Effect of CD40/CD40L signaling on IL-10-producing regulatory B cells in Chinese children with Henoch-Schönlein purpura nephritis

The aim of the present study was to examine the role and mechanism of interleukin-10 (IL-10)-producing regulatory B cells (B10 cells) in the pathogenesis of Henoch-Schönlein purpura nephritis (HSPN). We examined the percentage of B10 cells, CD19⁺CD24^hiCD38^hi B cells, CD19⁺CD24^hiCD27⁺ B cells, Th17 cells, and T regulatory (Treg) cells within the peripheral blood mononuclear cell (PBMC) population in healthy subjects and HSP/HSPN patients. The percentage of B10 cells and CD19⁺CD24^hiCD38^hi B cells was reduced in HSPN patients and that of CD19⁺CD24^hiCD27⁺ B cells was decreased only in HSPN patients with hematuria and proteinuria or massive proteinuria. The expression of IL-10 by B10 cells and their subsets was decreased in HSPN patients and returned to normal levels in HSP/HSPN patients in remission. B10 cells and their subsets negatively correlated with the Th17/Treg ratio. There was no difference in B10pro + B10 cells, Th17 cells, Treg cells, and the Th17/Treg ratio between children with HSP/HSPN and healthy controls after CD40L stimulation. On the other hand, the level of IL-10 expressed by CD19⁺CD40⁺ B cells was decreased in HSPN, and the percentage of B10pro + B10 cells and Treg cells was reduced and that of Th17 cell was increased in the presence of anti-CD40L monoclonal antibody (mAb). Thus, decreased B10 cells and CD19⁺CD24^hiCD38^hi B cells may function as an early marker of renal impairment in HSPN. The dysfunction of B10 cells may play a role in the pathogenesis of HSPN by regulating the Th17/Treg balance. Moreover, the CD40/CD40L signaling pathway may play a role in B10 cell differentiation and functional maturation.     

CD43−, but not CD43+, IL-10-producing CD1dhiCD5+ B cells suppress type 1 immune responses during Chlamydia muridarum genital tract infection

Regulatory B (Breg) cells are known to modulate immune responses through predominantly interleukin-10 (IL-10)-dependent mechanisms and can be hypothetically divided into innate and adaptive subsets based on the nature of their activating signals. However, the specific role of different Breg subsets in modulating immune responses remains ambiguous. Here we have shown that Chlamydia induces IL-10-producing splenic B-cell populations consisting of CD43⁺ and CD43⁻ subsets of IgM^hiIgD^lo innate-like B (ILB) cells in vitro. While CD43⁺IL-10-producing B cells displayed innate type features and were readily induced by Chlamydia via Toll-like-receptor (TLR) signaling, CD43⁻IL-10-producing B cells required additional B-cell activating factor (BAFF)-mediated signals from dendritic cells (DCs) for their differentiation and activation, thereby classifying them as adaptive type Bregs. Importantly, CD43⁻, but not CD43⁺, IL-10-producing ILB cells displayed bona fide Breg activity by potently suppressing interferon-γ (IFN-γ) production in vitro in an IL-10-dependent manner. Furthermore, a novel CD43⁻CD1d^hiCD5⁺ IL-10-producing Breg population was predominantly induced by Chlamydia genital infection in vivo. Correspondingly, mixed bone marrow chimeric mice with B-cell-specific IL-10 deficiency exhibited significantly increased type 1 immune responses, decreased bacterial burden, and reduced oviduct pathology upon infection. Our data demonstrate for the first time a distinct role for CD43⁻CD1d^hiCD5⁺-adaptive Bregs over CD43⁺ innate counterparts in controlling mucosal responses against intracellular bacterial infection.     

IgM+CD27+ B cells possessed regulatory function and represented the main source of B cell-derived IL-10 in the synovial fluid of osteoarthritis patients

