Divergent effects of BAFF on human memory B cell differentiation into Ig-secreting cells

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation, yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study, we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface, whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation, because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore, the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead, our data suggest that accessibility to soluble BAFF is limited within GCs, perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept, whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation, we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation, a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events, which protect the balance between MB cells and ISCs outside GCs. Taken together, these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation.     

TLR3- and MyD88-Dependent Signaling Differentially Influences the Development of West Nile Virus-Specific B Cell Responses in Mice following Immunization with RepliVAX WN,a Single-Cycle Flavivirus Vaccine Candidate

Recognition of conserved pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) results in the activation of innate signaling pathways that drive the innate immune response and ultimately shape the adaptive immune response. RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine candidate derived from West Nile virus (WNV), is intrinsically adjuvanted with multiple PAMPs and induces a vigorous anti-WNV humoral response. However, the innate mechanisms that link pattern recognition and development of vigorous antigen-specific B cell responses are not completely understood. Moreover, the roles of individual PRR signaling pathways in shaping the B cell response to this live attenuated SCFV vaccine have not been established. We examined and compared the role of TLR3- and MyD88-dependent signaling in the development of anti-WNV-specific antibody-secreting cell responses and memory B cell responses induced by RepliVAX WN. We found that MyD88 deficiency significantly diminished B cell responses by impairing B cell activation, development of germinal centers (GC), and the generation of long-lived plasma cells (LLPCs) and memory B cells (MBCs). In contrast, TLR3 deficiency had more effect on maintenance of GCs and development of LLPCs, whereas differentiation of MBCs was unaffected. Our data suggest that both TLR3- and MyD88-dependent signaling are involved in the intrinsic adjuvanting of RepliVAX WN and differentially contribute to the development of vigorous WNV-specific antibody and B cell memory responses following immunization with this novel SCFV vaccine.     

The agonists of TLR4 and 9 are sufficient to activate memory B cells to differentiate into plasma cells in vitro but not in vivo

Memory B cells can persist for a lifetime and be reactivated to yield high affinity, isotype switched plasma cells. The generation of memory B cells by Ag immunization requires adjuvants that generally contain TLR agonists. However, requirements for memory B cell activation and the role of TLRs in this activation are not well understood. In this study, we analyzed the response of memory B cells from immunized mice to TLR9 and 4 agonists CpG oligodeoxynucleotides (ODN) and LPS. Mouse memory B cells express both TLR9 and 4, and respond to both CpG ODN and LPS in vitro by differentiating into high affinity IgG secreting plasma cells. In contrast, neither CpG ODN nor LPS alone is sufficient to activate memory B cells in vivo. Ag is required for the clonal expansion of Ag-specific memory B cells, the differentiation of memory B cells to high affinity IgG secreting plasma cells, and the recall of high affinity Ab responses. The Ag-specific B cells that have not yet undergone isotype switching showed a relatively higher expression of TLR4 than memory B cells, which was reflected in a heightened response to LPS, but in both cases yielded mostly low affinity IgM secreting plasma cells. Thus, although memory B cells are sensitive to TLR agonists in vitro, TLR agonists alone appear to have little affect on B cell memory in vivo.     

CD40 stimulation of human peripheral B lymphocytes: distinct response from naive and memory cells

During secondary immune response, memory B lymphocytes proliferate and differentiate into Ig-secreting cells. In mice, the binding of CD40 by CD154 clearly enhances the activation and differentiation of memory B lymphocytes. In humans, the role of CD40-CD154 in the stimulation of memory B lymphocytes is not as obvious since in vitro studies reported positive and negative effects on their proliferation and differentiation in Ig-secreting cells. In this study, we examine the response of peripheral memory and naive cells in relation to the duration of CD40-CD154 interaction. We measured the proliferation and differentiation of both subsets stimulated with CD154 and IL-4 for short- (4-5 days) and long-term (>7 days) periods. Following short-term stimulation, memory B lymphocytes did not expand but represented the only subset differentiating into IgG- and IgM-secreting cells. A longer stimulation of this population led to cell death, while promoting naive B lymphocyte proliferation, expansion, and differentiation into IgM- or IgG-secreting cells. This prolonged CD40 stimulation also triggered naive B lymphocytes to switch to IgG and to express CD27 even in absence of somatic hypermutation, suggesting that these latter events could be independent. This study suggests that naive and memory B lymphocytes have distinct requirements to engage an immune response, reflecting their different roles in humoral immunity.     

Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets--neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells--into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7- T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.     

Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.     

Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.     

Plexin-D1 is a novel regulator of germinal centers and humoral immune responses

Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Igλ(+) and Igκ(+) B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1(-/-) mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1(-/-) B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1(-/-) mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory.     

Adaptive features of natural killer cells,lymphocytes of innate immunity

Natural killer cells (NK cells) are traditionally attributed to the innate immune system. It is considered that previously received stimuli have little effect on the functioning of these immune cells. Indeed, NK cells even without prior sensitization provide a rapid effector response against tumor, virus-infected, or otherwise damaged cells. They have a limited repertoire of receptors, the expression of which does not require somatic recombination. However, recent data indicate that NK cells may acquire the properties specific to adaptive immune cells. In particular, NK cells have been shown to possess the features of immunological memory, namely, the ability to more quickly and effectively respond to a repeated stimulus. The mechanisms of memory acquisition in NK cells differ from those implemented in T and B lymphocytes and are still rather vague. Presumably, some of these mechanisms underlie the significant phenotypic and functional NK cell diversity emerging during their differentiation. The recent data accumulated in this area considerably change the existing immunology paradigm, which postulates a clear delineation of the adaptive and innate immune cells. The published data on phenotypic and functional characteristics of NK cells and particular changes in these characteristics during NK cell differentiation and generation of memory-like NK cells are reviewed.     

IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-alpha, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-alpha, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-alpha, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-gamma production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.     

Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs.

Immune stimulatory oligodeoxynucleotides (ODN) with unmethylated CpG motifs are potent inducers of both innate and adaptive immunity. It initially appeared that a single type of optimal CpG motif would work in all applications. We now report that specific motifs of CpG ODN can vary dramatically in their ability to induce individual immune effects and that these differences impact on their antitumor activity in different tumor models. In particular, a distinct type of CpG motif, which has a chimeric backbone in combination with poly(G) tails, is a potent inducer of NK lytic activity but has little effect on cytokine secretion or B cell proliferation. One such NK-optimized CpG ODN (1585) can induce regression of established melanomas in mice. Surprisingly, no such therapeutic effects were seen with CpG ODN optimized for activation of B cells and Th1-like cytokine expression (ODN 1826). The therapeutic effects of CpG 1585 in melanoma required the presence of NK but not T or B cells and were not associated with the induction of a tumor-specific memory response. In contrast, CpG 1826, but not CpG 1585, was effective at inducing regression of the EL4 murine lymphoma; this rejection was associated with the induction of a memory response and although NK cells were necessary, they were not sufficient. These results demonstrate that selection of optimal CpG ODN for cancer immunotherapy depends upon a careful analysis of the cellular specificities of various CpG motifs and an understanding of the cellular mechanisms responsible for the antitumor activity in a particular tumor.     

Imprinting the fate of antigen-reactive B cells through the affinity of the B cell receptor

Long-lived plasma cells (PCs) and memory B cells (B(mem)) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short- or long-lived PCs and B(mem). Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither B(mem) nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but B(mem) remain extinct. In contrast, lower affinity interactions show tempered GCs, producing B(mem) and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity.     

The microenvironment of human Peyer's patches inhibits the increase in CD38 expression associated with the germinal center reaction

Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer's patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin alpha(4)beta(7), is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-gamma. However, coculture of tonsil naive B cells with an Ab directed against integrin beta(7) inhibits IFN-gamma-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of beta(7) can inhibit the induction of CD38 expression, suggests that ligation of alpha(4)beta(7) in vivo may contribute to a PP-specific GC phenotype.     

Generation of CD8 T cell memory is regulated by IL-12

Various signals during infection influence CD8 T cell memory generation, but these factors have yet to be fully defined. IL-12 is a proinflammatory cytokine that has been shown to enhance IFN-gamma-producing T cell responses and has been widely tested as a vaccine adjuvant. In this study, we show that IL-12-deficient mice generate a weaker primary CD8 T cell response and are more susceptible to Listeria monocytogenes infection, but have substantially more memory CD8 T cells and greater protective immunity against reinfection. Kinetic analyses show that in the absence of IL-12 there is a reduced contraction of Ag-specific CD8 T cells and a gradual increase in memory CD8 T cells as a result of increased homeostatic renewal. By signaling directly through its receptor on CD8 T cells, IL-12 influences their differentiation to favor the generation of fully activated effectors, but hinders the formation of CD8 T cell memory precursors and differentiation of long-term CD8 T cell memory(.) These results have implications for understanding memory T cell development and enhancing vaccine efficacy, and offer new insight into the role of IL-12 in coordinating the innate and adaptive immune response.     

