Regulatory B cells: TIM-1, transplant tolerance, and rejection.

Regulatory B cells (Bregs) ameliorate autoimmune disease and prevent allograft rejection. Conversely, they hinder effective clearance of pathogens and malignancies. Breg activity is mainly attributed to IL-10 expression, but also utilizes additional regulatory mechanisms such as TGF-ß, FasL, IL-35, and TIGIT. Although Bregs are present in various subsets defined by phenotypic markers (including canonical B cell subsets), our understanding of Bregs has been limited by the lack of a broadly inclusive and specific phenotypic or transcriptional marker. TIM-1, a broad marker for Bregs first identified in transplant models, plays a major role in Breg maintenance and induction. Here, we expand on the role of TIM-1+  Bregs in immune tolerance and propose TIM-1 as a unifying marker for Bregs that utilize various inhibitory mechanisms in addition to IL-10. Further, this review provides an in-depth assessment of our understanding of Bregs in transplantation as elucidated in murine models and clinical studies. These studies highlight the major contribution of Bregs in preventing allograft rejection, and their ability to serve as highly predictive biomarkers for clinical transplant outcomes.

Systemic immunosuppression depletes peripheral blood regulatory B cells in patients with immune thrombocytopenia.

Regulatory B (Breg) cells are potentially implicated in the pathogenesis of immune thrombocytopenia (ITP). We analysed a prospective cohort of newly diagnosed steroid naïve ITP patients enrolled in the multicentre FLIGHT trial and found that the numbers of Bregs in their peripheral blood were similar to healthy controls. In contrast, Breg numbers were significantly reduced in ITP patients treated with systemic immunosuppression (glucocorticoids or mycophenolate mofetil). We also demonstrate that glucocorticoid treatment impairs Breg interleukin-10 production via an indirect T-cell-mediated mechanism.

Therapeutic targeting of gut-originating regulatory B cells in neuroinflammatory diseases.

Regulatory B cells (Bregs) are immunosuppressive cells that support immunological tolerance by the production of IL-10, IL-35, and TGF-ß. Bregs arise from different developmental stages in response to inflammatory stimuli. In that regard, mounting evidence points towards a direct influence of gut microbiota on mucosal B cell development, activation, and regulation in health and disease. While an increasing number of diseases are associated with alterations in gut microbiome (dysbiosis), little is known about the role of microbiota on Breg development and induction in neuroinflammatory disorders. Notably, gut-originating, IL-10- and IgA-producing regulatory plasma cells have recently been demonstrated to egress from the gut to suppress inflammation in the CNS raising fundamental questions about the triggers and functions of mucosal-originating Bregs in systemic inflammation. Advancing our understanding of Bregs in neuroinflammatory diseases could lead to novel therapeutic approaches. Here, we summarize the main aspects of Breg differentiation and functions and evidence about their involvement in neuroinflammatory diseases. Further, we highlight current data of gut-originating Bregs and their microbial interactions and discuss future microbiota-regulatory B cell-targeted therapies in immune-mediated diseases.

The imbalance between Bregs, Tfh, and Tregs in patients with anti-N-methyl-D-aspartate receptor encephalitis.

OBJECTIVE: To detect the alteration of regulatory B cells (Bregs), follicular helper T cells (Tfh), and regulatory T cells (Tregs) frequencies in patients with anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis. Analyze their association with clinical severity and activity, and explore the effects of different immunotherapies on those immune cell subsets. METHODS: We enrolled 21 patients with anti-NMDAR encephalitis, 22 patients with neuromyelitis optica spectrum disorder (NMOSD), 14 patients with idiopathic intracranial hypertension (IIH), and 20 healthy controls (HC) in our study. The frequencies of various immune cell subsets were determined using flow cytometry. RESULTS: Compared to patients with IIH and HC, the frequencies of CD24hiCD38hi transitional B cells as well as Tregs were significantly lower while the frequency of Tfh was significantly higher in patients with anti-NMDAR encephalitis. The frequency of CD24hiCD38hi transitional B cells was significantly lower in the acute stage than in the recovery stage, and was negatively correlated with the modified Rankin scale (mRS) and the clinical assessment scale for autoimmune encephalitis (CASE). The frequency of CD24hiCD38hi transitional B cells at the last follow-up after rituximab (RTX) treatment was significantly higher than those treated with oral immunosuppressants or untreated. There was no clear difference between anti-NMDAR encephalitis and NMOSD in the above immune cell subsets. CONCLUSION: We suggested that the frequencies of CD24hiCD38hi transitional B cells and Tregs were decreased while the frequency of Tfh was increased in patients with anti-NMDAR encephalitis. CD24hiCD38hi transitional B cells frequency may be a potential indicator to estimate the disease activity and severity.

