Effects of regulatory B cells on intracranial aneurysms by mediating IL-1ß/IL-1R pathways.

The purpose of this study was to explore the effects of regulatory B-cells (Breg) on intracranial aneurysms by mediating IL-1ß/IL-1R pathways.  The study involved 60 patients undergoing angiography in a hospital from January to June 2022, divided into two groups: 30 with intracranial aneurysms (observation group) and 30 without (control group). Researchers extracted peripheral blood mononuclear cells (PBMC) to analyze the proportion of CD19+CD24hiCD38hiB cells using flow cytometry. These cells, along with T-cells and regulatory T-cells (Treg), were isolated through magnetic bead cell sorting. Following co-culture, the proliferation of T-cells and their related secretory factors were assessed. Additionally, Breg cells, treated with an IL-1R receptor blocker or IL-1R expression adenovirus, were studied to evaluate the levels of IL-10 and TGF-ß. In the study, the observation group showed lower levels of CD19+CD24hiCD38hiB cells, IL-10, and TGF-ß in PBMC than the control group (P<0.05). T-cell proportions were similar in both groups pre and post co-culture (P>0.05). Post co-culture, IFN-γ decreased while IL-4 increased in both groups. The observation group had higher IFN-γ and lower IL-4 than the control group (P<0.05). TNF-α in CD8+T cells, and granzyme B and perforin mRNA levels decreased post co-culture but were higher in the observation group (P<0.05). IL-10 and TGF-ß in Treg cells increased in both groups post co-culture but were lower in the observation group (P<0.05). The observation group also had fewer CD19+IL-1R+IL-10+B cells (P<0.05). After IL-1R blocker addition, IL-10 and TGF-ß in the supernatant decreased in the observation group (P<0.05). Following transfection, IL-1 and TGF-ß levels increased compared to the blank group (P<0.05). The function of peripheral blood CD19+CD24hiCD38hiB cells is impaired in patients with intracranial aneurysms, which may be related to IL-1ß/IL-1R pathways disorder.

Proinflammatory phenotype of B10 and B10pro cells elicited by TNF-α in rheumatoid arthritis.

OBJECTIVES: B10 and B10pro cells suppress immune responses via secreting interleukin (IL)-10. However, their regulators and underlying mechanisms, especially in human autoimmune diseases, are elusive. This study aimed to address these questions in rheumatoid arthritis (RA), one of the most common highly disabling autoimmune diseases. METHODS: The frequencies and functions of B10 and B10pro cells in healthy individuals and patients with RA were first analysed. The effects of proinflammatory cytokines, particularly tumour necrosis factor (TNF)-α on the quantity, stability and pathogenic phenotype of these cells, were then assessed in patients with RA before and after anti-TNF therapy. The underlying mechanisms were further investigated by scRNA-seq database reanalysis, transcriptome sequencing, TNF-α-/- and B cell-specific SHIP-1-/- mouse disease model studies. RESULTS: TNF-α was a key determinant for B10 cells. TNF-α elicited the proinflammatory feature of B10 and B10pro cells by downregulating IL-10, and upregulating interferon-γ and IL-17A. In patients with RA, B10 and B10pro cells were impaired with exacerbated proinflammatory phenotype, while anti-TNF therapy potently restored their frequencies and immunosuppressive functions, consistent with the increased B10 cells in TNF-α-/- mice. Mechanistically, TNF-α diminished B10 and B10pro cells by inhibiting their glycolysis and proliferation. TNF-α also regulated the phosphatidylinositol phosphate signalling of B10 and B10pro cells and dampened the expression of SHIP-1, a dominant phosphatidylinositol phosphatase regulator of these cells. CONCLUSIONS: TNF-α provoked the proinflammatory phenotype of B10 and B10pro cells by disturbing SHIP-1 in RA, contributing to the disease development. Reinstating the immunosuppressive property of B10 and B10pro cells might represent novel therapeutic approaches for RA.

Myeloid-derived suppressor cells from tumour-bearing mice induce the population expansion of CD19hiFcγRIIbhi regulatory B cells via PD-L1.

