TLR9 ligand sequestration by chemokine CXCL4 negatively affects central B cell tolerance.

Central B cell tolerance is believed to be regulated by B cell receptor signaling induced by the recognition of self-antigens in immature B cells. Using humanized mice with defective MyD88, TLR7, or TLR9 expression, we demonstrate that TLR9/MYD88 are required for central B cell tolerance and the removal of developing autoreactive clones. We also show that CXCL4, a chemokine involved in systemic sclerosis (SSc), abrogates TLR9 function in B cells by sequestering TLR9 ligands away from the endosomal compartments where this receptor resides. The in vivo production of CXCL4 thereby impedes both TLR9 responses in B cells and the establishment of central B cell tolerance. We conclude that TLR9 plays an essential early tolerogenic function required for the establishment of central B cell tolerance and that correcting defective TLR9 function in B cells from SSc patients may represent a novel therapeutic strategy to restore B cell tolerance.

TACI and endogenous APRIL in B cell maturation.

While many of the genes and molecular pathways in the germinal center B cell response which initiate protective antibody production are known, the contributions of individual molecular players in terminal B cell differentiation remain unclear. We have previously investigated how mutations in TACI gene, noted in about 10% of patients with common variable immunodeficiency, impair B cell differentiation and often, lead to lymphoid hyperplasia and autoimmunity. Unlike mouse B cells, human B cells express TACI-L (Long) and TACI-S (Short) isoforms, but only TACI-S promotes terminal B cell differentiation into plasma cells. Here we show that the expression of intracellular TACI-S increases with B cell activation, and colocalizes with BCMA and their ligand, APRIL. We show that the loss of APRIL impairs isotype class switch and leads to distinct metabolic and transcriptional changes. Our studies suggest that intracellular TACI-S and APRIL along with BCMA direct long-term PC differentiation and survival.

Corrigendum: TACI Isoforms Regulate Ligand Binding and Receptor Function.

[This corrects the article DOI: 10.3389/fimmu.2018.02125.].

TACI Isoforms Regulate Ligand Binding and Receptor Function.

TACI signals activate B cell proliferation, isotype switch and antibody production in both normal immunity and autoimmune states. In contrast to murine TACI, the human TACI gene undergoes alternative splicing to produce short and long isoforms (TACI-S and TACI-L). In previous studies, we showed that transduction of the short, but not long isoform, into murine B cells or human pre-B cells lacking TACI, caused them to become transcriptional and morphologically identical to plasma cells. These data suggest that the expression of different isoforms in humans provides unique controls on B cell maturation. In these studies we show that TACI-S and TACI-L form complexes in a ligand-independent manner, not dependent on a single extracellular domain. Both TACI isoforms are detectable in the endosomal cellular compartment where they co-localize with MyD88, TRAF6, and the activated 65 kDa form of TLR9, depending on a conserved intracellular TACI sequence. In contrast to TACI-L expressing cells, or cells bearing both isoforms, TACI-S binds ligands BAFF and APRIL with substantially greater affinity and promotes enhanced NF-kB activation. Using isoform-specific monoclonal antibodies, we show that while TACI-L is predominant as a surface receptor surface on human B cells, significantly more TACI-S is noted in the intracellular compartment and also in marginal zone, isotype switched and plasmablast in resting B cells. TACI-S is increased in tonsillar B cells and also in the intracellular compartment of activated peripheral B cells. These data shows that alternative splicing of the human TACI gene leads to two isoforms both of which intersect with MyD88 and TRAF6 and form complexes with TLR9, but the two isoforms have different ligand binding capacities, subcellular locations and activation capabilities.

mTOR intersects antibody-inducing signals from TACI in marginal zone B cells.

Mechanistic target of rapamycin (mTOR) enhances immunity in addition to orchestrating metabolism. Here we show that mTOR coordinates immunometabolic reconfiguration of marginal zone (MZ) B cells, a pre-activated lymphocyte subset that mounts antibody responses to T-cell-independent antigens through a Toll-like receptor (TLR)-amplified pathway involving transmembrane activator and CAML interactor (TACI). This receptor interacts with mTOR via the TLR adapter MyD88. The resulting mTOR activation instigates MZ B-cell proliferation, immunoglobulin G (IgG) class switching, and plasmablast differentiation through a rapamycin-sensitive pathway that integrates metabolic and antibody-inducing transcription programs, including NF-κB. Disruption of TACI-mTOR interaction by rapamycin, truncation of the MyD88-binding domain of TACI, or B-cell-conditional mTOR deficiency interrupts TACI signaling via NF-κB and cooperation with TLRs, thereby hampering IgG production to T-cell-independent antigens but not B-cell survival. Thus, mTOR drives innate-like antibody responses by linking proximal TACI signaling events with distal immunometabolic transcription programs.