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.

Marginal Zone B-Cell Populations and Their Regulatory Potential in the Context of HIV and Other Chronic Inflammatory Conditions.

Inflammation in the context of Human Immunodeficiency Virus (HIV) establishes early and persists beyond antiretroviral therapy (ART). As such, we have shown excess B-cell activating factor (BAFF) in the blood of HIV-infected progressors, as soon as in the acute phase, and despite successful ART. Excess BAFF was associated with deregulation of the B-cell compartment; notably, with increased frequencies of a population sharing features of both transitional immature (TI) and marginal zone (MZ) B-cells, we termed Marginal Zone precursor-like (MZp). We have reported similar observations with HIV-transgenic mice, Simian Immunodeficiency Virus (SIV)-infected macaques, and more recently, with HIV-infected Beninese commercial sex workers, which suggests that excess BAFF and increased frequencies of MZp B-cells are reliable markers of inflammation in the context of HIV. Importantly, we have recently shown that in healthy individuals, MZps present an important regulatory B-cell (Breg) profile and function. Herein, we wish to review our current knowledge on MZ B-cell populations, especially their Breg status, and that of other B-cell populations sharing similar features. BAFF and its analog A Proliferation-Inducing Ligand (APRIL) are important in shaping the MZ B-cell pool; moreover, the impact that excess BAFF-encountered in the context of HIV and several chronic inflammatory conditions-may exert on MZ B-cell populations, Breg and antibody producing capacities is a threat to the self-integrity of their antibody responses and immune surveillance functions. As such, deregulations of MZ B-cell populations contribute to autoimmune manifestations and the development of MZ lymphomas (MZLs) in the context of HIV and other inflammatory diseases. Therefore, further comprehending the mechanisms regulating MZ B-cell populations and their functions could be beneficial to innovative therapeutic avenues that could be deployed to restore MZ B-cell immune competence in the context of chronic inflammation involving excess BAFF.

Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination.

The unmet medical need of patients with multiple sclerosis (MS) is the inexorable loss of CNS myelin and latterly neurons leading to permanent neurologic disability. Solicitation of endogenous oligodendrocytes progenitor cells, the precursor of oligodendrocytes, to remyelinate axons may abort the onset of disability. In female mice with experimental autoimmune encephalomyelitis (EAE), a murine model of MS, adoptive transfer of IL-10+ regulatory B cells (Bregs) has been shown to reverse EAE by promoting the expansion of peripheral and CNS-infiltrating IL-10+ T cells. Here, we examined whether Bregs treatment and its bystander effect on regulatory T cells are associated with CNS repair as reflected by oligodendrogenesis and remyelination. We have found that transfusion of Bregs reverses established clinical EAE and that clinical improvement is associated with a significant increase in spinal cord remyelination as reflected by g-ratio analysis within the thoracic and lumbar spine. We further observed in the spinal cords of EAE Bregs-treated mice that CNS resident CD11b/CD45intLy6C- microglia, and infiltrating CD11b+/CD45high monocytes/macrophages content reverts to normal and polarize to a M2-like CD206+ phenotype. Concurrently, there was a substantial increase in neo-oligodendrogenesis as manifest by an increase in CD45-/low CNS cells expressing A2B5, an early marker in oligodendrocytes progenitor cell differentiation as well as GalC+/O1+ premyelinating and myelin basic protein+/myelin oligodendrocyte glycoprotein+ mature oligodendrocytes with reciprocal downregulation of paired related homeobox protein 1. These results demonstrate that the clinical benefit of Bregs is associated with normalization of CNS immune milieu and concurrent activation of oligodendrocyte progenitor cells with subsequent remyelination.SIGNIFICANCE STATEMENT In multiple sclerosis patients, demyelination progresses with aging and disease course, leading to irreversible disability. In this study, we have discovered, using a mouse model of multiple sclerosis, that the transfusion of autologous regulatory B cells (Bregs) is able to ameliorate, cure, and sustain the durable remission of the disease. We show that the adoptive transfer of Bregs dramatically decreased the frequency of myeloid-derived cells, both infiltrating monocytes/macrophages and resident microglia, and converted their phenotype to an immunosuppressive-like phenotype. Moreover, we showed that CNS oligodendrocyte progenitor cells are activated following Bregs treatment and differentiate into myelinating oligodendrocytes, which results in neo-oligodendrogenesis and remyelination of spinal cords.