Synovial inflammation is observed in patients with osteoathritis (OA) and likely contributed to its exacerbation. Regulatory B (Breg) cells are shown to suppress inflammation in various diseases, including rheumatoid arthritis (RA). To examine whether Breg cells also participated in OA, we examined the synovial fluid from OA patients, and compared with that in RA patients. In OA synovial fluid, IL-10-producing B cells were present directly ex vivo and were increased upon stimulation, indicating that B cells were a source of IL-10 directly at the affected site of OA patients. Interestingly, the frequency of IL-10⁺ cells in synovial B cells was higher in OA patients than in RA patients, but the total number of IL-10⁺ B cells in OA was lower than that in RA, suggesting that OA patients presented lower B cell infiltration than RA patients. Phenotypical analysis demonstrated that the IL-10⁺ B cells were IgM⁺ and CD27⁺, but not CD24^hi or CD38^hi. To allow functional analysis of IgM⁺CD27⁺ B cells, the IgM⁺CD27⁺ B cells in the blood of OA patients were examined. These blood IgM⁺CD27⁺ B cells expressed more IL-10, but less CD80 and CD86 than non-IgM⁺CD27⁺ B cells. Blood IgM⁺CD27⁺ B cells suppressed the proliferation and IFN-γ expression of autologous T cells, and this effect could be reverted if IL-10 was inhibited. Furthermore, we found that patients with more severe OA presented lower levels of IL-10⁺ B cells in the synovial fluid. Together, our study described an IgM⁺CD27⁺ B cell subset in OA patients, which represented the major IL-10-secreting B cell type in the synovial fluid of OA patients and possessed regulatory function.     

TLR4 supports the expansion of FasL+CD5+CD1dhi regulatory B cells,which decreases in contact hypersensitivity

Certain B cells termed as “regulatory B cells” (Bregs) can suppress the ongoing immune responses and a splenic CD5⁺CD1d^hi Breg subset identified earlier was shown to exert its regulatory functions through secretion of IL-10. Though FasL expression is an alternative mechanism of immune suppression used by B cells, little is known about the FasL expressing CD5⁺CD1d^hi Bregs. In this study, we isolated splenocytes or splenic CD19⁺ B cells and compared the efficiency of toll-like receptor(TLR)4 ligand (lipopolysaccharide) with TLR9 ligand (CpG), anti-CD40 and TLR9 ligand (CpG) plus anti-CD40 on the FasL expression of splenic CD5⁺CD1d^hi Bregs by flow cytometry. FasL expression in CD5⁺CD1d^hi B cells was rapidly increased after TLR4 ligation. Intriguingly, anti-CD40 and CpG plus anti-CD40 combinations failed to stimulate FasL expression in CD5⁺CD1d^hi B cells although the IL-10 production was up-regulated in this subset. In addition, LPS and other B10-cell inducers increased the expression of surface molecules like CD86 and CD25, which are correlated to the regulatory functions of B cells. Furthermore, NF-κB and NF-AT inhibitors decreased the TLR4-activated FasL expression in CD5⁺CD1d^hi B cells. Then we sorted splenic CD5⁺CD1d^hi Bregs using flow cytometry and found that TLR4-activated CD5⁺CD1d^hi Bregs suppressed the proliferation of CFSE-labeled CD4⁺ T cells in vitro, which was partly blocked by anti-FasL antibody. In oxazolone-sensitized mice having contact hypersensitivity, FasL expression in splenic CD5⁺CD1d^hi B cells was decreased compared to the control group after TLR4 ligation. Our findings suggest that the regulatory function of CD5⁺CD1d^hi B cells could be partly mediated by Fas-FasL pathway and this FasL expressing CD5⁺CD1d^hi Bregs might participate in the regulation of inflammatory diseases.     

Increased resistance to CD4+CD25hi regulatory T cell‐mediated suppression in patients with type 1 diabetes

Type I diabetes (T1D) is a T cell‐mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin‐producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4⁺CD25^hi naturally occurring regulatory T cells (T_regs), defects in which could contribute to loss of self‐tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4⁺CD25^hi T_regs of autologous CD4⁺CD25^‐ responder cells. Here we demonstrate that this defective regulation is also present in subjects with long‐standing T1D (> 3 years duration; P = 0·009). No difference was observed in forkhead box P3 or CD127 expression on CD4⁺CD25^hi T cells in patients with T1D that could account for this loss of suppression. Cross‐over co‐culture assays demonstrate a relative resistance to CD4⁺CD25^hi T_reg‐mediated suppression within the CD4⁺CD25^‐ T cells in all patients tested (P = 0·002), while there appears to be heterogeneity in the functional ability of CD4⁺CD25^hi T_regs from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.     