Myeloid-derived suppressor cells (MDSCs) increase in number and gain immunosuppressive functions in tumours and many other pathological conditions. MDSCs are characterized by their strong T-cell immunosuppressive capacity. The effects that MDSCs may have on B cells, especially within the tumour microenvironment, are less well understood. Here, we report that either monocytic MDSCs or polymorphonuclear MDSCs can promote increases in interleukin (IL)-10-expressing CD19hiFcγRIIbhi regulatory B cells in vitro and in vivo. Splenic transitional-1, -2, and -3 cells and marginal zone B cells, but not follicular B cells, differentiate into IL-10-expressing CD19hiFcγRIIbhi regulatory B cells. The adoptive transfer of CD19hiFcγRIIbhi regulatory B cells via tail vein injection can promote subcutaneous 3LL tumour growth in mice. The expression of programmed death-ligand 1 on MDSCs was found to be strongly associated with CD19hiFcγRIIbhi regulatory B cell population expansion. Furthermore, the frequency of circulating CD19+FcγRIIhi regulatory B cells was significantly increased in advanced-stage lung cancer patients. Our results unveil a critical role of MDSCs in regulatory B-cell differentiation and population expansion in lung cancer patients.

Co-expression of regulatory B-cell markers, transforming growth factor ß and interleukin-10 as a prognostic factor in diffuse large B-cell lymphoma.

Regulatory B cells (Bregs) suppress antitumor immunity by producing anti-inflammatory cytokines such as transforming growth factor ß (TGF-ß) and interleukin-10 (IL-10) and promoting tumor growth. It is unknown whether diffuse large B-cell lymphoma (DLBCL), a common subtype of B-cell malignancy, exhibits characteristics similar to those of Bregs. This study aimed to clarify the features of DLBCLs carrying Breg markers. In 123 DLBCL cases, we evaluated TGF-ß and IL-10 expression in tumor biopsy samples using immunohistochemical staining and retrospectively analyzed their clinicopathological characteristics. Fifteen cases (12.2 %) classified as Breg-type DLBCL were positive for both TGF-ß and IL-10. Breg-type DLBCL is mainly classified as having activated B cell-like cells of origin. Breg-type DLBCL cases showed significantly worse progression-free survival and overall survival (OS) than other DLBCL cases (P = 0.0016 and P = 0.042, respectively). In multivariate analysis, Breg-type DLBCL significantly affected OS (hazard ratio, 3.13; 95 % confidence interval 1.15-8.55; P = 0.025). Gene expression analysis showed that the expression of follicular dendritic cell-associated genes (FCER2, PIK3CD, FOXO1) was downregulated in Breg-type DLBCLs compared to other DLBCLs. These results suggest that the double expression of Breg markers, TGF-ß and IL-10, in tumor cells indicates a poor prognosis in DLBCL patients. Further studies evaluating genomic abnormalities could confirm the characteristics of Breg-type DLBCL.

The dichotomy of regulatory B cells in cancer versus allergic disease.

Regulatory B cells (Bregs) are an immunosuppressive cell phenotype that affects the immune system by limiting the inflammatory cascade. Dysregulation of Bregs can interestingly play a dichotomous role in the pathophysiology of many diseases and is especially highlighted when examining cancer pathology compared to allergic disease. This study reviews the existing literature on Bregs and compares their role in allergic disease in contrast to cancer development. Upregulation of Bregs in cancer states has been associated with poor prognostic outcomes across various cancer types, and Breg proliferation was associated with chronic interferon signaling, activation of the BCR-BTK (B cell receptor-Bruton's tyrosine kinase) pathway, and release of C-X-C motif ligand 13. In contrast, Breg dysfunction has been identified as a key mechanism in many allergic diseases, such as allergic asthma, allergic rhinitis, atopic dermatitis, and contact dermatitis. Development of Breg-targeted immunotherapies is currently at the preclinical level, but strategies differentially focus on Breg depletion in cancer versus Breg stimulation in allergy. Our review highlights the divergent functions that Bregs play in cancer compared to allergy. We conclude that natural homeostasis hinges on a fine balance between the dichotomous role of Bregs-over or underactivation can result in a pathological state.

IL-10+CD19+ regulatory B cells induce CD4+Foxp3+regulatory T cells in serum of cervical cancer patients.

Increase of regulatory T cells (Tregs) in the tumour microenvironment predicts worse survival of patients with various types of cancer. Recently, B cells play a significant role in the maintenance of Treg cells. However, the relevance of regulatory B cells (Bregs) to tumour immunity in humans remains elusive. Flow cytometry analysis was used to detect the Bregs and Tregs. Double staining results illustrated that the proportion of Bregs and Tregs were prominently higher in cervical cancer than normal tissues. Increase of Bregs and Tregs in cervical cancer microenvironment was associated with poor survival. Furthermore, Bregs cocultured with cervical cancer cell lines increased and induced Tregs. To sum up, the increased expression of Bregs contributes to the differentiation of CD4+ T cells into Tregs in the cervical cancer.

