Lower proportion of intra-thyroidal B lymphocytes CD20+ associated to methimazole and lack of influence of iodide on lymphocyte subpopulations in Graves' disease.

Graves' disease (GD), an autoimmune thyroid disease, is one of the main autoimmune diseases in the general population. It is known that the pathophysiology of this disease may be related to immunological mechanisms dysregulation. These mechanisms can be influenced by GD therapies, such as iodide or antithyroid drugs (ATD). OBJECTIVE: Verify relation between clinical, biochemical and treatment modalities used prior to surgery and histopathological characteristics observed in total thyroidectomy products from patients previously diagnosed with Graves' disease. Furthermore, these data were related to composition of lymphocytic infiltrate in terms of proportions of lymphocytes CD4+, CD8+, CD25+ and CD20+. We aim to contribute to the understanding of the evolution pattern of GD, whose pathophysiology is not yet completely understood. METHODS: Cross-sectional study assessing thyroidectomy products for the presence of lymphocytic infiltrate, as well as the proportion and intensity of CD4+, CD8+, CD25+ and CD20+ markers. We selected 50 patients who underwent total or partial thyroidectomy in a tertiary service between 1996 and 2013 due to GD with histopathological confirmation. The control group (non-autoimmune disease group) consisted of 12 patients with histopathological data compatible with normal perilesional thyroid parenchyma. The intensity of lymphocytic infiltrate and immunohistochemical expression of the markers CD4+ (helper T lymphocytes), CD8+ (cytotoxic T lymphocytes), CD25+ (regulatory T lymphocytes) and CD20+ (B lymphocytes) were retrospectively evaluated and relationship with ultrasound, laboratory and clinical data was assessed. RESULTS: No differences were found in intensity, presence of lymphoid follicles, and expression of CD4+/CD8+/CD25+ in patients with GD who did or did not use ATD or iodide. In the group that did not use ATD, a higher proportion of CD20+ expression was found. The GD group was associated with hyperplastic epithelium and the control group was associated with simple epithelium. There was no difference in ultrasound thyroid volume between the groups. In GD patients with mild lymphocytic infiltrate, higher free thyroxin (FT4) levels were observed than those in patients with no infiltrate or moderate infiltrate. CONCLUSION: We found a lower proportion of intrathyroidal CD20+ B lymphocytes in patients under use of methimazole. However, no difference was observed in intrathyroidal lymphocyte subpopulations related to the short-term use of iodide. The understanding of thyroid autoimmunity, as well as identifying points of pharmacological modulation, are very important for advancement and improvement in treatments for these diseases.

Soluble MIC-A, IPI, and response to treatment strongly predict survival in patients with germinal center diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoma. MIC-A and MIC-B are the natural ligands for NKG2D, a receptor expressed in NK cells. MIC-A soluble isoforms (sMICA) have been described in different malignancies. OBJECTIVES: To analyze lymphocyte subsets and sMIC-A in germinal center DLBCL. MATERIALS AND METHODS: sMICA, sMICB, and peripheral blood lymphocyte subsets (CD4+, CD8+, NK, NKT, γδ T cells, and dendritic cells) were analyzed in 59 patients and 60 healthy donors. RESULTS: Patients had decreased numbers of type 1 and type 2 dendritic cells, NK, iNKT, CD4 T, and CD8 T cells, and higher levels of sMIC-A. The 2-year PFS for high IPI scores and high sMIC-A was 24% and 28%, respectively. The 2-year OS for high IPI scores and high sMIC-A was 42% and 33%. The 2-year PFS and OS for patients not achieving response to treatment were 0% and 10%, respectively. The MICPI score (one point each for high IPI score and high sMIC-A) showed that those patients summing two points had worse PSF and OS. CONCLUSIONS: Patients with DLBCL have decreased numbers of peripheral lymphocyte subsets and high levels of sMIC-A. The addition of sMIC-A to IPI could improve its prognostic relevance.

Comprehensive Immune Profiling Reveals CD56+ Monocytes and CD31+ Endothelial Cells Are Increased in Severe COVID-19 Disease.