Alterations in regulatory T-cells: Rediscovered pathways in immunotoxicology

In addition to the effector T-cells subsets, T-cells can also differentiate into cells that play a suppressive or regulatory role in adaptive immune responses. The cell types currently identified as regulatory T-cells (T_regs) include natural or thymic-derived T_regs, T-cells which express Foxp3⁺CD25⁺CD4⁺ and can suppress immune responses to autoreactive T-cells, as well as inducible T_regs, that are generated from naïve T-cells in the periphery after interaction with antigens presented by dendritic cells. Inducible T_regs include T_H3 cells, T_r1 cells, and Foxp3⁺-inducible T_regs. T_regs have been shown to be critical in the maintenance of immune responses and T-cell homeostasis. These cells play an important role in suppressing responses to self-antigens and in controlling inappropriate responses to non-self-antigens, such as commensal bacteria or food in the gut. For example, depletion of CD4⁺CD25⁺ T_regs from mice resulted in the development of multi-organ autoimmune diseases. CD4⁺CD25⁺ T_regs and/or IL-10-producing T_r1 cells are capable of suppressing or attenuating T_H2 responses to allergens. Moreover, adoptive transfer of CD4⁺CD25⁺ T_regs from healthy to diseased animals resulted in the prevention or cure of certain autoimmune diseases, and was able to induce transplantation tolerance. Clinical improvement seen after allergen immunotherapy for allergic diseases such as rhinitis and asthma is associated with the induction of IL-10- and TGFβ-producing T_r1 cells as well as FoxP3-expressing IL-10 T-cells, with resulting suppression of the T_H2 cytokine milieu. Activation, expansion, or suppression of CD4⁺CD25⁺ T_regs in vivo by xenobiotics, including drugs, may therefore represent a relevant mechanism underlying immunotoxicity, including immunosuppression, allergic asthma, and autoimmune diseases.     

Th17, synchronically increased with Tregs and Bregs,promoted by tumour cells via cell‐contact in primary hepatic carcinoma

Documented reports about T helper type 17 (Th17) cells have revealed that Th17 plays a critical role in inflammation and autoimmunity diseases. However, the role of Th17 in cancer remains contradictory. The interplay between Th17 and tumour cells in the tumour microenvironment of primary hepatic carcinoma (PHC) needs to be explored further and the relationship between Th17, regulatory T cells (T_regs) and regulatory B cells (B_regs) has not been defined completely. In this study, numerous experiments were undertaken to elucidate the interaction of Th17 and T_reg/B_reg cells involved in PHC. Our work demonstrated that an increased Th17 was detected in the peripheral circulation and in tumour tissues in PHC patients. In addition, increases in peripheral blood Th17 corresponded with tumour–node–metastasis (TNM) stage progression. Also, further studies indicated that Th17 cells were promoted by tumour cells in the PHC tumour microenvironment through both contact‐dependent and ‐independent mechanisms, but cell‐contact played the major important role in promoting the production and proliferation of Th17. When isolated CD4⁺CD25⁺CD127^low T_regs and CD4⁺CD25^–CD127⁺ non‐T_regs were cultured with autologous tumour cells, it implied that the phenotype of Th17 and T_regs was modified by tumour cells in the tumour microenvironment. As well as this, Th17 cells were also found to correlate positively with CD4⁺forkhead box protein 3⁺ T_regs and CD19⁺CD5⁺CD1d^hi B_regs in PHC. Notably, Th17 increased synchronically with T_regs and B_regs in PHC. These findings may provide new clues to reveal the mechanisms of immune escape in PHC.     

Increased CD4+CXCR5+T follicular helper cells in diabetic nephropathy

Background: T follicular helper (Tfh) cells are known to regulate humoral immune response. In this study we examined the correlation of different subsets of peripheral blood Tfh cells in patients with diabetic nephropathy (DN). Methods: A total of 23 DN patients and 15 healthy controls (HC) were investigated for various subsets of Tfh cells by flow cytometry. The molecules ICOS⁺, PD-1⁺, CD28⁺, CD154⁺, IL-21⁺, IFN-γ⁺, IL-4⁺, IL-17⁺ Tfh cells were examined. The subsets of B cells were investigated by flow cytometry. The levels of 24?h urinary protein and estimated glomerular filtration rate (eGFR) were calculated. A potential correlation between the number of different subsets of Tfh cells, B cells and DN, was assessed. Results: The circulating CD4⁺CXCR5⁺PD-1⁺, PD-1⁺CD154⁺, PD-1⁺CD28⁺, PD-1⁺IL-21⁺, PD-1⁺IL-4⁺, PD-1⁺-IL-17⁺-Tfh cell counts, CD38⁺CD19⁺, CD38⁺CD19⁺CD40⁺ B cells and plasma levels of IL-21 were significantly increased in DN patients (p?+CXCR5⁺PD-1⁺ Tfh cell counts negatively correlated with eGFR; Tfh cell counts positively correlated with 24?h urinary protein concentration in DN patients. Post-treatment, there was a significant reduction in the CD4⁺CXCR5⁺PD-1⁺ Tfh cell counts and its subsets, with a corresponding decrease in plasma levels of IL-6 and IL-17A (p?Conclusion: An increased number of CD4⁺CXCR5⁺PD-1⁺ Tfh cells were observed in DN patients, which may be new targets for intervention in DN.     

