B-cell Research Articles

Early life stress (ELS) promotes B cell expansion and aortic pathology in a mouse model of systemic lupus erythematosus (SLE)

SLE is an autoimmune disease that primarily affects young women and is associated with high rates of cardiovascular disease (CVD). Exposure to ELS (adversity in childhood such as verbal and physical abuse and/or familial dysfunction) is an independent risk factor for CVD and SLE. ELS heightens the risk of developing SLE and SLE patient-reported disease activity, however the underlying mechanism is unclear. We previously showed in a mouse model of ELS, maternal separation with early weaning (MSEW), that mice with pristane-induced SLE develop autoantibodies and increased aortic stiffness earlier in disease progression compared to normally reared (NR) SLE mice. We hypothesized that disease acceleration in MSEW+SLE mice compared to NR+SLE mice is associated with an expansion of B-cells and aortic structural changes. The pristane-induced model of SLE uses an intraperitoneal injection (i.p.) to induce SLE-like symptoms in female mice. The MSEW protocol separates pups from the dam 4h/day (postnatal, PD 2 to 5) and 8h/day (PD 6 to 16) with weaning at PD17. NR litters are weaned at PD21. At 6-8 weeks of age, female mice were randomly assigned to control (CON) or SLE groups: CON received an i.p. PBS injection, while NR+SLE and MSEW+SLE received an i.p. pristane injection. Spleen and thymus were weighed and collected for B-cell subset analysis with flow cytometry and aorta were dissected for histological assessments. At 4 months-post pristane, spleen weights did not differ between groups, however, we observed that MSEW+SLE mice had significantly larger thymus/body weight (BW) compared to CON and NR+SLE mice (p=0.0004 F=7.5 n=4). At 7 months post-pristane or PBS injection, MSEW+SLE mice and NR+SLE mice had significantly higher splenic weights/BW compared to CON mice (p=0.03 F=4.36, n=6) as expected. Using flow cytometry, we found increased thymic %age of CD19+ B-cells in both NR+SLE and MSEW+SLE compared to CON (p<0.0001 F=57.33, n=8,4,4), whereas we found similar %age of CD19+ B-cells in the aorta or spleen of mice in all groups. MSEW+SLE mice had increased germinal center B-cells compared to CON (CON: 0.77%, NR+SLE: 6.0%, MSEW+SLE 7.4%, p=0.008 F=7.08 n=8,4,4) while NR+SLE mice had increased plasma cells compared to CON (CON: 0.08%, NR+SLE: 1.3%, MSEW+SLE 0.68%, p=0.015 F=5.92 n=8,4,4). At 7 months post-pristane, MSEW+SLE mice had significantly greater aortic wall thickness (um) compared to NR+SLE and NR+CON mice (p=0.008 F=11.99, n=3,3,3), however, aortic collagen content (picrosirius red) was similar. These data suggest a difference in B-cell subset expansion and aortic structural changes may contribute to the MSEW driven acceleration of disease activity. Understanding how ELS accelerates autoimmunity and CVD risk is crucial to preventing worsened outcomes in vulnerable populations of individuals exposed to ELS with SLE. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

B cell subsets in thymus from infants with Down syndrome

B lymphocytes are still poorly characterized in the human thymus, and due to the immunological peculiarities of Down syndrome, a detailed investigation of thymic B lymphocytes and their subsets was performed in <2-year-old infants. This is a period of intense thymic activity, and here we compared thymuses from infants with Down syndrome (DG) and age-matched control group (CG) with heart disease as the only described malformation, no clinical signs of immunodeficiency and normal blood lymphocyte numbers. MethodsThis study, approved by the institutional ethics committee, included 10 thymic samples from DG infants, and 20 from CG infants. Depending on thymus size and the type of surgical approach, babies were submitted to total thymectomy or only the removal of a small thymic fragment (mainly in the DG), always during cardiac surgery in order to correct congenital defects. Flow cytometry was used for immunophenotyping of cell suspension obtained from thymic tissue; and the B lymphocyte topographic localization was investigated by immunohistochemistry, and a quantitative evaluation of B-cells in each region was carried out using methods of digitized images analysis. ResultsFlow cytometry revealed that CD20+ cells represent around 1% of total lymphocytes in both groups, with higher naive and lower unswitched memory B lymphocyte frequencies in DG when compared to the CG, while B1 cells were present in much higher frequency in DG than in CG. All the other B-cell subpopulations (transitional, switched memory, plasmablasts and plasmocytes) were statistically equivalent between groups. Immunohistochemistry showed higher frequencies of CD20+ cells in medullary region compared to cortex, without differences between the groups (DG = 16.8% and CG = 17.1% of total cell number), while the cortex region had few B cells in both groups, with significantly lower frequency in DG group, reaching 0.25%, compared with the CG, which showed 0.6%. Conclusion: Flow cytometry revealed a different distribution of B-cell subtypes in DG thymus compared to CG and, thus, data obtained from such rare material could be relevant in the characterization and further analysis and understanding of the immunological mechanisms that involve the participation of B lymphocyte subpopulations in the thymus. FAPESP (grant 2014/50489-9).

