94/NKG2A is a heterodimeric receptor commonly found on natural killer (NK) and T cells, and its interaction with its ligand HLA-E on adjacent cells leads to inhibitory signaling and cell suppression. We have identified several killer cell lectin–like receptor (KLR)C1 (NKG2A) single nucleotide polymorphisms (SNPs) that are associated with NKG2A expression on NK cells, CD8+ T cells, and Vγ9/Vδ2+ T cells. Additionally, due to strong linkage disequilibrium, polymorphisms in KLRC2 (NKG2C) and KLRK1 (NKG2D) are also associated with NKG2A surface density and frequency. NKG2A surface expression correlates with single-cell NK responsiveness, and NKG2A+ NK cell frequency is associated with total NK repertoire response and inhibitability, making the identification of SNPs responsible for expression and frequency important for predicting the innate immune response. Because HLA-E expression is dependent on HLA class I signal peptides, we analyzed the relationship between peptide abundance and HLA-E expression levels. Our findings revealed a strong association between peptide availability and HLA-E expression. We identified the HLA-C killer immunoglobulin–like receptor ligand epitope as a predictive marker for HLA-ABC expression, with the HLA-C1 epitope associated with high HLA-E expression and the HLA-C2 epitope associated with low HLA-E expression. The relationship between HLA-C epitopes and HLA-E expression was independent of HLA-E allotypes and HLA-B leader peptides. Although HLA-E expression showed no significant influence on NKG2A-mediated NK education, it did affect NK cell inhibition. In summary, these findings underscore the importance of NKG2A SNPs and HLA-C epitopes as predictive markers of NK cell phenotype and function and should be evaluated as prognostic markers for diseases that express high levels of HLA-E.

Immunological characterization of IgG subclass deficiency reveals decreased Tregs and increased circulating costimulatory and regulatory immune checkpoints.

Immunoglobulin G subclass deficiencies (IgGsd) comprise a wide clinical spectrum from no symptoms to repeated respiratory infections and risk for the development of lung damage. Our aims were to investigate whether the immunological phenotype of IgGsd patients on and off immunoglobulin replacement therapy (IgRT) was reflected in the clinical features of IgGsd. Thirty patients with IgGsd were included in this prospective study of 18 months of IgRT, followed by 7-18 months of IgRT discontinuation. Blood samples were collected when patients were on and off IgRT and compared with samples from 34 cross-sectional healthy controls. An in-depth lymphocyte phenotyping was performed by flow cytometry and plasma levels of immune checkpoints were assessed. IgG3 subclass deficiency was most common. Patients with IgGsd had decreased levels of activated T cells and B cells and plasma levels of negative immune checkpoint molecules correlated negatively with T cell and B cell activation. The decreased T cell activation level was unaffected by IgRT, while the B cell activation was partly restored. Of note, decreased levels of activated regulatory T cells (Tregs) were found in IgGsd patients and was partly restored during IgRT. The profile of comorbidities did not associate with Treg levels. IgGsd is associated with decreased B cell and T cell activation including Tregs, and increased plasma levels of negative immune checkpoint molecules. The consequence of reduced activated Tregs in IgGsd remains unclear. Decreased immune cell activation was partly restored during IgRT, demonstrating that IgRT may contribute to improved immune function in patients with IgGsd.

Detection and consistency of mucosal fluid T lymphocyte phenotypes and their relationship with blood, age and gender

Innate and adaptive immune responses at mucosal surfaces play a role in protection against most infectious diseases. However, the relative importance either of mucosal versus systemic, or of cellular versus humoral immunity in protection against such infections remains unclear. We aimed to determine the relative percentages and reproducibility of detection of five major T lymphocyte phenotypes in stimulated whole mouth fluid (SWMF); to compare matched mucosal and blood phenotypes; to evaluate the consistency of phenotypes in SWMF over time; and to determine any associations with age or gender. Peripheral blood and SWMF samples were collected from 194 participants and sequential concomitant samples were collected from 27 of those and from 12 subjects living with HIV. CD3, CD4, CD8, Th1 and Th2 T lymphocyte phenotypes were determined by FACS. All the five T lymphocyte phenotypes were detected consistently by FACS in PBMC and SWMF with experimental replicates (N = 10; PBMC CV: 3–30%; SWMF CV: 12–36%). In longitudinal samples detection rates were reproducible in both fluids but variations were higher in SWMF (CV: 23–79.6%) than PBMC (CV: 9.7–75%). Statistically significant correlations of the percentages of all the T lymphocyte phenotypes except CD8 was seen between the two fluids. In PBMCs a negative correlation with age was found with CD3, CD4 and CD8 phenotypes, whilst a positive correlation was found in both SWMF and PBMC with the Th2 phenotype. CD3, CD4 and CD8 phenotypes in SWMF and PBMCs from an HIV-positive cohort were not significantly correlated in contrast with the HIV-negative controls. Our study provides a robust FACS protocol for the detection of the five major T lymphocyte phenotypes in SWMF which should prove useful for research with other mucosal fluids.

