Lymphocyte Phenotype Research Articles

The role of serum and bronchoalveolar lavage fluid chitotriosidase activity on diagnosis, disease characteristics and prognosis of sarcoidosis.

Sarcoidosis is a multisystem granulomatous disease with an unpredictable clinical course. Chitotriosidase is a chitinase mainly expressed by activated macrophages. Increased chitotriosidase activity has been reported in serum and bronchoalveolar lavage (BAL) of sarcoidosis patients compared to healthy controls. This study aims to evaluate the role of serum and BAL chitotriosidase activity on diagnosis, disease characteristics, and prognosis of sarcoidosis. Patients referred with suspected sarcoidosis or other interstitial lung disease were prospectively included in the study. All patients underwent bronchoscopy with BAL. Serum and BAL chitotriosidase activity, BAL differential cell counts, and lymphocyte phenotypes were determined. Sarcoidosis patients were followed up regularly. Forty-two sarcoidosis and 28 non-sarcoidosis patients were included in the study. Serum chitotriosidase activity was higher in sarcoidosis group 247.5 (2.78-461) vs 108 (2.78-272) nmol/h/mL (p< 0.001). BAL chitotriosidase activity tended to be higher in sarcoidosis group 11 (2-308) vs 6.95 (2.27-44) nmol/h/mg but was not found to be statistically significant (p= 0.11). Serum and BAL chitotriosidase activities were correlated with each other (p= 0.023, r= 0.355). No significant difference was found between the diagnostic performance of BAL CD4/CD8 ratio and serum chitotriosidase activity (p= 0.079). Serum chitotriosidase and ACE activities were correlated with each other (p= 0.004, r= 0.457). No significant difference was found between serum or BAL chitotriosidase activity and stage or extrapulmonary involvement. Serum chitotriosidase activity was higher in patients who needed systemic therapy at diagnosis (p= 0.046). However, no significant difference was found between serum or BAL chitotriosidase activities and disease progression (p= 0.395 and p= 0.723, respectively). Serum chitotriosidase activity can be helpful in the differential diagnosis of sarcoidosis with a similar diagnostic performance with BAL CD4/CD8 ratio. Although serum chitotriosidase activity at diagnosis does not predict progressive disease, it is associated with the need for systemic therapy at diagnosis. Serial chitotriosidase measurements may be useful in monitoring disease progression during follow-up.

CD8-Lymphocytic Phenotype Significance in Primary Multiple and Familial Melanoma with Various CDKN2A Mutational Status.

Background and Objectives: In the realm of the rising incidence of cutaneous and mucous melanoma, CDKN2A mutations characterize familial and multiple primary melanoma cases. The involvement of tumor-infiltrating lymphocytes (TILs) is interconnected with survival rates, but may extend even further. The aim of this study is to verify the accuracy of the classical "naked eye" count of CD8-positive T cells comprised within the tumoral population and peritumoral infiltrate versus that obtained via a special software run by the aid of artificial intelligence (AI), used to determine the percentage of CD8-positive TILs. Materials and Methods: The present retrospective cross-sectional study conducted over a period of 5 years (2018-2022) focused on patients diagnosed with mucous and/or cutaneous melanoma, with a positive family history for melanoma, or personal antecedents of primary malignant melanocytic lesions. The 23 selected cases were diagnosed histopathologically, tested for CDKN2A mutations through fluorescent hybridization in situ, and CD8 immunohistochemistry was performed. The included slides were evaluated both manually (naked-eye examination) and automatically (via QuPath platform) for quantifying the CD8-positive TILs. Results: The number of CD8-positive TILs in melanoma samples has been more accurately identified through the use of an AI-mediated software as compared to the human-eye evaluation performed by experimental pathologists. A higher percentage of CD8-positive intratumoral lymphocytes versus stromal lymphocytes was positively associated with more numerous metastatic sites. Conclusions: The CD8 lymphocytic phenotype harbors major significance in the context of familial and multiple primary melanoma and may comprise a cost-effective investigation meant to help in the establishment of melanoma prognosis and response to immunotherapy.

