Reduced CD5+CD24hiCD38hi and interleukin-10+ regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies

Pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis is B cell-dependent, although how particular B cell subsets modulate immunopathogenesis remains unknown. Although their phenotype remains controversial, regulatory B cells (B_regs), play a role in immunological tolerance via interleukin (IL)-10. Putative CD19⁺CD24^hiCD38^hi and CD19⁺CD24^hiCD27⁺ B_regs were evaluated in addition to their CD5⁺ subsets in 69 patients with ANCA-associated vasculitis (AAV). B cell IL-10 was verified by flow cytometry following culture with CD40 ligand and cytosine–phosphate–guanosine (CpG) DNA. Patients with active disease had decreased levels of CD5⁺CD24^hiCD38^hi B cells and IL-10⁺ B cells compared to patients in remission and healthy controls (HCs). As IL-10⁺ and CD5⁺CD24^hiCD38^hi B cells normalized in remission within an individual, ANCA titres decreased. The CD5⁺ subset of CD24^hiCD38^hi B cells decreases in active disease and rebounds during remission similarly to IL-10-producing B cells. Moreover, CD5⁺ B cells are enriched in the ability to produce IL-10 compared to CD5^neg B cells. Together these results suggest that CD5 may identify functional IL-10-producing B_regs. The malfunction of B_regs during active disease due to reduced IL-10 expression may thus permit ANCA production.     

Higher frequencies of circulating ICOS+, IL-21+ T follicular helper cells and plasma cells in patients with new-onset membranous nephropathy

Background: T follicular helper (TFH) cells and B cells are known to regulate humoral immune responses. This study is aimed at examining the putative contribution of different subsets of circulating of TFH cells and B cells to membranous nephropathy (MN).

Methods: A total of 45?MN patients and 19 healthy controls (HCs) were examined for the number of TFH cells and B cells by flow cytometry. The level of 24-h urinary protein and eGFR were calculated, and the level of serum cytokines was examined. The potential association among these measures was analyzed.

Results: Compared to the HCs, MN patients had significantly higher numbers of circulating CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, and CD4⁺CXCR5⁺CD28⁺ TFH cells, as well as IgD⁺CD27^?CD19⁺ and CD138⁺CD19⁺ B cells. However, the number of IgD⁺CD27⁺CD19⁺ B cells was significantly lower in MN patients than in the HC. The levels of serum IL-21, IL-2, IL-4, IL-10, IL-17A, and IFN-γ were significantly higher in MN patients than in the HC. Furthermore, the numbers of CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, CD4⁺CXCR5⁺CD28⁺ TFH cells, CD138⁺CD19⁺ B cells, and the level of sera IL-21 were negatively correlated with the values of eGFR, but positively correlated with the levels of 24-h urinary proteins. Following treatment, the numbers of CD4⁺CXCR5⁺, CD4⁺CXCR5⁺ICOS⁺, CD4⁺CXCR5⁺CD154⁺, CD4⁺CXCR5⁺IL-21⁺, CD4⁺CXCR5⁺CD28⁺ TFH cells, CD138⁺CD19⁺ B cells, and the levels of IL-21 were significantly reduced. In contrast, IL-4 and IL-10 levels were noticeably increased after treatment.

Conclusions: Data suggest that activated TFH and plasma cells may contribute to the pathogenesis of MN.     

Increased CD45RO CD62L CD4 T-cell subpopulation responsible for Th2 response in Kimura’s disease

Kimura’s disease is characterized by subcutaneous masses, eosinophilia, and markedly elevated serum immunoglobulin E, suggesting that T helper (Th)2 cells may play a role in the pathogenesis. We investigated Th2 cytokine synthesis by mononuclear cells and possible Th1/Th2 subpopulations in Kimura’s disease. Peripheral blood samples were obtained from seven patients with Kimura’s disease and CD4⁺ T-cell subpopulations separated by CD45RO and CD62L were isolated. Purified cells were stimulated with PHA or anti-CD3 mAb, and the cytokine levels were measured by Cytometric Bead Array kit. Peripheral blood mononuclear cells in the majority of the patients produced Th2 cytokines such as interleukin (IL)-3, IL-4, IL-5, IL-13 or GM-CSF higher than those of controls. The ratio of CD45RO⁺ CD62L⁺ cells in CD4⁺ T cells was increased in six out of seven patients compared to age-matched controls. Especially, patient 1 had remarkably increased levels of CD45RO⁺ CD62L⁺ population in CD4⁺ T cells. In addition, IL-4 production levels by CD45RO⁺ CD62L⁺ CD4⁺ T cells of patients 1 and 2 were higher than those of their CD45RO⁺ CD62L⁻ CD4⁺ T cells, in the same manner as those by a normal control. Taken together, the synthesis of Th2 cytokines and CD62L-positive subpopulation in CD45RO⁺ CD4⁺ T cells, which may represent characteristics of Th2, are increased in patients with Kimura’s disease, suggesting that deviation to Th2 may involve in pathogenesis of the disease.     

