调节性B细胞的发现和研究进展

B细胞主要通过呈递抗原和产生抗体发挥免疫调节作用.新近研究表明,一种全新的B细胞亚群--调节性B细胞(regulatory B cell,Bregs),可通过产生白细胞介素10(IL-10)或转化生长因子β1(TGF-β1)等抑制性细胞因子介导免疫耐受,抑制过度炎症反应.Bregs在一些慢性炎性疾病包括肠炎、类风湿性关节炎、实验性自身免疫脑脊髓炎、多发性硬化症、感染和肿瘤等发生、发展和转归过程起重要调节作用.Bregs的发现和作用机制的阐明,将为全面、深入了解免疫耐受的机制,寻找和开发更合理治疗慢性炎性疾病的策略提供理论依据.本文综述了Bregs的发现、生物学特征、发育调节及其参与炎性疾病发病的作用和机制.     

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.     

Acquisition of humoral transplantation tolerance upon de novo emergence of B lymphocytes

A major obstacle to transplantation tolerance is humoral immunity. In this paper, we demonstrate that the intrinsic developmental propensity of the B lymphocyte compartment for acquisition of self-tolerance can be harnessed to induce humoral unresponsiveness to transplanted alloantigens. In the current study, when transitional B cells developed in the presence of donor lymphoid cells, the mature B lymphocyte compartment failed to mount a donor-specific alloantibody response to an organ transplant--despite unrestrained acute T cell-mediated allograft rejection. Specifically, we generated an experimental system wherein a B6 strain B cell compartment developed de novo in the presence of F1 (B6xBALB/c) lymphoid cells and in a T cell-deficient setting. Following establishment of a steady-state B cell compartment, these B6 mice were transplanted with heterotopic cardiac allografts from allogeneic BALB/c donors. The mice were then inoculated with purified syngeneic B6 T cells. As expected, all cardiac allografts were acutely rejected. However, the B lymphocyte compartment of these mice was completely inert in its capacity to form a BALB/c-specific alloantibody response. Using an alloantigen-specific Ig transgenic system, we demonstrated that this profound degree of humoral tolerance was caused by clonal deletion of alloreactive specificities from the primary B cell repertoire. Thus, de novo B cell compartment development at the time of transplantation is of critical importance in recipient repertoire "remodeling" to a humoral tolerant state.     

Notch信号途径在Bregs发育及口服耐受中的作用

目的:调节性B细胞(Bregs)是近年来确认的一类具有负向免疫调节功能的B细胞亚群,在免疫反应中通过IL-10发挥调节功能,又称为B10细胞,其表型为CD19+CD5+CD1dhigh,在多种疾病中发挥重要的调节作用,但关于Bregs的发育及发挥调节功能的具体机制尚不明确。Notch信号途径是调控细胞双向亚群和双向功能分化的重要信号途径,在T/B细胞分化、CD4/CD8细胞等细胞发育中发挥重要的作用,但其在B10细胞发育及其免疫应答中调控作用尚不明确。本研究利用特异性在B细胞剔除Notch信号分子RBP-J基因的小鼠,分析Notch信号对Bregs发育及口服耐受的影响。方法:将CD19-Cre小鼠与RBP-Jflox小鼠交配,获得CD19-Cre/RBP-Jf/f基因型小鼠,流式细胞仪分析该小鼠CD19+CD5+CD1dhighB10细胞及体外刺激后产生IL-10的B10细胞数量;制备口服免疫耐受模型,分析Notch信号缺失对B10细胞发育和功能的影响。结果:B细胞特异性敲除RBP-J基因的小鼠的CD19+CD5+CD1dhighBregs数量及体外刺激后产生IL-10的B10细胞数量比杂合子小鼠显著降低;且在口服免疫耐受模型中血清IgE水平显著升高。结论:Notch信号敲除后,脾脏Bregs及其产生IL-10减少,在口服耐受中不能产生耐受,Notch信号可能促进Bregs发育,并通过IL-10参与调控口服耐受。     

Mice lacking endogenous IL-10-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Th1/Th17 but a decrease in regulatory T cells