IL-10-producing memory B regulatory cells as a novel target for HLA-G to prolong human kidney allograft survival

Despite the growing interest in the role of regulatory B cells (Bregs) in autoimmunity, their distinct role and function in kidney transplant outcomes remain elusive. Here, we retrospectively analyzed the proportion of Bregs, transitional Bregs (tBregs) and memory Bregs (mBregs) and their capacity to produce IL-10 in non-rejected (NR) versus rejected (RJ) kidney transplant recipients. In the NR group, we observed a significant increase in the proportion of mBregs (CD19⁺CD24^hiCD27⁺) but no difference in tBregs (CD19⁺CD24^hiCD38⁺), as compared to the RJ group. We also observed a significant increase in IL-10-producing mBregs (CD19⁺CD24^hiCD27⁺IL-10⁺) in the NR group. As our group and others have previously reported a potential role of the human leukocyte antigen G (HLA-G) in human renal allograft survival, notably through IL-10, we then investigated possible crosstalk between HLA-G and IL-10⁺ mBregs. Our ex vivo data suggest a role of HLA-G in enhancing IL-10⁺ mBreg expansion upon stimulation, which further decreased CD3⁺ T cell proliferation capability. Using RNA-sequencing (RNA-seq), we identified potential key signaling pathways involved in HLA-G-driven IL-10⁺ mBreg expansion, such as the MAPK, TNF and chemokine signaling pathways. Together, our study highlights a novel HLA-G-mediated IL-10-producing mBreg pathway that may serve as a therapeutic target to improve kidney allograft survival.     

Acquisition of CD24 expression by Lin−CD43+B220lowckithi cells coincides with commitment to the B cell lineage

The present study describes three subsets of bone marrow-derived Lin⁻CD43⁺B220^low (B220^low) progenitor cells which represent distinct stages in hematopoietic development. These populations differ in their expression of CD24 and ckit and the occurrence of Ig_H gene rearrangement. B220^low CD24⁻ckit^hi progenitors have their Ig_H loci in germ-line configuration and are multipotent since they can give rise to B cells, T cells and myeloid cells. B220^lowckit^hi cells which have acquired CD24 expression have retained Ig_H loci in germ-line configuration and the ability to generate B cells, however, they have lost the ability to generate T cells and myeloid cells. Thus acquisition of CD24 by B220^low cells occurs concurrently with the transition from a multipotent to a lineage-restricted progenitor population. B220^lowCD24⁺ cells expressing low levels of ckit are also lineage restricted, giving rise to only B cells and have begun D-J_H rearrangement, implying that initiation of Ig_H rearrangement coincides with the down-regulation of ckit expression.     

Decrease in the proportion of CD24hiCD38hi B cells and impairment of their regulatory capacity in type 1 diabetes patients