Abstract 443: BAFF Neutralization Impairs The Regression Of Insulin Resistance In Diabetic Mice By Modulating The Innate Immune Response

Adults with type 2 diabetes have a two-to-three-fold increased risk of cardiovascular diseases. B cell activating factor (BAFF), a tumor necrosis factor superfamily member, is implicated both in diabetes and cardiovascular diseases, and hence a therapeutic target of interest. High levels of BAFF are known to promote autoantibody synthesis and autoimmune diseases. We examined if BAFF neutralization with an anti-BAFF antibody improves glucose intolerance in a model of regression of insulin resistance in diabetic mice. Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks and segregated into two groups based on their glucose intolerance determined by a glucose tolerance test. Each group was simultaneously switched from HFD to a normal chow diet and received injections of anti-BAFF or an isotype control antibody every 2 nd week for a total of 6 weeks. As expected, body weights decreased in both groups parallel with the increase in insulin sensitivity. Unexpectedly, the BAFF-neutralized mice had impaired regression of insulin resistance compared to the control mice. Extensive cellular phenotyping of immune cells indicated systemic depletion of only the B2 cells and lower levels of circulating autoantibodies in the BAFF-neutralized mice. Gonadal white adipose tissue (gWAT) was the primary source of BAFF, and no differences in the M1 and M2 macrophages, eosinophils, natural killer cells, helper T cells, and cytotoxic T cell populations were found. RNA sequencing analysis of stromal vascular fraction from gWAT revealed impaired B cell receptor signaling, immunoglobulin production, complement activation, and phagocytosis pathways in the BAFF-neutralized mice. In this line, (i) an increased number of dead adipocytes, (ii) decreased levels of IgG2b and IgM, (iii) increased citrullinated histone H3, neutrophils, and DNA extracellular traps were found in the gWAT of BAFF-neutralized mice. These results suggest attenuated humoral response and a dominant and harmful role of innate response in the gWAT following BAFF neutralization in late-stage obesity. Since anti-BAFF biologics are approved for the treatment of lupus patients, our results highlight the need for further studies on the role of BAFF in late-stage obesity and diabetes.

Heterozygous TCF3-Related Disease Presenting as X-linked Agammaglobulinemia Mimicry in a Male Toddler with B-cell Aplasia, Agammaglobulinemia, and Severe Neutropenia