IL-10-producing B cells, also known as regulatory B cells (Bregs), play a key role in controlling autoimmunity. In this study, we report that chimeric mice specifically lacking IL-10-producing B cells (IL-10(-/-)B cell) developed an exacerbated arthritis compared with chimeric wild-type (WT) B cell mice. A significant decrease in the absolute numbers of Foxp3 regulatory T cells (Tregs), in their expression level of Foxp3, and a marked increase in inflammatory Th1 and Th17 cells were detected in IL-10(-/-) B cell mice compared with WT B cell mice. Reconstitution of arthritic B cell deficient (μMT) mice with different B cell subsets revealed that the ability to modulate Treg frequencies in vivo is exclusively restricted to transitional 2 marginal zone precursor Bregs. Moreover, transfer of WT transitional 2 marginal zone precursor Bregs to arthritic IL-10(-/-) mice increased Foxp3(+) Tregs and reduced Th1 and Th17 cell frequencies to levels measured in arthritic WT mice and inhibited inflammation. In vitro, IL-10(+/+) B cells established longer contact times with arthritogenic CD4(+)CD25(-) T cells compared with IL-10(-/-) B cells in response to Ag stimulation, and using the same culture conditions, we observed upregulation of Foxp3 on CD4(+) T cells. Thus, IL-10-producing B cells restrain inflammation by promoting differentiation of immunoregulatory over proinflammatory T cells.     

Primary B-cell deficiencies reveal a link between human IL-17-producing CD4 T-cell homeostasis and B-cell differentiation

IL-17 is a pro-inflammatory cytokine implicated in autoimmune and inflammatory conditions. The development/survival of IL-17-producing CD4 T cells (Th17) share critical cues with B-cell differentiation and the circulating follicular T helper subset was recently shown to be enriched in Th17 cells able to help B-cell differentiation. We investigated a putative link between Th17-cell homeostasis and B cells by studying the Th17-cell compartment in primary B-cell immunodeficiencies. Common Variable Immunodeficiency Disorders (CVID), defined by defects in B-cell differentiation into plasma and memory B cells, are frequently associated with autoimmune and inflammatory manifestations but we found no relationship between these and Th17-cell frequency. In fact, CVID patients showed a decrease in Th17-cell frequency in parallel with the expansion of activated non-differentiated B cells (CD21(low)CD38(low)). Moreover, Congenital Agammaglobulinemia patients, lacking B cells due to impaired early B-cell development, had a severe reduction of circulating Th17 cells. Finally, we found a direct correlation in healthy individuals between circulating Th17-cell frequency and both switched-memory B cells and serum BAFF levels, a crucial cytokine for B-cell survival. Overall, our data support a relationship between Th17-cell homeostasis and B-cell maturation, with implications for the understanding of the pathogenesis of inflammatory/autoimmune diseases and the physiology of B-cell depleting therapies.     

Insight into lymphoid development by gene expression profiling of avian B cells

The avian immune system provides an excellent model to track B-cell development from prebursal stem cells throughout B-cell differentiation and maturation. Bursal B cells are uniquely positioned at the crossroads of B-cell development, having properties of both stem cells and of mature B cells, as demonstrated by their ability to reconstruct the bursal B-cell compartment and to express and diversify the B-cell receptor at their cell surface. To understand avian B-cell development better, we determined the gene expression profile of different B-cell stages using a bursal expressed sequence tag array. The expression profile of bursal B cells reveals the presence of factors associated with B-cell signaling and defines novel B-cell-specific genes. Genes associated with proliferation, apoptosis, DNA repair and recombination are abundantly expressed. The expression profile of the DT40 cell line is most similar to bursal B cells rather than to other stages of B-cell development, confirming the suitability of DT40 for studies of B-cell physiology. Interestingly, prebursal stem cells express genes involved in B-cell receptor signaling, although they express only low levels of immunoglobulin genes. This suggests that B-cell receptor-mediated selection is present before bursal colonization. The gene expression signatures of germinal centers and cells of the Harderian gland indicate that evolutionarily conserved genetic programs regulate B-cell activation and terminal differentiation.Electronic Supplementary Material Supplementary material is available in the online version of this article atK. Koskela, P. Kohonen and P. Nieminen contributed equally to this work     

Induction of IL-10-producing regulatory B cells following Toxoplasma gondii infection is important to the cyst formation