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.     

CCR1 antagonist J-113863 corrects the imbalance of pro- and anti-inflammatory cytokines in a SJL/J mouse model of relapsing-remitting multiple sclerosis

Multiple sclerosis (MS), an immune-mediated and neurodegenerative disorder of the central nervous system (CNS), is characterized by infiltrating myelin-reactive T lymphocytes and demyelinating lesions. Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model used to study MS. To explore the impact of chemokine receptor CCR1 blockade in EAE and the underlying mechanisms, we used CCR1 antagonist J-113863 in PLP_139-151-induced EAE in SJL/J mice. Following EAE induction, mice were treated with J-113863 (10 mg/kg) daily from day 14 until day 25. We investigated the effect of J-113863 on expression levels of GM-CSF, IL-6, IL-10, IL-27 in CD4⁺ spleen cells, using flow cytometry. We also analyzed the effect of J-113863 on GM-CSF, IL-6, IL-10, IL-27 mRNA and protein expression levels using RT-PCR and Western blot analysis in brain tissues. J-113863 treatment decreased the populations of CD4⁺GM-CSF⁺ and CD4⁺IL-6⁺ cells and increased CD4⁺IL-27⁺ and CD4⁺IL-10⁺ cells in the spleen. J-113863 had a suppressive effect on the mRNA and protein expression levels of GM-CSF, and IL-6 in the brain tissue. On the other hand, J-113863 treatment increased the mRNA and protein expression of IL-10 and IL-27 in the brain tissue. Our results highlighted J-113863′s potential role in suppressing pro-inflammatory expression and up-regulating anti-inflammatory mediators, which could represent a beneficial alternative approach to MS treatment.     

The immune potential and immunopathology of cytokine-producing B cell subsets: A comprehensive review

B lymphocytes are generally recognized for their potential to mediate humoral immunity by producing different antibody isotypes and being involved in opsonization and complement fixation. Nevertheless, the non-classical, antibody-independent immune potential of B cell subsets has attracted much attention especially in the past decade. These B cells can release a broad variety of cytokines (such as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-α, IFN-γ, TNF-α, TGF-β, LT), and can be classified into distinct subsets depending on the particular cytokine profile, thus emerging the concept of cytokine-producing B cell subsets. Although there is still controversy surrounding the key cell surface markers, intracellular factors and cellular origins of cytokine-producing B cell subsets, accumulating evidence indicates that these B cells are endowed with great potential to regulate both innate and adaptive arms of immune system though releasing cytokines. On the one hand, they promote immune responses through mounting Th1/Th2/Th17 and neutrophil response, inducing DC maturation and formation of lymphoid structures, increasing NK cell and macrophage activation, enhancing development of themselves and sustaining antibody production. On the other hand, they can negatively regulate immune responses by suppressing Th cell responses, inhibiting Tr1 cell and Foxp3⁺ Treg differentiation, impairing APC function and pro-inflammatory cytokine release by monocytes, and inducing CD8⁺ T cell anergy and CD4⁺ T cell apoptosis. Therefore, cytokine-producing B cell subsets have multifunctional functions in health and diseases, playing pathologic as well as protective roles in autoimmunity, infection, allergy, and even malignancy. In this review, we revisit the history of discovering cytokine-producing B cells, describe the identification of cytokine-producing B cell subsets, introduce the origins of cytokine-producing B cell subsets as well as molecular and cellular mechanisms for their differentiation, and summarize the recent progress made toward understanding the unexpectedly complex and potentially opposing roles of cytokine-producing B cells in immunological disorders.     