Transcriptional meta-analysis of regulatory B cells.

Regulatory B cells (Bregs) have the ability to regulate inflammation in various pathological situations, making them key players in immune regulation. Several mechanisms have been described and we recently identified a GZMB expressing Breg population in kidney transplanted patients who tolerate a kidney graft. To further investigate their biology and mechanisms, we conducted a transcriptomic analysis by RNAseq of these cells and we performed the first weighted meta-analysis of publicly available transcriptomic data from published Breg studies both in humans and mice. We identified two distinct and unique transcriptional signatures of 126 and 93 genes, respectively, associated with these Bregs. While we highlighted genes coding for proteins with potent involvement in regulatory functions, proliferation, and coding for transcription factors, the comparison between humans and mice did not allow identifying a common pattern. Thus, our results suggest distinct species-restricted Breg transcriptional signatures in humans and mice.

Targeting immunosuppressor cells with nanoparticles in autoimmunity: How far have we come to?

Autoimmunity is the assault of immune response towards self-antigens, resulting to inflammation and tissue injury. It is staged into three phases and caused by malfunction of immune tolerance. In our body, immune tolerance is synchronized by several immunosuppressor cells such as regulatory T cells and B cells as well as myeloid-derived suppressor cells, which are prominently dysregulated in autoimmunity. Hence, targeting these cell populations serve as a significant potential in the therapy of autoimmunity. Nanotechnology with its advantageous properties is shown to be a remarkable tool as drug delivery system in this field. This review focused on the development of therapeutics in autoimmune diseases utilizing various nanoparticles formulation based on two targeting approaches in autoimmunity, passive and active targeting. Lastly, this review outlined the approved present nanomedicines as well as in clinical evaluations and issues regarding the lack of translation of these nanomedicines into the market, despite the abundant of positive experimental observations.

Higher CD19+CD25+ Bregs are independently associated with better graft function in renal transplant recipients.

BACKGROUND: The Identification of B cell subsets with regulatory functions might open the way to new therapeutic strategies in the field of transplantation, which aim to reduce the dose of immunosuppressive drugs and prolong the graft survival. CD25 was proposed as a marker of a B-cell subset with an immunosuppressive action termed Bregs. The effect of CD19 + CD25 + Bregs on graft function in renal transplant recipients has not yet been elucidated. We investigated a potential impact of CD19 + CD25 + Bregs on renal graft function as well as a possible interaction of CD19 + CD25 + Bregs with peripheral Tregs in healthy controls, end-stage kidney disease patients (ESKD), and renal transplant recipients. Moreover, we aimed to investigate the association of CD19 + CD25 + Bregs with serum IL-10, TGF-ß1, and IFN-γ in the same study groups. METHOD: Thirty-one healthy controls, ninety renal transplant recipients, and eighteen ESKD patients were enrolled. We evaluated the CD19 + CD25 + Bregs and Treg absolute counts. Next, we investigated CD19 + CD25 + Bregs as predictors of good graft function in multiple regression and ROC analyses. Finally, we evaluated the association between CD19 + CD25+ Bregs and serum IL-10, TGF-ß, and IFN-γ. RESULTS: ESKD patients and renal transplant recipients showed lower counts of CD19 + CD25+ Bregs compared to healthy controls (p < 0.001). Higher CD19 + CD25+ Breg counts were independently associated with a better GFR in renal transplant recipients (unstandardized B coefficient = 9, p = 0.02). In these patients, higher CD19 + CD25+ Bregs were independently associated with higher Treg counts (unstandardized B = 2.8, p = 0.004). In ROC analysis, cut-offs for CD19 + CD25 + Breg counts and serum TGF-ß1 of 0.12 cell/µl and 19,635.4 pg/ml, respectively, were shown to provide a good sensitivity and specificity in identifying GFR ≥ 30 ml/min (AUC = 0.67, sensitivity 77%, specificity 43%; AUC = 0.65, sensitivity 81%, specificity 50%, respectively). Finally, a significant positive association between CD19 + CD25+ Bregs and TGF-ß1 was shown in renal transplant recipients (r = 0.255, p = 0.015). CONCLUSIONS: Our findings indicate that higher counts of CD19 + CD25+ Bregs are independently associated with better renal function and higher absolute Treg counts in renal transplant recipients.