CYP1A1 (RS4646421) gene polymorphism and peculiarities of immune profile in children under aerogenic exposure to benzo(a)pyrene

Introduction. The benzo(a)pyrene exposure forms the special phenotype of the immune profile in children. The study of immune system features associated with the polymorphism of detoxification genes is relevant in the conditions of health risks linked with environmental pollution.&#x0D; Materials and methods. There was performed clinical and laboratory examination of four hundred seventy nine children aged of 3–6 years. The observation group consisted of 308 children living in conditions of aerogenic exposure to benzo(a)pyrene. The comparison group included 171 children from a relatively clean territory. Determination of benzo(a)pyrene concentration in an atmospheric air and in blood was carried out by HPLC. The study of polymorphism of CYP1A1 (rs4646421) gene was carried out by real-time PCR. CD3+CD4+-lymphocyte phenotyping was performed by flow cytometry, IgG to benzo(a)pyrene – by allergosorbent testing.&#x0D; Results. Aerogenic exposure to benzo(a)pyrene at an average daily dose of 8.76•10–2 µg/(kg•day) causes an increase in the level of blood contamination with PAH, forms an imbalance of the immune profile (IgG to benzo(a)pyrene hyperproduction, CD3+CD4+-lymphocyte expression deficiency and CD4+/CD8+decrease) associated with T-allele and CT-genotype of the CYP1A1 gene (rs4646421) (OR(CI)=2.35–6.65; p&lt;0.05). Children with the CT-genotype of the CYP1A1 gene (rs4646421) are characterized by the most pronounced changes in the immune profile (excess IgG to benzo(a)pyrene; reduction of CD3+CD4+ and CD4+/CD8+ against the background of maximum blood contamination with benzo(a)pyrene in relation to other genotypic groups (OR(CI)=1.64–3.08; p&lt;0.05). &#x0D; Limitations. The limitations are related to the need to increase the sample and verify the results obtained in subsequent observations.&#x0D; Conclusion. The peculiarities of the immune profile in CT-genotype carriers of the CYP1A1 (rs4646421) gene under the conditions of aerogenic exposure to benzo(a)pyrene at a dose of 8.76•10–2 mcg/(kg•day) should include the formation of specific sensitization to benzo(a)pyrene, deficiency of cellular differentiation clusters: CD3+CD4+-lymphocytes, CD4+/CD8+ reduction associated with blood contamination with benzo(a)pyrene, which indicate the likelihood of hereditary predisposition realization and the formation of immune regulation disorders associated with exposure to benzo(a)pyrene.

Positive impact of sulfamethoxazole-trimethoprim prophylaxis on prevention of severe infections in a patient with glycogen storage disease type Ib

ObjectiveGlycogen storage disease type Ib (GSD-Ib) is a very rare disease complicated by neutropenia with consequent recurrent bacterial infections. Treatment with Filgrastim is not always effective.The low CD4 count observed in our patient is likely the underlying cause of this treatment failure. This low value was previously examined in a small-scale study. Therefore, adding sulfamethoxazole-trimethoprim (SMX-TMP) might be a good strategy.MethodsWe present the case of a male patient with GSD-Ib. He has severe neutropenia (380/mm3) with recurrent infections. Despite neutrophil improvement with Filgrastim, he developed three severe infections requiring hospitalization. Lymphocyte phenotyping showed a deficit in T CD4 + cells (280/mm3) which led us to HIV testing returning negative. Based on this finding we initiated prophylaxis with SMX-TMP.ResultsSince the start of SMX-TMP along with Filgrastim, the patient was not admitted to the hospital for any bacterial infection.ConclusionTo date, no study has examined the significance of incorporating antibiotic prophylaxis for neutropenic patients with GSD-Ib based on CD4 count, akin to the approach of adding SMX-TMP to antiretroviral therapy for HIV patients. Cotrimoxazole is empirically prescribed without assessing the CD4 count or conducting a comparative analysis of the advantages of its addition to Filgrastim. Such practices could potentially exert a significant influence on the disease’s presentation and severity.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

IImmunological parameters in patients with chronic opisthorchiasis bearing gene mutations associated with osteoporosis-predisposition

To identify the features of the immune system functioning in patients with chronic opisthorchiasis bearing mutations in loci associated with predisposition to developing osteoporosis, comprehensive studies of cellular and humoral arms were carried out. The state of the phagocytic system was assessed by assessing absorption, metabolic activity and reactive oxygen species formation to restore nitrosine tetrazolium (spontaneous and stimulated NBT test). The phenotype of lymphocytes was determined by flow cytometry. The humoral immune arm was evaluated by the number of immunoglobulin classes M, G, A and E. Differences in the functional state of various arms of the immune system were revealed. In patients with chronic opisthorchiasis in the presence of rs1544410 polymorphism of the gene encoding the intracellular vitamin D receptor, the relative number of T-helper cells is significantly lower than in the group with the normal allele. In the presence of rs1800012 polymorphism of the gene encoding the α1-chain of type I collagen, the absolute lymphocyte count is significantly higher, spontaneous and stimulated NBT test were lower, the number of DN-T lymphocytes is significantly lower (both in relative and absolute values). In the presence of the rs3736228 mutation of the gene encoding the transmembrane low-density lipoprotein receptor, the level of myeloperoxidase and the neutrophil stimulation index are lower, the absorption activity of neutrophils is higher. The presence of the rs2234693 mutation for estrogen receptor gene leads to significantly increased level of stimulated NBT test and IgG concentration. Thus, patients with chronic opisthorchiasis bearing mutations in the COL1 A1, LRP5, ESR1(rs2234693) genes, have altered both nonspecific innate reactions and parameters of the adaptive immune arm; mutation of the VDR gene solely affects adaptive immunity. Analysis of the results suggests that the presence of mutations associated with the development of osteoporosis has a modulating effect on the immune response in chronic opisthorchiasis invasion. The identification of polymorphic genes associated with metabolic disorders of bone tissue and the study of the immunological profile in patients with chronic opisthorchiasis invasion will allow to implement an individual approach in the treatment of such patients.