B10 cells restore immune balance by producing interleukin (IL)-10. Impaired B10 cell responses are related to numerous autoimmune diseases. However, the function of B10 cells in type 1 diabetes (T1D) patients is controversial. We hypothesized that there are numerical and functional defects of B10 cells in T1D. Sixty-two patients with T1D and 74 healthy volunteers were included in our study. We showed that B10 cells in human peripheral blood belong to a CD24^hiCD38^hi B cell subpopulation. CD24^hiCD38^hi B cells from healthy individuals possessed regulatory capacity, suppressed interferon (IFN)-γ, tumor necrosis factor (TNF)-α and IL-17A production and promoted IL-4 production and forkhead box protein 3 (FoxP3) expression in CD4⁺ T cells through an IL-10-dependent mechanism. Compared to healthy controls, B10 cell percentages in T1D were significantly lower (5·6 ± 3·5 versus 6·9 ± 3·3%; P < 0·05), produced less IL-10 (15·4 ± 4·3 versus 29·0 ± 4·5%; P < 0·001) and lacked regulatory capacity. In addition, Pearson’s correlation analysis showed that the frequency of circulating B10 cells was negatively correlated with the frequency of CD4⁺IFN-γ⁺ and CD4⁺TNF-α⁺ T cells (r = −0·248 and r = −0·283, P = 0·008 and P = 0·017, respectively), positively correlating with the frequency of CD4⁺CD25⁺FoxP3⁺ T cells (r = 0·247, P = 0·001). These data offer direct proof that there is a deficiency of circulating CD24^hiCD38^hi B cells in peripheral blood of patients with T1D, which participate in the T1D immune imbalance involved in the development of T1D.     

Expression of selectin-binding epitopes and cytokines by CD4+ T cells repopulating scid mice with colitis

Recruitment into the gut of CD4⁺ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4⁺ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4⁺ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-γ and TNF-α but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-γ- and TNF-α-producing effector CD4⁺ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4⁺ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4⁺ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-L^hi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-L^hi CD4⁺ T cells were enriched in IFN-γ-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-L^lo CD4⁺ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4⁺ T blasts that were almost exclusively P-L^hi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4⁺ T cells residing in inflamed and non-inflamed tissues during the development of colitis.     

Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4⁺ T regulatory cells (T_regs) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2⁺ cells increased over time for both CD4⁺ and CD8⁺ T cells. The T_reg subsets CD25^highCD127^low, CD25^highCD147⁺⁺ and CD25^highCD127^lowCD161⁺ expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25^highCD127^lowCD39⁺ T_regs remained unchanged. The fraction of CD25^highCD127^low T_regs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of T_regs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4⁺ and CD8⁺ T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.     

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications

Although CD4⁺/CD25⁺ T regulatory cells (T_regs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell‐separation strategy complying with good manufacturing practice guidelines. We isolated T_regs from standard leukapheresis products using double‐negative selection (anti‐CD8 and anti‐CD19 monoclonal antibodies) followed by positive selection (anti‐CD25 monoclonal antibody). The final cell fraction (CD4⁺/CD25⁺) showed a mean purity of 93·6% ± 1·1. Recovery efficiency was 81·52% ± 7·4. The CD4⁺/CD25^+bright cells were 28·4% ± 6·8. The CD4⁺/CD25⁺ fraction contained a mean of 51·9% ± 15·1 FoxP3 cells and a mean of 18·9% ± 11·5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4⁺CD25⁺FoxP3⁺ cells were in line with flow cytometric results. In Vβ spectratyping the complexity scores of CD4⁺/CD25⁺ cells and CD4⁺/CD25^‐ cells were not significantly different, indicating that T_regs had a broad T cell receptor repertoire. The inhibition assay showed that CD4⁺/CD25⁺ cells inhibited CD4⁺/CD25^‐ cells in a dose‐dependent manner (mean inhibition percentages: 72·4 ± 8·9 [ratio of T responder (T_resp) to T_regs, 1:2]; 60·8% ± 20·5 (ratio of T_resp to T_regs, 1:1); 25·6 ± 19·6 (ratio of T_resp to T_regs, 1:0·1)). Our study shows that negative/positive T_reg selection, performed using the CliniMACS device and reagents, enriches significantly CD4⁺CD25⁺FoxP3⁺ cells endowed with immunosuppressive capacities. The CD4⁺CD25⁺FoxP3⁺ population is a source of natural T_reg cells that are depleted of CD8⁺ and CD4⁺/CD25^‐ reacting clones which are potentially responsible for triggering graft‐versus‐host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft‐versus‐tumour effect.     

Lower proportion of CD19+IL-10+ and CD19+CD24+CD27+ but not CD1d+CD5+CD19+CD24+CD27+ IL-10+ B cells in children with autoimmune thyroid diseases

Abstract

Introduction: As it is generally known, regulatory B cells (Bregs) control inflammation and autoimmunity. The significance of Bregs in the population of children with autoimmune thyroid diseases (AITD) still offers plenty of potential to explore. The aim of this study was to estimate the expression of Bregs (phenotype CD19⁺CD24⁺CD27⁺IL-10⁺, CD19⁺IL-10⁺, CD1d⁺CD5⁺CD19⁺IL-10⁺ and CD1d⁺CD5⁺CD19⁺CD24⁺CD27⁺) in a paediatric cohort with AITD and in health controls.