Altered Monocyte and Lymphocyte Phenotypes Associated with Pathogenesis and Clinical Efficacy of Progestogen Therapy for Peritoneal Endometriosis in Adolescents.

Background: Immunological imbalances characteristic of endometriosis may develop as early as the primary manifestations of the disease in adolescence. Objective: To evaluate subpopulation dynamics of monocytes and lymphocytes in peripheral blood and peritoneal fluid of adolescents with peritoneal endometriosis at diagnosis and after 1-year progestogen therapy. Methods: This study included 70 girls, 13-17 years old, diagnosed laparoscopically with peritoneal endometriosis (n = 50, main group) or paramesonephric cysts (n = 20, comparison group). Phenotypes of monocytes and lymphocytes of the blood and macrophages of the peritoneal fluid were analyzed by flow cytometry at diagnosis and during progestogen therapy. Results: Differential blood counts of CD16+ (p < 0.001) and CD86+ (p = 0.017) monocytes were identified as independent risk factors for peritoneal endometriosis in adolescents. During the treatment, cytotoxic lymphocytes CD56dimCD16bright (p = 0.049) and CD206+ monocytes (p < 0.001) significantly increased while CD163+ monocytes decreased in number (p = 0.017). The CD56dimCD16bright blood counts before (p < 0.001) and during progestogen therapy (p = 0.006), as well as CD206+ blood counts during the treatment (p = 0.038), were associated with the efficacy of pain relief after 1-year progestogen therapy. Conclusions: Adolescents with peritoneal endometriosis have altered counts of pro- and anti-inflammatory monocytes and lymphocytes both before and after 1-year progestogen therapy, correlating with treatment efficacy and justifying long-term hormonal therapy.

P-015 Impaired spermatogenesis in colitis: the role of interferon-γ mediated ferroptosis in sertoli cells by colitic CD4+ T cells

Abstract Study question Does colitis have an impact on spermatogenesis and what could be the potential mechanism? Summary answer Colitis causes sertoli cell ferroptosis and disrupts spermatogenesis in mice due to the migration of colitic CD4+ T cells that produce interferon-γ. What is known already In recent years, there has been a significant increase in rheumatic and inflammatory diseases. Epidemiological data reveals that dysregulated immune conditions can directly or indirectly impact male fertility and sexual function in patients of reproductive age. Inflammatory bowel disease (IBD), which encompasses Crohn’s disease and ulcerative colitis, is a chronic, recurring gastrointestinal inflammatory disorder characterized by bloody stools and weight loss. A study from a Swedish national cohort showed that IBD patients had diminished sperm fertility and increased sperm DNA fragmentation compared to healthy individuals. However, the precise mechanisms underlying male fertility problems in IBD remain unclear. Study design, size, duration In this study, C57BL/6 mice were kept under specific pathogen-free conditions. A murine colitis model that simulates human colitis histological features and symptoms was created using Dextran Sulfate Sodium (DSS). To establish the model, 2-2.5% DSS was added to the mice’s drinking water from days 0 to 7, followed by a three-day recovery period. Participants/materials, setting, methods Testis, epididymis and colon sections were stained with hematoxylin and eosin for histological analysis. The localization and phenotype of infiltrated lymphocytes in epididymis and testis were studied by immunostaining and flow cytometry. Sperm functions including motility, viability, and DNA integrity were assessed by standard assays. Iron, malondialdehyde, and reactive oxygen species (ROS) levels were analysed by commercial kit. Main results and the role of chance Colitis was found to impair male fertility in DSS-induced colitic mice, causing testis and epididymis tissue damage along with inflammatory cell infiltration. Colitis altered the immune cell phenotypes in the testis and epididymis. The proportion and absolute cell count of CD4+ T cells and CD19+ B cells increased after colon inflammation. Testicular sertoli cells in colitic mice exhibited ferroptosis, characterized by increased levels of iron deposition, malondialdehyde, and ROS. We assessed sperm parameters from the cauda epididymis of mice. Compared to the control group, there were significant reduction in sperm concentration, motility, viability and DNA integrity in the group with murine colitis induction. Consistently, we observed lower numbers of active pups from the fathers of the colitis group compared to control mice. CD4+ T cells with interferon-γ producing capabilities accumulated in the testes of colitis mice. Leukocyte flow cytometric analysis and parabiotic mouse model further demonstrated that colitis promoted pathogenic CD4+ T cells migration to the testis through circulation. The treatment of anti-CD4 antibody, anti-interferon-γ antibody or ferroptosis inhibitor can attenuate the colitis-associated testis pathologies. Limitations, reasons for caution Our study was conducted exclusively in mice. The pathogenic effect and the corresponding mechanism of colitis on male fertility in humans still needs to be confirmed. Wider implications of the findings Our team identified the mechanism linking dysregulated gut immune cell activity to testicular dysfunction. By identifying specific molecules on colitic immune cells, which allow their infiltration into the testis, may pave the way for a new therapeutic approach potentially improving the prognosis of colitis and related fertility impairments. Trial registration number not applicable