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.

Flow Cytometry Analysis Reveals a Wide Cytologic and Immunophenotypic Spectrum of Peripheral B Lymphocytes in Angioimmunoblastic T-Cell Lymphoma.

INTRODUCTION: Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive T-cell lymphoma commonly associated with B-cell dysregulation. Correlations involving B-cell dysregulation and clinicopathological features remain unclear. METHODS: We prospectively collected blood samples from 11 AITL patients and 17 healthy controls. The percentages of B-cell subpopulations and lymphocytes with IL-21 production were assessed using flow cytometry. Peripheral blood lymphocyte morphology was evaluated microscopically. RESULTS: Six of 11 (54.5%) patients presented with polyclonal hypergammaglobulinemia. Three of 11 (27.3%) tumor biopsies showed monoclonal immunoglobulin gene rearrangement. The patients exhibited significantly lower levels of naive (p < 0.001) and class-switched (p < 0.001) B cells than controls. The percentages of IgD-CD27- B cells (p = 0.007) and antibody-secreting cells (ASCs) (p = 0.001) were increased. Blood smears revealed atypical lymphocytes and immature plasma cells with morphological diversity. In comparison to normal controls, IL-21 production significantly increased in CD4+ (p < 0.001) and CD8+ (p = 0.020) T cells. B-cell clonality, RHOA G17V mutation, and the presence of sheets of clear cells and immature/mature plasma cells in lymph nodes were significantly associated with percentages of class-switched B cells and ASCs. The patients with circulating EBV DNA had a lower percentage of naive B cells (p = 0.009). CONCLUSIONS: Our results demonstrated a wide spectrum of peripheral B-cell morphologies and immunophenotypes of peripheral B cells in AITL. These findings correspond to dysregulated B-cell immunity and heterogeneous clinicopathological features.

High Pretransplant BAFF Levels and B-cell Subset Polarized towards a Memory Phenotype as Predictive Biomarkers for Antibody-Mediated Rejection.

Antibody-mediated rejection (AbMR) is one of the leading causes of graft loss in kidney transplantation and B cells play an important role in the development of it. A B-cell activating factor (BAFF) is a cytokine involved in B cell ontogeny. Here, we analyzed whether B cell maturation and the effect of B cell soluble factors, such as BAFF could be involved in AbMR. Serum BAFF levels and B and T cell subpopulations were analyzed 109 kidney transplant patients before transplantation and at 6 and 12 months after kidney transplantation. Pretransplant serum BAFF levels as well as memory B cell subpopulations were significantly higher in those patients who suffered clinical AbMR during the first 12 months after kidney transplantation. Similar results were observed in the prospective analysis of patients with subclinical antibody-mediated rejection detected in the surveillance biopsy performed at 12 months after kidney transplantation. A multivariate analysis confirmed the independent role of BAFF in the development of AbMR, irrespective of other classical variables. Pretransplant serum BAFF levels could be an important non-invasive biomarker for the prediction of the development of AbMR and posttransplant increased serum BAFF levels contribute to AbMR.

JAK inhibitors attenuate hyperactivation of nonswitched memory B cells in rheumatoid arthritis patients in remission.

OBJECTIVE: To investigate the distribution and activation of B-cell subpopulations in rheumatoid arthritis (RA) patients treated with Janus kinase inhibitors (JAKis) and to analyze their correlation with disease remission. METHODS: Peripheral blood samples were collected from 23 adult healthy controls and 58 RA patients, 31 of whom were treated with JAKis and assessed during a 24-month follow-up. The number of peripheral B-cell subpopulations (including naive B cells, nonswitched memory B (NSMB) cells, switched memory B cells, and double-negative B cells), their activation, and phosphorylation of SYK and AKT upon B-cell receptor (BCR) stimulation in each population were analyzed by flow cytometry. RESULTS: Compared with that in healthy controls, the frequency of NSMB cells was significantly lower in new-onset untreated RA patients. However, expression of CD40, CD80, CD95, CD21low and pAKT significantly increased in these NSMB cells. Additionally, the number of NSMB cells correlated negatively with DAS28-ESR and IgG and IgA levels in these patients; expression of CD80, CD95 and CD21low on NSMB cells correlated positively with DAS28-ESR and IgG and IgA levels. After treatment with JAKis, the serum IgG concentration significantly decreased in RA patients in remission, but CD40, CD95 and pAKT levels in NSMB cells significantly decreased. CONCLUSION: RA patients present different B-cell subpopulations, in which the frequency of NSMB cells is negatively associated with disease activity. However, treatment with JAKis can inhibit activation of NSMB cells, restore the balance of kinase phosphorylation, and facilitate disease remission in RA patients.

