Class-switched Memory Research Articles

Elevated frequencies of activated memory B cells in multiple sclerosis are reset to healthy control levels after B cell depletion with Ocrelizumab

In multiple sclerosis (MS) the B cell depleting drug ocrelizumab has shown high efficacy in reducing inflammatory activity. Its mechanism of action is unclear due to B cell subset complexity and unknown roles in pathogenesis. Here, we comprehensively phenotyped and quantitated peripheral blood B cell subsets before and after ocrelizumab infusion to gain insight into the fate of B cell subsets with pathogenic potential. Peripheral blood B cells were collected from treatment naïve patients at baseline and months one, three, and six following the first course of ocrelizumab treatment; at 6 months following the second treatment cycle; ~14 months following their last infusion; and from healthy controls. Flow cytometry combined with cluster analysis was used to track depletion and repletion of naïve, memory, and antibody secreting cells. By month one, naïve B cells were depleted, but a small subset of memory B cells were retained with no depletion of antibody secreting cells. Uniform manifold approximation and projection for dimension reduction analysis of flow cytometry data revealed two non-class switched naïve clusters and two class switched memory clusters. One class switched cluster was activated in MS patients but largely absent in healthy controls. Both memory B cell subsets underwent depletion after a single six-month course of ocrelizumab treatment after which their proportions were reset to heathy control levels. These observations suggest that activated class-switched memory B cells could serve as a biomarker of recent or ongoing MS disease activity to guide redosing.

Mass cytometry reveals cellular correlates of immune response heterogeneity to SARS-CoV-2 vaccination in the elderly

Heterogeneity in vaccine response, particularly in vulnerable populations like the elderly, represents a significant public health challenge. We conducted an in-depth examination of immune cell profiles before and after SARS-CoV-2 vaccination utilizing mass cytometry in a cohort of healthy Norwegian seniors (65–80 years). We have demonstrated that higher pre-vaccination frequencies of CD27+IgD- class-switched memory B cells and subsets of CD27-CD24+CD38+ transitional B cells were associated with a robust vaccine response. Post-vaccination, high responders exhibited increased frequencies of IFN-γ+CD4+ T cells with antigen recall and a concurrent decrease in CCR6(+) TH cell subset frequencies compared to low responders. The presence of a γδ T cell subset displaying polyfunctional cytokine responses was also associated with better vaccine response in the elderly. This in-depth profiling sheds light on inherent differences in immune cell frequencies and functions that may offer insights for targeted vaccination strategies in older populations.

Maintenance and functional regulation of immune memory to COVID-19 vaccines in tissues.

Memory T and B cells in tissues are essential for protective immunity. Here, we performed a comprehensive analysis of the tissue distribution, phenotype, durability, and transcriptional profile of COVID-19 mRNA vaccine-induced immune memory across blood, lymphoid organs, and lungs obtained from 63 vaccinated organ donors aged 23-86, some of whom experienced SARS-CoV-2 infection. Spike (S)-reactive memory Tcells were detected in lymphoid organs and lungs and variably expressed tissue-resident markers based on infection history, and S-reactive B cells comprised class-switched memory cells resident in lymphoid organs. Compared with blood, S-reactive tissue memory Tcells persisted for longer times post-vaccination and were more prevalent with age. S-reactive Tcells displayed site-specific subset compositions and functions: regulatory cell profiles were enriched in tissues, while effector and cytolytic profiles were more abundant in circulation. Our findings reveal functional compartmentalization of vaccine-induced Tcell memory where surveilling effectors and in situ regulatory responses confer protection with minimal tissue damage.