Immune response dysregulation plays a key role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. In this study, we evaluated immune and endothelial blood cell profiles of patients with coronavirus disease 2019 (COVID-19) to determine critical differences between those with mild, moderate, or severe COVID-19 using spectral flow cytometry. We examined a suite of immune phenotypes, including monocytes, T cells, NK cells, B cells, endothelial cells, and neutrophils, alongside surface and intracellular markers of activation. Our results showed progressive lymphopenia and depletion of T cell subsets (CD3+, CD4+, and CD8+) in patients with severe disease and a significant increase in the CD56+CD14+Ki67+IFN-γ+ monocyte population in patients with moderate and severe COVID-19 that has not been previously described. Enhanced circulating endothelial cells (CD45-CD31+CD34+CD146+), circulating endothelial progenitors (CD45-CD31+CD34+/-CD146-), and neutrophils (CD11b+CD66b+) were coevaluated for COVID-19 severity. Spearman correlation analysis demonstrated the synergism among age, obesity, and hypertension with upregulated CD56+ monocytes, endothelial cells, and decreased T cells that lead to severe outcomes of SARS-CoV-2 infection. Circulating monocytes and endothelial cells may represent important cellular markers for monitoring postacute sequelae and impacts of SARS-CoV-2 infection during convalescence and for their role in immune host defense in high-risk adults after vaccination.

Type 1 Innate Lymphoid Cell and Natural Killer Cells Are Sources of Interferon-γ and Other Inflammatory Cytokines Associated With Distinct Clinical Presentation in Early Dengue Infection.

BACKGROUND: Increased levels of inflammatory cytokines are associated with severe dengue evolution, but the source of such hypercytokinemia is elusive. We investigated the contribution of innate lymphocytes, innate lymphoid cells (ILCs), and natural killer (NK) cells in cytokine production in early dengue infection. METHODS: Peripheral blood mononuclear cells of individuals with dengue without warning signs (DWS-) and dengue with warning signs and severe dengue (SD) presentation combined (DWS+) were obtained between 2 and 7 days since fever onset and submitted to flow cytometry without specific antigen stimulation to evaluate cytokines in ILC and NK cell subpopulations. RESULTS: ILCs and NK cells were found to be important sources of cytokines during dengue. ILCs of the DWS+/SD group displayed higher production of interferon gamma (IFN-γ) and interleukin (IL) 4/IL-13 when compared to DWS- individuals. On the other hand, NK Eomes+ cells of DWS- patients displayed higher IFN-γ production levels compared with the DWS+/SD group. Interestingly, when NK cells were identified by CD56 expression, DWS+/SD displayed higher frequency of IL-17 production compared with the DWS- group. CONCLUSIONS: These results indicate that ILCs and NK cells are important sources of inflammatory cytokines during acute dengue infection and display distinct profiles associated with different clinical forms.

Signatures of GVHD and relapse after posttransplant cyclophosphamide revealed by immune profiling and machine learning.

The key immunologic signatures associated with clinical outcomes after posttransplant cyclophosphamide (PTCy)-based HLA-haploidentical (haplo) and HLA-matched bone marrow transplantation (BMT) are largely unknown. To address this gap in knowledge, we used machine learning to decipher clinically relevant signatures from immunophenotypic, proteomic, and clinical data and then examined transcriptome changes in the lymphocyte subsets that predicted major posttransplant outcomes. Kinetics of immune subset reconstitution after day 28 were similar for 70 patients undergoing haplo and 75 patients undergoing HLA-matched BMT. Machine learning based on 35 candidate factors (10 clinical, 18 cellular, and 7 proteomic) revealed that combined elevations in effector CD4+ conventional T cells (Tconv) and CXCL9 at day 28 predicted acute graft-versus-host disease (aGVHD). Furthermore, higher NK cell counts predicted improved overall survival (OS) due to a reduction in both nonrelapse mortality and relapse. Transcriptional and flow-cytometric analyses of recovering lymphocytes in patients with aGVHD identified preserved hallmarks of functional CD4+ regulatory T cells (Tregs) while highlighting a Tconv-driven inflammatory and metabolic axis distinct from that seen with conventional GVHD prophylaxis. Patients developing early relapse displayed a loss of inflammatory gene signatures in NK cells and a transcriptional exhaustion phenotype in CD8+ T cells. Using a multimodality approach, we highlight the utility of systems biology in BMT biomarker discovery and offer a novel understanding of how PTCy influences alloimmune responses. Our work charts future directions for novel therapeutic interventions after these increasingly used GVHD prophylaxis platforms. Specimens collected on NCT0079656226 and NCT0080927627 https://clinicaltrials.gov/.

