Identification And Functional Characteristics Of Novel Subsets Of B Lymphocytes

More and more new subsets of immune cells have been identified and their functions have been extensively investigated in recent years. For example, T cell subsets, such as regulatory T cells (Treg), Th17, Th9, Tfh, have been successfully identified and shown to be important in the initiation and regulation of immune response, thus contributing to the better understanding of the cellular and molecular mechanisms for the regulation of immune responses. In addition, new subsets of macrophages and dendritic cells (DC) have also been identified. However, subpopulations of B cells, the most important immune cells in humoral immunity, were poorly studied. As we know, B lymphocytes protect host from pathogens by producing antibodies, as well as opsonization, and complement fixation, thus playing critical role in humoral immune responses against pathogens. Moreover, B cells are also implicated in cellular immunity. For example, they can promote naive CD4+T cell differentiation into T helper 1(Th1) or Th2 subsets. B cells can also serve as antigen-presenting cells, providing co-stimulatory signals for T cell activation. Additionally, they are capable of producing cytokines and participate in the regulation of immune responses. Therefore, identification of new B cell subsets and their functional characteristics will contribute to the comprehensive understanding of B cells in the immune network and present new concepts of underlying mechanisms of immune response and immune regulation.B lymphocytes are divided into B-1 and B-2 B cells according to the different membrane molecules, distribution and function. B-1 B cells, which are characterized by their unique phenotype of sIgMhisIgDloCDllb+, constitute most of the B cells existing in the peritoneal and pleural cavities in mice. CD5+ B-1a B cells recognize high-molecular-weight polymeric antigens and produce'natural'antibodies which are critical in the early responses to encapsulated extracellular bacteria, whereas CD5- B-1b B cells produce antibodies required for long-lasting protective immunity to pathogens such as Streptococcus pneumoniae. Conventional B-2 cells function as antibody-producing cells or antigen-presenting cells, promote T helper cell differentiation and provide co-stimulatory signals for T cell activation and thus are implicated in the humoral and cellular immunity. Recently, a subset of CDldhiCD5+CD19hi B10 cells have been identified and proved to negatively regulate T cell-mediated inflammatory responses through IL-10 secretion. Massive evidences from clinical observations and basic researches prove that B cells are of great heterogeneity, indicating.they may differentiate into different subsets with various activities under different physiological and pathological conditions. So, identification and functional analysis of new subsets of B cells are one of challenging and hot topics in the froniters of immunology nowadays.Innate immune response is the first line of immune defense against pathogens. Well-established components of the innate immune system include natural killer (NK) cells, macrophages, eosinophils, neutrophils and mast cells. A recent study in teleost fish demonstrated that B cells have potent phagocytic activities. Activation of B cells by a wide range of stimuli independently of the B cell receptor (BCR) also suggests that B cells may play a role in innate immunity. Components of bacteria, fungi and virus selectively activate Toll-like receptors (TLR), leading to the shared and unique responses in innate immune cells. So, TLRs (e.g., TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9), which are widely expressed on B cells, may facilitate B cells to sense and respond to microbial antigens, thus proposing a possibility that B cells may participate in innate immune responses aganist pathogenic infections.Considering the heterogeneity and multiple functions of B cells in innate and adaptive immune responses, we, in the following three parts of our work, analyzed the composition of B cells in the mie after microbial infection and different inflammatory stimulation, investigated the functions and the underlying mechanisms of new B cell subsets we identified, as well as studied the transcriptional regulation of B cell function. The primary aim of this study is to identify new subsets of B cells and investigate the role of the B cell subsets in the innate immunity and regulation of adaptive immune response.1. Identification and functional characteristics of CD19+DX5+ B cellsTo study the role of B cells in the anti-microbial immune responses, we analyzed the B cell subpopulations of different lymph organs from the mice infected with L. monocytogene-, E. coli-or VSV, as well as challenged with LPS-, CpG-ODN-or poly I:C. We found one new subset of B cells with unique phenotype (CD19+DX5+) in the bone marrow, spleen and mesenteric lymph nodes. Then we analyzed the number and proportion of these CD19+ DX5+ cells generated in the infected mice in the following 21 days and found that CD19+ DX5+ cells in the spleen started to increase on the third day post-infection, and reached the peak on the seventh day, then drop gradually and back to the