Whole transcriptome sequencing indicated the Anti-tumor immunity of NLRP3 in breast cancer

Breast cancer (BC) is a prevalent cancer of the female reproductive system and a major contributor to cancer-related mortality. The activation of NLRP3, a key inflammasome, has been extensively associated with tumor-related molecular and cellular processes; however, the regulatory mechanisms and specific role of NLRP3 in breast cancer remain incompletely elucidated. This study aimed to evaluate the molecular mechanisms of NLRP3-related genes in BC. Utilizing bioinformatics methods, the present research analyzed the TCGA-BRCA dataset, which included four groups of transcriptome sequencing data as follows, normal (WT), NLRP3 knockout (KO), non-knockout-BRCA (BC-WT), and NLRP3-knockout-BRCA (BC-KO). Results indicated that NLRP3 was significantly down-regulated in TCGA-BRCA. Key module genes were mainly enriched in leukocyte cell-cell adhesion and cytokine-cytokine receptor interaction. Moreover, correlation analysis showed that NLRP3 was positively associated with cancer-associated fibroblasts and negatively associated with CD4+ Th1 T-cells. In addition, the DEGs1 and DEGs2 overlapping indicated 505 feature genes, with Chac1 (negative) and Ugt8a (positive) had the strongest correlation with differential immune cells (class-switched memory B cells). Pathway intersection revealed 13 co-KEGG pathways. The BC-KO group indicated markedly reduced levels of four genes (Ccl19, Ccl20, Ccl21a, and H2-Oa) and increased levels of two genes (Il2ra and H2-Ob). This study delved into the role of NLRP3 in BC, exploring its regulatory mechanisms and the impact gene knockout. Bioinformatics approaches identified NLRP3-associated genes, their enriched pathways, and interactions within the tumor microenvironment (TME), providing novel insights into NLRP3 function, TME dynamics, and potential targets for BC prevention and treatment.

Imbalance of B-Cell Subpopulations in the Microenvironment of Sarcoidosis or Lung Cancer.

Although the role of T lymphocytes in sarcoidosis (SA) and lung cancer (LC) is quite well reported, the occurrence of B cells in disease microenvironments may suggest their potential role as natural modifiers of the immune response. The aim of this study was to investigate the B-cell profile and lymphocyte-related hematological parameters between patients with SA, LC and healthy controls (HCs). The cells were assessed by flow cytometry and a hematological analyzer in peripheral blood (PB) and material from lymph nodes (LNs) obtained by the EBUS/TBNA method. We showed that in SA patients, there were higher percentages of naïve B and CD21low B cells and a lower percentage of class-switched memory B cells than LC patients in LNs. We observed a higher median proportion of non-switched memory and transitional B cells in the PB of SA patients than in LC patients. We noticed the lowest median proportion of class-switched memory B cells in the PB from SA patients. LC patients had a higher percentage of RE-LYMP and AS-LYMP than SA patients. Our study presented a different profile of B-cell subpopulations in SA and LC patients, distinguishing dominant subpopulations, and showed the relocation from distant compartments of the circulation to the disease microenvironment, thus emphasizing their role.

Integrative analysis of blood transcriptome profiles in small-cell lung cancer patients for identification of novel chemotherapy resistance-related biomarkers.

Chemoresistance constitutes a prevalent factor that significantly impacts thesurvival of patients undergoing treatment for smal-cell lung cancer (SCLC). Chemotherapy resistance in SCLC patients is generally classified as primary or acquired resistance, each governedby distinct mechanisms that remain inadequately researched. In this study, we performed transcriptome screening of peripheral blood plasma obtainedfrom 17 patients before and after receiving combined etoposide and platinum treatment. We firs testimated pseudo-single-cell analysis using xCell and ESTIMATE and identified differentially expressed genes (DEGs), then performed network analysis to discover key hub genes involved in chemotherapy resistance. Our analysis showed a significant increase in class-switched memory B cell scores acrossboth chemotherapy resistance patterns, indicating their potential crucial role in mediatingresistance. Moreover, network analysis identifed PRICKLE3, TNFSFI0, ACSLl and EP300 as potential contributors to primary resistance, with SNWl, SENP2 and SMNDCl emerging assignificant factors in acquired resistance, providing valuable insights into chemotherapy resistancein SCLC. These findings offer valuable insights for understanding chemotherapy resistance and related gene signatures in SCLC, which could help further biological validation studies.

