Plasma Cell Development Research Articles

B-cell survival factors in autoimmune rheumatic disorders.

Autoimmune rheumatic disorders have complex etiopathogenetic mechanisms in which B cells play a central role. The importance of factors stimulating B cells, notably the B-cell activating factor (BAFF) and A proliferation inducing ligand (APRIL) axis is now recognized. BAFF and APRIL are cytokines essential for B-cell proliferation and survival from the immature stages to the development of plasma cells. Their levels are increased in some subsets of patients with autoimmune disorders. Several recent biologic drugs have been developed to block this axis, namely belimumab [already licensed for systemic lupus erythematosus (SLE) treatment], tabalumab, atacicept and blisibimod. Many clinical trials to evaluate the safety and efficacy of these drugs in several autoimmune disorders are ongoing, or have been completed recently. This review updates the information on the use of biologic agents blocking BAFF/APRIL for patients with SLE, rheumatoid arthritis, Sjögren's syndrome and myositis.

T-bet regulates long-lived plasma cell development in an influenza model (IRM10P.604)

Abstract Long-lived plasma cells (LLPC) arise from germinal center B cells (GCB). The transcription factor Bcl6, which is required for GCB cell survival, inhibits LLPC development by repressing the transcription factor Blimp1, which normally initiates LLPC development. To date, it is not clear how these opposing transcription factors are regulated in GCB cells. In T lymphocytes, the transcription factor T-bet modulates the balance between Blimp1 and Bcl6 and controls the subsequent differentiation of the T cells into memory and effector cells. When naïve B cells are activated in vitro with T helper 1 (Th1) cells, the B cells differentiate into antibody (Ab) secreting cells in a T-bet and IFNγ dependent manner. Therefore, we hypothesized that B cell intrinsic expression of T-bet would also be necessary for LLPC development following influenza A infection. We found that T-bet expression increased in GCB cells formed after flu infection and was highest within GCB cells expressing low levels of the PC marker CD138, suggesting that T-bet supports PC differentiation from GCB cells. In chimeric mice in which 50% of cells are T-bet-/-, T-bet-/- GCB cells expressed lower levels of Blimp1 and IRF4 than WT GCB cells. Furthermore, flu-specific Ab titers were attenuated in chimeric animals lacking T-bet selectively within the B lineage cells compared to control chimeras with WT B cells. These results support a role for T-bet in the development of LLPC following influenza infection.

B cell memory but not antibody response is maintained in the elderly after repeated influenza vaccine immunizations and aged murine plasma cell generation is defective (VAC11P.1066)

Abstract The success of a vaccine depends on both plasma cells and memory B cells. We previously showed that serum antibodies and the in vitro AID response to a new influenza vaccine are decreased in elderly individuals. We hypothesized that even though AID is decreased in the elderly, the ability to generate memory B cells might be improved by a repeated vaccine. To test this hypothesis, we recruited young and elderly individuals who had been immunized in at least two consecutive influenza vaccine seasons in which the H1N1 strain was the same. Results showed that frequencies of influenza vaccine-specific memory B cells were similar in young and elderly individuals but the levels of serum antibodies were lower in the elderly. These findings suggest that multiple re-exposures to the vaccine cause a divergence between memory B cell frequencies and antibody levels with age, which could be due to impaired memory B cell to plasma cell differentiation in the elderly. To test the hypothesis of defective plasma cell development in old mice, we measured the percentage of plasma cells and Blimp-1 in LPS-stimulated B cells and found them significantly lower in old than in young cultures. The average fold reduction in the secreted amount of IgM and IgG3 in aged cultures compared to young cultures is higher than the average reduction in CD138+ plasma cells (10-fold vs 2-fold) suggesting qualitative as well as quantitative plasma cell defects with age.

UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development, Linking Linear Ubiquitination to Multiple Autoimmune Diseases