IntroductionDisease associated with TCF3 variants are rare and associated with autosomal dominant inheritance agammaglobulinemia. Patients present with profound reduction in CD19+ cells and a block in B cell development at the common lymphoid precursor to pro–B cell phase of differentiation. Severe neutropenia is frequently described in X-linked agammaglobulinemia (XLA) patients but not yet described as a presenting finding in TCF3-related disease. Case ReportThis case describes a 13-month-old full term male with a history of reactive airway disease and recurrent infections including multiple bouts of acute otitis media, hand -foot-mouth, and viral upper respiratory illnesses who presented with fever and persistent severe neutropenia (absolute neutrophil count <100 cell/uL) necessitating hospital admission. Given his infectious history and severe neutropenia, he underwent evaluation for an underlying inborn error of immunity with the following pertinent findings: undetectable IgG (<16 mg/dL), IgM (<10 mg/dL), IgA (<2 mg/dL) and IgE (<2 kU/L), negative pneumococcal, tetanus and diphtheria vaccine titers despite vaccination, lymphocyte subsets with normal CD3, CD4, CD8, and CD16/56 absolute values but extremely low CD19 level (38 cells/cmm), and negative anti-neutrophil antibodies. Patient Bruton tyrosine kinase (Btk) protein flow cytometry results in monocytes were similar to an experimental control, while B-cell subset analysis was unable to be obtained given low B-cell numbers. Comprehensive genetic testing subsequently identified a heterozygous pathogenic TCF3 variant, c.1663G>A, p. (Glu555Lys). Following initiation of intravenous immunoglobulin (IVIG) replacement, our patient's neutropenia resolved quickly and completely. He continues to receive monthly IVIG with goal IgG troughs >800 mg/dL and resultant significant improvement in infections. ConclusionAs in XLA, our patient's severe neutropenia rapidly resolved with initiation of immunoglobulin replacement. While it has been shown that neutrophil development is somewhat dependent on the Btk protein, further study into the importance of the TCF3 protein and additionally adequate immunoglobulin levels in neutrophil expansion is needed. Finally, germline inborn errors of immunity, including TCF3 variants, should be considered in children with a history of recurrent infections and persistent neutropenia.

B Cell Targeting mAb–siRNA Conjugates Reduced Autoantibody in Experimental Myasthenia

Abstract Conjugates of monoclonal antibodies (mAbs) and siRNA have been used for target cell-specific gene repression. B cells are integrally involved in the anti-Acetylcholine receptor autoantibody-mediated pathogenic development of autoimmune myasthenia gravis (MG). Here we show that simultaneous targeting of the critical receptors expressed on pathogenic B-cell subsets with the optimized dose of mAb-siRNA conjugates markedly depleted target B cells and substantially reduced pathogenic autoantibody in a mouse model of MG. The results show the potential of mAb-conjugates in the treatment of myasthenia gravis. Supported by grants from AFM-Telethon, France

STAT5-regulated autocrine IL-6 signaling dictates IL-10-dependent regulatory functions of neonatal B10 cells

Abstract Neonates have suboptimal immune responses to immunization, resulting in higher susceptibility to microbial infection. B cells have a central role in humoral immunity to immunization, and antigen recognition by BCRs is essential for the activation and differentiation of B cells. Neonatal B cells, however, do not respond to BCR activation as robustly as adult cells do. Our hypothesis is that unique features of neonatal BCR signaling may contribute to suboptimal humoral responses. The aim of this project is to decipher the neonatal BCR signaling pathways and identify those that may be responsible for suboptimal B cell functions. Purified adult and neonatal B cells were stimulated with anti-IgM. RNA-seq analysis revealed that STAT3 and STAT5 signaling pathways were activated in neonatal B cells. STAT3 and STAT5 were phosphorylated in neonatal B cells. BCR-mediated STAT5 phosphorylation induces IL-6 production, which in turn activates STAT3 in an autocrine/paracrine manner. Moreover, IL-6-induced STAT3 activation led to the production of anti-inflammatory cytokine IL-10. Further underscoring the role of IL-6 in IL-10 production, neonatal B cells from IL-6-KO mice did not secrete IL-10. To assess whether the autocrine IL-6 is essential for IL-10-dependent immunosuppression, we incubated peritoneal macrophages with conditioned medium (CM) from adult, wild-type neonatal, or IL-6-KO neonatal B cells. We found that CM from wild-type neonatal B cells suppressed TNF-α production by macrophages in an IL-10-dependent manner, whereas CM from IL-6-KO neonatal B cells or adult B cells did not. Our studies unveiled the essential role for STAT5-induced autocrine IL-6 signaling in IL-10-mediated immunosuppressive functions of neonatal B cells.

B1 B cells contribute to inflammation and metabolic dysfunction in aged adipose tissue during endotoxemia.