Integrative Analysis by Mendelian Randomization and Large-Scale Single-Cell Transcriptomics Reveals Causal Links between B Cell Subtypes and Diabetic Kidney Disease

Introduction: The increasing incidence of diabetic kidney disease (DKD) and the challenges in its management highlight the necessity for a deeper understanding of its pathogenesis. While recent studies have underscored the substantial impact of circulating immunity on the development of diabetic microvascular complications such as retinopathy and neuropathy, research on circulating immunity in DKD remains limited. Methods: This study utilized Mendelian randomization analysis to explore the potential independent causal relationships between circulating immune cells and DKD pathogenesis. Additionally, a combination of single-cell disease relevance score (scDRS) and immune cell infiltration analysis was employed to map the circulating immunity landscape in DKD patients. Results: Ten immune traits, including 5 of B cells, 2 of T cells, 2 of granulocytes, and one of monocytes, were defined to be associated with the pathogenesis of DKD. Notably, IgD-CD27-B cell Absolute Count (IVW: OR, 1.102 [1.023–1.189], p = 0.011) and IgD-CD24-B cell Absolute Count (IVW: OR, 1.106 [1.030–1.188], p = 0.005) were associated with promoting DKD pathogenesis, while CD24+CD27+B cell %B cell (IVW: OR, 0.943 [0.898–0.989], p = 0.016) demonstrated a protective effect against DKD onset. The presence of B cell-activating factor receptor (BAFF-R) on CD20−CD38−B cell (IVW: OR, 0.946 [0.904–0.989], p = 0.015) and BAFF-R on IgD-CD38+B cell (IVW: OR, 0.902 [0.834–0.975], p = 0.009) also indicated a potential role in preventing DKD. scDRS analysis revealed that two main subsets of B cells, naïve B and memory B cells, had a higher proportion of DKD-related cells or a higher scDRS score of DKD phenotype, indicating their strong association with DKD. Furthermore, immune infiltrate deconvolution analysis showed a notable decrease in the circulating memory B cells and class-switched memory B cells in DKD patients compared to those of DM patients without DKD. Conclusion: Our study revealed the causal relations between circulating immunity and DKD susceptibility, particularly highlighted the potential roles of B cell subtypes in DKD development. Further studies addressing the related mechanisms would broaden the current understanding of DKD pathogenesis.

The role of adenoid immune phenotype in polysensitized children with allergic rhinitis and adenoid hypertrophy.

There is increasing interest in elucidating the relationship between adenoid hypertrophy (AH) and allergic rhinitis (AR). However, the impact of aeroallergen sensitization patterns on children with AH and AR remains unclear. Patients aged 2-8 years (recruited from January 2019 to December 2022) with nasal symptoms were assessed for allergies, adenoid size, and respiratory viral infection history. The serum total immunoglobulin E (IgE) and specific IgE levels were measured, and flexible nasal endoscopy was performed. The relationship between AH, aeroallergen sensitization patterns, and lymphocyte subpopulations in adenoid samples was analyzed using flow cytometry. In total, 5281 children were enrolled (56.5% with AR; and 48.6% with AH). AH was more prevalent in children with AR. Compared to nonsensitized individuals, those polysensitized to molds had a higher prevalence of AH (adjusted OR 1.61, 95% CI 1.32-1.96) and a greater occurrence of two or more respiratory viral infections, particularly in adenoidectomy patients. The percentages and corrected absolute counts of regulatory T (Treg) cells, activated Tregs, class-switched memory B cells (CSMBs), natural killer (NK) T cells, and NK cell subpopulations were reduced in the adenoid tissues of children with both AH and AR (AH-AR) compared to AH-nAR children. Polysensitization in AH-AR children correlated with lower CSMB percentages. Polysensitivity to molds is associated with an increased risk of AH in children with AR. Fewer B cells, NK cells, and Treg cells with an effector/memory phenotype were detected in the adenoids of AR children, and these lower percentages of immune cells, particularly CSMBs, were closely linked to aeroallergen sensitization models and respiratory viral infection.