B-Cell-Activating Factor Contributes to Elevation of the Content of Regulatory B Cells in Neonatal Sepsis.

We studied the role of B cell-activating factor (BAFF) in PI3K/AKT/mTOR signaling pathway in promoting proliferation and maintaining survival of regulatory B lymphocytes (Breg) in newborns with sepsis. The peripheral blood samples were collected from preterm neonates (n=40) diagnosed with sepsis on the day of diagnosis and on days 7, 14, and 21 after diagnosis, as well as from the matched preterm neonates without sepsis (n=40; control group). The peripheral blood mononuclear cells and B cells were isolated, cultured, and stimulated with LPS and immunostimulant CpG-oligodeoxynucleotide (CpG-ODN). Proliferation and differentiation of B-cells into CD19+CD24hiCD38hi Breg cells and the role of the PI3K/AKT/mTOR signaling pathway in these processes were studied by flow cytometry, real-time quantitative reverse transcription PCR (qRT-PCR), and Western blotting. BAFF levels in the peripheral blood of neonates with sepsis were significantly increased at one week after diagnosis in parallel with increasing trend of expression of BAFF receptor. When applied with LPS and CpG-ODN, BAFF promoted differentiation of B cells into CD19+CD24hiCD38hi Breg cells. Phosphorylation of 4E-BP1 factor and 70S6K kinase located downstream in PI3K/AKT/mTOR signaling pathway was significantly up-regulated when stimulated with BAFF in combination with LPS and CpG-ODN. Thus, increased level of BAFF activates PI3K/AKT/mTOR signaling pathway and induces in vitro differentiation of peripheral blood B cells into CD19+CD24hiCD38hi Breg cells.

Kidney allograft rejection is associated with an imbalance of B cells, regulatory T cells and differentiated CD28-CD8+ T cells: analysis of a cohort of 1095 graft biopsies.

Introduction: The human immune system contains cells with either effector/memory or regulatory functions. Besides the well-established CD4+CD25hiCD127lo regulatory T cells (Tregs), we and others have shown that B cells can also have regulatory functions since their frequency and number are increased in kidney graft tolerance and B cell depletion as induction therapy may lead to acute rejection. On the other hand, we have shown that CD28-CD8+ T cells represent a subpopulation with potent effector/memory functions. In the current study, we tested the hypothesis that kidney allograft rejection may be linked to an imbalance of effector/memory and regulatory immune cells. Methods: Based on a large cohort of more than 1000 kidney graft biopsies with concomitant peripheral blood lymphocyte phenotyping, we investigated the association between kidney graft rejection and the percentage and absolute number of circulating B cells, Tregs, as well as the ratio of B cells to CD28-CD8+ T cells and the ratio of CD28-CD8+ T cells to Tregs. Kidney graft biopsies were interpreted according to the Banff classification and divided into 5 biopsies groups: 1) normal/subnormal, 2) interstitial fibrosis and tubular atrophy grade 2/3 (IFTA), 3) antibody-mediated rejection (ABMR), 4) T cell mediated-rejection (TCMR), and 5) borderline rejection. We compared group 1 with the other groups as well as with a combined group 3, 4, and 5 (rejection of all types) using multivariable linear mixed models. Results and discussion: We found that compared to normal/subnormal biopsies, rejection of all types was marginally associated with a decrease in the percentage of circulating B cells (p=0.06) and significantly associated with an increase in the ratio of CD28-CD8+ T cells to Tregs (p=0.01). Moreover, ABMR, TCMR (p=0.007), and rejection of all types (p=0.0003) were significantly associated with a decrease in the ratio of B cells to CD28-CD8+ T cells compared to normal/subnormal biopsies. Taken together, our results show that kidney allograft rejection is associated with an imbalance between immune cells with effector/memory functions and those with regulatory properties.

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-ß, 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.

Regulatory B cells are reduced in the blood in patients with granulomatosis with polyangiitis, and fail to regulate T-cell IFN-γ production.