UBE2L3 is associated with increased susceptibility to numerous autoimmune diseases, but the underlying mechanism is unexplained. By using data from a genome-wide association study of systemic lupus erythematosus (SLE), we observed a single risk haplotype spanning UBE2L3, consistently aligned across multiple autoimmune diseases, associated with increased UBE2L3 expression in B cells and monocytes. rs140490 in the UBE2L3 promoter region showed the strongest association. UBE2L3 is an E2 ubiquitin-conjugating enzyme, specially adapted to function with HECT and RING-in-between-RING (RBR) E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC). Our data demonstrate that UBE2L3 is the preferred E2 conjugating enzyme for LUBAC in vivo, and UBE2L3 is essential for LUBAC-mediated activation of NF-κB. By accurately quantifying NF-κB translocation in primary human cells from healthy individuals stratified by rs140490 genotype, we observed that the autoimmune disease risk UBE2L3 genotype was correlated with basal NF-κB activation in unstimulated B cells and monocytes and regulated the sensitivity of NF-κB to CD40 stimulation in B cells and TNF stimulation in monocytes. The UBE2L3 risk allele correlated with increased circulating plasmablast and plasma cell numbers in SLE individuals, consistent with substantially elevated UBE2L3 protein levels in plasmablasts and plasma cells. These results identify key immunological consequences of the UBE2L3 autoimmune risk haplotype and highlight an important role for UBE2L3 in plasmablast and plasma cell development.

Factors regulating immunoglobulin production by normal and disease-associated plasma cells.

Immunoglobulins are molecules produced by activated B cells and plasma cells in response to exposure to antigens. Upon antigen exposure, these molecules are secreted allowing the immune system to recognize and effectively respond to a myriad of pathogens. Immunoglobulin or antibody secreting cells are the mature form of B lymphocytes, which during their development undergo gene rearrangements and selection in the bone marrow ultimately leading to the generation of B cells, each expressing a single antigen-specific receptor/immunoglobulin molecule. Each individual immunoglobulin molecule has an affinity for a unique motif, or epitope, found on a given antigen. When presented with an antigen, activated B cells differentiate into either plasma cells (which secrete large amounts of antibody that is specific for the inducing antigen), or memory B cells (which are long-lived and elicit a stronger and faster response if the host is re-exposed to the same antigen). The secreted form of immunoglobulin, when bound to an antigen, serves as an effector molecule that directs other cells of the immune system to facilitate the neutralization of soluble antigen or the eradication of the antigen-expressing pathogen. This review will focus on the regulation of secreted immunoglobulin by long-lived normal or disease-associated plasma cells. Specifically, the focus will be on signaling and transcriptional events that regulate the development and homeostasis of long-lived immunoglobulin secreting plasma cells.

Amyloidogenicity of Immunoglobulin Light Chains

Systemic light chain amyloidosis (AL) is a rare protein aggregation disease. It usually strikes in the wake of myeloma, which affects plasma cells in the adaptive immune system. During plasma cell development, the immunoglobulin light chain (LC) genes undergo several rearrangements that leave each clone with a unique protein sequence. The produced monoclonal light chains (LC) are deposited as amyloid in AL but not in Multiple Myeloma (MM) patients. We aim to elucidate the biophysical basis of this difference. In both diseases large amounts of soluble LC are secreted into circulation and excreted with urine. Hence we hypothesize that amyloidogenicity depends on the amyloid formation propensity of the individual LC sequences rather than being a result of different LC concentrations being present in both diseases, which may also alter susceptibilities to the green tea phenol Epigallocatechin-3-gallate (EGCG).To test this hypothesis we used a simple diafiltration approach to isolate LC from AL and MM patient¯s urine, including only cases with albuminuria less than 5% of total proteinuria. We monitored their aggregation under physiological conditions in presence and absence of EGCG over a time course of three weeks in a Thioflavin T assay and compared the aggregate sizes at different time points by semi-denaturing SDS-PAGE and filter retardation assay. We probed stabilities of native and of aggregated LC by Guanidine and thermal denaturation and imaged aggregate morphologies by atomic force microscopy (AFM).Each individual LC displayed unique characteristic aggregation kinetics. However, there were no systematic differences between proteins from MM and AL patients. EGCG treatment accelerated the formation of large aggregates that are partially stable against SDS denaturation. By determining the sequence of the LC protein via MS/MS we hope to establish a correlation between sequences, aggregation propensities and clinical parameters in AL and MM.

Circulating Bcma Binding to Its Ligand BAFF Prevents Normal Antibody Production in Multiple Myeloma Patients