Tissue-Specific Molecular Markers and Heterogeneity in Type 2 Innate Lymphoid Cells.

Innate lymphoid cells (ILCs) are the most recently described group of lymphoid subpopulations. These tissue-resident cells display a heterogeneity resembling that observed on different groups of T cells, hence their categorization as cytotoxic NK cells and helper ILCs type 1, 2 and 3. Each one of these groups is highly diverse and expresses different markers in a context-dependent manner. Type 2 innate lymphoid cells (ILC2s) are activated in response to helminth parasites and regulate the immune response. They are involved in the etiology of diseases associated with allergic responses as well as in the maintenance of tissue homeostasis. Markers associated with their identification differ depending on the tissue and model used, making the study and understanding of these cells a cumbersome task. This review compiles evidence for the heterogeneity of ILC2s as well as discussion and analyses of molecular markers associated with their identity, function, tissue-dependent expression, and how these markers contribute to the interaction of ILC2s with specific microenvironments to maintain homeostasis or respond to pathogenic challenges.

Induction of food tolerance is dependent on intestinal inflammatory state.

Food allergies are usually managed by food avoidance. Hidden allergens in food, due to cross-contamination and/or allergenic additives added during production, place an important concern in today's increasing food allergy cases worldwide. Previous studies showed that the introduction of unacquainted food components, in an inflamed intestine, results in sensitization to this food. Thus, our aim was to evaluate the kinetics of multiple food allergy induction. Adult male C57BL/6 mice were divided into five groups, four of which were submitted to an intestinal inflammation induction protocol to peanuts. Egg white (OVA) diluted 1:5 v/v in distilled water was instilled by gavage 6h-before (PRIOR), concomitant (AT) and 6h-after (DURING) the onset of the peanut challenge diet. Positive control (POS CONT) and NEG CONT received saline per gavage. Finally, animals were challenged with subcutaneous injections of OVA. Results showed no changes in diet intake were observed. Anti-OVA polyisotypic IgG antibody titers significantly increased in AT. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in AT. Histomorphometrically, AT and DURING were classified as Infiltrative and Partial Destruction stages. PRIOR was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. Together, our results confirm that the introduction of unfamiliar food only a few hours before the initiation of a gut inflammation process is able to induce oral tolerance, however the introduction of a dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.

Hypoxia enhances ILC3 responses through HIF-1α-dependent mechanism.

Group 3 innate lymphoid cells (ILC3) have a prominent role in the maintenance of intestine mucosa homeostasis. The hypoxia-inducible factor (HIF) is an important modulator of immune cell activation and a key mechanism for cellular adaptation to oxygen deprivation. However, its role on ILC3 is not well known. In this study, we investigated how a hypoxic environment modulates ILC3 response and the subsequent participation of HIF-1 signaling in this process. We found increased proliferation and activation of intestinal ILC3 at low oxygen levels, a response that was phenocopied when HIF-1α was chemically stabilized and was reversed when HIF-1 was blocked. The increased activation of ILC3 relied on a HIF-1α-dependent transcriptional program, but not on mTOR-signaling or a switch to glycolysis. HIF-1α deficiency in RORyt compartment resulted in impaired IL-17 and IL-22 production by ILC3 in vivo, which reflected in a lower expression of their target genes in the intestinal epithelium and an increased susceptibility to Clostridiodes difficile infection. Taken together, our results show that HIF-1α activation in intestinal ILC3 is relevant for their functions in steady state and infectious conditions.