A Longitudinal Single-Cell Atlas of Treatment Response in Pediatric AML

Introduction: Pediatric acute myeloid leukemia (pAML) is a heterogeneous disease in terms of driver alterations, treatment response and patient outcomes. Although approximately 90% of pAML patients respond to initial treatment, tumours relapse frequently. Despite large-scale genomic sequencing efforts, it remains elusive why tumors relapse. Previously, mutations have been identified that affect transcriptional regulation, indicating that epigenetic processes may contribute to treatment resistance in pAML. We therefore studied how tumor epigenetic patterns, transcriptional regulation and cell populations change over the course of treatment using single-cell ATAC sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) in pediatric populations. Methods: We profiled 330,047 cells using scRNA-seq and 353,984 cells using scATAC-seq derived from 28 uniformly treated pAML patients, driven by either MLL fusions, CBFB fusions, RUNX1 fusions, FLT3-ITD or other aberrations (i.e. CEBPA mutations). All patients were enrolled in the AAML1031 trial (https://childrensoncologygroup.org/aaml1031), a randomized phase 3 clinical trial that investigated the addition of Bortezomib (BTZ) and Sorafenib to standard treatment modalities. Samples were obtained serially at diagnosis, at remission and at relapse and single-cell profiles were compared between these stages. Results: We identified malignant and non-malignant populations in the scATAC-seq and scRNA-seq data, showing that in all cases, malignant cells from patients at diagnosis were distinct from those taken at relapse. Major differences between diagnosis and relapse were associated with differentiation, and by inferring distinct cell populations we identified that relapsed cell populations appeared to shift towards a more primitive state. The extent of this shift was dependent on the genetic subtype and the effects were more pronounced in MLL-rearranged tumors. To identify the candidate mechanisms and transcription factors involved in this shift we studied the monocytic, lymphocytic and erythrocytic lineages in non-malignant cells and compared these to the tumor at diagnosis and relapse. Based on motif enrichment we found that across all different subgroups, transcription factors (TFs) that drive monocytic differentiation (i.e. CEBPA) have a lower activity while TFs driving erythrocytic and lymphocytic differentiation (i.e. TCF3) have a higher activity. Analysis of hematopoietic stem-like and progenitor-like cells (HSPC-like) confirmed this change, indicating that the “priming” towards other lineages is not only a result of shifts in cell populations but also a result of rewiring of early progenitors. To investigate this rewiring, we analysed regions of open chromatin that were enriched in malignant HSPCs compared to normal HSPCs across different subgroups. We found that those regions were highly specific to the driving alteration and were enriched for MEF2C binding sites in MLL-driven tumors and AP-1 binding sites in other tumors. Overall, upon relapse MEF2C expression and inferred TF activity appeared to increase, coinciding with an apparent increase in lymphoid priming and early lymphoid factors including LMO2, LYL1 and TCF3, among others. Two cases in the cohort appeared to switch lineage from a monocytic to lymphocytic phenotype and were enriched in CD19 and CD79A positive malignant cells upon relapse. Both cases shared the characteristic that MEF2C was upregulated upon relapse and showed upregulation of typical B-lymphoid transcription factors such as PAX5. Discussion: By generating a large dataset of pediatric AML patient samples collected at diagnosis, remission and relapse, we have identified changes in cellular hierarchies and transcriptional networks that occur upon relapse. Our study shows that shifts in cell populations readily occur upon treatment and are dependent on subgroup-defining alterations. Particularly MLL-rearranged tumors show a high level of plasticity that could explain the poor outcome of this group. Furthermore, we show that tumors appear to adapt their transcriptional programs towards a more “stem-like” state, with a likely higher propensity to differentiate towards other lineages such as the lymphoid lineage. We have identified MEF2C as a possible regulator of these processes, opening a new perspective to target the plasticity of these tumors.

