Total B-cell Research Articles

Obinutuzumab as a viable therapeutic strategy in rituximab-refractory childhood frequently relapsing, steroid-dependent nephrotic syndrome that relapsed during B-cell depletion.

A subgroup of children with frequently-relapsing, steroid-dependent nephrotic syndrome relapse during B-cell depletion after rituximab. A 15-year-old boy with focal segmental glomerulosclerosis became rituximab-refractory after 5 courses of treatments, with a relapse-free period shortened to 1month. Circulating total and memory B-cells were undetectable at the time of relapse. A single infusion of obinutuzumab sustained relapse-free remission up to the last follow-up at 18months. There was persistent hypogammaglobulinemia but no infection was observed. Obinutuzumab may be a viable option for attaining long-term remission with reasonable side effect profiles in patients who relapse during B-cell depletion after rituximab.

Aconitate decarboxylase 1 mediates the acute airway inflammatory response to environmental exposures.

Environmental lipopolysaccharide (LPS) and microbial component-enriched organic dusts cause significant lung disease. These environmental exposures induce the recruitment and activation of distinct lung monocyte/macrophage subpopulations involved in disease pathogenesis. Aconitate decarboxylase 1 (Acod1) was one of the most upregulated genes following LPS (vs. saline) exposure of murine whole lungs with transcriptomic profiling of sorted lung monocyte/macrophage subpopulations also highlighting its significance. Given monocyte/macrophage activation can be tightly linked to metabolism, the objective of these studies was to determine the role of the immunometabolic regulator ACOD1 in environmental exposure-induced lung inflammation. Wild-type (WT) mice were intratracheally (i.t.) instilled with 10 μg of LPS or saline. Whole lungs were profiled using bulk RNA sequencing or sorted to isolate monocyte/macrophage subpopulations. Sorted subpopulations were then characterized transcriptomically using a NanoString innate immunity multiplex array 48 h post-exposure. Next, WT and Acod1-/- mice were instilled with LPS, 25% organic dust extract (ODE), or saline, whereupon serum, bronchoalveolar lavage fluid (BALF), and lung tissues were collected. BALF metabolites of the tricarboxylic acid (TCA) cycle were quantified by mass spectrometry. Cytokines/chemokines and tissue remodeling mediators were quantitated by ELISA. Lung immune cells were characterized by flow cytometry. Invasive lung function testing was performed 3 h post-LPS with WT and Acod1-/- mice. Acod1-/- mice treated with LPS demonstrated decreased BALF levels of itaconate, TCA cycle reprogramming, decreased BALF neutrophils, increased lung CD4+ T cells, decreased BALF and lung levels of TNF-α, and decreased BALF CXCL1 compared to WT animals. In comparison, Acod1-/- mice treated with ODE demonstrated decreased serum pentraxin-2, BALF levels of itaconate, lung total cell, neutrophil, monocyte, and B-cell infiltrates with decreased BALF levels of TNF-α and IL-6 and decreased lung CXCL1 vs. WT animals. Mediators of tissue remodeling (TIMP1, MMP-8, MMP-9) were also decreased in the LPS-exposed Acod1-/- mice, with MMP-9 also reduced in ODE-exposed Acod1-/- mice. Lung function assessments demonstrated a blunted response to LPS-induced airway hyperresponsiveness in Acod1-/- animals. Acod1 is robustly upregulated in the lungs following LPS exposure and encodes a key immunometabolic regulator. ACOD1 mediates the proinflammatory response to acute inhaled environmental LPS and organic dust exposure-induced lung inflammation.

Essential roles of B-cell subsets in the progression of MASLD and HCC

Essential roles of B-cell subsets in the progression of MASLD and HCC

Multi-dimensional analysis of B cells reveals the expansion of memory and regulatory B cell clusters in humans living in rural tropical areas.

B-cells play a critical role in the formation of immune responses against pathogens by acting as antigen-presenting cells, by modulating immune responses and by generating immune memory and antibody responses. Here, we studied B-cell subset distributions between regions with higher and lower microbial exposure, i.e. by comparing peripheral blood B-cells from people living in Indonesia or Ghana to those from healthy Dutch residents using a 36-marker mass cytometry panel. By applying an unbiased multidimensional approach, we observed differences in the balance between the naïve and memory compartments, with higher CD11c+ and double negative (DN-IgDnegCD27neg) memory (M)B-cells in individuals from rural tropical areas, and conversely lower naïve B-cells compared to residents from an area with less pathogen exposure. Furthermore, characterization of total B-cell populations, CD11c+, DN and Breg cells showed the emergence of specific memory clusters in individuals living in rural tropical areas. Some of these differences were more pronounced in children compared to adults and suggest that a higher microbial exposure accelerates memory B cell formation, which 'normalizes' with age.