Toxoplasma gondii is a protozoan parasite that infects humans and animals via congenital or postnatal routes. During parasite infection, IL-10-producing Bregs are stimulated as part of the parasite-induced host immune responses that favor infection. In this study, we investigated whether T. gondii infection induces immune regulatory cells including IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and whether Breg induction is critical for the development of chronic infection of T. gondii. Furthermore, B cell-deficient (μMT) mice revealed that the IL-10-producing B cells might be associated with the development of chronic T. gondii infection. To better understand the mechanism underlying the accumulation of IL-10-producing B cells upon T. gondii infection, we determined the effect of products released by T. gondii on the induction and differentiation of IL-10-producing B cells during the acute stage of infection using transgenic green fluorescent protein (GFP)-expressing T. gondii strain. We demonstrated that products secreted at the stage of cell lysis by fully replicated tachyzoites induced the differentiation of naive B cells to IL-10-producing Bregs. Our results indicated that the downregulation of the immune response via Bregs during T. gondii infection is related to cyst formation in the host brain and to the establishment of chronic infection.     

Interleukin 35 Regulatory B Cells

B lymphocytes play a central role in host immunity. They orchestrate humoral immune responses that modulate activities of other immune cells and produce neutralizing antibodies that confer lasting immunity to infectious diseases including smallpox, measles and poliomyelitis. In addition to these traditional functions is the recent recognition that B cells also play critical role in maintaining peripheral tolerance and suppressing the development or severity of autoimmune diseases. Their immune suppressive function is attributed to relatively rare populations of regulatory B cells (Bregs) that produce anti-inflammatory cytokines including interleukin 10 (IL-10), IL-35 and transforming growth factor-β. The IL-35-producing B cell (i35-Breg) is the newest Breg subset described. i35-Bregs suppress central nervous system autoimmune diseases by inducing infectious tolerance whereby conventional B cells acquire regulatory functions that suppress pathogenic Th17 responses. In this review, we discuss immunobiology of i35-Breg cell, i35-Breg therapies for autoimmune diseases and potential therapeutic strategies for depleting i35-Bregs that suppress immune responses against pathogens and tumor cells.     

Altered B cell homeostasis is associated with type I diabetes and carriers of the PTPN22 allelic variant

The PTPN22 genetic variant 1858T, encoding Lyp620W, is associated with multiple autoimmune disorders for which the production of autoantibodies is a common feature, suggesting a loss of B cell tolerance. Lyp620W results in blunted BCR signaling in memory B cells. Because BCR signal strength is tightly coupled to central and peripheral tolerance, we examined whether Lyp620W impacts peripheral B cell homeostasis in healthy individuals heterozygous for the PTPN221858T variant. We found that these subjects display alterations in the composition of the B cell pool that include specific expansion of the transitional and anergic IgD(+)IgM(-)CD27(-) B cell subsets. The PTPN22 1858T variant was further associated with significantly diminished BCR signaling and a resistance to apoptosis in both transitional and naive B cells. Strikingly, parallel changes in both BCR signaling and composition of B cell compartment were observed in type 1 diabetic subjects, irrespective of PTPN22 genotype, revealing a novel immune phenotype and likely shared mechanisms leading to a loss of B cell tolerance. Our combined findings suggest that Lyp620W-mediated effects, due in part to the altered BCR signaling threshold, contribute to breakdown of peripheral tolerance and the entry of autoreactive B cells into the naive B cell compartment.     

Discovery and Function of B-Cell IgD Low (BDL) B Cells in Immune Tolerance

It is now appreciated that in addition to their role in humoral immunity, B cells also exert regulatory mechanisms that lead to attenuation of inflammatory responses. The concept of B-cell regulation became well recognized when mice deficient in B cells due to genetic disruption were shown to be refractory to recovery from the signs of experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. This seminal study spurred the search for B-cell regulatory phenotypes and mechanisms of action. Our approach was to utilize differential B-cell depletion with anti-CD20 to retain B cells whose presence were required to achieve EAE recovery. Utilizing flow cytometry, adoptive cell therapy and genetic approaches, we discovered a new B-cell subset that, upon adoptive transfer into B cell-deficient mice, was sufficient to promote EAE recovery. This B-cell subset is IgM⁺, but due to low/negative IgD cell surface expression, it was named B-cell IgD low (BD_L). Mechanistically, we found that in the absence of BD_L, the absolute cell number of CD4⁺ Foxp3⁺ T regulatory cells (Treg), essential for immune tolerance, was significantly reduced. Furthermore, we found that BD_L expression of glucocorticoid-induced tumor necrosis factor ligand (GITRL) was essential for induction of Treg proliferation and maintenance of their homeostasis. Thus, we have identified a new B-cell subset that is critical for immunological tolerance through interactions with Treg.     