Changes in natural killer and T lymphocyte phenotypes in response to cardiovascular risk management

The pro-inflammatory and regulatory roles of T lymphocytes in atherosclerosis are well established but less is known about natural killer (NK) cells and natural killer T (NKT)-like cells. The effects of cardiovascular risk management on the phenotypes of these cells are unknown. To assess changes in NK cell and lymphocyte phenotypes and circulating inflammatory proteins in response to cardiovascular risk management in patients with carotid atherosclerosis. Fifty patients were included in a prospective clinical study. Measurements were at baseline and after 12 months of cardiovascular risk management. Circulating NK, NKT-like and T lymphocyte subpopulations were phenotyped by multi-colour flow cytometry. Proximity extension assay was performed for 176 plasma proteins associated with inflammation and cardiovascular disease. At 12 months there were significant reductions in LDL (P = 0.001) and blood pressure (P = 0.028). NK cells responded with a reduction in pro-inflammatory (NKG2C+) cells (P = 0.0003), an increase in anti-inflammatory (NKG2A+) cells (P = 0.032), and a reduction in terminally differentiated (CD57+) NK cells. NKT-like cells showed a similar decrease in terminally differentiated subpopulations (P = 0.000002). Subpopulations of T helper cells exhibited a significant reduction in central memory (P = 1.09 × 10−8) and a significant increase in CD4+ naïve- (P = 0.0008) and effector memory T cells (P = 0.006). The protein analysis indicated that cardiovascular risk management affects proteins involved in the inflammatory NF-κB pathway. The consistent decrease in senescent phenotypes of NK, NKT-like and CD4+ cells with a concomitant increase in more naïve, phenotypes suggests a change towards a less pro-inflammatory lymphocyte profile in response to cardiovascular risk management.Trial registry name: CARotid MRI of Atherosclerosis (CARMA). ClinicalTrials.gov identifier NCT04835571 (08/04/2021). https://www.clinicaltrials.gov/study/NCT04835571.

Profound and selective lymphopaenia in primary lymphatic anomaly patients demonstrates the significance of lymphatic-lymphocyte interactions.

The lymphatic system has a pivotal role in immune homeostasis. To better understand this, we investigated the impact of Primary Lymphatic Anomalies (PLA) on lymphocyte numbers and phenotype. The study comprised (i) a retrospective cohort: 177 PLA subjects from the National Primary Lymphatic Anomaly Register with clinical and laboratory data, and (ii) a prospective cohort: 28 patients with PLA and 20 healthy controls. Patients were subdivided using established phenotypic diagnostic categories and grouped into simplex (localised tissue involvement only) and systemic (involvement of central lymphatics). Further grouping variables included genital involvement and the likelihood of co-existent intestinal lymphangiectasia. Haematology laboratory parameters were analysed in both cohorts. In the prospective cohort, prospective blood samples were analysed by flow cytometry for markers of proliferation, differentiation, activation, skin-homing, and for regulatory (CD4+Foxp3+) T cells (Treg). In patients with PLA, lymphopaenia was frequent (22% of subjects), affected primarily the CD4+ T cell subset, and was more severe in subjects with systemic versus simplex patterns of disease (36% vs 9% for lymphopaenia; 70% vs 33% for CD4+ cells). B cells, NK cells and monocytes were better conserved (except in GATA2 deficiency characterised by monocytopaenia). Genital oedema and likelihood of concomitant intestinal lymphangiectasia independently predicted CD4+ T cell depletion. Analysing CD4+ and CD8+ T cells by differentiation markers revealed disproportionate depletion of naïve cells, with a skewing towards a more differentiated effector profile. Systemic PLA conditions were associated with: increased expression of Ki67, indicative of recent cell division, in naïve CD4+, but not CD8+ T cells; increased levels of activation in CD4+, but not CD8+ T cells; and an increased proportion of Treg. Skin-homing marker (CCR10, CLA and CCR4) expression was reduced in some patients with simplex phenotypes. Patients with PLA who have dysfunctional lymphatics have a selective reduction in circulating lymphocytes which preferentially depletes naïve CD4+ T cells. The presence of systemic disease, genital oedema, and intestinal lymphangiectasia independently predict CD4 lymphopaenia. The association of this depletion with immune activation and increased circulating Tregs suggests lymphatic-lymphocyte interactions and local inflammatory changes are pivotal in driving immunopathology.