Characteristics of Early Antibody Mediated Rejection in Antibody Incompatible Living Donor Kidney Transplantation.

Antibody incompatible transplantation (AIT) may be an only option for highly sensitized patients. Severe form of early antibody mediated rejection (AMR) adversely affects graft survival after AIT. The aim of this study was to identify individuals at risk of AMR. We analyzed 213 living donor AITs performed at our center. Among 120 ABOi, 58 HLAi and 35 DSA + FCXM-negative cases, the rates of early AMR were 6%, 31%, and 9%, respectively (p < 0.001). On multivariate analysis for graft loss, early AMR had a HR of 3.28 (p < 0.001). The HLAi group had worse death-censored graft survival (p = 0.003). In the HLAi group, Patients with aggressive variant AMR (AAMR) had greater percentage of C3d complement fixing DSA, higher baseline class I and total DSA MFI levels and B-cell FCXM RMF. C1q and C3d complement fixing DSA and strong positivity of baseline B- or T-cell FXCM as predictors of AAMR had 100% sensitivity. Early AMR is of significant clinical concern in AIT as it results in poor graft survival and is not well described in literature. An aggressive variant is characterized by massive rise in DSA levels at rejection. Baseline DSA, C1q, and C3d and baseline FCXM values can be used to risk-stratify candidates for AIT.

Glycolysis Changes in Alloreactive Memory B Cells in Highly Sensitized Kidney Transplant Recipients Undergonig Desensitization Therapy.

Despite the growing use of desensitization strategies, hyperimmune patients remain at high risk of antibody-mediated rejection suggesting that, even when donor-specific antibodies (DSA) are effectively depleted, anti-donor specific B cells persist. We included 10 highly sensitized recipients that underwent desensitization with plasmapheresis and B cell depletion prior to kidney transplantation. We quantified changes in DSA (luminex), total B-cell subsets (flow cytometry), anti-donor HLA B cells (fluorospot), and single-cell metabolism in serially collected samples before desensitization, at the time of transplant, and at 6 and 12months thereafter. Desensitization was associated with a decrease in DSA and total memory B cell and naive B cell percentage, while plasma cells and memory anti-donor HLA circulating B cells persisted up to 12months after transplant. At 12-month post-transplantation, memory B cells increased their glycolytic capacity, while proliferative KI67+ plasma cells modified their metabolism by increasing fatty acid and amino acid oxidation capacity and decreasing their glucose dependence. Despite effective DSA depletion, anti-donor B cells persist in kidney transplant recipients. Due to the reliance of these cells on glycolysis, glycolysis-targeting therapies might represent a valuable treatment strategy.

Increased Natural Killer (NK)-cell cytotoxicity and Trypanosoma cruzi-specific memory B cells in subjects with discordant serology for Chagas disease

The presence of memory T cell specific for Trypanosoma cruzi in subjects with discordant serology for Chagas disease supports a cleared infection in these subjects. Using high-dimensional flow cytometry, ELISPOT assays and quantitative PCR, antibody-secreting cells and memory B cells specific for T. cruzi, total B-cell phenotypes, innate immune responses and parasite DNA were evaluated in serodiscordant, seropositive and seronegative subjects for T. cruzi infection. T. cruzi-specific memory B cells but no antibody-secreting cells specific for T. cruzi, increased proportion of nonclassical monocytes and increased levels of polyfunctional NK cells were found in serodiscordant compared with seropositive subjects. None of the serodiscordant subjects evaluated showed detectable parasite DNA, most of them did not show cardiac abnormalities and a group of them had had confirmed positive serology for Chagas disease. The unique immune profiles in serodiscordant subjects support that T. cruzi infection was cleared or profoundly controlled in these subjects.

B-cells in pulmonary arterial hypertension: friend, foe or bystander?