Vaccine-induced tumor-specific immunity despite severe B-cell depletion in mantle cell lymphoma

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell-dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody-based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4(+) and CD8(+) antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.     

Myeloid-derived suppressor cells regulate the immunosuppressive functions of PD-1−PD-L1+ Bregs through PD-L1/PI3K/AKT/NF-κB axis in breast cancer

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells that are closely related to tumor immune escape, but the mechanism by which MDSCs regulate B cells has not been elucidated. Our previous studies revealed that breast cancer-derived MDSCs could induce a group of PD-1⁻PD-L1⁺ Bregs with immunosuppressive functions. Here, we reported that blocking PD-1/PD-L1 interaction between MDSCs and B cells could reverse the immunosuppressive functions of PD-1⁻PD-L1⁺ Bregs. The activation of PI3K/AKT/NF-κB signaling pathway is essential for PD-1⁻PD-L1⁺ Bregs to exert immunosuppressive effects. MDSCs activated the PI3K/AKT/NF-κB pathway in B cells via the PD-1/PD-L1 axis. Furthermore, inhibition of PD-1/PD-L1 or PI3K/AKT signaling suppressed both tumor growth and the immunosuppressive functions of PD-1⁻PD-L1⁺ Bregs. Dual suppression of PD-1/PD-L1 and PI3K/AKT exerted better antitumor effect. Finally, MDSCs and PD-1⁻PD-L1⁺ Bregs were colocalized in breast cancer tissues and PD-1⁻PD-L1⁺ Bregs were positively correlated with poor prognosis. Thus, MDSC-educated PD-1⁻PD-L1⁺ Bregs and their regulatory mechanisms could contribute to the immunosuppressive tumor microenvironment. Our study proposes a novel mechanism for MDSC-mediated regulation of B cell immunity, which might shed new light on tumor immunotherapy.⁺Subject terms: Breast cancer, Cancer microenvironment     

Skewed abrogation of tolerance to a neo self-antigen in double-transgenic mice coexpressing the antigen with interleukin-1beta or interferon-gamma

Transgenic (Tg) mice expressing hen egg lysozyme (HEL) under the control of the alphaA-crystallin promoter exhibit tolerance to HEL by both their T- and B-cell compartments. Here, we show that double-Tg mice, coexpressing HEL with either interleukin-1beta or interferon (IFN)-gamma, demonstrated unresponsiveness to HEL by their T-cell compartment, but most of them developed antibodies against HEL following a challenge with the antigen. The abrogation of humoral tolerance was more pronounced in the HEL/IL-1 double-Tg mice than in the HEL/IFN-gamma mice. Unlike their controls, double-Tg mice exhibited remarkable levels of variability in their antibody levels. The skewed abrogation of tolerance in the double-Tg mice is proposed to be due to the cytokines' capacity to rescue from clonal deletion small numbers of T cells, which provide help to antibody producing B cells. This notion is supported by the finding that adoptive transfer of small numbers of Th1 or Th2 cells into HEL-Tg mice made possible antibody production similar to that seen in the double-Tg mice.     

Cognate T cell help is sufficient to trigger anti-nuclear autoantibodies in naive mice

The mechanisms involved in the initiation of anti-nuclear autoantibodies are unknown. In this study, we show that one factor allowing anti-nuclear autoantibodies to develop is the incomplete nature of immune tolerance to many of these proteins. Immune responses in mice toward the ubiquitous nuclear autoantigen La/SS-B are much weaker than responses to the xenoantigen, human La (hLa; 74% identical). However, in transgenic (Tg) mice expressing hLa, the Ab response to this neo-autoantigen was reduced to a level resembling the weak autoimmune response to mouse LA: Partial tolerance to endogenous La autoantigen was restricted to the T compartment because transfer of CD4(+) T cells specific for one or more hLa determinants into mice bearing the hLa transgene was sufficient to elicit production of anti-hLa autoantibodies. Notably, only hLa- specific T cells from non-Tg mice, and not T cells from hLa Tg mice, induced autoantibody production in hLa Tg mice. These findings confirm partial Th tolerance to endogenous La and indicate the existence in normal animals of autoreactive B cells continuously presenting La nuclear AG: Therefore, the B cell compartment is constitutively set to respond to particular nuclear autoantigens, implicating limiting Th responses as a critical checkpoint in the development of anti-nuclear autoantibodies in normal individuals.     