Introduction: A hallmark of multiple myeloma (MM) is the low levels of uninvolved immunoglobulin (Ig) levels. B-cell maturation antigen (BCMA) is a receptor expressed in mature non-malignant and malignant B lymphocytes, including plasma cells. Its ligands are BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand). We previously demonstrated that BCMA is present in the serum of MM patients (pts) and that its levels predict survival (Sanchez et al. Br J Haematol 2012). We hypothesized that circulating BCMA binds it ligands, preventing normal plasma cell development in MM patients which may explain their reduction in uninvolved Ig levels.Methods: BCMA-Fc and control Ig were obtained and reconstituted in PBS (R&D Systems). Retro-orbital bleeds were performed on SCID mice implanted with the human xenografts LAGλ-1, LAGk-1A or LAGk-2 following BCMA treatment. Human BCMA and mouse BAFF, IgM and IgA levels were measured with ELISA (R&D Systems & Bethyl Laboratories). Human IgA and IgG levels were determined in MM patients using nephelometry (Immage 800, Beckman Coulter). Hevylite® Assays (Binding Site) were used to quantify the levels of heavy-light chain isoform pairs in MM patients.Results: Recombinant mouse BAFF (rmBAFF) was mixed with recombinant human BCMA (rhBCMA) and incubated on plates coated with anti-mouse BAFF antibody (Ab). An anti-BCMA detection Ab was added, and BCMA-BAFF complexes were easily detected at concentrations of human BCMA and BAFF present in MM pts. We determined if mBAFF from SCID mice would also bind rhBCMA. SCID mice were bled; their plasma isolated and incubated with rhBCMA, and showed that rhBCMA formed complexes with mBAFF.To determine what effect human BCMA had on Ig levels in immune competent mice, rhBCMA-Fc or control Ig-Fc (100 mg) was injected into C57 Bl and Balb/c mice, and plasma IgA and IgM levels were measured. Following rhBCMA-Fc injection, a marked decrease in both antibody classes was observed in both mouse strains. Decreases in IgA levels were observed following BCMA treatment when compared to baseline plasma IgA on days 4 and 6 (P = 0.0031 and P = 0.0064, respectively), and the control groups (P = 0.0087 and P = 0.0221). Samples were also analyzed for mouse IgM levels with similar marked reductions when compared to the untreated (P = 0.0001) and Ig-Fc (P = 0.0088) groups. To determine if rhBCMA-mBAFF complexes formed in vivo, an ELISA was performed. Plates were pre-coated with a monoclonal mouse anti-BAFF capture Ab. Plasma samples were incubated and an anti-human-BCMA detection Ab was added. rhBCMA-mBAFF complexes were detected at high levels in plasma samples from mice dosed 4 and 6 days previously with BCMA-Fc whereas no complexes were found in samples in control Ig-Fc or untreated mice. We evaluated mBAFF levels in SCID mice with and without human MM. We have previously shown high levels of human BCMA in the plasma of MM tumor-bearing mice (Sanchez et al. Br J Haematol 2012). On days 14, 21 and 28 post-tumor implantation, mice implanted with LAGλ-1 had markedly lower plasma levels of mBAFF (P = 0.0143, P = 0.0002 and P = 0.0008, respectively) when compared to age and sex-matched mice not bearing any tumors. We obtained similar results using two additional human MM xenograft models (LAGk-1A and LAGk-2). Next, we determined whether serum BCMA levels inversely correlated with uninvolved Ig levels in MM pts. For pts with IgA (n = 134) or IgG (n = 313) MM, higher BCMA levels (> 100 ng/ml) correlated with below normal levels of uninvolved IgG in IgA MM and uninvolved IgA in IgG MM, whereas lower BCMA levels (< 100 ng/ml) correlated with normal uninvolved levels (P < 0.0001). Using the Hevylite Assay, similar results were observed for the levels of BCMA compared to uninvolved IgG isoforms in both pts with involved IgG lambda (n = 62, P = 0.0006) and IgG kappa (n = 117, P < 0.0001) MM.Conclusions: Our laboratory previously reported that BCMA is present in the serum of MM patients, correlates with response to treatment and predicts survival. We now demonstrate the formation of circulating BCMA-BAFF complexes in MM, and administration of recombinant BCMA to normal mice results in marked reductions in their antibody levels. We also show that BCMA levels inversely correlate with uninvolved Ig levels in MM pts. Thus, the lack of normal antibody production in MM pts results in part from circulating BCMA binding its ligands, preventing production of normal antibody-producing cells. DisclosuresNo relevant conflicts of interest to declare.

The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells.

The unfolded protein response (UPR) is a conserved stress-signaling pathway activated after accumulation of unfolded proteins within the endoplasmic reticulum (ER). Active UPR signaling leads to unconventional, enzymatic splicing of XBP1 mRNA enabling expression of the transcription factor XBP1s to control ER homeostasis. While IRE1 has been identified as the endoribonuclease required for cleavage of this mRNA, the corresponding ligase in mammalian cells has remained elusive. Here, we report that RTCB, the catalytic subunit of the tRNA ligase complex, and its co-factor archease mediate XBP1 mRNA splicing both in vitro and in vivo. Depletion of RTCB in plasma cells of Rtcbfl/fl Cd23-Cre mice prevents XBP1s expression, which normally is strongly induced during plasma cell development. RTCB-depleted plasma cells show reduced and disorganized ER structures as well as severe defects in antibody secretion. Targeting RTCB and/or archease thus represents a promising strategy for the treatment of a growing number of diseases associated with elevated expression of XBP1s.