There is an unmet need for new therapeutic strategies that target alternative pathways to improve the prognosis of patients with pulmonary arterial hypertension (PAH). As immunity has been involved in the development and progression of vascular lesions in PAH, we review the potential contribution of B-cells in its pathogenesis and evaluate the relevance of B-cell-targeted therapies. Circulating B-cell homeostasis is altered in PAH patients, with total B-cell lymphopenia, abnormal subset distribution (expansion of naïve and antibody-secreting cells, reduction of memory B-cells) and chronic activation. B-cells are recruited to the lungs through local chemokine secretion, and activated by several mechanisms: 1) interaction with lung vascular autoantigens through cognate B-cell receptors; 2) costimulatory signals provided by T follicular helper cells (interleukin (IL)-21), type 2 T helper cells and mast cells (IL-4, IL-6 and IL-13); and 3) increased survival signals provided by B-cell activating factor pathways. This activity results in the formation of germinal centres within perivascular tertiary lymphoid organs and in the local production of pathogenic autoantibodies that target the pulmonary vasculature and vascular stabilisation factors (including angiotensin-II/endothelin-1 receptors and bone morphogenetic protein receptors). B-cells also mediate their effects through enhanced production of pro-inflammatory cytokines, reduced anti-inflammatory properties by regulatory B-cells, immunoglobulin (Ig)G-induced complement activation, and IgE-induced mast cell activation. Precision-medicine approaches targeting B-cell immunity are a promising direction for select PAH conditions, as suggested by the efficacy of anti-CD20 therapy in experimental models and a trial of rituximab in systemic sclerosis-associated PAH.

Роль клеточного иммунитета в развитии злокачественных новообразований у лиц, подвергшихся хроническому облучению

Some long-term effects of radiation could be related to changes in the immune system resulting from radiation exposure. Immunity disorders caused by radiation can influence carcinogenesis. Cellular immunity factors were investigated in peripheral blood of workers chronically exposed to occupational combined radiation (external gamma-rays and internal alpha-particles), with malignant neoplasms diagnosed after blood samples were taken or without them, and in the control group. The aim of this study was examine effects of radiation on the cellular immunity status in individuals chronically exposed to ionizing radiation who had malignant neoplasms developed after blood sampling. The relative and absolute number of lymphocyte subpopulations (total T-cells, T-helpers, T-cytotoxic, total B-cells, NK-cells, NKT-cells and activated T-cells) was detected by flow cytofluorometry. The absolute number of T-cells was significantly reduced in workers chronically exposed to occupational combined irradiation, with or without malignancies, compared to the control, which may contribute to tumor progression at an early stage of its onset. At the same time, workers without malignancies had a significant increase in the relative number of T-cytotoxic lymphocytes, which may be a factor preventing tumor development. A significant increase in the relative number of natural killer cells (NK cells) was detected in individuals with malignant neoplasms chronically exposed to occupational combined irradiation, compared with the control, which may indicate enhanced antitumor defense that developed in response to exposure to tumor antigens. In addition, a significant decrease in the absolute and relative number of T- and B-lymphocytes was found in the group of workers with malignant neoplasm, compared with the control. A significant increase in the relative number of T-helpers was found in both groups of workers. Since the role of T-helpers in the antitumor response is ambiguous, additional research on types of T-helpers is planned to clarify the results of the present study

Role of cellular immunity in malignant tumors development in individuals chronically ex-posed to ionising radiation

Some long-term effects of radiation could be related to changes in the immune system resulting from radiation exposure. Immunity disorders caused by radiation can influence carcinogenesis. Cellular immunity factors were investigated in peripheral blood of workers chronically exposed to occupational combined radiation (external gamma-rays and internal alpha-particles), with malignant neoplasms diagnosed after blood samples were taken or without them, and in the control group. The aim of this study was examine effects of radiation on the cellular immunity status in individuals chronically exposed to ionizing radiation who had malignant neoplasms developed after blood sampling. The relative and absolute number of lymphocyte subpopulations (total T-cells, T-helpers, T-cytotoxic, total B-cells, NK-cells, NKT-cells and activated T-cells) was detected by flow cytofluorometry. The absolute number of T-cells was significantly reduced in workers chronically exposed to occupational combined irradiation, with or without malignancies, compared to the control, which may contribute to tumor progression at an early stage of its onset. At the same time, workers without malignancies had a significant increase in the relative number of T-cytotoxic lymphocytes, which may be a factor preventing tumor development. A significant increase in the relative number of natural killer cells (NK cells) was detected in individuals with malignant neoplasms chronically exposed to occupational combined irradiation, compared with the control, which may indicate enhanced antitumor defense that developed in response to exposure to tumor antigens. In addition, a significant decrease in the absolute and relative number of T- and B-lymphocytes was found in the group of workers with malignant neoplasm, compared with the control. A significant increase in the relative number of T-helpers was found in both groups of workers. Since the role of T-helpers in the antitumor response is ambiguous, additional research on types of T-helpers is planned to clarify the results of the present study.