initial number during the 17-21 days post-infection. Besides, number of CD19+DX5+ cells in the stimulus-injected mice increased to the peak five days post-infection and decreased gradually. The data indicated that pathogens and their components could induce the in vivo generation of CD19+DX5+ cells, which may be involved in the regulation of innate and adaptive immune responses against microbial infection.To further confirm the characteristics of CD19+DX5+ cells, we observed their morphology, and analyzed their phenotype, cytokine profiles. We found that CD19+DX5+ cells had smooth compact nuclei and rare cytoplasm, expressed markers of both NK cells and B cells, such as mIgD, mIgM, B220, IA-e, CD40, CD 154, CD86, Ly49A, Ly49C/I, Ly49D, NKG2D, NKG2A/C/E, CD43, CD25, CD122, CD11b. Thus we named these CD19+DX5+ cells as NKB cells. By intracellular staining and ELISA, we confirmed that NKB cells expressed IFN-γ, IL-1α, IL-1β, IL-6 and Granzyme-B, but not IL-4 or IL-10. In addition, LPS-stimulated NKB cells secreted IgG2a, IgG2b, IgG3, IgM. Importantly, CD19+DX5+ cells could not be inducible generated in Btk-/- mice but could be generated in NK cell-depleted mice, indicating these CD19+DX5+ cells are derived from B cells but not from NK cells. Therefore, we identified one new subset of B cells with NK-like phenotype and secretion of IFN-y and other Thl cytokines.But how are NKB cells inducibly generated? What are the progenitors of NKB cells? By using co-culture system of CD19+DX5- cells with different kinds of lymphocytes and immune cells, we found that dendritic cells (DC) could induce the differentiation of NKB cells in vitro in the presence of the stimulation with Heat-killed L. monocytogenes (HKLM). To understand the mechanism of NKB cell generation, we used 0.4μM transwell system or added neutralizing antibodies against IL-6, IL-1β, IL-12, CD40, CD40L in the coculture system, and found that DC induced NKB differentiation through CD40-CD40L pathway, while IL-6 and IL-1βproduced by DC could promote the generation of NKB cells. We also confirmed this conclusion in the CD11c-DTR, Cd40-/-, Cd40l-/-, Il1r-/- and Il6-/- mice with L.monocytogenes, in which NKB cells could not be efficiently generated. Then we investigated the functions of NKB cells in innate defense against listeria, and found that IFN-γproduced by NKB cells could activate macrophages and subsequently promote bacterial clearance. Adoptively transfer of NKB cells from wild type mice but not from Ifnγ-/- mice significantly reduced the bacterial burden in the liver and spleen of the infected mice, with increased serum IFN-y level. So, upon bacterial infection of the mice, NKB cells produced IFN-y to activate macrophages and contributed to the clearance of listeria, suggesting that NKB cells, just like the NK cells, can participate in the innate responses during the early period of listeria infection.Considering that the number of NKB cells could be induced to reach the peak on the seventh day post-infection, we adoptively transferred CD45.1+ OT-I CD8+T cells into C57BL/6 mice before LM-OVA infection, then purified OT-I T cells and NKB cells from the mice infected with listeria for seven days (LM-7d) and investigated the role of NKB cells in adaptive immune responses. Data showed that NKB cells could induce apoptosis of antigen-specific OT-I CD8+T cells derived from listeiria-infected mice on day 7. Then we went further to study the underlying mechanisms and found that IFN-y produced by NKB cells could upregulate FasL expression on NKB cells and Fas. expression on CD8+T cells in the NKB-CD8+T co-culture system in vitro. Once signals of IFN-y or Fas-FasL were blocked, the apoptosis of antigen-specific OT-I CD8+T cells induced by NKB cells significantly reduced. The data demonstrate that NKB cells can induce the apoptosis of antigen-specific CD8 T cells through IFN-y and FasL-Fas pathway at the late stage of immune response, thus controlling the immune response in an approariate state. So, in other side, NKB cells may exert their immunoregulatory function in the regulation of adaptive immune response, just like regulatory B cells.Next, the in vivo experiments were designed as follows. We adoptively transferred CD45.1+OT-I CD8+T and LM-7d NKB cells, NK cell or B cells into Btk-/- mice before or after LM-OVA infection. Compared to the control groups with NK cell or B cell transfer, the mice transferred with NKB cells showed decreased numbers of OT-I CD8+T cells seven days post-infection but the expression of CD44, CD62L and CD69 on CD8+T cells remained unchanged. However, the expansion of OT-I CD8+T cells after NKB cell transfer was comparable to that in the control groups, indicating that NKB cells did not affect the generation of antigen-specific CD8+T cells. In addition, adoptive transfer of Ifnγ-/- or Fasl-/- NKB cells had no effect on the number and activation of OT-I CD8+T cells. In brief, NKB cells, generated from the mice infected with listeria on day 7, can induce the apoptosis of antigen-specific CD8+ T cells through IFN-y and Fas-FasL pathway, thus negatively regulating the antigen-specific T cell adaptive immunity appropriately and stabilizing the immune responses once the pathogen infection is cleared.2. Identification