The prevalence of immunodeficiency in a special population: intern doctors

Background: This study analyzes the immune system parameters of intern doctors working actively during the COVID-19 pandemic. When an intern doctor failed to respond to the treatment during a severe COVID-19 condition, the immune system panel was examined and as a result, it revealed an underlying immunodeficiency. The death of the intern doctor caused several concerns among other intern doctors, and for this reason, their immune systems were also considered to be examined. This study aims to demonstrate that immunodeficiencies might be more common than is known among the general population. Methods: In this single-center study, the demographic characteristics and European Society for Immunodeficiencies (ESID) diagnostic criteria of 92 intern doctors have been examined retrospectively. For the study, immune system parameters (complete blood count, serum immunoglobulins and subgroup levels, specific vaccine responses, isohemagglutinin titers, lymphocyte subgroups, and class-switched memory B cell (cSMB) levels have been evaluated. Results: When the demographic characteristics have been analyzed it is seen that the median age is 23.6 (21-28) years, and 64 (70%) of the intern doctors are female. In immune system parameters, one or more are found to be low in 51.08% of the doctors. Among the immunoglobulin subgroups, low IgG4 has been the most common. Selective IgA deficiency has been detected in 2.17% and selective IgM deficiency has been detected in also 2.17% of them. Low B cells (CD19+) are detected in 10.9% and low levels of class-switched memory B cells are found in 35.7% of them. Conclusion: This study reveals that deficient immunological parameters, especially selective Ig A, selective IgM deficiency, and low IgG4, might be more frequent than known. Depending on the data, it can be concluded that immunodeficiency might be more common than it is known among the general population; however, low immunological parameters alone do not lead to immunodeficiency.

Qualitative Immunoglobulin Deficiency Causes Bacterial Infections in Patients with STAT1 Gain-of-Function Mutations.

STAT1 is a transduction and transcriptional regulator that functions within the classical JAK/STAT pathway. In addition to chronic mucocutaneous candidiasis, bacterial infections are a common occurrence in patients with STAT1 gain-of-function (GOF) mutations. These patients often exhibit skewing of B cell subsets; however, the impact of STAT1-GOF mutations on B cell-mediated humoral immunity remains largely unexplored. It is also unclear whether these patients with IgG within normal range require regular intravenous immunoglobulin (IVIG) therapy. Eleven patients (harboring nine different STAT1-GOF mutations) were enrolled. Reporter assays and immunoblot analyses were performed to confirm STAT1 mutations. Flow cytometry, deep sequencing, ELISA, and ELISpot were conducted to assess the impact of STAT1-GOF on humoral immunity. All patients exhibited increased levels of phospho-STAT1 and total STAT1 protein, with two patients carrying novel mutations. In vitro assays showed that these two novel mutations were GOF mutations. Three patients with normal total IgG levels received regular IVIG infusions, resulting in effective control of bacterial infections. Four cases showed impaired affinity and specificity of pertussis toxin-specific antibodies, accompanied by reduced generation of class-switched memory B cells. Patients also had a disrupted immunoglobulin heavy chain (IGH) repertoire, coupled with a marked reduction in the somatic hypermutation frequency of switched Ig transcripts. STAT1-GOF mutations disrupt B cell compartments and skew IGH characteristics, resulting in impaired affinity and antigen-specificity of antibodies and recurrent bacterial infections. Regular IVIG therapy can control these infections in patients, even those with normal total IgG levels.