CD91 Derived Treg Epitope Modulates Regulatory T Lymphocyte Response, Regulates Expression of Costimulatory Molecules on Antigen-Presenting Cells, and Rescues Pregnancy in Mouse Pregnancy Loss Model.

The loss of immune tolerance to fetal antigens may result in reproductive failure. The downregulated number and activity of T regulatory lymphocytes, which are critical for the establishment of immune tolerance to fetal antigens, during pregnancy may lead to miscarriage. The adoptive transfer of Tregs prevents fetal loss in abortion-prone mice. Recently, we demonstrated that the administration of tregitopes, which are short peptides found in human and mouse immunoglobulins (IgGs), decreased the incidence of abortions in female CBA/J mice mated with DBA/2J mice. Here, two non-IgG source peptides (SGS and LKD) that can potentially bind to the major histocompatibility complex II (MHC II) with high affinity and induce Treg expansion were designed in silico. The immune dysregulation-induced pregnancy failure mouse model was used to evaluate the effect of SGS and LKD on immune response and pregnancy outcome. The fetal death rate in the SGS-treated group was lower than that in the phosphate-buffered saline-treated group. SGS and LKD upregulated the splenic pool of Tregs and modulated the T-helper cell (Th1)/Th2-related cytokine response at the preimplantation stage. Additionally, SGS and LKD downregulated the expression of CD80 and MHC class II molecules in splenic CD11c+ antigen-presenting cells. Thus, SGS treatment can result in beneficial pregnancy outcomes. Additionally, SGS peptide-mediated immunomodulation can be a potential therapeutic strategy for immune dysregulation-induced pregnancy failure.

Assessing the expression of immunosuppressive cytokines in the newly diagnosed systemic lupus Erythematosus patients: a focus on B cells.

BACKGROUND: The immunosuppressive effects of regulatory B-cells (Bregs) and their immunosuppressive cytokines on immune responses in autoimmune disorders, mainly systemic lupus erythematosus (SLE), have been recently established. Therefore, the purpose of this article has been the exploration of the expressions of cytokines produced by B cells in newly diagnosed SLE patients. RESULTS: The findings demonstrated that the gene expression of IL-10, TGF-ß, IL-35, PD-L1, and FasL was significantly up-regulated in SLE patients compared to healthy subjects (P < 0.05). Additionally, the results revealed that serum levels of IL-10, TGF-ß, IL-35, PD-L1 were remarkably increased in patients with SLE compared to healthy subjects (P < 0.0001). However, serum levels of IL-10 and TGF-ß decreased significantly with increasing SLEDAI score in studied patients (P < 0.05). CONCLUSION: It was concluded that the release of anti-inflammatory cytokines, particularly IL-10 and TGF-ß, might inhibit immune responses and autoreactive immune cells in a compensatory manner in SLE patients with mild to moderate disease activity.

The blockade of PD-1/PD-L1 pathway promotes the apoptosis of CD19+ CD25+ Bregs and suppresses the secretion of IL-10 in patients with allergic rhinitis.

PD-1/PD-L1 pathway is crucial to immune regulation by controlling the balance between T cell tolerance and activation. However, the association between PD-1/PD-L1 pathway and regulatory B cells has not been fully investigated in allergic rhinitis. In this study, we detected the number of peripheral CD19+ CD25+ Bregs and the expression of IL-10 on this cell subset in healthy control and patients with allergic rhinitis using flow cytometry. Then, we evaluated the level of PD-L1 in CD19+ CD25+ Bregs and investigated the correlation between PD-L1 and CD4+ follicular T helper cells. Finally, we studied the effects of anti-PD-L1 on the apoptosis of Bregs and the production of IL-10. Comparing with healthy controls, the percentage of CD19+ CD25+ Bregs and the expression of IL-10 were both significantly decreased in AR group. In addition, the expression of PD-L1 on CD19+ CD25+ Bregs was also lower in allergic rhinitis patients. Interestingly, a negative correlation was found between the expression of PD-L1+ Bregs and CD4+ CXCR5+ follicular T helper cells. In vitro assay revealed that anti-PD-L1 promoted Bregs apoptosis and inhibited the expression of IL-10 in CD19+ CD25+ Bregs. Collectively, these results suggest that PD-L1 expressed on CD19+ CD25+ Bregs may be a potential regulator in the treatment of allergic rhinitis. Blockade of PD-1/PD-L1 pathway might be a valuable pathogenic target for allergic rhinitis through inhibiting the secretion of immunosuppressive cytokine and promoting CD19+ CD25+ Bregs apoptosis.