Delphinidin diminishes in vitro interferon-γ and interleukin-17 producing cells in patients with psoriatic disease

The anthocyanidin delphinidin reduces psoriasiform lesions and inflammatory mediators in human cell culture systems. Its role in psoriatic disease has not yet been investigated. We assessed delphinidin’s in vitro immunomodulatory effect on ex vivo stimulated peripheral blood mononuclear cells (PBMCs) from 50 individuals [26 with psoriasis, 10 with psoriatic arthritis (PsA) and 14 healthy controls (HCs)]. Cells were either left untreated or stimulated with PMA plus ionomycin in the presence or absence of delphinidin. Intracellular production of interferon-γ (IFNγ), interleukin-17A (IL-17A), and interleukin-10 (IL-10) was measured flow cytometrically. Delphinidin dose-dependently reduced IFNγ⁺ T cells from patients and HCs. The mean IFNγ decrease in CD4⁺ T subpopulations was 42.5?±?28% for psoriasis patients, 51.8?±?21.5% for PsA patients and 49?±?17% for HCs (p?<?0.001 for all). Similarly, IFNγ reduction in CD8⁺ T cells was 34?±?21.6% for psoriasis patients, 47.1?±?22.8% for PsA and 44.8?±?14.3% for HCs (P <?0.001 for all). An inhibitory effect of delphinidin was also noted in IFNγ producing NKs and NKTs from psoriasis individuals. Delphinidin also significantly decreased IL-17⁺ CD4⁺ T cells in all tested subjects, with marginal effect on the increase of IL-10-producing T regulatory subsets. In conclusion, delphinidin exerts a profound in vitro anti-inflammatory effect in psoriasis and psoriatic arthritis by inhibiting IFNγ⁺ innate and adaptive cells and T helper (Th) 17 cells. If this effect is also exerted in vivo, delphinidin may be regarded as a nutraceutical with immunosuppressive potential.

     

CD5+ B lymphocytes secrete IL-10 rather than TGF-β1 which control the immune response in autoimmune haemolytic anaemia/Evans syndrome

Objective: To investigate the quantity and secretion function of cytokines-secreted CD5⁺ B lymphocytes in Autoimmune Haemolytic Anaemia (AIHA)/Evans syndrome (ES) patients.

Methods: Twenty-five untreated AIHA/ES patients, 28 remission AIHA/ES patients and 25 healthy controls (HCs) were enrolled in this study. The quantity of CD5⁺B lymphocytes which produce interleukin-10 (IL-10) (CD5⁺IL-10⁺) and transforming growth factor (TGF-β1) (CD5⁺TGF-β1⁺) were detected by flow cytometry (FCM). CD5⁺ B lymphocytes were sorted from peripheral blood (PB) by FCM and the expression of IL-10 and TGF-β1 mRNA in CD5⁺ B cells were measured by real-time PCR (RT-PCR).

Results: The percentage of CD5⁺IL-10⁺B cells in CD5⁺ B lymphocytes in newly diagnosed patients was 82.18?±?14.78%, which being significantly higher than that of remission AIHA/ES patients (56.68?±?24.39%) and HCs (51.90?±?22.95%) (p?<?.05). The percentage of CD5⁺IL-10⁺ B cells in CD5⁺ B lymphocytes in newly diagnosed patients was negatively correlated with haemoglobin (Hb), complement 3 (C3) (p?<?.05) and positively correlated with lactate dehydrogenase (LDH), total bilirubin (TBIL) and indirect unconjugated bilirubin (IBIL) (p?<?.05). The expression level of IL-10 mRNA in CD5⁺ B lymphocytes of newly diagnosed patients (49.34?±?22.84) was higher than that of remission patients (3.97?±?3.83) and HCs (1.78?±?1.66) (p?<?.05). There was no significant difference among three groups with the proportion of CD5⁺TGF-β1⁺ B lymphocytes and the expression level of TGF-β1 mRNA in CD5⁺B lymphocytes (p?>?.05).

Conclusions: CD5⁺ B lymphocytes could secrete IL-10 rather than TGF- β1 which control the immune response in AIHA/ES.     

