Marginal zone, but not follicular B cells, are potent activators of naive CD4 T cells

The early involvement of marginal zone (MZ) B lymphocytes in T-independent immune responses is well established. In this study we compared the abilities of MZ and follicular (FO) B cells to collaborate with T cells. After immunization with soluble hen egg lysozyme, both MZ and FO B cells captured Ag and migrated to T cell areas in the response to hen egg lysozyme. MZ B cells were far superior to FO B cells in inducing CD4+ T cell expansion both in vitro and in vivo. MZ, but not FO, B cells, after interaction with T cells, differentiated into plasma cells, and in addition they stimulated Ag-specific CD4+ T cells to produce high levels of Th1-like cytokines upon primary stimulation in vitro. These results indicate that MZ B cells rapidly and effectively capture soluble Ag and activate CD4+ T cells to become effector T cells. The enhanced capacity of MZ B cells to prime T cells in this study appeared to be intrinsic to MZ B cells, as both MZ and FO B cell populations express an identical Ag receptor.     

Uncoupling of anergy from developmental arrest in anti-insulin B cells supports the development of autoimmune diabetes

Loss of tolerance is considered to be an early event that is essential for the development of autoimmune disease. In contrast to this expectation, autoimmune (type 1) diabetes develops in NOD mice that harbor an anti-insulin Ig transgene (125Tg), even though anti-insulin B cells are tolerant. Tolerance is maintained in a similar manner in both normal C57BL/6 and autoimmune NOD mice, as evidenced by B cell anergy to stimulation through their Ag receptor (anti-IgM), TLR4 (LPS), and CD40 (anti-CD40). Unlike B cells in other models of tolerance, anergic 125Tg B cells are not arrested in development, and they enter mature subsets of follicular and marginal zone B cells. In addition, 125Tg B cells remain competent to increase CD86 expression in response to both T cell-dependent (anti-CD40) and T cell-independent (anti-IgM or LPS) signals. Thus, for anti-insulin B cells, tolerance is characterized by defective B cell proliferation uncoupled from signals that promote maturation and costimulator function. In diabetes-prone NOD mice, anti-insulin B cells in this novel state of tolerance provide the essential B cell contribution required for autoimmune beta cell destruction. These findings suggest that the degree of functional impairment, rather than an overt breach of tolerance, is a critical feature that governs B cell contribution to T cell-mediated autoimmune disease.     

Murine B1 B cells require IL-5 for optimal T cell-dependent activation

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L(-/-) mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.     

Recruitment of a distinct but related set of VH sequences into the murine CD21hi/CD23- marginal zone B cell repertoire to that seen in the class-switched antibody response

Development and maintenance of cells in the murine follicular and marginal zone compartments is thought to involve differing levels of stimulation of the BCR, although it is still not clear which BCR ligands mediate these events. How the delineation between naive and Ag experienced B cell populations relates to cell phenotype and how precise or blurred this delineation is, is also not well understood. In this study, using PCR to analyze the Ab response to phenyl-oxazolone in the mouse, we show that the Ab repertoire of CD21(hi)/CD23(-) marginal zone B cells shows persistent increase in levels of particular IgM after immunization with foreign Ag. Further, we show that these IgMs have different but related VH/CDR3 sequences from those seen in the class-switched response to oxazolone that we have also analyzed. We also detect an effect of Ag on the follicular B cell repertoire that is less persisting. These results provide evidence consistent with the signal-strength model of mature B cell development being extended to include stimulation by foreign Ag, and also further the known zone of influence of foreign Ag on the B cell compartment.     

CD36 is differentially expressed on B cell subsets during development and in responses to antigen

Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.     

CD9 is a unique marker for marginal zone B cells,B1 cells,and plasma cells in mice

Marginal zone (MZ), follicular (FO), and B1 B cells form the long-lived naive B cell compartment. To identify surface markers that define MZ B cells in mice, we generated a panel of mAbs reactive with MZ but not FO B cells. One of these mAbs, MZ3, was found to recognize the tetraspanin CD9. CD9 expression not only distinguishes MZ B cells from FO B cells but also divided peritoneal cavity B1 cells into smaller subsets. After short-term in vitro stimulation with various mitogens, FO B cells failed to induce CD9 protein, while MZ B cells up-regulated the level of CD9 protein. However, after prolonged culture of FO B cells with LPS, surface CD9 was induced, together with syndecan 1, indicative of plasma cell differentiation. Following immunization with a T-independent-2 Ag, R36A, or a T-dependent Ag, SRBC, we found that CD9 is not expressed by germinal center B cells but is eventually expressed on plasma cells in response to both T-independent-2 and T-dependent Ags. Collectively, these results suggest that MZ B cells and B1 cell subsets are the immediate precursors of plasma cells in the primary response and that CD9 is acquired by T-dependent plasma cells.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Danger and OX40 receptor signaling synergize to enhance memory T cell survival by inhibiting peripheral deletion

