Danielle T. Avery

BAFF selectively enhances the survival of plasmablasts generated from human memory B cells

The generation of Ig-secreting cells (ISCs) from memory B cells requires interactions between antigen-specific (Ag-specific) B cells, T cells, and dendritic cells. This process must be strictly regulated to ensure sufficient humoral immunity while avoiding production of pathogenic autoantibodies. BAFF, a member of the TNF family, is a key regulator of B cell homeostasis. BAFF exerts its effect by binding to three receptors - transmembrane activator of and CAML interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R). To elucidate the contribution of BAFF to the differentiation of B cells into ISCs, we tracked the fate of human memory B cells stimulated with BAFF or CD40L. BAFF and CD40L significantly increased the overall number of surviving B cells. This was achieved via distinct mechanisms. CD40L induced proliferation of nondifferentiated blasts, while BAFF prevented apoptosis of ISCs without enhancing proliferation. The altered responsiveness of activated memory B cells to CD40L and BAFF correlated with changes in surface phenotype such that expression of CD40 and BAFF-R were reduced on ISCs while BCMA was induced. These results suggest BAFF may enhance humoral immunity in vivo by promoting survival of ISCs via a BCMA-dependent mechanism. These findings have wide-ranging implications for the treatment of human immunodeficiencies as well as autoimmune diseases.

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.

B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans.

Engagement of cytokine receptors by specific ligands activate Janus kinase–signal transducer and activator of transcription (STAT) signaling pathways. The exact roles of STATs in human lymphocyte behavior remain incompletely defined. Interleukin (IL)-21 activates STAT1 and STAT3 and has emerged as a potent regulator of B cell differentiation. We have studied patients with inactivating mutations in STAT1 or STAT3 to dissect their contribution to B cell function in vivo and in response to IL-21 in vitro. STAT3 mutations dramatically reduced the number of functional, antigen (Ag)-specific memory B cells and abolished the ability of IL-21 to induce naive B cells to differentiate into plasma cells (PCs). This resulted from impaired activation of the molecular machinery required for PC generation. In contrast, STAT1 deficiency had no effect on memory B cell formation in vivo or IL-21–induced immunoglobulin secretion in vitro. Thus, STAT3 plays a critical role in generating effector B cells from naive precursors in humans. STAT3-activating cytokines such as IL-21 thus underpin Ag-specific humoral immune responses and provide a mechanism for the functional antibody deficit in STAT3-deficient patients.

Early commitment of naïve human CD4 + T cells to the T follicular helper (T FH ) cell lineage is induced by IL-12

T follicular helper (TFH) cells are a specialized subset of CD4+ T cells that localize to B‐cell follicles, where they are positioned to provide help for the induction of optimal humoral immune responses. Key features of TFH cells are the expressions of CXCR5, ICOS, interleukin (IL)‐21 and BCL‐6. The requirements for human TFH cell development are unknown. Here we show that IL‐6, IL‐12, IL‐21 and IL‐23 are capable of inducing IL‐21 expression in naïve CD4+ T cells isolated from human tonsils, peripheral blood and cord blood. However, only IL‐12 induced sustained expressions of CXCR5 and ICOS on these activated naïve CD4+ T cells, and endowed them with the ability to provide increased help to B cells for their differentiation into immunoglobulin‐secreting cells. The effects of IL‐12 were independent of interferon‐γ and T‐bet, and associated with upregulation of BCL‐6 expression. Thus, these cytokines, particularly IL‐12, are likely to act at an early stage during dendritic cell‐mediated priming of naïve CD4+ T cells into a TFH cell fate, and thus underpin antibody‐mediated immunity.

Intrinsic Differences in the Proliferation of Naive and Memory Human B Cells as a Mechanism for Enhanced Secondary Immune Responses

Humoral immune responses elicited after secondary exposure to immunizing Ag are characterized by robust and elevated reactivity of memory B cells that exceed those of naive B cells during the primary response. The mechanism underlying this difference in responsiveness of naive vs memory B cells remains unclear. We have quantitated the response of naive and memory human B cells after in vitro stimulation with T cell-derived stimuli. In response to stimulation with CD40 ligand alone or with IL-10, both IgM-expressing and Ig isotype-switched memory B cells entered their first division 20–30 h earlier than did naive B cells. In contrast, the time spent traversing subsequent divisions was similar. Consistent with previous studies, only memory cells differentiated to CD38+ blasts in a manner that increased with consecutive division number. These differentiated CD38+ B cells divided faster than did CD38− memory B cell blasts. Proliferation of CD40 ligand-stimulated naive B cells as well as both CD38+ and CD38− cells present in cultures of memory B cells was increased by IL-10. In contrast, IL-2 enhanced proliferation of CD38− and CD38+ memory B cell blasts, but not naive cells. Thus, memory B cells possess an intrinsic advantage over naive B cells in both the time to initiate a response and in the division-based rate of effector cell development. These differences help explain the accelerated Ab response exhibited by memory B cells after secondary challenge by an invading pathogen, a hallmark of immunological memory.