T cell-specific BLIMP-1 deficiency exacerbates experimental autoimmune encephalomyelitis in nonobese diabetic mice by increasing Th1 and Th17 cells

Recently, we demonstrated that B lymphocyte-induced maturation protein 1 (BLIMP-1) has a role in regulating the differentiation and effector function of Th1 and Th17 cells. As these cells play critical roles in the induction and pathogenesis of experimental autoimmune encephalomyelitis (EAE), we investigated the potential role of T cell BLIMP-1 in modulating MOG_35–55-induced EAE. We established T cell-specific BLIMP-1 conditional knockout (CKO) NOD mice to dissect the role of BLIMP-1 in EAE using loss-of-function model. Our results indicate that EAE severity is dramatically exacerbated in CKO mice. The numbers of CNS-infiltrating Th1, Th17, IFN-γ⁺IL-17A⁺, and IL-21⁺IL-17A⁺ CD4⁺ T cells are remarkably increased in brain and spinal cord of CKO mice. Moreover, the ratio of Tregs/effectors and IL-10 production of Tregs are significantly downregulated in CNS of CKO mice. We conclude that BLIMP-1 suppresses autoimmune encephalomyelitis via downregulating Th1 and Th17 cells and impairing Treg cells.     

Increased Microchimeric CD4 T Lymphocytes in Peripheral Blood from Women with Systemic Sclerosis

Recent studies have demonstrated the presence of microchimeric cells in peripheral blood and skin lesions from patients with systemic sclerosis (SSc). In a previous study we found that some peripheral blood CD3⁺ cells from female patients with SSc contained male DNA. Here, peripheral blood samples from 47 patients with SSc (30 with diffuse cutaneous SSc and 17 with limited cutaneous SSc) and 22 healthy controls were sorted for CD4⁺ and CD8⁺ T cells. Both positively and negatively selected populations were analyzed for male DNA by quantitative PCR. Analysis of Y chromosome sequences in the sorted cells demonstrated the presence of microchimerism in 82.9% of SSc patients compared to 63.6% of controls. The numbers of CD4⁺ and CD8⁺ T cells were found to be significantly higher in the SSc patients than in controls. Furthermore, patients with dcSSc were observed to have significantly more CD4⁺ microchimeric T cells than the controls. In the CD8⁺ T-cell population, there was a trend toward more microchimeric cells in the patients but this did not reach significance. These results support the hypothesis that microchimeric CD4⁺ T cells may be involved in the pathogenesis of SSc.     

1,25‐dihydroxyvitamin D3‐induced dendritic cells suppress experimental autoimmune encephalomyelitis by increasing proportions of the regulatory lymphocytes and reducing T helper type 1 and type 17 cells

Dendritic cells (DCs), a bridge for innate and adaptive immune responses, play a key role in the development of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Administration of tolerogenic DCs has been used as an immunotherapy in autoimmune diseases. Deficiency of vitamin D is an environmental risk factor of MS. In this study, we induced tolerogenic DCs by 1,25‐dihydroxyvitamin D₃ and transferred the tolerogenic DCs (VD₃‐DCs) into EAE mice by adoptive transfer. We found that VD₃‐DCs inhibited the infiltrations of T helper type 1 (Th1) and Th17 cells into spinal cord and increased the proportions of regulatory T cells (CD4⁺ CD25⁺ Foxp3⁺), CD4⁺ IL‐10⁺ T cells and regulatory B cells (CD19⁺ CD5⁺ CD1d⁺) in peripheral immune organs, which resulted in attenuated EAE. However, the proportions of T helper type 1 (Th1) and Th17 cells in spleen and lymph nodes and the levels of pro‐inflammatory cytokines and IgG in serum also increased after transfer of VD₃‐DCs. We conclude that transfer of VD₃‐DCs suppressed EAE by increasing proportions of regulatory T cells, CD4⁺ IL‐10⁺ T cells and regulatory B cells in spleen and reducing infiltration of Th1 and Th17 cells into spinal cord, which suggests a possible immunotherapy method using VD₃‐DCs in MS.     

Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic <Emphasis Type="Italic">apoE</Emphasis><Superscript><Emphasis Type="Italic">−/−</Emphasis></Superscript> mice

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10⁺ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10⁺ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10⁺ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5⁺ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10⁺ B cells with T1-like and MZ, and decreased IL-10⁺ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4⁺CD25^? T cells. CD5⁺, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)⁺ and tumor necrosis factor alpha (TNF-α)⁺ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ⁺ and TNF-α⁺ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.     