Comprehensive molecular characterization of long-term glioblastoma survivors

Fewer than 5 % glioblastoma (GBM) patients survive over five years and are termed long-term survivors (LTS), yet their molecular background is unclear. The present cohort included 72 isocitrate dehydrogenase (IDH)-wildtype GBM patients, consisting of 35 LTS and 37 short-term survivors (STS), and we employed whole exome sequencing, RNA-seq and DNA methylation array to delineate this largest LTS cohort to date. Although LTS and STS demonstrated analogous clinical characters and classical GBM biomarkers, CASC5 (P = 0.002) and SPEN (P = 0.013) mutations were enriched in LTS, whereas gene-to-gene fusions were concentrated in STS (P = 0.007). Importantly, LTS exhibited higher tumor mutation burden (P < 0.001) and copy number (CN) increase (P = 0.013), but lower mutant-allele tumor heterogeneity score (P < 0.001) and CN decrease (P = 0.026). Additionally, LTS demonstrated hypermethylated genome (P < 0.001) relative to STS. Differentially expressed and methylated genes both enriched in olfactory transduction. Further, analysis of the tumor microenvironment revealed higher infiltration of M1 macrophages (P = 0.043), B cells (P = 0.016), class-switched memory B cells (P = 0.002), central memory CD4+ T cells (P = 0.031) and CD4+ Th1 cells (P = 0.005) in LTS. We also separately analyzed a subset of patients who were methylation class-defined GBM, contributing 70.8 % of the entire cohort, and obtained similar results relative to prior analyses. Finally, we demonstrated that LTS and STS could be distinguished using a subset of molecular features. Taken together, the present study delineated unique molecular attributes of LTS GBM.

Identification of m6A/m5C-related lncRNA signature for prediction of prognosis and immunotherapy efficacy in esophageal squamous cell carcinoma.

N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have garnered significant attention in the field of epigenetic research due to their close association with human cancers. This study we focus on elucidating the expression patterns of m6A/m5C-related long non-coding RNAs (lncRNAs) in esophageal squamous cell carcinoma (ESCC) and assessing their prognostic significance and therapeutic potential. Transcriptomic profiles of ESCC were derived from public resources. m6A/m5C-related lncRNAs were obtained from TCGA using Spearman's correlations analysis. The m6A/m5C-lncRNAs prognostic signature was selected to construct a RiskScore model for survival prediction, and their correlation with the immune microenvironment and immunotherapy response was analyzed. A total of 606 m6A/m5C-lncRNAs were screened, and ESCC cases in the TCGA cohort were stratified into three clusters, which showed significantly distinct in various clinical features and immune landscapes. A RiskScore model comprising ten m6A/m5C-lncRNAs prognostic signature were constructed and displayed good independent prediction ability in validation datasets. Patients in the low-RiskScore group had a better prognosis, a higher abundance of immune cells (CD4 + T cell, CD4 + naive T cell, class-switched memory B cell, and Treg), and enhanced expression of most immune checkpoint genes. Importantly, patients with low-RiskScore were more cline benefit from immune checkpoint inhibitor treatment (P < 0.05). Our findings underscore the potential of RiskScore system comprising ten m6A/m5C-related lncRNAs as effective biomarkers for predicting survival outcomes, characterizing the immune landscape, and assessing response to immunotherapy in ESCC.

Anti-synthetase syndrome is associated with a higher risk of hospitalization among patients with idiopathic inflammatory myopathy and COVID-19.

Data with fine granularity about COVID-19-related outcomes and risk factors were still limited in the idiopathic inflammatory myopathies (IIMs) population. This study aimed to investigate clinical factors associated with hospitalized and severe COVID-19 in patients with IIMs, particularly those gauged by myositis-specific antibodies. This retrospective cohort study was conducted in the Renji IIM cohort in Shanghai, China, under an upsurge of SARS-CoV-2 omicron variant infections from December 2022 to January 2023. Clinical data were collected and analyzed by multivariable logistic regression to determine risk factors. High-dimensional flow cytometry analysis was performed to outline the immunological features. Among 463 infected patients in the eligible cohort (n=613), 65 (14.0%) were hospitalized, 19 (4.1%) suffered severe COVID-19, and 10 (2.2%) died. Older age (OR=1.59/decade, 95% CI 1.18 to 2.16, p=0.003), requiring family oxygen supplement (2.62, 1.11 to 6.19, 0.028), patients with anti-synthetase syndrome (ASyS) (2.88, 1.12 to 7.34, 0.027, vs. other dermatomyositis), higher IIM disease activity, and prednisone intake >10mg/day (5.59, 2.70 to 11.57, <0.001) were associated with a higher risk of hospitalization. Conversely, 3-dose inactivated vaccination reduced the risk of hospitalization (0.10, 0.02 to 0.40, 0.001, vs. incomplete vaccination). Janus kinase inhibitor (JAKi) pre-exposure significantly reduced the risk of severe COVID-19 in hospitalized patients (0.16, 0.04 to 0.74, 0.019, vs. csDMARDs). ASyS patients with severe COVID-19 had significantly reduced peripheral CD4+ T cells, lower CD4/CD8 ratio, and fewer naive B cells but more class-switched memory B cells compared with controls. ASyS and family oxygen supplement were first identified as risk factors for COVID-19-related hospitalization in patients with IIMs. JAKi pre-exposure might protect IIM patients against severe COVID-19 complications.