Regulatory B (Breg) cells can dampen inflammation, autoreactivity, and transplant rejection. We investigated the frequencies, phenotypes, and function of Breg cells in granulomatosis with polyangiitis (GPA) to gain further knowledge as to whether there are numerical alterations or limitations of their ability to regulate T-cell function. Frequencies and phenotypes of CD24hiCD27+ and CD24hiCD38hi B-cells in the blood were determined with flow cytometry in 37 GPA patients (22 in remission and 15 with active disease) and 31 healthy controls (HC). A co-culture model was used to study the capacity of Breg cells to regulate T-cell activation and proliferation in cells from 10 GPA patients in remission and 12 HC. T-cell cytokine production in vitro and levels in plasma were determined with enzyme-linked immunosorbent assay. Frequencies of CD24hiCD27+ B-cells were reduced both during active disease and remission compared with HC (P = 0.005 and P = 0.010, respectively), whereas CD24hiCD38hi B-cells did not differ. Patient CD24hiCD27+ B-cells exhibited decreased expression of CD25 but increased expression of PD-L1 and PD-L2 during remission. B-cells from GPA patients regulated T-cell proliferation but failed to regulate interferon (IFN)-γ production (median T-cells alone 222 ng/ml vs. T-cells + B-cells 207 ng/ml, P = 0.426). IFN-γ was also elevated in patient plasma samples (P = 0.016). In conclusion, GPA patients exhibit altered numbers and phenotypes of CD24hiCD27+ B-cells. This is accompanied by a disability to control T-cell production of Th1-type cytokines during remission, which might be of fundamental importance for the granulomatous inflammation that characterizes the chronic phase of this disease.

Ten-eleven translocation-2 inactivation restrains IL-10-producing regulatory B cells to enable antitumor immunity in hepatocellular carcinoma.

BACKGROUND AND AIMS: IL-10-producing regulatory B cells (IL-10 + B cells), a dominant regulatory B cell (Breg) subset, foster tumor progression. However, the mechanisms underlying their generation in HCC are poorly understood. Ten-eleven translocation-2 (TET2), a predominant epigenetic regulatory enzyme in B cells, regulates gene expression by catalyzing demethylation of 5-methylcytosine into 5-hydroxymethyl cytosine (5hmC). In this study, we investigated the role of TET2 in IL-10 + B cell generation in HCC and its prospects for clinical application. APPROACH AND RESULTS: TET2 activation in B cells triggered by oxidative stress from the HCC microenvironment promoted IL-10 expression, whereas adoptive transfer of Tet2 -deficient B cells suppressed HCC progression. The aryl hydrocarbon receptor is required for TET2 to hydroxylate Il10 . In addition, high levels of IL-10, TET2, and 5hmc in B cells indicate poor prognosis in patients with HCC. Moreover, we determined TET2 activity using 5hmc in B cells to evaluate the efficacy of anti-programmed death 1 (anti-PD-1) therapy. Notably, TET2 inhibition in B cells facilitates antitumor immunity to improve anti-PD-1 therapy for HCC. CONCLUSIONS: Our findings propose a TET2-dependent epigenetic intervention targeting IL-10 + B cell generation during HCC progression and identify the inhibition of TET2 activity as a promising combination therapy with immune checkpoint inhibitors for HCC.

Effect of curcumin on regulatory B cells in chronic colitis mice involving TLR/MyD88 signaling pathway.

Curcumin (Cur) is a natural active phenolic compound extracted from the root of Curcuma Longa L. It has anti-inflammatory, anti-tumor and other pharmacological activities, and is commonly used to treat ulcerative colitis (UC). However, it is not clear whether curcumin regulates the function and differentiation of Breg cells to treat UC. In this study, mice with chronic colitis were induced by dextran sulfate sodium (DSS), and treated with curcumin for 12 days. Curcumin effectively improved the body weight, colonic weight, colonic length, decreased colonic weight index and pathological injury score under colonoscopy in mice with chronic colitis, and significantly inhibited the production of IL-1ß, IL-6, IL-33, CCL-2, IFN-γ, TNF-α, and promoted the secretion of IL-4, IL-10, IL-13 and IgA. Importantly, curcumin markedly upregulated CD3- CD19+ CD1d+ , CD3- CD19+ CD25+ , CD3- CD19+ Foxp3+ Breg cells level and significantly down-regulated CD3- CD19+ PD-L1+ , CD3- CD19+ tim-1+ , CD3- CD19+ CD27+ Breg cells level. In addition, our results also showed that curcumin observably inhibited TLR2, TLR4, TLR5, MyD88, IRAK4, p-IRAK4, NF-κB P65, IRAK1, TRAF6, TAB1, TAB2, TAK1, MKK3, MKK6, p38MAPK, p-p38MAPK and CREB expression in TLR/MyD88 signaling pathway. These results suggest that curcumin can regulate the differentiation and function of Breg cell to alleviate DSS-induced colitis, which may be realized by inhibiting TLR/MyD88 pathway.