Materials and methods: A total of 100 blood samples were obtained from 53 paediatric patients with Graves’ disease (GD) (N?=?12 newly diagnosed, mean age 12.5?±?3.5 and N?=?17 during methimazole therapy, mean age 12.7?±?4.4), Hashimoto’s thyroiditis (HT) (N?=?10 newly diagnosed, mean age 13.3?±?2.9 and N?=?10 during L-thyroxine therapy, mean age 13.7?±?3.4) and compared with healthy controls (C) (N?=?15, mean age 13.1?±?3.1). The expressions of the immune cell populations were analysed by four-color flow cytometry using a FASC Canto II cytometer (BD Biosciences).

Results: There was a decreasing tendency in the number of lymphocytes B producing IL-10 (B10) cells among all B lymphocytes and more widely, also among all lymphocytes, in each study group, as compared to C. We reported a reduction in IL-10 production in Bregs with the expression of CD19⁺CD24⁺CD27⁺IL-10 and CD1d⁺CD5⁺CD19⁺IL-10⁺ in both untreated and treated AITD.

Conclusions: Our data demonstrate that the reduction in the number of Bregs with CD19⁺CD24⁺CD27⁺IL-10⁺ and CD19⁺IL-10⁺ expression could be responsible for breaking immune tolerance and for AITD development in children.     

The imbalance of T helper 17/regulatory T cells and memory B cells during the early post‐transplantation period in peripheral blood of living donor liver transplantation recipients under calcineurin inhibitor‐based immunosuppression

There is limited clinical research regarding the changes in peripheral lymphocyte subsets during the early post-operative period of liver transplantation. Serial changes of T cells and B cells in living donor liver transplantation (LDLT) recipients during the early post-transplantion period were prospectively investigated. From June 2010 to February 2011, 27 consecutive LDLT recipients were enrolled. Percentages of T helper type 1 (Th1; interferon-γ-producing), Th2 (interleukin-4-producing), Th17 (interleukin-17-producing), regulatory T (Treg; CD4⁺ CD25⁺ FoxP3⁺), memory B (CD19⁺ CD24^hi CD38⁻) and mature B (CD19⁺ CD24^int CD38^int) cells were measured using fluorescence-activated cell sorting. Patients were followed up for a median of 9·9 months (range 6·8−15·5 months) after transplantation. Serial monitoring of immunological profiles showed no significant suppression of Th1, Th2, Th17, mature B or memory B cells, whereas frequencies of Treg cells significantly decreased. Interleukin-17 production by central and effector memory cells was not suppressed during the early post-operative period. The continuous production of interleukin-17 by the memory T cells may contribute to the persistence of Th17 cells. This prospective study demonstrated that current immunosuppression maintained the effector T or memory B cells during the early post-transplantation period but significantly suppressed Treg cells. Serial immune monitoring may suggest clues for optimal or individualized immunosuppression during the early post-operative period in clinical practice.     

Neutralization of IL-10 produced by B cells promotes protective immunity during persistent HCV infection in humanized mice

Chronic HCV infection can lead to cirrhosis and is associated with increased mortality. Interleukin (IL)-10-producing B cells (B10 cells) are regulatory cells that suppress cellular immune responses. Here, we aimed to determine whether HCV induces B10 cells and assess the roles of the B10 cells during HCV infection. HCV-induced B10 cells were enriched in CD19^hi and CD1d^hiCD5⁺ cell populations. HCV predominantly triggered the TLR2-MyD88-NF-κB and AP-1 signaling pathways to drive IL-10 production by B cells. In a humanized murine model of persistent HCV infection, to neutralize IL-10 produced by B10 cells, mice were treated with pcCD19scFv-IL-10R, which contains the genes coding the anti-CD19 single-chain variable fragment (CD19scFv) and the extracellular domain of IL-10 receptor alpha chain (sIL-10Ra). This treatment resulted in significant reduction of B10 cells in spleen and liver, increase of cytotoxic CD8⁺ T-cell responses against HCV, and low viral loads in infected humanized mice. Our results indicate that targeting B10 cells via neutralization of IL-10 may offer a novel strategy to enhance anti-HCV immunotherapy.