Assessing the causal relationship between 731 immunophenotypes and the risk of lung cancer: a bidirectional mendelian randomization study

BackgroundPrevious studies have observed a link between immunophenotypes and lung cancer, both of which are closely associated with genetic factors. However, the causal relationship between them remains unclear.MethodsBidirectional Mendelian randomization (MR) was performed on publicly available genome-wide association study (GWAS) summary statistics to analyze the causal relationships between 731 immunophenotypes and lung cancer. Sensitivity analyses were conducted to verify the robustness, heterogeneity, and potential horizontal pleiotropy of our findings.ResultsFollowing Bonferroni adjustment, CD14− CD16+ monocyte (OR = 0.930, 95%CI 0.900–0.960, P = 8.648 × 10− 6, PBonferroni = 0.006) and CD27 on CD24+ CD27+ B cells (OR = 1.036, 95%CI 1.020–1.053, P = 1.595 × 10 − 5, PBonferroni = 0.012) were identified as having a causal role in lung cancer via the inverse variance weighted (IVW) method. At a more relaxed threshold, CD27 on IgD+ CD24+ B cell (OR = 1.035, 95%CI 1.017–1.053, P = 8.666 × 10− 5, PBonferroni = 0.063) and CD27 on switched memory B cell (OR = 1.037, 95%CI 1.018–1.056, P = 1.154 × 10− 4, PBonferroni = 0.084) were further identified. No statistically significant effects of lung cancer on immunophenotypes were found.ConclusionsThe elevated level of CD14− CD16+ monocytes was a protective factor against lung cancer. Conversely, CD27 on CD24+ CD27+ B cell was a risk factor. CD27 on class-switched memory B cells and IgD+ CD24+ B cells were potential risk factors for lung cancer. This research enhanced our comprehension of the interplay between immune responses and lung cancer risk. Additionally, these findings offer valuable perspectives for the development of immunologically oriented therapeutic strategies.

Cholera toxin and O-specific polysaccharide immune responses after oral cholera vaccination with Dukoral in different age groups of Bangladeshi participants.

Vaccination is important to prevent cholera. There are limited data comparing anti-O-specific polysaccharide (OSP) and anti-cholera toxin-specific immune responses following oral whole-cell with cholera toxin B-subunit (WC-rBS) vaccine (Dukoral, Valneva) administration in different age groups. An understanding of the differences is relevant because young children are less well protected by oral cholera vaccines than older children and adults. We compared responses in 50 adults and 49 children (ages 2 to <18) who were administered two doses of WC-rBS at a standard 14-day interval. All age groups had significant IgA and IgG plasma-blast responses to the OSP and cholera toxin B-subunit (CtxB) antigens that peaked 7 days after vaccination. However, in adults and older children (ages 5 to <18), antibody responses directed at the OSP antigen were largely IgA and IgG, with a minimal IgM response, while younger children (ages 2 to <5) mounted significant increases in IgM with minimal increases in IgA and IgG antibody responses 30 days after vaccination. In adults, anti-OSP and CtxB memory B-cell responses were detected after completion of the vaccination series, while children only mounted CtxB-specific IgG memory B-cell responses and no OSP-memory B-cell responses. In summary, children and adults living in a cholera endemic area mounted different responses to the WC-rBS vaccine, which may be a result of more prior exposure to Vibrio cholerae in older participants. The absence of class-switched antibody responses and memory B-cell responses to OSP may explain why protection wanes more rapidly after vaccination in young children compared to older vaccinees.IMPORTANCEVaccination is an important strategy to prevent cholera. Though immune responses targeting the OSP of V. cholerae are believed to mediate protection against cholera, there are limited data on anti-OSP responses after vaccination in different age groups, which is important as young children are not well protected by current oral cholera vaccines. In this study, we found that adults mounted memory B-cell responses to OSP, which were not seen in children. Adults and older children mounted class-switched (IgG and IgA) serum antibody responses to OSP, which were not seen in young children who had only IgM responses to OSP. The lack of class-switched antibody responses and memory B-cell responses to OSP in younger participants may be due to lack of prior exposure to V. cholerae and could explain why protection wanes more rapidly after vaccination in young children.