Peripheral Blood B-Cell Subsets Frequency and Distribution and the BSF-2(IL-6) to CSIF:TGIF(IL-10) Ratio as Severity-Associated Signatures in Primary Open-Angle Glaucoma: A Case-Controlled Study.

Flow cytometry was used to determine B-cell subset frequencies from 30 POAG patients grouped by hierarchical cluster analysis or the mean deviation (MD) of the visual field (VF) and correlated with the patients' clinical and pathological data, as well as with BSF-2(IL-6) and CSIF:TGIF(IL-10), which were quantified in peripheral blood samples of patients and controls by ELISA. The total B-cell frequency was increased in the POAG group in comparison to the control group (n = 30). Frequencies of specific B-cell subsets, such as double-negative (DN) and naïve B-cell subsets, were increased in relation to the severity of the POAG disease. However, the unswitched memory B compartment subset decreased in the POAG group. Other non-typical B-cell subsets such as DN B cells also showed significant changes according to the POAG disease severity course. These differences allow us to identify POAG severity-associated inflammatory clusters in patients with specifically altered B-cell subsets. Finally, ocular parameters, biomarkers of inflammation, and other glaucoma-related or non-clinical scores exhibited correlations with some of these B-cell subpopulations. The severity of the POAG disease course is accompanied by changes in the B-cell subpopulation, namely, DN B cells. Furthermore, the existing relationship of the B-cell subset frequencies with the clinical and the inflammatory parameters BSF-2(IL-6), CSIF:TGIF(IL-10), and the BSF-2(IL-6) to CSIF:TGIF(IL-10) ratio suggests that these B lymphocyte cells could serve as potential molecular bio-markers for assessing POAG disease severity and/or progression.

Momordicine-I Suppresses Head and Neck Cancer Growth by Reprogrammimg Immunosuppressive Effect of the Tumor-Infiltrating Macrophages and B Lymphocytes.

Head and neck cancer (HNC) is prevalent worldwide, and treatment options are limited. Momordicine-I (M-I), a natural component from bitter melon, shows antitumor activity against these cancers, but its mechanism of action, especially in the tumor microenvironment (TME), remains unclear. In this study, we establish that M-I reduces HNC tumor growth in two different immunocompetent mouse models using MOC2 and SCC VII cells. We demonstrate that the anticancer activity results from modulating several molecules in the monocyte/macrophage clusters in CD45+ populations in MOC2 tumors by single-cell RNA sequencing. Tumor-associated macrophages (TAM) often pose a barrier to antitumor effects, but following M-I treatment, we observe a significant reduction in the expression of Sfln4, a myeloid cell differentiation factor, and Cxcl3, a neutrophil chemoattractant, in the monocyte/macrophage populations. We further find that the macrophages must be in close contact with the tumor cells to inhibit Sfln4 and Cxcl3, suggesting that these TAMs are impacted by M-I treatment. Coculturing macrophages with tumor cells shows inhibition of Agr1 expression following M-I treatment, which is indicative of switching from M2 to M1 phenotype. Furthermore, the total B-cell population in M-I-treated tumors is significantly lower, whereas spleen cells also show similar results when cocultured with MOC2 cells. M-I treatment also inhibits PD1, PD-L1, and FoxP3 expression in tumors. Collectively, these results uncover the potential mechanism of M-I by modulating immune cells, and this new insight can help to develop M-I as a promising candidate to treat HNCs, either alone or as adjuvant therapy.

Tobacco exposure, but not aging, shifts the frequency of peripheral blood B cell subpopulations.

Several disturbances in T-cell mediated immunity have been described during aging, but immunosenescence of the B-cell compartment is less well elucidated. The peripheral blood B-cell compartment (CD19+) can be split into six main subpopulations according to the cell surface markers IgD, CD27, CD24, and CD38: Transitional, naïve, unswitched, switched, double negative and plasmablasts. We thus aimed to verify whether shifts in these subsets occur during healthy and pathological aging. We recruited three groups of aged people (> 60 years old), healthy, COPD patients, and smokers without altered pulmonary function test, and a fourth group of individuals 18-40 years old (youngs). Total B-cells percentage and absolute number were similar among the healthy aged, COPD patients, and youngs, but the smokers showed significantly higher absolute numbers. While all six B-cell subset percentages were comparable among the healthy aged, COPD patients, and youngs, smokers showed significantly higher percentages of switched B-cells and reduced naïve B-cells than the other three groups, resulting in an inverted naive:switched ratio. Analysis of the cell subset absolute numbers showed a similar trend. Overall, our results suggest that aging drives milder alterations in the distribution of peripheral blood B-cell subpopulations than in the T-cell compartment. We suggest that it is the T-cell immunosenescence that most contributes to the poor humoral immune responses in the elderly, vaccine responses included. Surprisingly it was the smokers who showed significant alterations when compared with the youngs, healthy aged, and aged COPD patients, probably as a result of the chronic immune stimulation described in active smoking subjects.