Associations among cytokines, EGF and lymphocyte subpopulations in patients diagnosed with advanced lung cancer.

Aging is considered the single most significant risk factor for the majority of common malignances including lung cancer. Together immunosenescence, changes occurring with aging in the immune system, and inflammaging, characterizes by a chronic, subclinical accumulation of pro-inflammatory factors, are suggested to stand at the origin of most of the diseases of the elderly, such as cancer. The aim of this study was to determine associations among lymphocyte subpopulations, pro-inflammatory cytokines and epidermal growth factor (EGF) in patients diagnosed with non-small cell lung cancer (NSCLC). Forty-six advanced NSCLC patients were enrolled. Sixteen patients with newly diagnosed and before treatment and 30 patients after first-line platinum-based chemotherapy. Peripheral blood subpopulations were studied by flow cytometry and serum concentrations of soluble factors by ELISA. The frequency of naïve CD4+ T cells, naïve B cells and central memory CD8+ T cells were significantly lower in NSCLC patients after chemotherapy, while effector memory CD4+ T cells and terminally differentiated CD8+ T cells were significantly higher. IL-1ß and TNFα significantly correlated among them before and after platinum-based chemotherapy. Terminally differentiated T cells expressing CD57+ significantly correlated with TNFα and IL-1ß. For the first time, associations between EGF serum levels and terminally differentiated CD4+ T cells, and memory B cells were detected. This study confirms the association among terminally differentiated lymphocytes and pro-inflammatory cytokines in patients diagnosed with lung cancer, reinforcing the interconnection between terminally differentiated lymphocytes and pro-inflammatory cytokines. Clinical trial registration number: RPCEC00000205, http://registroclinico.sld.cu/.

Contribution of Gut Microbiota to Immune Tolerance in Infants.

The prevalence of food allergy has increased in recent years, especially among the pediatric population. Differences in the gut microbiota composition between children with FA and healthy children have brought this topic into the spotlight as a possible explanation for the increase in FA. The gut microbiota characteristics are acquired through environmental interactions starting early in life, such as type of delivery during birth and breastfeeding. The microbiota features may be shaped by a plethora of immunomodulatory mechanisms, including a predominant role of Tregs and the transcription factor FOXP3. Additionally, a pivotal role has been given to vitamin A and butyrate, the main anti-inflammatory metabolite. These observations have led to the study and development of therapies oriented to modifying the microbiota and metabolite profiles, such as the use of pre- and probiotics and the determination of their capacity to induce tolerance to allergens that are relevant to FA. To date, evidence supporting these approaches in humans is scarce and inconclusive. Larger cohorts and dose-titration studies are mandatory to evaluate whether the observed changes in gut microbiota composition reflect medical recovery and increased tolerance in pediatric patients with FA. In this article, we discuss the establishment of the microbiota, the immunological mechanisms that regulate the microbiota of children with food allergies, and the evidence in research focused on its regulation as a means to achieve tolerance to food allergens.

Immune Monitoring of Patients With Primary Immune Regulation Disorders Unravels Higher Frequencies of Follicular T Cells With Different Profiles That Associate With Alterations in B Cell Subsets.