Lymphocyte B Subtypes in Peripheral Blood: A Prognostic Biomarker for COVID-19 Patients.

In view of the scientific gap in knowledge of the involvement of the B-cell compartment and clinical prognostic in SARS-CoV-2 infection, this work aims to evaluate the B-cell subsets and the presence of specific IgM and IgG, as well as neutralizing antibodies against SARS-CoV-2, in unvaccinated patients diagnosed with COVID-19. This study included 133 patients with COVID-19. Cellular components were assessed by flow cytometry, and immunoglobulin levels and reactivity were measured by indirect enzyme-linked immunosorbent assay. Our results showed no changes in less differentiated B cells. However, non-switched memory B cells (NS-MBCs) and class-switched memory B cells (CS-MBCs) were reduced in the patients with moderate disease. Also, plasmablasts and double-negative (DN) or "atypical" memory B cells were increased in groups of patients with moderate to critical conditions. In addition, the production of IgM, IgG, and neutralizing antibodies against SARS-CoV-2 demonstrated a positive correlation between the positivity of antibodies against SARS-CoV-2 and disease severity. Besides being related to the development of a more severe course of the disease, the increase in DN B-cell count also contributed to a poorer disease outcome in patients with a higher percentage of these cells. On the other hand, we observed an increase in the absolute number of CS-MBCs in patients with greater chances of survival. This study demonstrates that the B-cell compartment may contribute to the development of clinical symptoms of COVID-19, with changes in B-cell subset counts linked to disease course and patient prognosis.

Causative mechanisms and clinical impact of immunoglobulin deficiencies in ataxia telangiectasia

BackgroundAtaxia Telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility, caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin (Ig)-deficiency, mainly IgA and IgG2, with a variable severity. So far, the exact mechanisms underlying the Ig-deficiency, especially the variable severity, remain unelucidated. ObjectiveTo characterize the clinical impact of Ig-deficiencies in AT and to elucidate the mechanisms of Ig-deficiencies in AT. MethodsWe analyzed long-term Ig-levels, immunophenotyping, and survival time in our cohort (n=87, median age 16 years; maximum 64 years). Somatic hypermutation (SHM) and class-switch junctions in B-cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed followed by RNAseq, to assess the effect of ATM-inhibition. ResultsOnly the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG2-deficiencies did not. The Ig-levels showed predominantly decreased IgG2 and IgA. Moreover, flowcytometric analysis demonstrated reduced naïve B- and T-lymphocytes and a deficiency of class-switched IgG2 and IgA memory B-cells. SHM frequencies were lowered in IgA and IgG2-deficient patients, indicating a hampered germinal center (GC) reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end-joining during class-switch DNA-repair. The in vitro class-switching and proliferation were negatively affected by ATM-inhibition. RNA-sequencing analysis showed that ATM-inhibitor influenced expression of GC reaction genes. ConclusionIg-deficiency in AT is caused by disturbed development of class-switched memory B-cells. This study suggests that ATM-deficiency affects both the GC reaction and the choice of DNA-repair pathways in class-switching.

"Deficiency in ELF4, X-Linked": a Monogenic Disease Entity Resembling Behçet's Syndrome and Inflammatory Bowel Disease.