Immunological biomarkers associated with survival in a cohort of Argentinian patients with common variable immunodeficiency.

Background: Common variable immunodeficiency (CVID) is the most common symptomatic syndrome among inborn errors of immunity. Although several aspects of CVID immunopathology have been elucidated, predictive factors for mortality are incompletely defined. A genetic cause can be identified only in approximately 30% of patients. Objective: We sought to develop a mortality predictive score on the basis of the immunophenotypes and genotypes of patients with CVID. Methods: Twenty-one patients diagnosed with CVID in Córdoba, Argentina, were analyzed for clinical and laboratory data. Immunophenotyping was done by flow cytometry. CVID-associated mutations were identified by whole-exome sequencing. Results: Alive (15) and deceased (6) patients were compared. Univariate analysis showed significant differences in CD4+ T cells (P = .002), natural killer (NK) cells (P = .001), and memory switched B cells (P = .001) between groups. Logistic regression analysis showed a negative correlation between CD4+, NK, and memory switched B-cell counts and probability of survival over a 10-year period (CD4+ T cells: odds ratio [OR], 1.01; 95% CI, 1.001-1.020; NK cells: OR, 1.07; 95% CI, 1.02-1.17; and memory switched B cells: OR, 26.23; 95% CI, 2.06-2651.96). Receiver-operating characteristic curve analysis identified a survival cutoff point for each parameter (CD4+ T cells: 546 cells/mL; AUC, 0.87; sensitivity, 60%; specificity, 100%; memory switched B cells: 0.84 cells/mL; AUC, 0.92; sensitivity, 100%; specificity, 85%; and NK cells: 45 cells/mL; AUC, 0.92; sensitivity, 83%; specificity, 100%). Genetic analysis on 14 (9 female and 5 male) patients from the cohort revealed mutations associated with inborn errors of immunity in 6 patients. Conclusions: A score to predict mortality is proposed on the basis of CD4+ T, NK, and memory switched B-cell counts in patients with CVID.

Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation in a pediatric-dominated cohort.

Introduction: It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions. Methods: Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. Results: We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine. Discussion: Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.

A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations.

Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability - together with the relatively easy quantification of phosphorylation - phosphoflow is increasingly and more commonly used, compared to classical western blot analysis. It can however be challenging to set-up a method that works for all targets of interest. Here, we present a step-by-step phosphoflow protocol allowing the evaluation of the phosphorylation status of signaling molecules in conjunction with extensive staining to identify various human and murine B cell subpopulations, as was previously published in the original paper by Rip et al. (2020). Next to a description of phosphoflow targets from the original paper, we provide directions on additional targets that play a pivotal role in BCR signaling. The step-by-step phosphoflow protocol is user-friendly and provides sensitive detection of phosphorylation of various BCR signaling molecules in human and murine B cell subpopulations.

Failure of B Cell Tolerance in CVID.