This report defines a cell surface receptor (OX40) expressed on effector CD4 T cells, which when engaged in conjunction with a danger signal, rescues Ag-stimulated effector cells from activation-induced cell death in vivo. Specifically, three signals were necessary to promote optimal generation of long-lived CD4 T cell memory in vivo: Ag, a danger signal (LPS), and OX40 engagement. Mice treated with Ag or superantigen (SAg) alone produced very few SAg-specific T cells. OX40 ligation or LPS stimulation, enhanced SAg-driven clonal expansion and the survival of responding T cells. However, when SAg was administered with a danger signal at the time of OX40 ligation, a synergistic effect was observed which led to a 60-fold increase in the number of long-lived, Ag-specific CD4 memory T cells. These data lay the foundation for the provision of increased numbers of memory T cells which should enhance the efficacy of vaccine strategies for infectious diseases, or cancer, while also providing a potential target (OX40) to limit the number of auto-Ag-specific memory T cells in autoimmune disease.     

Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.     

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.     

Annexin V binds to positively selected B cells

Recombinant annexin V (rAnV) has been used in flow cytometry to identify cells undergoing apoptosis, based on its ability to bind to phosphatidylserine, a negatively charged lipid normally restricted to the cytoplasmic face of the plasma membrane but externalized early during apoptosis. When we stained murine bone marrow (BM) cells with fluorescently labeled rAnV, we found that a surprisingly large fraction of BM B cells bearing selectable transgenic Ag receptors bind significant amounts of rAnV, but that these cells are not apoptotic. Here, we show that binding of rAnV to developing B cells in normal mice correlates with B cell receptor-dependent selection events at several stages of development within both B-1 and B-2 cell subsets. In fact, nearly all B-1 B cells and splenic marginal zone B cells bind rAnV, suggesting that the externalization of phosphatidylserine occurs once mature B cells are selected through BCR-mediated signaling. However, this plasma membrane alteration is apparently not shared by all lymphocytes, because we did not find a parallel population of rAnV-binding viable T cells in vivo in normal or TCR transgenic mice. We also show that BM stromal cell lines can influence the extent of rAnV binding by viable BM B cells during coculture in vitro. We suggest that rAnV detects a potentially important membrane alteration that occurs as B cells develop in the BM and are readied for export to the peripheral lymphoid organs and again among mature B cells recruited to the marginal zone or the B-1 compartment.     

Heterogeneity and function of human B lymphocytes

B lymphocytes represent an important arm of the immune system. Besides their main function of providing antibodies protecting against pathogens, they also exert some regulatory functions, in particular for secondary lymphoid tissue differentiation. Human B cells can be divided in various subsets representing different maturation stages and different pathways of humoral immune responses. Na?ve IgMlow IgDhigh CD27- B cells can participate in T-cell dependent immune responses leading to germinal center formation in follicles of secondary lymphoid organs. Interactions with follicular helper T cells, a recently identified CD185+ T cell population providing help to follicular B cell, involve costimulatory molecules including CD40, CD27, CD278 and SAP-recruiting receptors. B cell interaction with follicular helper T cells represents a critical step controlling the generation of plama cells that ultimately produce high affinity, somatically mutated, class-switched antibodies or of their memory B cell counterpart (identified as CD27+ Ig switched or IgMonly B cells). IgMhigh IgDlow CD27+ B cells are a puzzling population apparently specialized in T-independent responses to bacterial capsular polysaccharides. The extra-follicular, probably antigen-independent, differentiation pathway of these cells, allowing pre-immune repertoire diversification by somatic hypermutation, is not yet characterized. However, circulating IgMhigh IgDlow CD27+ B cells are similar to splenic marginal zone B cells. In addition to these subsets, minor populations can also be identified in peripheral blood, such as transitional B cells and plasma blasts. All together, deciphering human B cell heterogeneity provides tools for investigations of humoral immunodeficiencies and auto-immune diseases, that will in return shed more light on B cell biology.     

B7-h1 expressed by activated CD8 T cells is essential for their survival

An immunoinhibitory role of B7 homologue 1 (B7-H1) expressed by non-T cells has been established; however, the function of B7-H1 expressed by T cells is not clear. Peak expression of B7-H1 on Ag-primed CD8 T cells was observed during the contraction phase of an immune response. Unexpectedly, B7-H1 blockade at this stage reduced the numbers of effector CD8 T cells, suggesting B7-H1 blocking Ab may disturb an unknown function of B7-H1 expressed by CD8 T cells. To exclusively examine the role of B7-H1 expressed by T cells, we introduced B7-H1 deficiency into TCR transgenic (OT-1) mice. Naive B7-H1-deficient CD8 T cells proliferated normally following Ag stimulation; however, once activated, they underwent more robust contraction in vivo and more apoptosis in vitro. In addition, B7-H1-deficient CD8 T cells were more sensitive to Ca-dependent and Fas ligand-dependent killing by cytotoxic T lymphocytes. Activation-induced Bcl-x(L) expression was lower in activated B7-H1-deficient CD8 T cells, whereas Bcl-2 and Bim expression were comparable to the wild type. Transfer of effector B7-H1-deficient CD8 T cells failed to suppress tumor growth in vivo. Thus, upregulation of B7-H1 on primed T cells helps effector T cells survive the contraction phase and consequently generate optimal protective immunity.     