Polytypic B-Cells, B-Cell Lymphoproliferative Disorders/Lymphomas, and Neoplastic T-Cells Divergently Differentiate from TET2-/ DNMT3A-Mutated Clonal Hematopoiesis in Patients with Follicular Helper T-Cell Lymphomas/Lymphoproliferative Disorders

Introduction: Patients with follicular helper T-cell lymphomas/lymphoproliferative disorders (TFH-LPDs) commonly develop B-cell lymphoproliferative disorders and lymphomas (B-LPDs), but the pathophysiology is incompletely understood. TFH-LPDs often arise from TET2-/ DNMT3A-mutated clonal hematopoiesis (CH). Here we show TET2/ DNMT3A mutations are commonly shared by polytypic B-cells (PBCs), B-LPDs, and TFH-LPDs, with the latter two often carrying additional unique mutations. Methods: Pathology database search at Memorial Sloan Kettering identified patients with TFH-LPDs and PBCs or B-LPDs available for genotyping. If available, myeloid cells were also genotyped. PBCs lacked aberrancies by morphology and flow cytometry (FC) immunophenotyping (including polytypic light chain expression). TFH-LPDs and B-LPDs were defined using International Consensus Classification criteria. Genotyping assays included targeted next-generation sequencing (NGS) with (n=13) or without (n=8; cases 1, 4, 9, 10, 15, 16, 17, 22) a matched germline control and droplet digital PCR (n=4; cases 2, 3, 5, 6). Genotyping was performed on bulk samples or FC-sorted cells, including T-, B-, and myeloid as available. In bulk samples, mutations with allele frequencies (VAFs) &amp;gt;2x the percentage of neoplastic T- and myeloid cells combined in B-LPD samples and of neoplastic T- and total B-cells in bulk blood (PB)/bone marrow (BM) samples were considered present in B-LPD or myeloid compartments, respectively. A &amp;gt;2% VAF cutoff was utilized. EBV status was assessed using EBER in situ hybridization on tissues, serum EBV DNA by quantitative PCR, and/or evaluation of off target EBV reads by NGS. Statistical analyses utilized Mann-Whitney U tests. Results: In total, 25 patients (10 female, 15 male; median age 70 [range 38-82] years) with TFH-LPDs (19 TFH lymphoma, angioimmunoblastic type, 4 TFH lymphoma, NOS, 2 T-LPD with TFH phenotype [defined by morphologically and/or immunophenotypically atypical T-cells with both TFH-like phenotypes and genomic alterations that did not fulfill morphologic criteria for diagnosis of lymphoma]) were identified. The TFH-LPDs harbored TET2 (25/25, 100%), DNMT3A (12/25, 48%), RHOA p.G17 (15/25, 60%), and IDH2 p.R172 (9/25, 36%) mutations (Figure 1A). PBCs from 11 patients were evaluated (7 EBV+, 3 EBV-, 1 EBV unknown; 5 from lymph node, 4 PB, 2 BM). PBCs from 7/11 (64%) shared identical TET2 (n=6; median VAF 0.11 [range 0.02 - 0.27]) and/or DNMT3A (n=3; median VAF 0.05 [range 0.03-0.49]) mutations with corresponding TFH-LPDs. PBCs in 1 patient harbored a unique mutation ( TET2). Four of 8 (50%) patients in which myeloid cells were assessed harbored identical TET2 and/or DNMT3A mutations in the myeloid, PBC, and TFH-LPD compartments. B-LPDs were evaluated from 14 patients (5 diffuse large B-cell lymphoma, 5 polymorphic B-LPD, 2 follicular lymphoma, 1 B-LPD, NOS, 1 plasma cell myeloma; 11 EBV+, 3 EBV-; 9 from tissue, 4 PB, 1 BM). Polymorphic B-LPDs showed atypical polymorphic morphology and an abnormal B-cell immunophenotype by FC and/or a clonal IgH gene rearrangement. The B-LPD, NOS showed an abnormal CD5-/CD10- immunophenotype with light chain restriction by FC without available tissue for morphologic evaluation. Nine of 14 (64%) B-LPDs shared identical mutations with corresponding TFH-LPDs ( TET2 [n=9; median VAF 0.42 (range 0.10- 0.56)], DNMT3A [n=5; median VAF 0.27 (range 0.05-0.50)], TET3 [n=1; VAF 0.32]). Myeloid compartments in 9/12 (75%) patients shared identical TET2 and/or DNMT3A mutations with B- and/or T-LPDs. B-LPDs more commonly harbored additional mutations absent in corresponding TFH-LPDs than PBCs (12/14 [86%] vs 1/8 [13%] evaluated by NGS; p=0.001). Unique mutations in B-LPDs involved epigenetic/transcriptional regulation (n=14, e.g. TET2, KMT2D, SETD5), signaling pathways (n=22, e.g. DTX1, KRAS, EPHA5), and DNA damage response (n=3, e.g. ATM, CHEK2, BRCA2). Incidences of shared and unique mutations did not significantly differ among EBV+ and EBV- cases. Conclusions: Both PBCs and B-LPDs in TFH-LPD patients are commonly EBV+ and share TET2/ DNMT3A mutations with TFH-LPDs, consistent with common origin from CH. B-LPDs more often harbor additional unique mutations than PBCs. Thus, CH mutations and EBV activation in B-cells may predispose TFH-LPD patients to B-LPDs, which may develop with additional private genomic aberrations aiding transformation (Figure 1B).