The signature and predictive value of immune parameters in patients with secondary hemophagocytic lymphohistiocytosis

BackgroundSecondary hemophagocytic lymphohistiocytosis (sHLH) is a rare but fatal clinical syndrome, characterized by severe immune dysfunction and overwhelming inflammatory response. However, the host immune signature and also its role in predicting the clinical outcome are not fully described. ObjectiveThe present study aims to investigate the host immune status of sHLH patients in the early stage of the disease, including lymphocyte subsets, phenotypes and cytokines, and also to explore its clinical value in prognosis. MethodsSixty-four patients with sHLH admitted to a tertiary hospital in central China between 2018 and 2022 were enrolled, of which 21 were deceased. The subsets and phenotypes of lymphocytes, and the levels of cytokines in serum were analyzed. ResultsIn patients with sHLH, the percentages of total T cells, CD8+ T cells, HLA-DR+ T cells, HLA-DR+CD8+ T cells, CD45RO+CD4+ T cells, and the levels of IL-1β, IL-2R, IL-6, IL-8, IL-10 and TNF-α were significantly increased, while the percentages of CD4+ T cells, NK cells, CD45RA+CD4+ T cells, CD45RA+ regulatory T (Treg) cells, the counts of total T cells, total B cells, CD4+ T cells, CD8+ T cells, NK cells, and the ratio of CD4+ T/CD8+ T cells were significantly decreased, compared with healthy controls (HC). In addition, dysregulation of host immune response and high inflammatory status were more obvious in deceased patients than that of survivors. Kaplan-Meier survival analysis and multivariate logistic regression analysis demonstrated that lower levels of CD4+ T cells count and CD28+CD4+ T cells percentage, but higher levels of NK cells percentage and IL-1β were poor prognostic indicators of sHLH. ConclusionThe evaluation of immunological markers has critical value for selecting prognostic markers and potential treatment target among adults with sHLH.

Immunity and hemostasis system indicators in age groups of laboratory rats during the use of cyclosporine A

Dysregulation of the T-cell link increases the susceptibility to cardiovascular disease with age. Therefore, we modeled immunosuppression and experimentally compared the state of the hemostasis system in young and old animals. White outbred male rats were used for the experiment. Cyclosporine in Sandimmun-Neoral (Novartis, Switzerland) was administered intramuscularly to experimental rats. Complement system proteins C3, C4, immunoglobulins (Ig) A, M, G and interleukin-6 were examined by enzyme immunoassay. Phenotyping of lymphocytes was performed by indirect immunofluorescence using monoclonal antibodies (subpopulations CD3+, CD19+, CD4+, CD8+, CD16+). The data obtained indicate that inhibition of the T-lymphocyte link (CD3+) does not necessarily lead to a compensated increase in the number of B-lymphocytes (CD19+), but stimulates their function: immunoglobulins are elevated (M, G). Under conditions of a reduced reaction from the cellular level, components of the complement system (C3, C4) are activated. Despite a slight decrease in phagocytic function, there was an increase in the number of natural killers (CD16+), active consumption of T-helpers (CD4+), a decrease in T-suppressors (CD8+), and an increase in the level of interleukin-6 in the plasma of experimental rats. Increased activity of antithrombin in old animals prevents procoagulant shifts against the background of excessive growth of "areas" with tissue factor. We found desquamated endotheliocytes, that opens the subendothelial layer and its level was higher in the experimental groups. Thus, we see an activation of the humoral link of the immune system. Experimental excessive concentration of the calcineurin blocker exposes the first pathogenetic links from the hemostasis system. An increase in the coagulation time of the initial stages of coagulation is combined with an acceleration of the final one. Despite the growth of fibrinogen in young animals, the mismatch in the regulation of the hemostasis system manifested itself mainly in old specimens of laboratory rats.

Immune reconstitution following alemtuzumab therapy is characterized by exhausted T cells, increased regulatory control of proinflammatory T cells and reduced B cell control.

Alemtuzumab is a monoclonal antibody targeting CD52 on the surface of immune cells, approved for the treatment of active relapsing-remitting multiple sclerosis (RRMS). The purpose of this study was to analyze the repopulation of peripheral lymphocytes following alemtuzumab-induced lymphocyte depletion and investigate associations with disease activity and development of secondary autoimmunity. For this, blood samples were collected two years after initiation of alemtuzumab treatment and lymphocytes were subjected to a comprehensive flow cytometry analysis. Included in the study were 40 patients treated with alemtuzumab and 40 treatment-naïve patients with RRMS. Disease activity and development of secondary autoimmune disease was evaluated after three years of treatment. Our study confirms that alemtuzumab treatment profoundly alters the circulating lymphocyte phenotype and describes a reconstituted immune system characterized by T cell activation/exhaustion, an increased regulatory control of IL-17 producing effector T cells and CD20+ T cells, and a reduced control of B cells. There were no obvious associations between immune cell subsets and disease activity or development of secondary autoimmune disease during treatment with alemtuzumab. Our results indicate that the reconstituted immune response is skewed towards a more effective regulatory control of MS-associated proinflammatory T cell responses. Also, the enlarged pool of naïve B cells together with the apparent decrease in control of B cell activity may explain why alemtuzumab-treated patients retain the ability to mount a humoral immune response towards new antigens.