Abstract The elderly are at the greatest risk for severe endotoxemia and bacterial infections. The increase in low grade chronic inflammation and the decline of the immune response during aging contributes to this disease severity. Visceral adipose tissue (vWAT) exhibits immune recruitment and activation during pathogen challenge and metabolic diseases. Immune cells regulate lipolysis in vWAT which is required for the regulation of inflammation, but the intersection of bacterial inflammation and metabolism during aging is not clear. We investigated the contribution of B cells to metabolic dysfunction and inflammation during endotoxemia in vWAT. Old vWAT exhibited elevated inflammation and altered activation of resident B cells during LPS-induced endotoxemia. LPS also induced vWAT lipolysis in young mice but not in old mice. Diet induced obesity resulted in elevated LPS-induced inflammatory cytokines and B cells that failed to become activated by LPS. Deficiency of total B cells improved the homeostatic balance of immune cells within vWAT microenvironment, resulting in increased lipolysis and reduced Tnfα during endotoxemia in old mice. In contrast, B2 B cell depletion was insufficient to increase LPS-induced lipolysis or reduce inflammatory cytokines in vWAT from old mice, suggesting a driving role for B1 cells. B1 B cells from young and old vWAT were differentially activated by LPS indicating age-associated dysfunction. Our results demonstrate that endotoxemia results in immune and metabolic activation within the vWAT, both of which are dysfunctional during aging. These data reveal B cell subsets may differentially impair lipolysis and exacerbate the inflammatory response in vWAT following pathogenic challenge. Supported by grants from NIH (R00 AG058800, R01 AG079913)

Carteolol triggers senescence via activation of β-arrestin–ERK–NOX4–ROS pathway in human corneal endothelial cells in vitro

Carteolol is a commonly-used topical medication for primary open-angle glaucoma. However, long-term and frequent ocular application of carteolol entails its residuals at low concentration in the aqueous humor for a long duration and may exert latent toxicity in the human corneal endothelial cells (HCEnCs). Here, we treated the HCEnCs in vitro with 0.0117% carteolol for 10 days. Thereafter, we removed the cartelolol and normally cultured the cells for 25 days to investigate the chronical toxicity of carteolol and the underlying mechanism. The results exhibited that 0.0117% carteolol induces senescent features in the HCEnCs, such as increased senescence-associated β-galactosidase positive rates, enlarged relative cell area and upregulated p16INK4A and senescence-associated secretory phenotypes, including IL-1α, TGF-β1, IL-10, TNF-α, CCL-27, IL-6 and IL-8, as well as decreased Lamin B1 expression and cell viability and proliferation. Thereby, further exploration demonstrated that the carteolol activates β-arrestin–ERK–NOX4 pathway to increase reactive oxygen species (ROS) production that imposes oxidative stress on energetic metabolism causing a vicious cycle between declining ATP and increasing ROS production and downregulation of NAD+ resulting in metabolic disturbance-mediated senescence of the HCEnCs. The excess ROS also impair DNA to activate the DNA damage response (DDR) pathway of ATM–p53–p21WAF1/CIP1 with diminished poly(ADP-Ribose) polymerase (PARP) 1, a NAD+-dependent enzyme for DNA damage repair, resulting in cell cycle arrest and subsequent DDR-mediated senescence. Taken together, carteolol induces excess ROS to trigger HCEnC senescence via metabolic disturbance and DDR pathway.

Leveraging the tolerogenic potential of TNF-α and regulatory B cells in organ transplantation.

A subset of B-cells with tolerogenic functions, termed B-regulatory cells or Bregs, is characterized by the expression of anti-inflammatory/tolerogenic cytokines, namely IL-10, TGF-β, and IL-35, that contribute to their regulatory functions. Breg regulation favors graft acceptance within a tolerogenic milieu. As organ transplantation invariably triggers inflammation, new insights into the crosstalk between cytokines with dual properties and the inflamed milieu are needed to tailor their function toward tolerance. Using TNF-α as a proxy of dual-function cytokines involved in immune-related diseases and transplantation settings, the current review highlights the multifaceted role of TNF-α. It focuses on therapeutic approaches that have revealed the complexity of TNF-α properties tested in clinical settings where total TNF-α inhibition has proven ineffective and often detrimental to clinical outcomes. To improve the efficacy of current TNF-α inhibiting therapeutics, we propose a three-prong strategy to upregulate the tolerogenic pathway engaging the TNFR2 receptor while simultaneously inhibiting the inflammatory mechanisms associated with TNFR1 engagement. When combined with additional administrations of Bregs-TLR that activate Tregs, this approach may become a potential therapeutic in overcoming transplant rejection and promoting graft tolerance.