Mosaic Chromosomal Alterations and Monoclonal B-Cell Lymphocytosis: Relationship and Risk of Hematologic Malignancies

Background: Monoclonal B-cell lymphocytosis (MBL) is a precursor condition to chronic lymphocytic leukemia (CLL) characterized by the presence of circulating monoclonal B-cell populations in the peripheral blood of otherwise healthy individuals. MBL increases risk of several adverse clinical outcomes, including development of hematological malignancies. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis which affect large segments of DNA and include gains, losses, and copy number neutral loss of heterozygosity events. mCAs can be detected from SNP-array genotyping. Prior work has suggested mCAs as a potent risk factor for hematological malignancies. However, the nature of the relationship between mCAs and MBL is unknown. We sought to determine 1) the association between mCAs and MBL and 2) their combined effect on risk of hematological malignancies, in a large, prospectively collected, and well-annotated cohort of individuals with known MBL, mCA, and clinical outcomes. Methods: We examined the prevalence of mCA in a cohort of individuals from the Mayo Clinic Biobank who were screened for MBL using a sensitive eight-color flow cytometry assay. We classified MBL by the size of the clone relative to total B-cells, with high count (HC) defined as those individuals who had a percent clonal B-cell count ≥85% out of total B-cell count and low-count (LC) otherwise. Autosomal mCAs were detected from whole blood DNA collected at the time of MBL screening using a SNP-array-based assay. The MoChA pipeline was used to identify mCA events. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). To examine incident hematological cancers, we identified and validated all hematological cancers through medical record abstraction using the International Statistical Classification of Diseases codes. Cox regression was used to calculate hazard ratios (HR) and 95% CIs, stratified by MBL status or autosomal mCA status. All models were adjusted for age and sex. Results: We evaluated the prevalence of autosomal mCA in 3,764 individuals (792 individuals with MBL of whom 64 had HC-MBL and 2,972 without MBL). The prevalence of autosomal mCA was 7.7% ( n=288) with a median mCA cell fraction of 0.09 (range 0.01-1). Among those with and without MBL overall, the prevalence of mCA was 15.5% ( n=123) and 5.6% ( n=165), respectively (Figure 1). Compared to those without an autosomal mCA, individuals with an autosomal mCA were 2.4-fold more likely to have MBL (95% CI 1.9-3.2; P =2.0x10 -11; Figure 1). CLL-associated mCA (i.e., those commonly altered in CLL including trisomy 12 and deletion of 6q, 11q, 13q, and 17p) were present in 42 individuals, of whom 38 (90.5%) had MBL overall and 30 (71.4%) individuals had HC-MBL. Compared to those without a CLL-associated mCA, individuals with a CLL-associated mCA were 539.39-fold more likely to have HC-MBL (95% CI 190.3-1975.9; P &amp;lt;2.0x10 -16) and 6.7-fold more likely to have LC-MBL (95% CI 2.0-26.5; P =0.0031; Figure 1). Next, we evaluated the relationship between MBL and mCA with risk of developing a hematologic malignancy in 3,009 individuals with follow-up data (median 10 years). Forty-one individuals developed an incident hematological malignancy (68.3% lymphoid). When we evaluated the effect of autosomal mCA stratified by MBL status we observed elevated but non-significant risk of hematological malignancies (Figure 2). When we evaluated the effect of MBL stratified by autosomal mCA status we observed elevated and significant increased risk of developing a hematological malignancy (Figure 2). Conclusions: We found that the presence of an autosomal mCA is highly associated with having MBL. In particular, &amp;gt;90% of CLL-associated mCA carriers had MBL with ~7- and ~540-fold increased association for LC- and HC-MBL, respectively. These results suggest that almost all individuals in large biobanks with CLL-associated mCAs have undiagnosed MBL. However, not all individuals with MBL have an mCA. When evaluating risk of hematological malignancies, we found autosomal mCAs had &amp;gt;2-fold elevated but non-significant effects when stratified by MBL status while MBL remained a significant risk factor regardless of autosomal mCA status. Collectively these results support that MBL remains an important risk factor and suggests that mCAs hold potential to identify a subgroup of individuals at greater risk of developing a hematological malignancy.