GIF inhibits Th effector generation by acting on antigen-presenting B cells

Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50--95% reduction of IL-4 and IFN-gamma secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA(323--339) peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.     

Loss of IL-7 receptor alpha on CD4+ T cells defines terminally differentiated B cell-helping effector T cells in a B cell-rich lymphoid tissue

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Ralpha (CD127) and the common gamma-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage. In this study, we report that loss of CD127 defines terminally differentiated B cell-helping effector T cells in human tonsils. Although naive CD4(+) T cells uniformly express CD127, the memory/effector (non-FOXP3(+)) CD4(+) T cells are divided into CD127(+) and CD127(-) cells. The CD127(-) T cells are exclusively localized within the germinal centers where B cells become plasma and memory B cells, whereas CD127(+) T cells are found in T cell areas and the area surrounding B cell follicles. Consistently, the CD127(-) T cells highly express the B cell zone homing receptor CXCR5 with concomitant loss of CCR7. Compared with CD127(+) memory T cells, CD127(-) T cells have considerably shorter telomeres, do not proliferate in response to IL-7, and are prone to cell death. The CD127(-) T cells produce a large amount of the B cell follicle-forming chemokine CXCL13 upon stimulation with B cells and Ags. Most importantly, they are highly efficient in helping B cells produce Igs of all isotypes in a manner dependent on CD40L and ICOS and inducing activation-induced cytidine deaminase and Ig class switch recombination. The selective loss of CD127 on the B cell-helping effector T cells would have implications in regulation and termination of Ig responses.     

A division-linked mechanism for the rapid generation of Ig-secreting cells from human memory B cells

Memory B cells, when re-exposed to Ag and T cell help, differentiate into Ig-secreting cells (ISC) at the same time as maintaining a residual pool of non-Ig-secreting cells with memory capabilities. To investigate the mechanism underlying this dual process, we followed the fate of human B cells activated in vitro with the T cell-derived signals CD40 ligand (CD40L), IL-2, and IL-10 using CFSE to monitor cell division. A substantial number of ISCs detected by ELISPOT, intracellular Ig staining, and Ig secretion could be generated from memory but not naive B cells. The proportion of ISCs increased with successive cell divisions and was markedly enhanced by IL-10 at each division. Within ISCs, two distinct populations were detected after withdrawal of CD40L. The first had acquired the plasma cell marker CD38 and continued to proliferate despite the absence of CD40L. In contrast, the second population remained CD38(-), ceased dividing, and underwent rapid apoptosis. The former most likely represent the immediate precursors of long-lived plasma cells, which preferentially home to the bone marrow in vivo, whereas the latter contain short-lived ISCs responsible for the initial Ab response to stimulation with Ag and T cell help. Taken together, the results point to a division-based mechanism responsible not only for regulating differentiation of short- and long-lived ISCs from memory B cells, but for preserving the memory B cell pool for reactivation upon subsequent Ag exposure.     

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.     

TGF-beta 1 attenuates the acquisition and expression of effector function by tumor antigen-specific human memory CD8 T cells

TGF-beta1 is a potent immunoregulatory cytokine. However, its impact on the generation and effector function of Ag-specific human effector memory CD8 T cells had not been evaluated. Using Ag-specific CD8 T cells derived from melanoma patients immunized with the gp100 melanoma Ag, we demonstrate that the addition of TGF-beta1 to the initial Ag activation cultures attenuated the gain of effector function by Ag-specific memory CD8 T cells while the phenotypic changes associated with activation and differentiation into effector memory were comparable to control cultures. These activated memory CD8 T cells consistently expressed lower mRNA levels for T-bet, suggesting a mechanism for TGF-beta1-mediated suppression of gain of effector function in memory T cells. Moreover, TGF-beta1 induced a modest expression of CCR7 on Ag-activated memory CD8 T cells. TGF-beta1 also suppressed cytokine secretion by Ag-specific effector memory CD8 T cells, as well as melanoma-reactive tumor-infiltrating lymphocytes and CD8 T cell clones. These results demonstrate that TGF-beta1 suppresses not only the acquisition but also expression of effector function on human memory CD8 T cells and tumor-infiltrating lymphocytes reactive against melanoma, suggesting that TGF-beta1-mediated suppression can hinder the therapeutic benefits of vaccination, as well as immunotherapy in cancer patients.