Classification of Common Variable Immunodeficiency Using Measurement of B-cell Subsets in Moroccan Patients

Classification of Common Variable Immunodeficiency Using Measurement of B-cell Subsets in Moroccan Patients

Gingipains are the important virulence factors of Porphyromonas gingivalis downregulating B10 cells.

Porphyromonas gingivalis (P. gingivalis) is a keystone pathogen in periodontitis. Our previous study indicated that periodontitis induced by P. gingivalis increased the percentage of CD19+ B cells, but decreased the ratio of IL-10 producing regulatory B cells (B10) in collagen-induced arthritis (CIA) mice. It's still unclear which virulence factors of P. gingivalis are involved in these processes. Here, we compared the effects of different components of P. gingivalis on the biogenesis of B10 cells, and found that the decreased proportion of B10 cells was mainly resulted from the undenatured proteins other than the DNA, RNA or LPS of P. gingivalis. As gingipains are enzymes and virulence factors which play vital role in the progression in periodontitis through affecting innate and adaptive immune system, we then compared the influence of the wild-type (WT) strain of P. gingivalis (ATCC 33277) and its isogenic gingipain-null mutant (∆K∆RAB) on differentiation of splenic B cells into B10 cells. Interestingly, compared to WT strain, ∆K∆RAB treatment increased the frequency of B10 cells as well as expression of IL-6 in B cells. Furthermore, the acute peritonitis, an ideal model for quick evaluation of immune effects of agents, induced by ∆K∆RAB showed higher IL-6 production and proportion of B10 cells compared with WT. Finally, we performed transcriptomic analysis to better understand the effects and possible mechanism of gingipains on B cells. Compared with WT, ∆K∆RAB upregulated the PI3K-Akt pathway of B cells which is important for IL-10 production and B10 cell biogenesis, and more activated Jak-STAT pathway which is a classical signaling pathway mediated by IL-6. Cumulatively, this study preliminarily revealed that gingipains of P. gingivalis are vital virulence factors downregulating B10 cells and altering immune responses. This article is protected by copyright. All rights reserved.

Association of B cell Subsets and Aquaporin-4 antibody titers with Disease Activity in Participants in the N-MOmentum trial Receiving Inebilizumab Treatment (S5.005)

Association of B cell Subsets and Aquaporin-4 antibody titers with Disease Activity in Participants in the N-MOmentum trial Receiving Inebilizumab Treatment (S5.005)

CD5 knockout modulates effects of alcohol consumption in mouse models (P1-12.008)

<h3>Objective:</h3> We seek to elucidate the impact of cluster of differentiation 5 (CD5) expression on the effects of acute ethanol (EtOH) exposure. <h3>Background:</h3> CD5 is a transmembrane glycoprotein expressed on the surface of T and B1 cells. It has been previously shown that CD5 expression is altered by chronic EtOH consumption. Additionally, GABA neurons in the ventral tegmental area (VTA) are inhibited by EtOH, while dopamine neurons are excited. Given that some CD5+ lymphocytes may be involved in the regulation of inhibitory GABA synapses, we hypothesize that CD5 knockout may modulate the behavioral and physiological effects of EtOH consumption. <h3>Design/Methods:</h3> CD5 knockout (CD5KO) mice were compared to C57BL/6J wild type (WT) mice. CD5KO was confirmed with flow cytometry. Behavioral changes were measured via open field behavior, loss of righting reflex, and 24-hour access two bottle choice drinking. Neuronal dynamics were assessed using single-unit recordings, loose patch clamp recordings, fast scan cyclic voltammetry, and qRTPCR. <h3>Results:</h3> CD5KO mice displayed reduced EtOH consumption versus WT mice in two bottle choice drinking as well as decreased reduction in locomotor activity at low dose (0.5–2.0g/Kg) EtOH and decreased sedation at high dose (4.0g/Kg). Paired-pulse frequency response measure of dopamine release also differed between CD5KO and WT strains. Additionally, decreased levels of dopamine transporter (DAT) RNA were found in the nucleus accumbens. <h3>Conclusions:</h3> CD5KO mice may be less sensitive to some of the effects of EtOH, suggesting that CD5 may play a role in EtOH consumption and sedation. <b>Disclosure:</b> Mr. Amendolara has nothing to disclose. Dr. Payne has nothing to disclose. Dr. Obray has nothing to disclose. Mr. Williams has nothing to disclose. Ms. Small has nothing to disclose. The institution of Dr. Yorgason has received research support from American Osteopathic Association. Dr. Edwards has nothing to disclose. Dr. Weber has nothing to disclose. Dr. Steffensen has stock in Syzygy Innovations, LLC. Dr. Steffensen has received intellectual property interests from a discovery or technology relating to health care.