and functional characteristics of CD19+PDCA-1+B cells involved in the innate immune responsesBtk-/- mice were found to be more susceptible to L. monocyto genes infection, showing heavier bacterial burden and lower serum level compared to wild type mice. The data indicate that B cells are required for the immune response against L. monocytogenes infection. To investigate the role of B cells in innate immune responses, we analyzed the populations of splenic B cells from L. monocytogene- or E. coli-infected mice, and found one new subset of B cells with unique phenotype (CD19+PDCA-1+), which were inducibly generated in vivo, and existed in the bone marrow, spleen and mesenteric lymph nodes. Number of CD19+PDCA-1+cells in the spleen increased quickly and reached the peak 48 hours post-infection, then droped gradually and back to the initial number 8 days post-infection. The data indicated that CD19+PDCA-1+cells may be involved in the innate immune responses against microbial infection.To further confirm the characteristics of CD19+PDCA-1+cells, we observed their morphology, analyzed their phenotype and cytokine profiles. We found that CD19+PDCA-1+cells of diameter 6-8μM were morphologically similar to B cells with smooth compact nuclei and rare cytoplasm. The cells expressed membrane markers of B cells, such as mIgM,mIgD,B220,CD23. Intracellular staining showed that CD19+PDCA-1+ cells were positive for IL-6, IL12p40, TNF-a and IL-la intracellular expressions. ELISA results demonstrated higher concentration of IFN-a and IL-6 was found in the supernatants of PDCA-1+ B cells stimulated with HKLM. So, CD19+PDCA-1+ B cells were proved to be a new unique B cell subset with preferentially production of typeⅠIFN.We went further to investigate how these CD19+PDCA-1+ B cells were inducibly generated? HKLM stimulation alone failed to convert PDCA-1- B cells from wild-type mice into PDCA-1+ cells. HKLM stimulation generated PDCA-1+ B cells in co-culture of PDCA-1- B cells with macrophages, but not with NK cells, CD4+or CD8+T cells. Physical separation of macrophages from PDCA-1- B cells using a transwell system dramatically reduced the number of PDCA-1+ B cells converted. So, macrophages are required for the generation of PDCA-1+ B cells. What are the molecular mechanisms by which macrophages induce the generation of PDCA-1+ B cells? The generation of PDCA-1+ B cells induced by HKLM in macrophage/PDCA-1-B-cell co-culture was significantly reduced by neutralizing anti-CD40 or anti-CD40L antibody. PDCA-1- B cells from Cd40l-/- mice were unable to switch to the PDCA-1+ phenotype upon HKLM stimulation when co-cultured with macrophages. Also, macrophages from Cd40-/- mice did not induce the generation of PDCA-1+ B cells when cultured with wild type PDCA-1- B cells. Inclusion of neutralization IFN-y antibody in the macrophage-PDCA-1--B cell co-culture significantly reduced the ratio of PDCA-1+ B cells. L. monocytogenes infection of the Ifny-/- mice failed to induce generation of PDCA-1+ B cells. In vivo experiments in Cd40-/-or Cd40l-/- mice failed to demonstrate induction of PDCA-1+ B cell generation after listeria infection. Co-culture with macrophages isolated from Ifnγ-/- mice did not switch PDCA-1-B cells to the PDCA-1+ phenotype. Therefore, macrophages contribute to the generation of PDCA-1+ B cells though CD40-CD40L ligation and IFN-y signaling.We found that PDCA-1+ B cells facilitated NK cells to produce IFN-y though IFN-a. Deletion of NK cells in L. monocytogenes-infected Btk-/- mice attenuated the protective effects of adoptive transfer of PDCA-1+ B cells as well as the IFN-y response. Adoptive transfer of NK cells from wild-type mice, but not Ifnγ-/- NK cells from Ifnγ-/- mice into NK cell-deleted Btk-/- mice restored the protective action of PDCA-1+ B cells adoptively transferred and the IFN-y production. So, the data suggested that PDCA-1+ B cells facilitate NK cells to produce IFN-y though IFN-a, and IFN-y production by NK cells plays important role in innate protective function of PDCA-1+ B cells against L. monocytogenes infection in vivo. The existence of PDCA-1+ B cells explains more effective anti-listeria responses in WT mice compared to B cell deficient mice. Meanwhile, NK depleted mice also showed heavy bacterial burden and little IFN-y level in sera, which were not recovered after PDCA-1+ B cell/and afterward IFN-γ-/- NK transfusion, indicating that PDCA-1+ B cells could not perform their function without IFN-y-producing NK cell involvement. Therefore, these IFN-a-producing B cells with unique phenotype of PDCA-1+ have been confirmed for the first time as a novel B cell subset inducing an effective responses during the innate defense against L. monocytogenes.In sum, the results from the current study demonstrated that a novel subset of PDCA-1+IFN-a-producing B cells could trigger IFN-γresponse by NK cells, and by doing so, participate in the early innate immune responses to intracellular bacterial infection. Our results also stand as a proof of the positive feedback of IFN-αand IFN-γsignaling which provide a new mechanistic explanation for the rapid start-up of innate responses to pathogen invasion. Moreover, the identification and functional characterization of the IFN-a-producing