Excess BAFF Alters NR4As Expression Levels and Breg Function of Human Precursor-like Marginal Zone B-Cells in the Context of HIV-1 Infection.

We have reported excess B-cell activating factor (BAFF) in the blood of HIV-infected progressors, which was concomitant with increased frequencies of precursor-like marginal zone (MZp) B-cells, early on and despite antiretroviral therapy (ART). In controls, MZp possess a strong B-cell regulatory (Breg) potential. They highly express IL-10, the orphan nuclear receptors (NR)4A1, NR4A2 and NR4A3, as well as the ectonucleotidases CD39 and CD73, all of which are associated with the regulation of inflammation. Furthermore, we have shown MZp regulatory function to involve CD83 signaling. To address the impact of HIV infection and excessive BAFF on MZp Breg capacities, we have performed transcriptomic analyses by RNA-seq of sorted MZp B-cells from the blood of HIV-infected progressors. The Breg profile and function of blood MZp B-cells from HIV-infected progressors were assessed by flow-cytometry and light microscopy high-content screening (HCS) analyses, respectively. We report significant downregulation of NR4A1, NR4A2, NR4A3 and CD83 gene transcripts in blood MZp B-cells from HIV-infected progressors when compared to controls. NR4A1, NR4A3 and CD83 protein expression levels and Breg function were also downregulated in blood MZp B-cells from HIV-infected progressors and not restored by ART. Moreover, we observe decreased expression levels of NR4A1, NR4A3, CD83 and IL-10 by blood and tonsillar MZp B-cells from controls following culture with excess BAFF, which significantly diminished their regulatory function. These findings, made on a limited number of individuals, suggest that excess BAFF contributes to the alteration of the Breg potential of MZp B-cells during HIV infection and possibly in other situations where BAFF is found in excess.

IL-10 producing regulatory B cells are decreased in blood from smokers and COPD patients.

BACKGROUND: Two opposing B cell subsets have been defined based on their cytokine profile: IL-6 producing effector B cells (B-effs) versus IL-10 producing regulatory B cells (B-regs) that respectively positively or negatively regulate immune responses. B-regs are decreased and/or impaired in many autoimmune diseases and inflammatory conditions. Since there is increasing evidence that links B cells and B cell-rich lymphoid follicles to the pathogenesis of COPD, the aim of this study was to investigate the presence and function of B-regs in COPD. METHODS: First, presence of IL-10 producing regulatory B cells in human lung tissue was determined by immunohistochemistry. Secondly, quantification of IL-10 + B-regs and IL-6 + B-effs in peripheral blood mononuclear cells (PBMCs) from healthy controls, smokers without airflow limitation, and COPD patients (GOLD stage I-IV) was performed by flow cytometry. Thirdly, we exposed blood-derived B cells from COPD patients in vitro to cigarette smoke extract (CSE) and quantified IL-10 + B-regs and IL-6 + B-effs. Furthermore, we aimed at restoring the perturbed IL10 production by blocking BAFF. Fourthly, we determined mRNA expression of transcription factors involved in IL-10 production in FACS sorted memory- and naive B cells upon exposure to medium or CSE. RESULTS: The presence of IL-10 producing regulatory B cells in parenchyma and lymphoid follicles in lungs was confirmed by immunohistochemistry. The percentage of IL-10 + B-regs was significantly decreased in blood-derived memory B cell subsets from smokers without airflow limitation and patients with COPD, compared to never smokers. Furthermore, the capacity of B cells to produce IL-10 was reduced upon in vitro exposure to CSE and this could not be restored by BAFF-blockade. Finally, upon CSE exposure, mRNA levels of the transcription factors IRF4 and HIF-1α, were decreased in memory B cells. CONCLUSION: Decreased numbers and impaired function of B-regs in smokers and patients with COPD might contribute to the initiation and progression of the disease.

Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (µMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

Characterization of IL-10-producing regulatory B cells in thymoma.

BACKGROUND: Regulatory B cells (Bregs) are a subset of B cells that secrete interleukin 10 (IL-10) and play a vital role in suppressing the immune response. The aim of this study was to evaluate the proportion of Bregs in patients with thymoma. METHODS: The proportions of subgroups of Bregs in 23 patients with thymoma and 15 healthy controls were detected by flow cytometry. The serum IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α levels of the subjects were measured using a cytometric bead array (CBA). RESULTS: The proportions of circulating IL-10+ B cells, IL-10+CD24hiCD38hi Bregs, and IL-10+CD24hiCD27+ Bregs and the serum IL-10 level were significantly higher in patients with thymoma than in the control group and were negatively correlated with the Karnofsky Performance Scale (KPS) score. The serum levels of cytokines IL-2, IL-6, IFN-γ, and TNF-α were higher and serum IL-17A level was lower in patients with thymoma. Patients with advanced-stage thymoma exhibited significantly higher proportions of IL-10-producing Bregs and a higher serum IL-10 level. After tumour resection, the frequency of circulating IL-10+CD24hiCD38hi Bregs and the serum IL-10 level were significantly decreased in patients with thymoma. The serum IL-10 levels exhibited the best accuracy in assessing the risk of thymoma occurrence in this study. CONCLUSIONS: The expression of IL-10 produced by Bregs is increased in patients with thymoma, particularly those with advanced-stage disease, which may suggest that Bregs are involved in the pathogenesis and progression of thymoma.

Expression of PD-L1 on regulatory B cells in patients with acute myeloid leukaemia and its effect on prognosis.

Programmed death-ligand 1 (PD-L1) is involved in immunosuppression in variety of tumours. Regulatory B cells (Bregs) are critical immune regulatory cells, and it has been demonstrated that the number of regulatory B cells in patients with acute myeloid leukaemia (AML) is much higher than that in healthy donors (HDs), which is linked to a poor prognosis. This study aimed to determine whether increased expression of PD-L1, including in Bregs, is associated with a worse prognosis in individuals with AML. The proportion of Bregs, PD-L1 expression in Bregs and PD-1 expression in T cells were determined using flow cytometry using patient samples from 21 newly diagnosed AML patients at different stages of treatment and 25 HDs. We confirmed PD-L1 expression in Bregs, and PD-1 expression in CD3+ CD4+ T cells in bone marrow and peripheral blood samples from AML patients was higher than that in samples from HDs. The complete remission (CR) and progression-free survival (PFS) of Bregs with high PD-L1 expression were significantly decreased following induction chemotherapy. PD-L1 expression is indeed increased in Bregs from individuals with AML, and high PD-L1 expression is related to a poor prognosis.

c-Myc-driven glycolysis polarizes functional regulatory B cells that trigger pathogenic inflammatory responses.

B cells secreting IL-10 functionally are recognized as functional regulatory B (Breg) cells; however, direct evidence concerning the phenotype, regulation, and functional and clinical relevance of IL-10-secreting Breg cells in humans is still lacking. Here, we demonstrate that, although IL-10 itself is anti-inflammatory, IL-10+ functional Breg cells in patients with systemic lupus erythematosus (SLE) display aggressive inflammatory features; these features shift their functions away from inducing CD8+ T cell tolerance and cause them to induce a pathogenic CD4+ T cell response. Functional Breg cells polarized by environmental factors (e.g., CPG-DNA) or directly isolated from patients with SLE mainly exhibit a CD24intCD27-CD38-CD69+/hi phenotype that is different from that of their precursors. Mechanistically, MAPK/ERK/P38-elicited sequential oncogenic c-Myc upregulation and enhanced glycolysis are necessary for the generation and functional maintenance of functional Breg cells. Consistently, strategies that abrogate the activity of ERK, P38, c-Myc, and/or cell glycolysis can efficiently eliminate the pathogenic effects triggered by functional Breg cells.

Intraperitoneally Delivered Mesenchymal Stem Cells Alleviate Experimental Colitis Through THBS1-Mediated Induction of IL-10-Competent Regulatory B Cells.