Primary immune regulation disorders lead to autoimmunity, allergy and inflammatory conditions due to defects in the immune homeostasis affecting different T, B and NK cell subsets. To improve our understanding of these conditions, in this work we analyzed the T and B cell compartments of 15 PID patients with dysregulation, including 3 patients with STAT1 GOF mutation, 7 patients with CVID with dysregulation, 3 patients with mutations in CTLA4, 1 patient with CD25 mutation and 1 patient with STAT5b mutation and compared them with healthy donors and with CVID patients without dysregulation. CD4+ and CD8+ T cells from the patients exhibited a significant decreased frequency of naïve and regulatory T cells with increased frequencies of activated cells, central memory CD4+ T cells, effector memory CD8+ T cells and terminal effector CD8+ T cells. Patients also exhibited a significantly increased frequency of circulating CD4+ follicular helper T cells, with altered frequencies of cTfh cell subsets. Such cTfh cells were skewed toward cTfh1 cells in STAT1 GOF, CTLA4, and CVID patients, while the STAT5b deficient patient presented a skew toward cTfh17 cells. These alterations confirmed the existence of an imbalance in the cTfh1/cTfh17 ratio in these diseases. In addition, we unraveled a marked dysregulation in the B cell compartment, characterized by a prevalence of transitional and naïve B cells in STAT1 GOF and CVID patients, and of switched-memory B cells and plasmablast cells in the STAT5b deficient patient. Moreover, we observed a significant positive correlation between the frequencies cTfh17 cells and switched-memory B cells and between the frequency of switched-memory B cells and the serum IgG. Therefore, primary immunodeficiencies with dysregulation are characterized by a skew toward an activated/memory phenotype within the CD4+ and CD8+ T cell compartment, accompanied by abnormal frequencies of Tregs, cTfh, and their cTfh1 and cTfh17 subsets that likely impact on B cell help for antibody production, which likely contributes to their autoimmune and inflammatory conditions. Therefore, assessment of these alterations by flow cytometry constitutes a simple and straightforward manner to improve diagnosis of these complex clinical entities that may impact early diagnosis and patients' treatment. Also, our findings unravel phenotypic alterations that might be associated, at least in part, with some of the clinical manifestations observed in these patients.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria.

BACKGROUND: Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE: To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS: Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS: Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1ß and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS: Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

Lymphocyte responses to Mycobacterium tuberculosis and Mycobacterium bovis are similar between BCG-vaccinated patients with cystic fibrosis and healthy controls.

BACKGROUND: The low rate of nontuberculous mycobacteria (NTM) among Brazilian patients with cystic fibrosis (CF) may be due to cross-reactive Bacille Calmette-Guérin (BCG) vaccination. In the present pilot study, we aimed to compare the lymphocyte responses against Mycobacterium tuberculosis(Mtb) and Mycobacterium bovis (BCG) in BCG-vaccinated CF patients and healthy controls. METHODS: The lymphocyte responses of CF patients (n = 10) and healthy controls (n = 10) were assessed in terms of lymphocyte proliferation index (LPI), using flow cytometry. Median rates of each cell subtype - CD4, CD8, γδ T cells and CD19 (B) cells - were also determined. RESULTS: Median LPIs (CF vs. controls) were 22.9% vs. 13.0% (p = 0.481) and 23.1% vs. 17.6% (p = 0.481), upon stimulation with Mtb and BCG, respectively. Both groups had a predominant CD4 T cell response to Mtb (median rate = 82.5% vs. 79.7%; p = 0.796) and BCG (LPI = 84.3% vs. 83.0%; p = 0.853), which were significantly higher than the CD8, CD19 and γδ responses within both groups. CF patients tended to have a higher CD8 T cell response upon stimulation with the phytohemagglutinin mitogen than healthy controls (median rate = 42.8% vs. 31.7%, p = 0.075). CONCLUSION: The responses of BCG-vaccinated CF patients to Mtb and BCG are at least similar to those of healthy individuals. These are probably memory responses elicited by the BCG vaccination, which can cross-react with NTM and may explain the low frequency of NTM lung infection in our CF center.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria

BACKGROUND Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1β and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

Cocaine Use Disorder Is Associated With Changes in Th1/Th2/Th17 Cytokines and Lymphocytes Subsets.