Clinicopathological significance and potential drug target of p15INK4B in multiple myeloma.

Multiple myeloma (MM) is a clonal malignancy characterized by the proliferation of malignant plasma cells in the bone marrow and the production of monoclonal immunoglobulin. In addition to genetic changes, gene hypermethylation is an alternative mechanism of tumor suppressor gene inactivation in MM. The cyclin-dependent kinase inhibitor 1 (CDKN2B or p15INK4B) gene lies adjacent to the tumor suppressor gene, cyclin-dependent kinase inhibitor 2 (CDKN2A), and is frequently mutated and deleted in a wide variety of tumors, including MM. However, there is a lack of systematic analysis of p15 epigenetic modification such as methylation in MM from different studies that can provide more powerful estimation of an effect. In this study, we have systematically reviewed the studies of p15INK4B promoter methylation in MM and quantified the association between p15INK4B promoter methylation and MM using meta-analysis methods. We observed that the frequency of p15INK4B methylation is significantly higher in MM patients than in normal healthy controls. The pooled odds ratio (OR) from ten studies including 394 MM and 99 normal individuals is 0.08, while confidence interval (CI) is 0.03–0.21 (P<0.00001). This indicates that p15INK4B inactivation through methylation plays an important role in the pathogenesis of MM. In addition, the frequency of p15INK4B methylation was significantly higher in patients with MM than in those with asymptomatic monoclonal gammopathy of undetermined significance. The pooled OR from four studies is 0.40, 95% CI =0.21–0.78 (P=0.007). These results suggest that silencing of p15INK4B gene expression by epigenetic modification such as promoter hypermethylation plays a role not only in the initiation of MM but also in plasma cell malignant transformation, disease progression, and development.

Putting J Chain Back on the Map: How Might Its Expression Define Plasma Cell Development?

Joining chain (J chain) is a small polypeptide that regulates multimerization of secretory IgM and IgA, the only two mammalian Igs capable of forming multimers. J chain also is required for poly-Ig receptor-mediated transport of these Ig classes across the mucosal epithelium. It is generally assumed that all plasma cells express J chain regardless of expressed isotype, despite the documented presence of J chain(-) plasma cells in mammals, specifically in all monomeric IgA-secreting cells and some IgG-secreting cells. Compared with most other immune molecules, J chain has not been studied extensively, in part because of technical limitations. Even the reported phenotype of the J chain-knockout mouse is often misunderstood or underappreciated. In this short review, we discuss J chain in light of the various proposed models of its expression and regulation, with an added focus on its evolutionary significance, as well as its expression in different B cell lineages/differentiation states.

Abstract 3045: Defining an elusive multiple myeloma stem cell population in mice

Abstract Multiple Myeloma (MM) is a debilitating disease of proliferating and malignant plasma cells that is currently incurable. The ability of monoclonal recurrence of disease suggests it might arise from a stem cell-like population capable of self-renewal. The difficulty to isolate the cancer stem-like cell in MM in both mice and men has introduced confusion toward this hypothesis. XBP1s is a key positive regulator for unfolded protein response and a master transcription factor for plasma cell development. It was previously reported that transgenic overexpression of XBP1s in the B cell lineage of mice (XBP1s-Tg), induces a multiple myeloma phenotype resembling human MM. We have now defined a post-germinal center, pre-plasma B cell population that was significantly increased in XBP1s-Tg mice as the hallmarks of MM was demonstrated. This non-memory B cell population resembled a stem/progenitor cell population, with increased expression of aldehyde dehydrogenase (ALDH), Notch1 and c-kit. Importantly, utilizing an in vitro culture for differentiating plasma cells, the increased population began to differentiate into plasma cells, increasing in size and gaining CD138 expression, the hallmark marker for plasma cells. In conclusion, we have defined a transitional pre-plasma cell population that could potentially be a stem/progenitor population of multiple myeloma. These results provide a novel insight into stem cell biology with regard to multiple myeloma and the use of murine models of myeloma to study the disease. Citation Format: Joshua Kellner, Yunpeng Hua, Bei Liu, Zihai Li. Defining an elusive multiple myeloma stem cell population in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3045. doi:10.1158/1538-7445.AM2014-3045

Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits.

Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis.

The road to treating smoldering multiple myeloma.

The management of smoldering multiple myeloma (SMM) has been a challenge to clinicians, ever since the condition was first characterized in 1980. While the risk of progression to symptomatic myeloma is greater for SMM (10% per year) compared to MGUS (1% per year), several SMM patients remain asymptomatic for years without evidence of disease progression. Early clinical trials focusing on early treatment of SMM have been equivocal with no clear benefit. However, the last decade has seen a greater understanding of the pathogenesis of plasma cell disorders, including SMM, and development of better therapeutics. A recent randomized trial has provided evidence of clinical benefit with early treatment of high-risk SMM. In this review, we summarize issues related to the early treatment of SMM including risk stratification and possible outcomes with therapy initiation. In the context of reviewing recent clinical trial data supporting early treatment, we define challenges faced by clinicians and provide future directions to the road to treating SMM.

Gp49B-mediated negative regulation of antibody production by memory and marginal zone B cells.

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.

Preclinical validation of interleukin 6 as a therapeutic target in multiple myeloma.

Studies on the biologic and molecular genetic underpinnings of multiple myeloma (MM) have identified the pleiotropic, pro-inflammatory cytokine, interleukin-6 (IL-6), as a factor crucial to the growth, proliferation and survival of myeloma cells. IL-6 is also a potent stimulator of osteoclastogenesis and a sculptor of the tumor microenvironment in the bone marrow of patients with myeloma. This knowledge has engendered considerable interest in targeting IL-6 for therapeutic purposes, using a variety of antibody- and small-molecule-based therapies. However, despite the early recognition of the importance of IL-6 for myeloma and the steady progress in our knowledge of IL-6 in normal and malignant development of plasma cells, additional efforts will be required to translate the promise of IL-6 as a target for new myeloma therapies into significant clinical benefits for patients with myeloma. This review summarizes published research on the role of IL-6 in myeloma development and describes ongoing efforts by the University of Iowa Myeloma Multidisciplinary Oncology Group to develop new approaches to the design and testing of IL-6-targeted therapies and preventions of MM.

Distinct roles for TLR7 and TLR9 in regulation of B cell signal strength and differentiation (LYM6P.766)

Abstract BCR signal strength plays a key role in the fate decision of B cells. Strong BCR signals promote plasma cell differentiation and weaker BCR signals drive germinal center B cell fate. Signals through toll-like receptors (TLRs) also influence B cell differentiation, but it is unclear how the BCR and TLR signaling cascades interface to modulate B cell responses and differentiation. To address this question, we used a BCR sdTg mouse model, AM14, in which all mature B cells have a naïve follicular phenotype. The AM14 BCR recognizes IgG2a-bound immune complexes (ICs) and AM14 B cell activation depends on TLR7 and/or TLR9. ICs formed by the anti-nucleosome antibody PL2-3 can activate AM14 B cells through either TLR7 or TLR9, allowing us to dissect signaling pathways unique to each TLR. We find that activation of AM14 TLR7-deficient B cells leads to sustained activation of the NF-κB pathway and p38 phosphorylation, subsequently leading to cell death. In contrast, TLR9-deficient AM14 B cells activated with ICs are unable to sustain the initial NF-κB and p38 signaling response. Intriguingly, these TLR9-deficient AM14 B cells differentiate into short-lived plasmablasts as characterized by increased levels of plasma cell associated proteins such as IRF-4, Blimp-1 and CD138. Plasma cell development is not observed in IC activated TLR7-deficient AM14 B cells. Our results suggest divergent roles for TLR7 and TLR9 in B cell activation and plasma cell differentiation.

Naive Idiotope-Specific B and T Cells Collaborate Efficiently in the Absence of Dendritic Cells