COMPARISON OF CELL DEATH RATE IN VARIOUS CHICKEN LYMPHOCYTES POPULATIONS IN A RESPONSE TO THREE TEMPERATURE TREATMENTS

The goal of the experiment was to evaluate susceptibility of lymphocytes populations derived from bursa of Fabricius, spleen, thymus, and blood to temperature treatments in vitro. Temperatures used in study was 37 °C (thermoneutral), 41 °C (mild heat stress) and 45 °C (severe heat stress). Cells were incubated for 8 hours and after 3 and 8 hours were measured numbers of apoptotic, necrotic and CellROX positive cells by flow cytometry. Representation of live lymphocyte phenotypes (CD3, CD4, CD8, BU1) in cell populations were also measured. At all temperature treatments, thymus lymphocytes population had the lowest (p &lt; 0.05) percentage of apoptotic cells. On the other hand, significantly highest numbers (p &lt; 0.05) of apoptotic cells were found in bursal lymphocytes after 8 hours of incubation. Differences in numbers of necrotic cells were more pronounced at 45 °C, where after 3 and 8 hours of incubation were significantly highest numbers (p &lt; 0.05) of necrotic cells in spleen. Increasing temperature induce production of reactive oxygen species (ROS) in cells and highest proportion (p &lt; 0.05) of ROS-positive cells were found in bursal and spleen cell population after 8 hours of incubation. Transmission electron microscopy showed loss of mitochondrial matrix and disrupted mitochondrial membrane in mitochondria of lymphocytes at 45 °C as initial damage caused by ROS. From individual cell phenotypes, there were significantly decreased (p &lt; 0.05) B lymphocytes in bursa of Fabricius, CD4+ and CD8+ lymphocytes in thymus and whole T lymphocytes population in spleen at 45 °C. Various susceptibility to temperature can be predictive factor of worse function in some organs in stress conditions where body temperature increase.

#6158 UREMIC TOXINS ARE ASSOCIATED WITH IMMUNE-SENESCENCE AND IMMUNE-EXHAUSTION IN HEMODIALYSIS PATIENTS

Abstract Background and Aims Immune-senescence and exhaustion have been described in hemodialysis (HD) patients, with changes in lymphocyte phenotype possibly stimulated by the uremic environment, independent of age and comorbid conditions. We evaluated the potential association of uremic toxin accumulation with changes in lymphocyte count and phenotype in HD patients. Method In the peripheral blood of 54 HD patients (23 female, mean age 51.3±16.91 years) and 31 age-matched healthy controls, lymphocytes surface molecules were determined by flow cytometry: T cells (CD4, CD8, CD31, CD45RA, CCR7, CD28, CD57, PD1) and B cells (CD27, IgD). Plasma levels of the protein bound uremic toxins [hippuric acid (HA), indoxyl sulfate (IxS) and p-cresyl sulfate (pCS)] were measured by ultra-performance liquid chromatography. Lymphocyte phenotypes were correlated with uremic toxins levels using Spearman's correlation coefficient test. Patients with recent or active infection, malignancy, autoimmune disease and/or diabetes mellitus were excluded. Results A higher concentration of uremic toxins was observed in HD patients, compared to healthy controls, namely: total HA 3.05 (1.66-5.37) vs 0.102 (0.04-0.2)mg/dl (p&amp;lt;0.001), free HA 1.482 (0.7-2.8) vs 0.029 (0.03-0.04)mg/dl (p&amp;lt;0.001); total IxS 2.207 (1.27-3.34) vs 0.063 (0.04-0.09)mg/dl (p&amp;lt;0.001), free IxS 0.146 (0.09-0.27) vs 0.0004 (0.0004-0.0004)mg/dl (p&amp;lt;0.001); total pCS 1.248 (0.84-1.66) vs 0.066 (0.04-0.13)mg/dl (p&amp;lt;0.001), free pCS 0.089 (0.06-0.13) vs 0.004 (0.004-0.005)mg/dl (p&amp;lt;0.001); respectively. There was a positive correlation between hemodialysis vintage and total as well as free HA (p = 0.005, p = 0.003, respectively). No significant differences in uremic toxin levels were found in HD patients according to sex, age, dialysis prescription and method. Patients with residual urine output had reduced total and free HA levels 1.739 (3.5) vs 3.347 (4.16)mg/dl (p = 0.03), and 0.717 (1.9) vs 1.901 (3.5)mg/dl (p = 0.04), respectively and free IxS levels, 0.117 (0.1) vs 0.177 (0.2)mg/dl (p = 0.02), respectively. Total lymphocyte count and absolute number of CD4 cells were negatively correlated with total (p = 0.018 and p = 0.02 respectively) and free HA levels (p = 0.024 and p = 0.017 respectively). Several naïve and less differentiated CD4 subpopulations also demonstrated a negative correlation with total and free HA levels, most importantly CD4CD31+ (p = 0.037 and p = 0.027 respectively), CD8CD28+CD57- (p = 0.018 and p = 0,014 respectively) CD4CD45+CD57- (p = 0.039 and p = 0.027 respectively) and naïve B cells (CD19+IgD+CD27-) (p = 0.042 and p = 0.032 respectively). Advanced differentiated lymphocyte subtypes, CD4CD28+CD57+ and CD4CD45-CD57+ had a positive correlation with free ΗΑ levels (p = 0.036 and p = 0.045 respectively). Exhausted CD4 cells, defined as CD4+PD1+ had positive correlations with total and free IxS levels (p = 0.004 and p = 0.011 respectively) and total and free pCS (p = 0.027 and p = 0.018 respectively), but not with HA levels. Moreover, further divided exhausted lymphocytes, according to CD45RA expression, CD4CD45RA-PD1+ cells correlated positively with total and free IxS levels (p = 0.004 and p = 0.01 respectively) and CD4CD45RA+PD1+ with pCS total and free levels (p = 0.039 and p = 0.045 respectively). Conclusion Uremic toxins are increased in hemodialysis patients and may influence adaptive immunity. HA is associated with an immune-senescent, while IxS and pCS to an immune-exhausted lymphocyte phenotype.