PDCA-1+ B cell subset in this study may lead to further research of this B subset in humans that may similarly promote innate defense after bacterial infection. In brief, further defining the role and mechanism of PDCA-1+CD19+ cells in different infective diseases in vivo may provide new insights in innate function of B cells and find therapeutic approaches for treating infections in addition to previous ones.3. Functional regulation of B cells by transcriptional factor FoxplIt is well known that transcriptional factors paly critical roles in determining the differentiation and regulating function of immune cells. The subfamily members of Foxp (Foxp1, Foxp2, Foxp3) have attracted much attention in recent years in the field of immunology and cell biology. Altough discovered abd cloned from mouse B lymphoma cell line BCL110 years ago, few studies have been reported for the roel of Foxpl in the differentiation and functiona regulation of immune cells, especially B cells. To study the molecular mechanism of differentiation and function of B cells, we investigated regulation of B cell function by Foxpl. First, we confirmed Foxpl was widely expressed in the tissues, including heart, brain, lung, thymus, spleen, axillary and mesenteric lymph nodes. Then we found that Foxpl was expressed in B cells, CD4+T cells, CD8+T cells and macrophages. Furthermore, we confirmed that Foxpl expression in the B cells of Foxpl mutant mice decreased signicantly as compared to that of wild-type mice, identifying the mutant mice with Foxpl gene knockdown. B cells from Foxpl knockdown mice produced less IL-10 in vitro than B cells from wild type mice after LPS stimulation. Also, B cells from Foxpl knockdown mice produced less IL-10 than B cells from wild type mice after listeria infection. After LPS stimulation, the mRNA expression of inflammatory factors, such as IL-6, IFN-a, IFN-y, was upregulated. But IFN-γexpression in B cells from Foxpl knockdown mice were downregulated, while expression of IL-6 and IFN-a had no significant difference between mutant and wild type mice after liseria infection. Therefore, we suggest that Foxpl may facilitate the regulatory function of B cells, but how it regulate the inflammatory cytokine production and what role it plays in the innate function of B cells remain unclear, which need to be investigated in the future.4. SummaryIn PartⅠof our study, we have identified a new B cell subset (DX5+CD19+) with NK-like phenotype and cytokine profiles and named them as NKB cells. DX5+CD19+NKB cells have been found to produce IFN-γto activate macrophages and promote the clearance of intracellular bacteria in early period of listeria infection, indicating NKB cells could participate in the innate immune responses. Interestingly, we found large amounts of NKB cells generated in the late period of immune response (7 days after listeria infection), and NKB cells could upregulate FasL expression on themselves and Fas expression on antigen-specific CD8+ T cells by IFN-γand consequently induce apoptosis of antigen-specific CD8+ T cells. The dat suggest that NKB cells may negatively regulate the antigen-specific T cell adaptive immunity in the late period of immune response against pathogen and stabilizing the immune responses appropriately. Therefore, identification and functional chracterististics of DX5+CD19+ NKB cells provide new insights in innate responses, contribute to the better understanding of regulatory function of B cells, and outlines a new manner of negative regulation of adaptive T cell immunity by B cells.In PartⅡof our study, we identified a new subset of PDCA-1+CD19+ B cells inducibly generated in the mice infected with bacteria L. monocytogenes and E. coli. PDCA-1+CD19+ B cells secreted a large amount of IFN-αand thus facilitated IFN-γproduction and cytotoxicity function of NK cells via IFN-α. B cell-deficient Btk-/- mice were incapable of producing PDCA-1+CD19+ B cells, and more sensitive to L. monocytogenes infection. Adoptive transfer of PDCA-1+CD19+B cells to Btk-/- mice normalized their resistance to L. monocytogenes infection. Furthermore, we found that macrophages were essential for the inducible generation of PDCA-1+CD19+ B cells via CD40-CD40L ligation and IFN-γ. Therefore, we identify a new subset of PDCA-1+CD19+ B cells during the bacterial infection, which can enhance innate immune responses against bacterial infection by activating NK cells via secretion of IFN-α.In PartⅢof our study, we studied the transcriptional regulation of differentiation and function of B cells, and found that less IL-10 was produced by B cells from Foxp1 knockdown mice after LPS stimulation and listeria infection. The premilinary data suggest that Foxpl may facilitate the regulatory function of B cells, but how it regulates the inflammatory cytokine production and the innate function of B cells remain unclear to date.In brief, defining the roles and the underlying mechanisms of B cell subsets in the innate and adaptive immune response against pathogens may provide new insights in the initiation, maintenance and modulation of immune response, broaden our understanding of immune regulation network, and may be helpful to the design of new approaches to the immunotherapy of infectious diseases.