Defining monogenic drivers of autoinflammatory syndromes elucidates mechanisms of disease in patients with these inborn errors of immunity and can facilitate targeted therapeutic interventions. Here, we describe a cohort of patients with a Behçet's- and inflammatory bowel disease (IBD)-like disorder termed "deficiency in ELF4, X-linked" (DEX) affecting males with loss-of-function variants in the ELF4 transcription factor gene located on the X chromosome. An international cohort of fourteen DEX patients was assessed to identify unifying clinical manifestations and diagnostic criteria as well as collate findings informing therapeutic responses. DEX patients exhibit a heterogeneous clinical phenotype including weight loss, oral and gastrointestinal aphthous ulcers, fevers, skin inflammation, gastrointestinal symptoms, arthritis, arthralgia, and myalgia, with findings of increased inflammatory markers, anemia, neutrophilic leukocytosis, thrombocytosis, intermittently low natural killer and class-switched memory B cells, and increased inflammatory cytokines in the serum. Patients have been predominantly treated with anti-inflammatory agents, with the majority of DEX patients treated with biologics targeting TNFα.

Influence of Splenomegaly and Splenectomy on the Immune Cell Profile of Patients with Common Variable Immunodeficiency Disease.

About 25% of patients with common variable immunodeficiency disease (CVID) have splenomegaly, necessitating sometimes splenectomy whom consequences on the immunological profile of CVID patients have never been studied. We analyzed 11 CVID patients' comprehensive blood immune cell phenotypes pre- and post-splenectomy. Flow cytometry analyses of immune cell populations. Among 89 CVID cohort patients, 41 with splenomegaly, splenomegaly was strongly associated with granulomatous disease, autoimmune disorders, lymphoid hyperplasia, and/or portal hypertension. CVID patients with splenomegaly have significant peripheral lymphopenia (p = 0.001), and significantly fewer peripheral class-switched memory B cells (smBs) (p = 0.001), CD4+ T lymphocytes (p = 0.001), NK (p = 0.0001) and dendritic cells (p ≤ 0.01), and significantly more circulating CD4+ and CD8+ (p = 0.00001) T cell subset activation (p = 0.00005), than CVID patients without splenomegaly. Examination of splenectomy impact on circulating lymphocyte subset distributions demonstrated the drastically enhanced total circulating lymphocyte count post-splenectomy, predominantly B lymphocytes and CD8+ T cells. However, splenectomy did not change B cell distribution, with smBs remaining persistently low, in contrast to complete inversion of the circulating T cell composition, with reversal of the CD4+/CD8+ ratio suggesting that amplification of the CD8+ T cell compartment is a CVID characteristic in patients with splenomegaly. Our results highlight this CD8+ amplification in CVID-splenomegaly patients that might be explained by a homing effect to the spleen and/or possible chronic virus replication, which in turn could induce T cell expansions. Splenectomizing CVID patients with splenomegaly restores the absolute circulating lymphocyte count, suggesting that the decreased T cell count in the presence of splenomegaly cannot be used as an exclusive criterion for combined immunodeficiency.

Human IgM-expressing memory B cells.

A hallmark of T cell dependent (TD) humoral immune responses is the generation of long-lived memory B cells. The generation of these cells occurs primarily in the germinal center (GC) reaction, where antigen-activated B cells undergo affinity maturation as a major consequence of the combined processes of proliferation, somatic hypermutation of their immunoglobulin V (IgV) region genes, and selection for improved affinity of their B-cell antigen receptors. As many B cells also undergo class-switching to IgG or IgA in these TD responses, there was traditionally a focus on class-switched memory B cells in both murine and human studies on memory B cells. However, it has become clear that there is also a large subset of IgM-expressing memory B cells, which have important phenotypic and functional similarities but also differences to class-switched memory B cells. There is an ongoing discussion about the origin of distinct subsets of human IgM+ B cells with somatically mutated IgV genes. We argue here that the vast majority of human IgM-expressing B cells with somatically mutated IgV genes in adults is indeed derived from GC reactions, even though a generation of some mostly lowly mutated IgM+ B cells from other differentiation pathways, mainly in early life, may exist.