Dynamics of the innate immune response profile in patients with coronary heart disease at different terms after coronary artery stenting

Atherosclerosis is accompanied by damage to the vascular endothelium of arteries followed by development of inflammatory response and formation of atherosclerotic plaques. Innate immunity is an important component of this response being the earliest non-specific key mechanism. Our objective was to perform a comprehensive assessment of the cellular link of innate immunity, and to compare the results obtained at various terms after coronary stenting.The study involved 50 patients with coronary atherosclerosis (Group 1), who had clinical indications for stenting of coronary arteries, and 20 volunteers (Group 2), who have no signs of coronary artery disease. The study of immune parameters was carried out before surgery, at 4-5, 9-10 and 28-30 days after operation (during early postoperative period), as well as 6 and 12 months after stenting, i.e. over the late post-surgical period. Phenotyping of peripheral blood monocytes and lymphocytes was performed by flow cytometry using monoclonal antibodies (Beckman Coulter, USA). Intracellular content of Granzyme B was carried out with an FC500 flow laser cytofluorimeter. Metabolic activity of neutrophils was assessed by the NBT test. Alpha defensin was determined in blood plasma by ELISA technique (Hycult Biotech, USA). Statistical analysis was performed using the Statistica 12.0 program (StatSoft, USA). Statistical significance was considered significant at p ≤ 0.05.The numbers of natural killer cells and their activity, as well as those of monocytes, were increased in patients with coronary atherosclerosis. We have also shown a suppression of antigen presentation processes, an imbalance in microbicidal activity of neutrophils, with predominant secretion of antimicrobial peptides. Over the early post-surgical period, significant changes included only decreased content of intracellular Granzyme B on days 4-5, and expression of TLR4 and HLA-DR on days 4-5 and 9-10. During the late period, the patients with coronary artery disease exhibited a significant decrease in the content of some lymphocyte subsets: CD3+CD16+, CD16+Gr+ as well as amounts of monocytes: CD14+CD282+, CD14+CD284+, CD14+CD289+, along with HBT-test activity and α-defensin contents, and increased numbers of HLA-DR-expressing monocytes.There are changes in cellular component of innate immunity, indicating persistent inflammation in patients with coronary heart disease. The dynamics of revealed changes following coronary artery stenting may reflect a lability of assessed indicators mostly over the late postoperative period, thus serving a basis for predicting the outcome of coronary stenting.

Maternal Th17 Profile after Zika Virus Infection Is Involved in Congenital Zika Syndrome Development in Children.

Brazil is one of the countries that experienced an epidemic of microcephaly and other congenital manifestations related to maternal Zika virus infection which can result in Congenital Zika Syndrome (CZS). Since the Zika virus can modulate the immune system, studying mothers' and children's immune profiles become essential to better understanding CZS development. Therefore, we investigated the lymphocyte population profile of children who developed CZS and their mothers' immune response in this study. The study groups were formed from the Plaque Reduction Neutralization Test (PRNT) (CZS+ group) result. To evaluate the lymphocyte population profile, we performed phenotyping of peripheral lymphocytes and quantification of serum cytokine levels. The immunophenotyping and cytokine profile was correlated between CSZ+ children and their mothers. Both groups exhibited increased interleukin-17 levels and a reduction in the subpopulation of CD4+ T lymphocytes. In contrast, the maternal group showed a reduction in the population of B lymphocytes. Thus, the development of CZS is related to the presence of an inflammatory immune profile in children and their mothers characterized by Th17 activation.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies

TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating Bcells but overall normal humoral immune responses. Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

Effect of prophylactic antiviral intervention on T cell immunity in hepatitis B virus-infected pregnant women

BackgroundAntiviral intervention in hepatitis B virus (HBV)-infected pregnant women can effectively reduce mother-to-child transmission. However, the immunological characteristics of pregnant women with chronic HBV infection and the effects of antiviral intervention during pregnancy on maternal immune response remain unknown. We aimed to investigate these effects by comparing mothers who received antiviral intervention during pregnancy with those who did not.MethodsPregnant women positive for hepatitis B surface antigen and hepatitis B e-antigen (HBsAg+ HBeAg+) were enrolled at delivery, including 34 received prophylactic antiviral intervention during pregnancy (AVI mothers) and 15 did not (NAVI mothers). T lymphocyte phenotypes and functions were analysed using flow cytometry.ResultsAt delivery, maternal regulatory T cell (Treg) frequency in AVI mothers was significantly higher than that in NAVI mothers (P < 0.002), and CD4+ T cells in AVI mothers displayed a decreased ability to secrete IFN-γ (P = 0.005) and IL-21 (P = 0.043), but an increased ability to secrete IL-10 and IL-4 (P = 0.040 and P = 0.036), which represented a higher Treg frequency, enhanced Th2 response and suppressed Th1 response. Treg frequency among AVI mothers was correlated negatively with serum HBsAg and HBeAg levels. After delivery, the ability of CD4+ T cells or CD8+ T cells to secrete IFN-γ or IL-10 was similar and no significant difference in Treg frequency was found between the two groups.ConclusionsProphylactic antiviral intervention during pregnancy has an effect on T cell immunity in pregnant women, which was characterised by increased maternal Treg frequency, enhanced Th2 response and suppressed Th1 response at delivery.

Structural characterization of polysaccharide fractions in areca (Areca catechu L.) inflorescence and study of its immunological enhancement activity in vitro and in vivo

To obtain the structure–function relationship of the polysaccharides derived from areca (Areca catechu L.) inflorescences in the aspect of its immunomodulatory ability, the plant-based polysaccharide was isolated and purified on column chromatography. The purity, primary structure and immune activity of four polysaccharide fractions (AFP, AFP1, AFP2 and AFP2a) were characterized comprehensively. The main chain of AFP2a was confirmed to be composed of → 3,6)-β-D-Galp-(1→, with branch chains linked to the O-3 position on the main chain. The immunomodulatory activity of the polysaccharides was evaluated using the RAW264.7 cells and immunosuppression mice model. It was observed that AFP2a enabled greater NO release (49.72 μmol/L) than other fractions, significantly promoted the phagocytic activity of macrophages, and improved splenocyte proliferation and T lymphocyte phenotype in mice. The present results may shine a light on a new research direction in immunoenhancers and provide a theoretical foundation for the development and application of areca inflorescence.

Immune-Ageing Evaluation of Peripheral T and NK Lymphocyte Subsets in Chinese Healthy Adults

Ageing is often accompanied with a decline in immune system function, resulting in immune ageing. Numerous studies have focussed on the changes in different lymphocyte subsets in diseases and immunosenescence. The change in immune phenotype is a key indication of the diseased or healthy status. However, the changes in lymphocyte number and phenotype brought about by ageing have not been comprehensively analysed. Here, we analysed T and natural killer (NK) cell subsets, the phenotype and cell differentiation states in 43,096 healthy individuals, aged 20–88 years, without known diseases. Thirty-six immune parameters were analysed and the reference ranges of these subsets were established in different age groups divided into 5-year intervals. The data were subjected to random forest machine learning for immune-ageing modelling and confirmed using the neural network analysis. Our initial analysis and machine modelling prediction showed that naïve T cells decreased with ageing, whereas central memory T cells (Tcm) and effector memory T cells (Tem) increased cluster of differentiation (CD) 28-associated T cells. This is the largest study to investigate the correlation between age and immune cell function in a Chinese population, and provides insightful differences, suggesting that healthy adults might be considerably influenced by age and sex. The age of a person's immune system might be different from their chronological age. Our immune-ageing modelling study is one of the largest studies to provide insights into ‘immune-age’ rather than ‘biological-age’. Through machine learning, we identified immune factors influencing the most through ageing and built a model for immune-ageing prediction. Our research not only reveals the impact of age on immune parameter differences within the Chinese population, but also provides new insights for monitoring and preventing some diseases in clinical practice.