Mesenchymal stem cells (MSCs) show promising therapeutic potential in treating inflammatory bowel disease (IBD), and intraperitoneal delivery of MSCs have become a more effective route for IBD treatment. However, the underlying mechanisms are still poorly understood. Here, we found that intraperitoneally delivered MSCs significantly alleviated experimental colitis. Depletion of peritoneal B cells, but not macrophages, clearly impaired the therapeutic effects of MSCs. Intraperitoneally delivered MSCs improved IBD likely by boosting the IL-10-producing B cells in the peritoneal cavity, and a single intraperitoneal injection of MSCs could significantly prevent disease severity in a recurrent mouse colitis model, with lower proinflammation cytokines and high level of IL-10. The gene expression profile revealed that thrombospondin-1 (THBS1) was dramatically upregulated in MSCs after coculture with peritoneal lavage fluid from colitis mice. Knockout of THBS1 expression in MSCs abolished their therapeutic effects in colitis and the induction of IL-10-producing B cells. Mechanistically, THBS1 modulates the activation of transforming growth factor-ß (TGF-ß), which combines with TGF-ß receptors on B cells and contributes to IL-10 production. Blocking the interaction between THBS1 and latent TGF-ß or inhibiting TGF-ß receptors (TGF-ßR) significantly reversed the THBS1-mediated induction of IL-10-producing B cells and the therapeutic effects on colitis. Collectively, our study revealed that intraperitoneally delivered MSCs secreted THBS1 to boost IL-10+Bregs and control the progression and recurrence of colitis, providing new insight for the prevention and treatment of IBD.

Interleukin-35 promotes Breg expansion and interleukin-10 production in CD19+ B cells in patients with ankylosing spondylitis.

OBJECTIVE: IL-35 is a potent immunosuppressive and anti-inflammatory cytokine, consisting of a p35 subunit and an Epstein-Barr virus-induced gene 3 (EBI3) subunit, which suppresses CD4+ effector T cell proliferation and promotes regulatory T cell (Treg) expansion. However, the effects of IL-35 on regulatory B cells (Bregs) in ankylosing spondylitis (AS) have not been explored. The present study aimed (i) to measure serum IL-35 levels and the percentages of Bregs in the peripheral blood of patients with AS and (ii) to explore their relationships in the pathogenesis of AS. METHODS: A total of 77 patients with AS (AS group), including 47 inactive AS and 30 active AS cases, and 59 healthy controls (HCs) were enrolled into this study. The serum levels of IL-35 and IL-10 were detected by ELISA, and the mRNA levels of p35 and EBI3 were measured by RT-qPCR. The percentages of CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs and IL-35 receptor (IL-12Rß2, IL-27Rα and gp130), IL-10, p-STAT1, p-STAT3, and p-STAT4 in CD19+ B cells were detected by flow cytometry. The correlations between IL-35 levels and percentages of Bregs were analyzed by determining Pearson's correlation coefficient. The effect of IL-35 on Bregs was determined by mix-culture of recombinant (r) IL-35 with peripheral blood mononuclear cells (PBMCs). RESULTS: The serum IL-35 and IL-10 levels, p35 and EBI3 mRNA levels, and the percentages of CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs were significantly lower in AS patients than those in HCs. In addition, the percentages of CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs in active AS patients were significantly lower than those in inactive AS patients. The serum IL-35 levels were positively correlated with the percentages of CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs in AS patients. IL-12Rß2 and IL-27Rα, but not gp130 subunit, were expressed in CD19+ B cells in AS patients. RIL-35 could effectively promote CD19+CD24hiCD38hi Breg expansion and IL-10 production. Meanwhile, rIL-35 also promoted the expression of IL-12Rß2 and IL-27Rα and the phosphorylation of STAT1 and STAT3 in CD19+ B cells. CONCLUSION: These results demonstrated that reduced IL-35 production may be associated with Bregs defects in AS patients. RIL-35 induced the proliferation of CD19+CD24hiCD38hi Bregs and IL-10 production, suggesting that IL-35 may serve as a reference for further investigation to develop novel treatments for AS. Key Points • Our study investigated the effects of IL-35 on Bregs in AS patients. • We found the serum IL-35, IL-10 levels, and the percentages of CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs were significantly lower in AS patients. • The serum IL-35 levels were positively correlated with the percentages of CD19+CD24hiCD38hi and CD19+CD24hiCD27+ Bregs in AS patients. • Recombinant IL-35 could effectively promote CD19+CD24hiCD38hi Breg expansion and IL-10 production.