Immunological Signatures Associated With Ocrelizumab Treatment in Early RRMS: 12-Month Interim Analysis From the Ocrelizumab Phase IIIb (ENSEMBLE; NCT03085810) Immunological Substudy (S16.003)

Immunological Signatures Associated With Ocrelizumab Treatment in Early RRMS: 12-Month Interim Analysis From the Ocrelizumab Phase IIIb (ENSEMBLE; NCT03085810) Immunological Substudy (S16.003)

Transcriptomic analysis of B cells suggests that CD70 and LY9 may be novel features in patients with systemic lupus erythematosus

Dysfunction of B-cell subsets is critical in the development of systemic lupus erythematosus (SLE). There is a great diversity of B-lineage cells, and their features and functions in SLE need to be clarified. In this study, we analyzed single-cell RNA sequencing (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) and bulk transcriptomic data of isolated B-cell subsets from patients with SLE and healthy controls (HCs). We preformed scRNA-seq analysis focused on the diversity of B-cell subsets and identified a subset of antigen-presenting B cells in SLE patients that highly expressed ITGAX. A list of marker genes of each B-cell subset in patients with SLE was also identified. Comparison of bulk transcriptomic data of isolated B-cell subpopulations between SLE patients and HCs revealed the upregulated differentially expressed genes (DEGs) for each B-cell subpopulation in SLE. Common genes identified using these two methods were considered to be upregulated marker genes of B cells in SLE. The scRNA-seq data of SLE patients and HCs revealed that CD70 and LY9 were overexpressed in B cells vs. other cell types from SLE patients, and this pattern was validated by RT‒qPCR. Because CD70 is the cellular ligand of CD27, previous studies on CD70 have focused mainly on T cells from SLE patients. LY9 appears to have different functions in mice and humans: its expression is decreased in lupus-prone mice but is increased in T cells and some B-cell subpopulations in SLE patients. Here, we describe the overexpression of two costimulatory molecules, CD70 and LY9, which may be a novel feature of B cells in SLE patients.

Insights into the Roles of B Cells in Patients with Sepsis.

Sepsis is a life-threatening yet common disease, still posing high mortality worldwide. Sepsis-related deaths primarily occur during immunosuppression; the disease can hamper the numbers and function of B cells, which mediate innate and adaptive immune responses to maintain immune homeostasis. Dysfunction of B cells, along with aggravated immunosuppression, are closely related to poor prognosis. However, B cells in patients with sepsis have garnered little attention. This article focuses on the significance of B-cell subsets, including regulatory B cells, in sepsis and how the counts and function of circulating B cells are affected in patients with sepsis. Finally, potential B-cell-related immunotherapies for sepsis are explored.

B-1 cells in immunotoxicology: Mechanisms underlying their response to chemicals and particles.

Since their discovery nearly 40years ago, B-1 cells have continued to challenge the boundaries between innate and adaptive immunity, as well as myeloid and lymphoid functions. This B-cell subset ensures early immunity in neonates before the development of conventional B (B-2) cells and respond to immune injuries throughout life. B-1 cells are multifaceted and serve as natural- and induced-antibody-producing cells, phagocytic cells, antigen-presenting cells, and anti-/pro-inflammatory cytokine-releasing cells. This review retraces the origin of B-1 cells and their different roles in homeostatic and infectious conditions before focusing on pollutants comprising contact-sensitivity-inducing chemicals, endocrine disruptors, aryl hydrocarbon receptor (AHR) ligands, and reactive particles.