CXCR5 Expressing Human Central Memory CD4 T Cells and Their Relevance for Humoral Immune Responses

High expression of CXCR5 is one of the defining hallmarks of T follicular helper cells (TFH), a CD4 Th cell subset that promotes germinal center reactions and the selection and affinity maturation of B cells. CXCR5 is also expressed on 20–25% of peripheral blood human central memory CD4 T cells (TCM), although the definitive function of these cells is not fully understood. The constitutive expression of CXCR5 on TFH cells and a fraction of circulating TCM suggests that CXCR5+ TCM may represent a specialized subset of memory-type TFH cells programmed for homing to follicles and providing B cell help. To verify this assumption, we analyzed this cell population and show its specialized function in supporting humoral immune responses. Compared with their CXCR5− TCM counterparts, CXCR5+ TCM expressed high levels of the chemokine CXCL13 and efficiently induced plasma cell differentiation and Ig secretion. We found that the distinct B cell helper qualities of CXCR5+ TCM were mainly due to high ICOS expression and pronounced responsiveness to ICOS ligand costimulation together with large IL-10 secretion. Furthermore, B cell helper attributes of CXCR5+ TCM were almost exclusively acquired on cognate interaction with B cells, but not with dendritic cells. This implies that a preferential recruitment of circulating CXCR5+ TCM to CXCL13-rich B cell follicles is required for the promotion of a quick and efficient protective secondary humoral immune response. Taken together, we propose that CXCR5+ TCM represent a distinct memory cell subset specialized in supporting Ab-mediated immune responses.

IL-21-Induced Isotype Switching to IgG and IgA by Human Naive B Cells Is Differentially Regulated by IL-4

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-β. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1+ cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA+ cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1+ cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.

Functional STAT3 deficiency compromises the generation of human T follicular helper cells

T follicular helper (Tfh) cells are critical for providing the necessary signals to induce differentiation of B cells into memory and Ab-secreting cells. Accordingly, it is important to identify the molecular requirements for Tfh cell development and function. We previously found that IL-12 mediates the differentiation of human CD4(+) T cells to the Tfh lineage, because IL-12 induces naive human CD4(+) T cells to acquire expression of IL-21, BCL6, ICOS, and CXCR5, which typify Tfh cells. We have now examined CD4(+) T cells from patients deficient in IL-12Rβ1, TYK2, STAT1, and STAT3 to further explore the pathways involved in human Tfh cell differentiation. Although STAT1 was dispensable, mutations in IL12RB1, TYK2, or STAT3 compromised IL-12-induced expression of IL-21 by human CD4(+) T cells. Defective expression of IL-21 by STAT3-deficient CD4(+) T cells resulted in diminished B-cell helper activity in vitro. Importantly, mutations in STAT3, but not IL12RB1 or TYK2, also reduced Tfh cell generation in vivo, evidenced by decreased circulating CD4(+)CXCR5(+) T cells. These results highlight the nonredundant role of STAT3 in human Tfh cell differentiation and suggest that defective Tfh cell development and/or function contributes to the humoral defects observed in STAT3-deficient patients.

Expansion of Functionally Immature Transitional B Cells Is Associated with Human-Immunodeficient States Characterized by Impaired Humoral Immunity

X-linked lymphoproliferative disease (XLP) is a severe immunodeficiency associated with a marked reduction in circulating memory B cells. Our investigation of the B cell compartment of XLP patients revealed an increase in the frequency of a population of B cells distinct from those previously defined. This population displayed increased expression of CD10, CD24, and CD38, indicating that it could consist of circulating immature/transitional B cells. Supporting this possibility, CD10+CD24highCD38high B cells displayed other immature characteristics, including unmutated Ig V genes and elevated levels of surface IgM; they also lacked expression of Bcl-2 and a panel of activation molecules. The capacity of CD24highCD38high B cells to proliferate, secrete Ig, and migrate in vitro was greatly reduced compared with mature B cell populations. Moreover, CD24highCD38high B cells were increased in the peripheral blood of neonates, patients with common variable immunodeficiency, and patients recovering from hemopoietic stem cell transplant. Thus, an expansion of functionally immature B cells may contribute to the humoral immunodeficient state that is characteristic of neonates, as well as patients with XLP or common variable immunodeficiency, and those recovering from a stem cell transplant. Further investigation of transitional B cells will improve our understanding of human B cell development and how alterations to this process may precipitate immunodeficiency or autoimmunity.

Resting Human Memory B Cells Are Intrinsically Programmed for Enhanced Survival and Responsiveness to Diverse Stimuli Compared to Naive B Cells

Enhanced secondary Ab responses are a vital component of adaptive immunity, yet little is understood about the intrinsic and extrinsic regulators of naive and memory B cells that result in differences in their responses to Ag. Microarray analysis, together with surface and intracellular phenotyping, revealed that memory B cells have increased expression of members of the TNF receptor, SLAM (signaling lymphocytic activation molecule), B7, and Bcl2 families, as well as the TLR-related molecule CD180 (RP105). Accordingly, memory B cells exhibited enhanced survival, proliferation, and Ig secretion, and they entered division more rapidly than did naive B cells in response to both T cell-dependent and T cell-independent stimuli. Furthermore, both IgM and isotype-switched memory B cells, but not naive B cells, costimulated CD4+ T cells in vitro through a mechanism dependent on their constitutive expression of CD80 and CD86. This study demonstrates that up-regulation of genes involved in activation, costimulation, and survival provides memory B cells with a unique ability to produce enhanced immune responses and contributes to the maintenance of the memory B cell pool.