[Peripheral T and B lymphocytes in patients with occupational medicamentosa-like dermatitis due to trichloroethylene].

Objective: To investigate the changes in liver function and peripheral regulatory lymphocytes before and after treatment in patients with occupational medicamentosa-like dermatitis due to trichloroethylene (OMDT) . Methods: In December 2019, 16 patients with OMDT (8 patients with erythema multiforme and 8 with erythema multiforme major) who were admitted from February 2017 to February 2019 were enrolled as subjects. Liver function parameters and percentages of peripheral regulatory lymphocytes were measured before and after treatment, and the changes in liver function and peripheral regulatory T and B lymphocytes and their correlation were analyzed. Results: Before treatment, compared with the healthy control group, the experimental group had significantly higher levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , total bilirubin (TBIL) , direct bilirubin (DBIL) and gamma-glutamyl transpeptidase (GGT) and significantly lower levels of total protein (TP) , albumin (ALB) and cholinesterase (CHE) (P<0.05) . Compared with the healthy control group, the experimental group had significantly lower percentages of lymphocytes, CD4(+) T cells, CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+) Bregs and CD4(+)/CD8(+) ratio, as well as a significantly higher percentage of CD8(+) T cells (P<0.05) . Before treatment, the levels of ALT, AST, GGT and DBIL were negatively correlated with the percentages of CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+) Bregs, CD4(+) T cells and CD4(+)/CD8(+) ratio (r=-0.386 to -0.809, P<0.05) and was positively correlated with the percentage of CD8(+) T cells (except DBIL) (r=0.503-0.568, P<0.05) . The levels of TP and ALB were positively correlated with the percentages of CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+)Bregs and CD4(+) T cells (r= 0.351-0.784, P<0.05) , ALB was negatively correlated with the percentage of CD8(+) T cells (r=-0.315, P<0.05) . CHE was positively correlated with the percentages of CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+)Bregs and CD4(+)/CD8(+) ratio (r=0.390-0.527, P<0.05) . Conclusion: Immune dysfunction is observed in patients with OMDT, which may be caused by the imbalance of regulatory lymphocytes. And liver injury may be associated with the increase of CD8(+) T cells and the reductions of percentages of CD4(+) T cells, CD4(+)CD25(+) Tregs, CD19(+)CD24(+)CD27(+)Bregs and CD4(+)/CD8(+) ratio.

Regulatory impairment in untreated Parkinson's disease is not restricted to Tregs: other regulatory populations are also involved.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Various studies have suggested that the immune response plays a key role in this pathology. While a predominantly pro-inflammatory peripheral immune response has been reported in treated and untreated PD patients, the study of the role of the regulatory immune response has been restricted to regulatory T cells. Other immune suppressive populations have been described recently, but their role in PD is still unknown. This study was designed to analyze the pro and anti-inflammatory immune response in untreated PD patients, with emphasis on the regulatory response. METHODS: Thirty-two PD untreated patients and 20 healthy individuals were included in this study. Peripheral regulatory cells (CD4+Tregs, Bregs, CD8+Tregs, and tolerogenic dendritic cells), pro-inflammatory cells (Th1, Th2, and Th17 cells; active dendritic cells), and classical, intermediate, and non-classical monocytes were characterized by flow cytometry. Plasmatic levels of TNF-α, IFN-γ, IL-6, GM-CSF, IL-12p70, IL-4, IL-13, IL-17α, IL-1ß, IL-10, TGF-ß, and IL-35 were determined by ELISA. RESULTS: Decreased levels of suppressor Tregs, active Tregs, Tr1 cells, IL-10-producer CD8regs, and tolerogenic PD-L1+ dendritic cells were observed. With respect to the pro-inflammatory response, a decrease in IL-17-α and an increase in IL-13 levels were observed. CONCLUSION: A decrease in the levels of regulatory cell subpopulations in untreated PD patients is reported for the first time in this work. These results suggest that PD patients may exhibit a deficient suppression of the pro-inflammatory response, which could contribute to the pathophysiology of the disease.