Background: Cocaine is a psychostimulant drug with high addictive proprieties. Evidence suggests that cocaine use leads to critical changes in the immune system, with significant effects on T, B, and natural killer (NK) cells and influencing peripheral levels of cytokines. The presence of abstinence-related symptoms during detoxification treatment is known to influence the prognosis. Here, our aim was to investigate immune profiles in women with cocaine use disorder (CUD) according to withdrawal symptoms severity. Methods: Blood samples and clinical data were collected at onset of detoxification treatment of 50 women with CUD. The patients were stratified according to Cocaine Selective Severity Assessment (CSSA) scores in low withdrawal (L-W) and high withdrawal (H-W) categories. In addition, we also included a control group with 19 healthy women as reference to immune parameters. Peripheral blood was collected and lymphocyte subsets were phenotyped by multi-color flow cytometry (B cells, CD4+ T, CD8+ T, NK cells, and different stages of T-cell differentiation). PBMCs from patients and healthy controls were stimulated in vitro with phytohemagglutinin (1%) for 72 h to assess the production of Th1/Th2/Th17 cytokines. Results: Following stimulation, lymphocytes from women with CUD produced increased levels of Th1/Th2/Th17 cytokines. However, higher levels of IL-2 and IL-17 were observed only in the L-W group, while higher levels of IL-6 were detected in the H-W group compared to controls. H-W group showed lower percentage of early-differentiated Th cells (CD4+CD27+CD28+), elevated percentage of Th cells (CD3+CD4+), intermediate-differentiated Th cells (CD4+CD27-CD28+), and B cells (CD3-CD19+). Both CUD groups showed decreased percentages of naïve T cells (CD3+CD4+CD45RA+ and CD3+CD8+CD45RA+). Conclusion: Our data demonstrated that CUD can lead to increased production of Th1/Th2/Th17 cytokines and lymphocyte changes.

Normal Values of T, B and NK Lymphocyte Subpopulations in Peripheral Blood of Healthy Cuban Adults.

INTRODUCTION Quantification of lymphocyte subpopulations is useful for evaluating immune response in states of health and disease, including immunodeficiencies, autoimmunity, infections and cancer. Studies have found that concentrations and proportions of different cell subpopulations vary with geographic location, age, sex and ethnicity. Knowing the normal values of these cells and their variation in healthy populations will contribute to improved clinical practice and scientific research. OBJECTIVE Estimate normal absolute concentrations and percentages of the most abundant lymphocyte subpopulations in peripheral blood and their relation to sex and age. METHODS A cross-sectional analysis was conducted in 129 healthy adults, 61 men and 68 women aged 18-80 years; 89 aged <50 years and 40 ≥50 years. We included individuals who agreed to participate by written informed consent. Exclusion criteria were chronic disease, or use of tobacco, alcohol or medications that can alter immune system cell numbers and functions. Through dual platform flow cytometry, we determined absolute and percentage values for T lymphocyte subsets CD3+, CD3+/CD4+T, CD3+/CD8+T, CD19+ B cells and CD3-/CD56+ natural killer cells in peripheral blood, using an 8-color flow cytometer. We estimated medians and the 2.5 and 97.5 percentiles and calculated the Pearson correlation coefficient to evaluate associations. Significance tests were also used to compare groups. The significance threshold was p = 0.05 in all cases. RESULTS Ranges of absolute values and percentages (%) were: total lymphocytes: 1200-3475 cells/µL (20.2-49.3); CD3+ T cells: 880-2623 cells/µL (56.5-84.7); CD3+/CD4+ T cells: 479-1792 cells/µL (30.3-55.7); CD3+/CD8+ T cells: 248-1101 cells/µL (13.2-42.9); CD19+ B cells: 114-1491 cells/µL (5.4-49.5); CD3-/CD56+ natural killer cells: 70-652 cells/µL (3.7-28.0); and the CD4+:CD8+ index: 0.80-3.92. Absolute numbers--but not percentages--of lymphocytes and CD3+ T cells were higher in those <50 years (p = 0.025 and 0.020, respectively). Absolute values and relative percentages of CD3+/CD8+ and relative values of CD3+/ CD4+ T cells were significantly higher in the younger subgroup (p = 0.004 and p = 0.047). Age was not associated significantly with B lymphocytes or natural killer cells. Absolute and relative values ​​of CD3+/CD4+ T lymphocytes were significantly higher in women (p = 0.009 and 0.036, respectively). CONCLUSIONS. Absolute numbers of total lymphocytes and T and CD3+/CD8+ T lymphocytes are higher in younger individuals. In percentage values, CD3+/CD4+ T lymphocytes are lower in older persons. Absolute and percentage values ​​of CD3+/CD4+ T phenotype are higher in women. These differences justify adjusting clinical analyses to different values ​​by age and sex. KEYWORDS T lymphocytes, B lymphocytes, normal values, flow cytometry, age, sex, Cuba.