Flow Cytometric Analysis of Lymphocyte Subsets of Covid-19 Patients from A Single Centre in Turkey

Background:&#x0D; Lymphocyte counts have been shown to negatively correlate with the severity and different outcomes in Covid-19. The aim of this single centre study is to analyse the distribution of lymphocyte subsets in response to SARS-CoV-2 infection and its relation to the severity, course and prognosis of the disease.&#x0D; Methods:&#x0D; Blood samples were obtained from 67 consecutive patients between April 2020 and July 2020. Data on other laboratory parameters, and clinical course were collected retrospectively from patient files and patints were defined to have as severe or non-severe (mild/moderate) disease. Leukocyte subsets to be studied were identified by using flow cytometric analysis. Patients were allocated into 3 groups based on the day of blood sample collection for the flow cytometric analysis: Days 0-7, Days 8-14 and Days &gt;14 as Group I, Group II and Group III, respectively. In 10 available of 67 patients an additional flow cytometric analysis was done 7-10 days after the initial sampling.&#x0D; Results:&#x0D; Lower total lymphocyte, CD3 positive, CD4 positive and B-cell counts were identified in severe infection compared to non-severe infection group which were also correlated with high serum CRP, D-dimer and ferritin levels. NK and monocyte counts were not different between the two groups. Activation markers CD38 and HLA-DR on CD4 and CD8 positive lymphocytes also were not different in either group. &#x0D; Conclusion:&#x0D; CD3 and CD4 lymphopenia were lower in accordance with previous studies and were associated with severe disease. The expectancy of high activation markers was not met. Future studies with detailed subgroup analyses at different time-points, and immune profiling after vaccination as well as during new infection in vaccinated patients will shed more light on our general knowledge of the immune response to COVID-19 and viruses in general.

ASSESSMENT OF SARS-COV-2 SPECIFIC B-CELL IMMUNE MEMORY: EVIDENCE FOR PERSISTENCE UP TO 1 YEAR POST-INFECTION

Background: SARS-CoV-2, the virus responsible for COVID-19 pandemic, has posed huge global health challenges. Understanding the immune response to SARS-CoV-2 infection, and in particular – the role of B cells in the generation of immune memory is crucial for assessing the durability of protective immunity. Materials and Methods: In this longitudinal prospective study, individuals who had recovered from SARS-CoV-2 infection were included. Peripheral venous blood samples were collected at three time intervals post symptom onset (PSO): 1-3 mo, 4-8 mo, and 9-12 mo. The humoral immune response was evaluated by measuring anti-SARS-CoV-2 IgG, virus-neutralizing antibody activity, total S1-specific B-cells, and B cell subpopulations. Results: The levels of anti-SARS-CoV-2 specific IgG antibodies decreased from 390.3 to 204.5 BAU/ml in the first 6-8 months PSO but did not significantly decrease further until the 12 th mo (126.6 BAU/ml). Virus-neutralizing antibodies (activity decreased by 20.4% between the 1st and 6-8th mos but remained relatively stable thereafter and could be detected up to 12 months PSO. In peripheral blood, the amount of S1-specific plasmablasts was highest one month after COVID-19 infection, and the level of memory B cells at 6 months. Those were detected even 12 months PSO, albeit in smaller quantities. Conclusion: The study provides evidence for the persistence of SARS-CoV-2-specific B-cell immune memory up to 1year post-infection. The presence of virus-specific memory B cells and plasmablasts suggests potential for sustained protection against reinfection. Further research is needed to elucidate the role of B-cell immune memory in preventing infection and to understand the individual variations of immune response.

Frequency of B-Cell Subpopulations in Low Responders in Comparison with High Responders to Hepatitis B Vaccine Among Health Care Workers.