Phenotypic and functional alterations of regulatory B cell subsets in adult allergic asthma patients.

BACKGROUND: IL-10-producing regulatory B cells (Bregs) are widely ascribed immune regulatory functions. However, Breg subsets in human asthma have not been fully investigated. OBJECTIVE: We studied Breg subsets in adult allergic asthma patients by assessing two major parameters, frequency and IL-10 expression. We then investigated factors that affect these two parameters in patients. METHODS: Peripheral blood mononuclear cells (PBMCs) of adult allergic asthma patients (N = 26) and non-asthmatic controls (N = 28) were used to assess the frequency of five subsets of transitional B cells (TBs), three subsets of CD24high CD27+ B cells and B1 cells. In addition to clinical data, IL-10 expression by individual Breg subsets was assessed by flow cytometry. RESULTS: Asthma patients had decreases of CD5+ and CD1d+ CD5+ , but an increase of CD27+ TBs which was significant in patients with moderate asthma (60 < FEV1 < 80). Regardless of asthma severity, there was no significant alteration in the frequencies of 6 other Breg subsets tested. However, we found that oral corticosteroid (OCS) significantly affected the frequency of Bregs in Breg subset-specific manners. OCS decreased CD5+ and CD1d+ CD5+ TBs, but increased CD27+ TBs and CD10+ CD24high CD27+ cells. Furthermore, OCS decreased IL-10 expression by CD27+ TBs, all 3 CD24high CD27+ B cell subsets (CD5+ , CD10+ and CD1d+ ) and B1 cells. OCS-mediated inhibition of IL-10 expression was not observed in the other Breg subsets tested. CONCLUSION & CLINICAL RELEVANCE: Alterations in the frequency of Bregs and their ability to express IL-10 are Breg subset-specific. OCS treatment significantly affects the frequency as well as their ability to express IL-10 in Breg subset-specific manners.

Sexual dimorphism in Th17/Treg axis in lymph nodes draining inflamed joints in rats with collagen-induced arthritis.

Collagen type II-induced arthritis (CIA) in Dark Agouti rats, a model of rheumatoid arthritis (RA), reproduces sexual dimorphism in the incidence and severity of the human disease. Th17 cells are central in the induction/propagation of autoimmune inflammation in CIA and RA. To assess mechanisms underlying this dimorphism in CIA rats, in lymph nodes draining inflamed joints and adjacent tissues (dLNs) from CIA rats of both sexes Th17/CD25+Foxp3+CD4+ T-regulatory cell (Treg) ratio, Th17 cell redifferentiation in functionally distinct subsets and Treg transdifferentiation into IL-17-producing cells (exTregs) were examined. In female rats (developing more severe CIA than their male counterparts) the higher frequency of all Th17 cells (reflecting partly their greater proliferation), followed by the higher frequency of highly pathogenic IFN-γ/GM-CSF-co-producing cells, but lower frequency of less pathogenic/immunoregulatory IL-10-producing cells among them was found. Additionally, compared with male rats, in female rats the lower frequency of Tregs was observed. Moreover, Tregs from female rats exhibited diminished proliferative and suppressive capacity (judging by PD-1 expression) and enhanced conversion into IL-17-producing cells. Given that TGF-ß concentration was comparable in collagen-type II-stimulated dLN cell cultures from female and male rats, the shift in Th17/Treg ratio followed by augmented Th17 cell redifferentiation into IFN-γ/GM-CSF-co-producing cells and Treg transdifferentiation into IL-17-producing cells in female rats was associated with increased concentration of IL-6 in female rat dLN cell cultures, and the higher frequency of IL-1ß- and IL-23-producing cells among their dLN cells. The lower frequency of IL-10-producing B cells, presumably B regulatory cells (Bregs) could also contribute to the shift in Th17/Treg ratio in female rat compared with male rat dLNs. Consistently, the lower expression of IL-35 (the cytokine promoting Treg expansion directly and indirectly, by favoring Breg expansion and conversion into IL-10/IL-35-producing cells) in female rat dLN cells was detected. Thus, the study identified putative cellular and molecular substrates of the sexual dimorphism in the immunopathogenesis and clinical outcome of CIA and suggested mechanisms to be targeted in females to improve control of Th17 response, and consequently clinical outcome of CIA, and possibly RA.