TLR9 agonist adsorbed to alum adjuvant prevents asthma-like responses induced by Blomia tropicalis mite extract.

Blomia tropicalis mite is highly prevalent in tropical and subtropical regions and it is associated with allergic diseases such as rhinitis and asthma. By using an OVA-model of allergic lung disease, we have previously shown that sensitization in the presence of toll like receptors (TLRs) agonists attenuates subsequent OVA-induced allergic responses. Here, we evaluated the effect of CpG-ODN, a specific synthetic TLR-9 agonist, on the development of experimental asthma induced by Blomia tropicalis extract, a relevant source of aeroallergens. Among different protocols of Blomia tropicalis extract sensitization, the subcutaneous sensitization in the presence of alum adjuvant induced the highest Th2 responses, including high IgE levels. Adsorption of CpG to Blomia tropicalis extract/Alum attenuated the airway hyperreactivity, the infiltration of inflammatory cells including eosinophils, and the IL-5 content in BAL. In addition, lung peribronchial inflammatory infiltrate, mucus production and IL-5-producing CD3+ CD4+ T cells were significantly reduced in the Blomia tropicalis extract/Alum+CpG group. Importantly, CpG inhibited total IgE production as well as active systemic or cutaneous anaphylaxis reactions. Inhibition of pulmonary Th2 responses was associated with increased IL-10 production but not with IFN-γ production. Notably, in IL-10-deficient mice, sensitization with OVA/Alum+CpG resulted in intense lung neutrophilia and IFN-γ production, indicating that IL-10 is necessary to inhibit subsequent Th1 immunity. Our work highlights the mechanisms of allergy attenuation by CpG and it indicates the potential use of Alum-based formulation with CpG to treat allergic processes.

Managing costs in primary immunodeficiency: minimal immunophenotyping and three national references.

Our aim was to evaluate the cost-effectiveness of a minimal lymphocyte subset quantification (LSQ) by flow cytometry as the first screening in children with clinically suspected primary immunodeficiency (PID). Two hundred sixty-eight Brazilian patients (0-21 years old) were studied. They were divided by clinical and phenotypical features into those fulfilling criteria for PID (PID phenotype) according to the 2017 International Union of Immunological Societies (IUIS) classification and those not fulfilling these criteria (non-PID phenotype). We evaluated how many patients had values below the 10th percentile for five lymphocyte subsets in peripheral blood, (suggestive of PID) according to reference values for Brazil, Italy and USA. Three lymphocyte subsets (T CD3/CD4, B CD19 and NK CD16/CD56) had p-value < 0.05 and Odds Ratio (OR) indicating a risk at least two times higher for the diagnosis of a PID phenotype. The application of Kappa coefficient (k) on Brazilian vs Italian and Brazilian vs US data sets resulted in k compatible with strong or excellent level of agreement between the three classification systems. The authors conclude that a number of CD3+ /CD4+ , CD19+ and CD16+ /CD56+ (NK) cells in peripheral blood <10th percentile represented a significant risk for the diagnosis of PID in this cohort. Natural killer (NK) deficiency is quite rare and has a very specific clinical profile. So, the analysis of these cells could be requested only in some cases, saving even more costs. The minimal immunophenotyping, with quantification of T CD4+ , CD19+ and in some cases CD16+ /CD56+ cells, may be a useful tool for the first screening of PID, saving costs, especially in developing countries.

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage.

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL-17A+) and Th1 (T-bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal-periportal (P-P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P-P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg-positive patients and was associated with hepatitis severity and higher frequency of P-P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg-negative cases with less severe hepatitis grade and lower frequency of P-P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.