Vaccination is the most effective way to prevent Hepatitis B (HB) infection. The goal of vaccination is to induce immunological memory. Hence, determining the frequency of memory B-cell (MBC) subsets is an important indicator of vaccine efficacy. This study aimed to evaluate the frequency of different B-cell subpopulations and the expression of PD-1 on B-cell subsets in low responders (LR) and high responders (HR) to HB vaccine. According to our findings, the expression level of PD-1 was significantly higher on atypical MBC (atMBC) than that of naive B cell and classical MBC (cMBC) in LR and HR groups. Moreover, cMBCs had a significant higher PD-1 expression than naive B cells in LR group. No significant differences were found in the frequency of various B-cell subpopulations and the expression level of PD-1 on B-cell subsets between LR and HR groups. We observed a negative correlation between age and HBsAb titer and a positive correlation between age and PD-1 expression level on cMBC in LR group. It can be concluded that inadequate specific memory B-cell response, rather than total memory B-cell deficiency, may be implicated in low responsive rate to HB vaccine in healthy individuals.

Aberrant B-cell activation and B-cell subpopulations in rheumatoid arthritis: analysis by clinical activity, autoantibody seropositivity, and treatment.

Few studies analyze the role of B-cell subpopulations in rheumatoid arthritis (RA) pathophysiology. Therefore, this study aimed to analyze the differences in B-cell subpopulations and B-cell activation according to disease activity, RA subtype, and absence of disease-modifying antirheumatic drugs (DMARDs) therapy. These subgroups were compared with control subjects (CS). One hundred and thirty-nine subjects were included, of which 114 were RA patients, and 25 were controls. Patients were divided into 99 with seropositive RA, 6 with seronegative RA, and 9 without DMARDs. The patients with seropositive RA were subclassified based on the DAS28 index. A seven-color multicolor flow cytometry panel was used to identify B-cell immunophenotypes and cell activation markers. There were no changes in total B-cell frequencies between RA patients and controls. However, a lower frequency of memory B cells and pre-plasmablasts was observed in seropositive RA compared to controls (P < 0.0001; P = 0.0043, respectively). In contrast, a higher frequency of mature B cells was observed in RA than in controls (P = 0.0002). Among patients with RA, those with moderate activity had a higher percentage of B cells (P = 0.0021). The CD69+ marker was increased (P < 0.0001) in RA compared to controls, while the CD40+ frequency was decreased in patients (P < 0.0001). Transitional, naïve, and double-negative B-cell subpopulations were higher in seronegative RA than in seropositive (P < 0.01). In conclusion, in seropositive and seronegative RA patients, there are alterations in B-cell activation and B-cell subpopulations, independently of clinical activity and DMARDs therapy.

Age and memory B cells at baseline are associated with risk of relapse and memory B-cell reappearance following anti-CD20 treatment in pediatric frequently-relapsing/steroid-dependent nephrotic syndrome

B-cell depleting anti-CD20 monoclonal antibodies, mainly rituximab, have proven efficacy in children with frequently-relapsing/steroid-dependent nephrotic syndrome (FR/SDNS). However, drug-free remission is variable and specific baseline markers predictive of relapse after anti-CD20 treatment are still being defined. To clarify this, we performed a bicentric observational study in a large cohort of 102 children and young adults with FR/SDNS treated with anti-CD20 monoclonal antibodies (rituximab and ofatumumab). Sixty-two patients (60.8%) relapsed during a 24-month period (median [interquartile range] relapse-free survival, 14.4 months [7.9-24.0]). A higher risk of relapse was significantly associated with a younger age (under 9.8 years, hazard ratio, 0.44; 95% confidence interval, 0.26-0.74) and with significantly higher circulating levels of memory B cells (1.14; 1.09-1.32) at time of anti-CD20 infusion, independent of time elapsed from onset, previous anti-CD20 treatment, type of administered anti-CD20 monoclonal antibodies, previous or maintenance oral immunosuppression. Patients younger than 9.8 years at anti-CD20 infusion had a subsequent higher recovery of total, transitional, mature-naïve and memory B-cell subsets independent of previous anti-CD20 treatment and maintenance immunosuppression. Significantly, younger age and higher circulating levels of memory B cells at time of anti-CD20 infusion were also independently associated with the recovery of memory B cells by linear mixed-effects modelling. Thus, both younger age and higher circulating levels of memory B cells at time of infusion are independently associated with a higher risk of relapse and an earlier recovery of memory B cells following anti-CD20 treatment in children with FR/SDNS.