Low Expression of IL-10 in Circulating Bregs and Inverted IL-10/TNF-α Ratio in Tears of Patients with Perennial Allergic Conjunctivitis: A Preliminary Study.

Allergic conjunctivitis (AC) is one of the most common ophthalmological disorders seen in clinical practice. Growing evidence from recent years suggests that a subset of IL-10-expressing B cells is involved in inflammatory allergic diseases. In this study, we aimed to evaluate the potential involvement of blood Bregs cells in perennial allergic conjunctivitis (PAC), and interleukins (IL)-1ß, IL-6, IL-8, IL-10, and IL-12, and tumor necrosis factor (TNF)-α, were measured in tear samples and compared with healthy controls (HC) using flow cytometry. Non-significant differences in CD19⁺IL-10⁺ cell frequency between PAC patients and healthy controls (HC) were observed. Nevertheless, when we analyzed the mean fluorescence intensity (MFI) of IL-10 on CD19⁺CD38Lo/Med/Hi-gated cells, we observed a significant decrease in MFI in all Bregs subsets in PAC patients. Additionally, tear cytokines showed 2.8 times lower levels of IL-10 than TNF-α in PAC patients when compared to HC. Our findings demonstrate an immunological dysregulation in patients with allergic conjunctivitis, characterized by the low expression of IL-10 in circulating CD19⁺CD38⁺ Bregs subsets and an inverted tear IL-10/TNF-α ratio, promoting a local pro-inflammatory microenvironment. These findings highlight the novel pathologic changes involved in ocular allergic diseases. Understanding systemic and local mechanisms will aid the design of immunomodulating therapeutics at different levels.

Multiple Functions of B Cells in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by excessive autoantibody production and multi-organ involvement. Although the etiology of SLE still remains unclear, recent studies have characterized several pathogenic B cell subsets and regulatory B cell subsets involved in the pathogenesis of SLE. Among pathogenic B cell subsets, age-associated B cells (ABCs) are a newly identified subset of autoreactive B cells with T-bet-dependent transcriptional programs and unique functional features in SLE. Accumulation of T-bet+ CD11c+ ABCs has been observed in SLE patients and lupus mouse models. In addition, innate-like B cells with the autoreactive B cell receptor (BCR) expression and long-lived plasma cells with persistent autoantibody production contribute to the development of SLE. Moreover, several regulatory B cell subsets with immune suppressive functions have been identified, while the impaired inhibitory effects of regulatory B cells have been indicated in SLE. Thus, further elucidation on the functional features of B cell subsets will provide new insights in understanding lupus pathogenesis and lead to novel therapeutic interventions in the treatment of SLE.

Chronic lymphocytic leukemia cells acquire regulatory B-cell properties in response to TLR9 and CD40 activation.

Circulating chronic lymphocytic leukemia (CLL) cells share phenotypic features with certain subsets of regulatory B-cells (Bregs). The latter cells have been reported to negatively regulate immune cell responses, mostly by provision of IL-10. The purpose of the current study was to identify and delineate Breg properties of CLL cells. B-cells and T-cells were obtained from the peripheral blood of untreated CLL patients diagnosed according to the 2008 Guidelines of the International Workshop on Chronic Lymphocytic Leukemia. Co-culture assays were used to examine the ability of CLL cells to suppress autologous T-cell immune responses. IL-10 potency of CLL cells was assessed following stimulation with activators of the toll-like receptor 9 (TLR9) or CD40 and was correlated with the inhibitory activity of the cells. TLR9-activated CLL cells were found to increase the frequency of CD4+CD25hiFOXp3+ regulatory T-cells (Tregs) and to inhibit autologous CD4+ T-cell proliferation. This signaling cascade proved to control IL-10 generation in CLL cells, which in turn promoted the inhibition of T-cell proliferation by CLL cells. However, CD40 activation of CLL cells, while exhibiting a similar ability to augment Treg frequency, did not either affect IL-10 generation or T-cell proliferation. In conclusion, CLL cells demonstrate a unique clonal quality of adopting Breg properties which promote modulation of T-cell characteristics. TLR9 appears to be a potent activator of regulatory abilities in CLL cells, possibly contributing to preferential immune escape of TLR9-responsive cells.