Th17 responses to pneumococcus in blood and adenoidal cells in children

Pneumococcal infections cause a large global health burden, and the search for serotype-independent vaccines continues. Existing conjugate vaccines reduce nasopharyngeal colonization by target serotypes. Such mucosal effects of novel antigens may similarly be important. CD4⁺ Th17 cell-dependent, antibody-independent reductions in colonization and enhanced clearance have been described in mice. Here we describe the evaluation of T helper type 17 (Th17) cytokine responses to candidate pneumococcal protein vaccine antigens in human cell culture, using adenoidal and peripheral blood mononuclear cells. Optimal detection of interleukin (IL)-17A was at day 7, and of IL-22 at day 11, in these primary cell cultures. Removal of CD45RO⁺ memory T cells abolished these responses. Age-associated increases in magnitude of responses were evident for IL-17A, but not IL-22, in adenoidal cells. There was a strong correlation between individual IL-17A and IL-22 responses after pneumococcal antigen stimulation (P < 0·015). Intracellular cytokine staining following phorbol myristate acetate (PMA)/ionomycin stimulation demonstrated that  > 30% CD4⁺ T cells positive for IL-22 express the innate markers γδT cell receptor and/or CD56, with much lower proportions for IL-17A⁺ cells (P < 0·001). Responses to several vaccine candidate antigens were observed but were consistently absent, particularly in blood, to PhtD (P < 0·0001), an antigen recently shown not to impact colonization in a clinical trial of a PhtD-containing conjugate vaccine in infants. The data presented and approach discussed have the potential to assist in the identification of novel vaccine antigens aimed at reducing pneumococcal carriage and transmission, thus improving the design of empirical clinical trials.     

Human Th1 responses driven by IL-12 are associated with enhanced expression of CD40 ligand

IL- 12 is the prominent inducer of Th1 responses in humans and in the mouse. CD40 ligand (CD40L) plays important roles in regulation of immune responses, including T cell-dependent activation of B cells and cytokine production by monocytes and dendritic cells. The present study examined the influences of IL-12 on the CD40L expression of activated human CD4⁺ T cells. IL-12 enhanced CD40L expression on CD4⁺ T cells stimulated with immobilized anti-CD3 in the complete absence of accessory cells, whereas IL-4 and IL-10 decreased it. Exogenous interferon-gamma (IFN-γ) did not increase CD40L expression on immobilized anti-CD3 stimulated CD4⁺ T cells at any time up to 168 h of culture. The IL-12-induced enhancement of CD40L expression on anti-CD3 activated CD4⁺ T cells was not influenced in the presence of a metalloproteinase inhibitor KB8301, which up-regulated CD40L expression by preventing the processing of membrane-bound CD40L, or B cells, which down-regulated CD40L expression by receptor-mediated endocytosis. These results indicate that IL-12 enhances the CD40L expression of activated CD4⁺ T cells independently of the IFN-γ production. The data thus suggest that Th1 responses induced by IL-12 might play an important role in the regulation of humoral immune responses through up-regulated CD40L expression.     

Antigen-independent IL-17A production by bystander-activated CD4+IL-1R1+ cells in patients with multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease caused by auto-antigen recognizing CD4⁺ T cells. However, IL-17A-producing CD4⁺ T cells that are bystander-activated by IL-1β and IL-23, and T cell receptors independently, could contribute to experimental autoimmune encephalomyelitis. Here, we studied the differences in the frequency and function of bystander-activated CD4⁺ T cells in patients with MS. A significantly higher frequency of CD4 + IL-1Rl + T cells was found in memory than in naïve CD4⁺ T cells and in Th17/Th17.1 than in Th1/Th2 subtypes in both MS and healthy controls (HC). Following IL-1β and IL-23 stimulation, IL-1Rl expression was markedly increased in both memory and Th17/Th17.1 cells, and their IL-17A-production was increased after bystander-activation, which was significantly higher in MS compared with HC. Our study suggests a potential role of IL-17A-producing bystander-activated CD4⁺IL-1Rl⁺ T cells in MS.