Regulatory B Cells Profile in Kidney Transplant Recipients With Chronic-Active Antibody-Mediated Rejection

This study aims to reveal the relationship between regulatory B cell (Breg) subsets and chronic-active antibody-mediated rejection (c-aABMR) in renal transplant recipients. Our study involved 3 groups of participants: renal transplant recipients with biopsy-proven c-aABMR as the chronic rejection group (c-aABMR, n=23), recipients with stable graft functions as the patient control group (PC; n=11), and healthy volunteers (HV; n=11). Breg subsets, immature/transitional B cells, plasmablastic cells, B10 cells, and BR1 cells were isolated from venous blood samples by flow cytometry. The median values of Breg frequencies in the total lymphocyte population were analyzed. There were no significant differences between the study groups for immature and/or transitional B cell frequencies. Plasmablastic cell frequencies of the c-aABMR group (7.80 [2.10-27.40]) and the PC group (6.00 [1.80-55.50]) were similar, but both of these values were significantly higher than the HVs' (3.40 [1.20-8.50]), (respectively, P=.005 and P=.039). B10 cell frequencies were also similar, comparing the c-aABMR (4.20 [0.10-7.40]) and the PC groups (4.10 [0.10-5.90]), whereas the HVs (5.90 [2.90-8.50]) had the highest B10 cell frequency with an only statistical significance against the PC group (respectively, P=.09 and P=.028). The c-aABMR and the PC groups were similar regarding BR1 cell frequencies. However, the HV group significantly had the highest frequency of BR1 cells (5.50 [2.80-10.80]) than the other groups (P < .001 for both). We demonstrated that frequencies of B10 and BR1 cells were higher in HVs than in transplant recipients, regardless of rejection state. However, there was no significant relation between Breg frequencies and the c-aABMR state.

Dandelion uses the single-cell adaptive immune receptor repertoire to explore lymphocyte developmental origins.

Assessment of single-cell gene expression (single-cell RNA sequencing) and adaptive immune receptor (AIR) sequencing (scVDJ-seq) has been invaluable in studying lymphocyte biology. Here we introduce Dandelion, a computational pipeline for scVDJ-seq analysis. It enables the application of standard V(D)J analysis workflows to single-cell datasets, delivering improved V(D)J contig annotation and the identification of nonproductive and partially spliced contigs. We devised a strategy to create an AIR feature space that can be used for both differential V(D)J usage analysis and pseudotime trajectory inference. The application of Dandelion improved the alignment of human thymic development trajectories of double-positive T cells to mature single-positive CD4/CD8 T cells, generating predictions of factors regulating lineage commitment. Dandelion analysis of other cell compartments provided insights into the origins of human B1 cells and ILC/NK cell development, illustrating the power of our approach. Dandelion is available at https://www.github.com/zktuong/dandelion .

A Subset of Breg Cells, B10, Contributes to the Development of Radiation-Induced Pulmonary Fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a serious side effect of radiotherapy, but the underlying mechanisms are unknown. B10 cells, as negative B regulatory cells, play important roles in regulating inflammation and autoimmunity. However, the role of B10 cells in RIPF progression is unclear. The aim of this study was to determine the role of B10 cells in aggravating RIPF and the underlying mechanism. The role of B10 cells in RIPF was studied by constructing mouse models of RIPF and depleting B10 cells with an anti-CD22 antibody. The mechanism of B10 cells in RIPF was further explored through cocultivation of B10 cells and MLE-12 or NIH3T3 cells and administration of an IL-10 antibody to block IL-10. B10 cell numbers increased significantly during the early stage in the RIPF mouse models compared with the controls. In addition, depleting B10 cells with the anti-CD22 antibody attenuated the development of lung fibrosis in mice. Subsequently, we confirmed that B10 cells induced EMT and the transformation of myofibroblasts via activation of STAT3 signaling in vitro. After blockade of IL-10, it was verified that IL-10 secreted by B10 cells mediates the EMT of myofibroblasts, thereby promoting RIPF. Our study uncovers a novel role for IL-10-secreting B10 cells that could be a new target of research for relieving RIPF.