Common variable immunodeficiency (CVID) comprises a group of related disorders defined by defects in B cell function and antibody production. Concurrent autoimmune features are common, but the underlying pathogenic mechanisms of autoimmunity in CVID are poorly understood. Overlap in some clinical and laboratory features suggests a shared pathogenesis, at least in part, with systemic lupus erythematosus (SLE). One important part of SLE pathogenesis is loss of B cell tolerance, an aspect that warrants further study in CVID. The study of inherently autoreactive 9G4+ B cells has led to a greater understanding of B cell tolerance defects in lupus. Study of these B cells in CVID has yielded conflicting results, largely due to differences in methodological approaches. In this study, we take a comprehensive look at 9G4+ B cells throughout B cell development in CVID patients and compare patients both with and without autoimmune features. Using flow cytometry to examine B cell subpopulations in detail, we show that only those CVID patients with autoimmune features demonstrate significant expansion of 9G4+ B cells, both in naïve and multiple memory populations. Examination of two autoreactive B cell subsets recently characterized in SLE, the activated naïve (aNAV) and double negative 2 (DN2) B cells, reveals an expanded 9G4+ DN2 population to be common among CVID patients. These results reveal that both multiple central and peripheral B cell tolerance defects are related to autoimmunity in CVID. Furthermore, these data suggest that the autoreactive DN2 B cell population, which has not previously been examined in CVID, may play an important role in the development of autoimmunity in patients with CVID.

An Exponential Regression Model Reveals the Continuous Development of B Cell Subpopulations Used as Reference Values in Children.

B lymphocytes are key players in humoral immunity, expressing diverse surface immunoglobulin receptors directed against specific antigenic epitopes. The development and profile of distinct subpopulations have gained awareness in the setting of primary immunodeficiency disorders, primary or secondary autoimmunity and as therapeutic targets of specific antibodies in various diseases. The major B cell subpopulations in peripheral blood include naïve (CD19+ or CD20+IgD+CD27-), non-switched memory (CD19+ or CD20+IgD+CD27+) and switched memory B cells (CD19+ or CD20+IgD-CD27+). Furthermore, less common B cell subpopulations have also been described as having a role in the suppressive capacity of B cells to maintain self-tolerance. Data on reference values for B cell subpopulations are limited and only available for older age groups, neglecting the continuous process of human B cell development in children and adolescents. This study was designed to establish an exponential regression model to produce continuous reference values for main B cell subpopulations to reflect the dynamic maturation of the human immune system in healthy children.

Antiretroviral naive and treated patients: Discrepancies of B cell subsets during the natural course of human immunodeficiency virus type 1 infection.

AIM: To evaluate alterations of memory B cell subpopulations during a 48-wk period in human immunodeficiency virus type 1 (HIV-1) patients. METHODS: Forty-one antiretroviral naïve and 41 treated HIV-1 patients matched for age and duration of HIV infection were recruited. All clinical, epidemiological and laboratory data were recorded or measured. The different B cell subsets were characterized according to their surface markers: Total B cells (CD19+), memory B cells (CD19+CD27+, BMCs), resting BMCs (CD19+CD27+CD21high, RM), exhausted BMCs (CD19+CD21lowCD27-, EM), IgM memory B (CD19+CD27+IgMhigh), isotype-switched BMCs (CD19+CD27+IgM-, ITS) and activated BMCs (CD19+CD21low+CD27+, AM) at baseline on week 4 and week 48. RESULTS: Mean counts of BMCs were higher in treated patients. There was a marginal upward trend of IgM memory B cell proportions which differed significantly in the treated group (overall trend, P = 0.004). ITS BMC increased over time significantly in all patients. Naive patients had of lower levels of EM B cells compared to treated, with a downward trend, irrespectively of highly active antiretroviral therapy (HAART) intake. Severe impairment of EM B cells was recorded to both treated (P = 0.024) and naive (P = 0.023) and patients. Higher proportions of RM cells were noted in HAART group, which differed significantly on week 4th (P = 0.017) and 48th (P = 0.03). Higher levels of AM were preserved in HAART naive group during the whole study period (week 4: P = 0.018 and 48: P = 0.035). HIV-RNA viremia strongly correlated with AM B cells (r = 0.54, P = 0.01) and moderately with RM cells (r = -0.45, P = 0.026) at baseline. CONCLUSION: HIV disrupts memory B cell subpopulations leading to impaired immunologic memory over time. BMC, RM, EM and ITS BMC were higher in patients under HAART. Activated BMCs (AM) were higher in patients without HAART. Viremia correlated with AM and RM. Significant depletion was recorded in EM B cells irrespectively of HAART intake. Perturbations in BMC-populations are not fully restored by antiretrovirals.