Anti-idiotope (anti-Id) Abs have a role in therapy against B cell lymphomas, as inhibitors of pathogenic autoantibodies, and as surrogate Ags for immunization. Despite these observations, the mechanism by which Id(+) Ig generates anti-Id Abs is essentially unknown. To address this issue, we generated a double knock-in mouse that expresses V regions of a somatically mutated anti-Id mAb with intermediate affinity (affinity constant [Ka] = 0.77 × 10(7) M(-1)) for the myeloma protein M315. The anti-Id mice have normal peripheral B cell populations, and allelic exclusion is efficient. Anti-Id B cells from BCR knock-in mice, together with Id-specific CD4(+) T cells from previously established TCR-transgenic mice, enabled us to study Id-specific T cell-B cell collaboration by dilution of transferred cells into syngeneic BALB/c recipients. We show that previously unstimulated (naive) Id-specific B and T cells collaborate efficiently in vivo, even at low frequencies and in the presence of low amounts of Id(+) Ig, resulting in germinal center formation, plasma cell development, and secretion of isotype-switched anti-Id Abs. We further demonstrate that Id-specific T cell-B cell collaboration occurs readily in the absence of adjuvant and is not dependent on Id-presentation by dendritic cells. The results underscore the potency of anti-Id B cells in MHC class II-restricted presentation of Id(+) Ig and suggest that Id-specific T cell-B cell collaboration is of physiological relevance.

Defining aspects of noncognate splenic iNKT cell help which contribute to humoral memory (INC6P.403)

Abstract Successful induction of pathogen-specific humoral B cell memory responses depends upon help from CD4+ T cells. We find that invariant natural killer T (iNKT) cells, glycolipid-specific, CD1d-restricted innate lymphocytes, can provide both cognate (direct) and noncognate (or indirect) helper signals to enhance B cell humoral responses. In fact, a subset of iNKT cells, termed iNKT follicular helper cells, are the cells which specifically act to promote germinal centers, facilitate optimal B cell memory, and plasma cell differentiation. While both cognate and noncognate iNKT cell help induce class-switched, antigen-specific humoral immune responses, only noncognate iNKT cell help drives formation of humoral memory and plasma cell development. We find that differences in the strength of the TcR signal received by iNKT cells, very early effector cytokine production by iNKT cells, T follicular helper cells, and T follicular regulatory cells all contribute to the observed differences in the ultimate humoral outcome. These findings help to identify the optimal requirements for iNKT induction of humoral memory, which has important implications for the application of glycolipid molecules as humoral adjuvants.

T follicular helper cells contribute to autoimmunity through the BCMA-BAFF axis (BA2P.117)

Abstract T follicular helper (TFH) cells are critical for the development of protective antibodies via germinal center B-cell responses; however, uncontrolled TFH cell expansion activates autoreactive B-cells to produce antibodies that cause autoimmunity. Although increased circulating level of the cytokine BAFF has been linked to loss of B-cell tolerance in systemic autoimmunity, the potential role for BAFF in TFH cell homeostasis is not known. B-cell maturation antigen (BCMA) is a receptor for BAFF and is critical for the development and survival of bone marrow plasma cells (PCs). Paradoxically, BCMA deficiency exacerbates the formation of autoantibody-secreting PCs in spleens of autoimmune-prone mice. Here, we show that TFH cells uniquely express BCMA and that the absence of BCMA in TFH cells enhances their numbers and responsiveness to BAFF, and in turn, drive autoantibody production. Moreover, circulating CD4+ T cells and TFH-like cells that express another BAFF receptor BR3 (but not BCMA) are elevated in SLE patients and positively correlate with disease severity. These results identify a new BCMA-BAFF axis in controlling TFH cell homeostasis and suggest that the balance between BCMA and BR3 signaling in TFH cells serves as an additional checkpoint of immune tolerance.

Long-lived bone marrow plasma cells are inhibited by protein A in Staphylococcus aureus infection. (VAC6P.955)

Abstract Long-lived plasma cells are now recognized as major contributors to protection against bacterial infection through their constitutive secretion of antibody. Staphylococcus aureus is a growing concern due to expansion of antibiotic resistance and causes recurring infections without inducing long-lived immunity. Little is known about the nature and longevity of the adaptive response to S. aureus. Protein A on the surface of S. aureus promotes virulence and inflammation which can alter B cell function and hinder long-lived plasma cell development. Although similar B cell responses were induced by WT and protein A-deficient infection and secondary challenge, long-term antigen-specific antibody levels waned after challenge with WT S. aureus, but were maintained after challenge with the protein A-deficient mutant. This suggests that long-lived B cell immunity is disrupted by protein A. We developed an ELISpot assay to detect S. aureus-specific antibody secreting cells and found that they accumulated in the bone marrow niche after protein A-deficient, but not WT challenge. The absence of bone marrow plasma cells after WT challenge was not a result of defects in germinal center or memory B cell formation. Our work indicates that protein A interferes with migration and/or survival of long-lived bone marrow plasma cells after S. aureus infection. It also provides insight into the requirements for successful development of long-lived immunity to bacterial pathogens.