Enhancement of CD8+ T cell immunity in the lung by CpG oligodeoxynucleotides increases protective efficacy of a modified vaccinia Ankara vaccine against lethal poxvirus infection even in a CD4-deficient host

Immunostimulatory CpG oligodeoxynucleotides (ODN) have proven effective as adjuvants for protein-based vaccines, but their impact on immune responses induced by live viral vectors is not known. We found that addition of CpG ODN to modified vaccinia Ankara (MVA) markedly improved the induction of longer-lasting adaptive protective immunity in BALB/c mice against intranasal pathogenic vaccinia virus (Western Reserve; WR). Protection was mediated primarily by CD8(+) T cells in the lung, as determined by CD8-depletion studies, protection in B cell-deficient mice, and greater protection correlating with CD8(+) IFN-gamma-producing cells in the lung but not with those in the spleen. Intranasal immunization was more effective at inducing CD8(+) T cell immunity in the lung, and protection, than i.m. immunization. Addition of CpG ODN increased the CD8(+) response but not the Ab response. Depletion of CD4 T cells before vaccination with MVA significantly diminished protection against pathogenic WR virus. However, CpG ODN delivered with MVA was able to substitute for CD4 help and protected CD4-depleted mice against WR vaccinia challenge. This study demonstrates for the first time a protective adjuvant effect of CpG ODN for a live viral vector vaccine that may overcome CD4 deficiency in the induction of protective CD8(+) T cell-mediated immunity.     

rC5a directs the in vitro migration of human memory and naive tonsillar B lymphocytes: implications for B cell trafficking in secondary lymphoid tissues.

Human C5a is a potent chemoattractant for granulocytes, monocytes, and dendritic cells. In mice C5a has been shown to be chemotactic for germinal center (GC) B cells. To date, no information is available on the effects of C5a on human B cell locomotion. Here we demonstrate that rC5a increases polarization and migration of human tonsillar B cells. The locomotory response was due to both chemokinetic and chemotactic activities of rC5a. Moreover, memory and, at a lesser extent, naive B cell fractions from purified tonsillar populations displayed rC5a-enhanced migratory properties, whereas GC cells did not. Flow cytometry revealed C5aR (CD88) on approximately 40% memory and 10% naive cells, respectively, whereas GC cells were negative. Immunohistochemistry showed that a few CD88+ cells were of the B cell lineage and localized in tonsillar subepithelial areas, where the majority of memory B cells settle. Pretreatment of memory B cells with the CD88 mAb abolished their migratory responsiveness to rC5a. Finally, the C5 gene was found to be expressed in naive, GC, and memory B lymphocytes at both the mRNA and the protein level. This study delineates a novel role for C5a as a regulator of the trafficking of human memory and naive B lymphocytes and supports the hypothesis that the B cells themselves may serve as source of C5 in secondary lymphoid tissues.     

The Vici syndrome protein EPG5 regulates intracellular nucleic acid trafficking linking autophagy to innate and adaptive immunity

Vici syndrome is a human inherited multi-system disorder caused by recessive mutations in EPG5, encoding the EPG5 protein that mediates the fusion of autophagosomes with lysosomes. Immunodeficiency characterized by lack of memory B cells and increased susceptibility to infection is an integral part of the condition, but the role of EPG5 in the immune system remains unknown. Here we show that EPG5 is indispensable for the transport of the TLR9 ligand CpG to the late endosomal-lysosomal compartment, and for TLR9-initiated signaling, a step essential for the survival of human memory B cells and their ultimate differentiation into plasma cells. Moreover, the predicted structure of EPG5 includes a membrane remodeling domain and a karyopherin-like domain, thus explaining its function as a carrier between separate vesicular compartments. Our findings indicate that EPG5, by controlling nucleic acids intracellular trafficking, links macroautophagy/autophagy to innate and adaptive immunity.