Molecular characterization of the cervical and systemic B-cell repertoire: Unique,yet overlapping,immune compartments of an HIV-1 resistant individual

The cervical mucosa of women who are highly exposed to HIV-1, yet remain persistently seronegative (HEPS), presents a unique opportunity to study the dynamics of an immune compartment potentially capable of preventing HIV-1 infection. Herein, we provide a detailed characterization of the immunoglobulin repertoire of cervical and systemic B cells from one such HEPS individual from Nairobi, Kenya. Analysis was done on 512 V_H sequences that were RT-PCR amplified from B cells in a paired sample from the cervix and peripheral blood. The V_H3 and D_H repertoire of class switched cervical B cells differs significantly from that of systemic B cells, indicating that the cervical environment affects local B-cell populations and hence V_H gene expression. Six networks of clonally related, heavily mutated B cells were identified that spanned the systemic and cervical B-cell compartments. Analysis of somatic mutations suggests this is likely the result of systemic, class switched B cells homing to the cervical mucosa. Multiple networks of somatically mutated V-gene sequences, unique to the cervical mucosa, were also identified. This supports the notion that site specific responses occur and have unique regulation of tolerance and recruitment into local memory or blast B-cell compartments. We conclude that while the nature of the cervical environment shapes the local B cell repertoire, the infusion of post germinal center B cells to the human cervix is a common occurrence, and represents a means by which systemic immunization could provide the local antibodies necessary to prevent HIV-1 at the site of initial contact.Key words: immunogenetics, HIV-1, B cells, cervical mucosa, somatic mutation, HIV resistance     

HSP70, a Novel Regulatory Molecule in B Cell-Mediated Suppression of Autoimmune Diseases

B cells have recently emerged as playing regulatory role in autoimmune diseases. We have previously demonstrated that human peripheral blood CD19⁺ CD24^hiCD27⁺ B cells have regulatory function both in healthy donors and in patients with autoimmune disease. However, the mechanism of this regulation is still not fully understood. In this study, microarrays were utilized to compare gene expression of CD19⁺ CD24^hiCD27⁺ B cells (regulatory B cells, Bregs) with CD19⁺ CD24^loCD27⁻ B cells (non-Bregs) in human peripheral blood. We found that heat shock protein 70 (HSP70) expression was significantly upregulated in Bregs. In vitro studies explored that HSP70 inhibition impaired the regulatory function of peripheral blood Bregs. In mouse models of autoimmune disease, using HSP70-deficient mice or HSP70 inhibitors, Bregs suppressed effector cells and rescued disease-associated phenotypes that were dependent on HSP70. Mechanistically, Bregs secreted HSP70, directly suppressing effector cells, such as T effect cells. These findings reveal that HSP70 is a novel factor that modulates Breg function and suggest that enhancing Breg-mediated production of HSP70 could be a viable therapy for autoimmune disease.     

Glioma cell-derived placental growth factor induces regulatory B cells

Tumor specific immune regulatory cells play an important role in the pathogenesis of glioma. The mechanisms have not been fully understood yet. It is suggested that placenta growth factor (PlGF) is involved in the generation of immune regulatory cells. This study aims to investigate the role of glioma cell-derived PlGF in the generation of regulatory B cells (Breg). Glioma cells were isolated from surgically removed glioma tissue. Cytokines were measured by enzyme-linked immunosorbent assay, quantitative real time RT-PCR and Western blotting. Immune suppressor functions of Bregs were assessed by T cell proliferation assay. The results showed that glioma cells expressed PlGF, which was increased after a non-specific activation. Naïve B cells captured the PlGF to differentiate into transforming growth factor-β positive Bregs. The Bregs were activated upon exposure to protein extracts of glioma tissue to suppress the CD8⁺ T cell proliferation and the release of perforin and granzyme B. We conclude that glioma cell-released PlGF can induce Bregs to suppress CD8⁺ T cell activities.