Expansion of inflammatory innate lymphoid cells in patients with common variable immune deficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is an antibody deficiency treated with immunoglobulin; however, patients can have noninfectious inflammatory conditions that lead to heightened morbidity and mortality. OBJECTIVES: Modular analyses of RNA transcripts in whole blood previously identified an upregulation of many interferon-responsive genes. In this study we sought the cell populations leading to this signature. METHODS: Lymphoid cells were measured in peripheral blood of 55 patients with CVID (31 with and 24 without inflammatory/autoimmune complications) by using mass cytometry and flow cytometry. Surface markers, cytokines, and transcriptional characteristics of sorted innate lymphoid cells (ILCs) were defined by using quantitative PCR. Gastrointestinal and lung biopsy specimens of subjects with inflammatory disease were stained to seek ILCs in tissues. RESULTS: The linage-negative, CD127(+), CD161(+) lymphoid population containing T-box transcription factor, retinoic acid-related orphan receptor (ROR) γt, IFN-γ, IL-17A, and IL-22, all hallmarks of type 3 innate lymphoid cells, were expanded in the blood of patients with CVID with inflammatory conditions (mean, 3.7% of PBMCs). ILCs contained detectable amounts of the transcription factors inhibitor of DNA binding 2, T-box transcription factor, and RORγt and increased mRNA transcripts for IL-23 receptor (IL-23R) and IL-26, demonstrating inflammatory potential. In gastrointestinal and lung biopsy tissues of patients with CVID, numerous IFN-γ(+)RORγt(+)CD3(-) cells were identified, suggesting a role in these mucosal inflammatory states. CONCLUSIONS: An expansion of this highly inflammatory ILC population is a characteristic of patients with CVID with inflammatory disease; ILCs and the interferon signature are markers for the uncontrolled inflammatory state in these patients.

Differential induction of plasma cells by isoforms of human TACI.

Subjects with common variable immune deficiency may have mutations in transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI). Unlike the murine gene, human TACI undergoes alternative messenger (m)RNA splicing to produce isoforms with 1 or 2 ligand-binding domains. Because both isoforms are found in human B cells, we compared their functions in transduced murine B and human pre-B cells. Although murine cells and pre-B cells transduced with the long TACI isoform retained surface CD19 and immunoglobulin G, cells transduced with the short TACI isoform completely lost these B-cell characteristics. Expression of the short TACI isoform produced intense nuclear factor κB activation, nuclear p65 translocation, and colocalization with myeloid differentiation factor 88 and calcium-modulating cyclophilin ligand. The short TACI-transduced cells became larger and CD138 positive, demonstrated upregulated BLIMP1 and XBP1 mRNA, and acquired the morphology of plasma cells. In contrast, cells bearing the long isoform had significantly less BLIMP1 and XBP1 mRNA and, for human pre-B cells, remained CD138 negative. Although human B cells express both isoforms, the short isoform predominates in CD27(+) B cells, toll-like receptor 9-activated peripheral B cells, and splenic marginal zone B cells. Although the transcriptional controls for alternative splicing of isoforms remain unknown, differential signals via isoforms may control plasma-cell generation in humans.

IL-15 expression on RA synovial fibroblasts promotes B cell survival.

INTRODUCTION: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival. METHODS: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib. RESULTS: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures. CONCLUSION: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

A dual action of rheumatoid arthritis synovial fibroblast IL-15 expression on the equilibrium between CD4+CD25+ regulatory T cells and CD4+CD25- responder T cells.

We previously described that fibroblast-like cells from the synovium of rheumatoid arthritis patients (RASFib) constitutively express intracellular and surface IL-15, which induces activation of cocultured T cells. Our objective was to study the effect of RASFib IL-15 expression on the function of human CD4(+)CD25(+) regulatory T cells (Treg). RASFib, through their constitutive IL-15 expression, were able to induce the proliferation of human Tregs stimulated through their TCR, and at the same time potentiated their suppressive action on the cytokine secretion of CD4(+)CD25(-) responder T cells (Tresp). In parallel, constitutive RASFib IL-15 expression mediated an up-regulated response of Tresp. Subsequently, total CD4(+) T cells, containing natural proportions of Treg and Tresp, secreted an increased amount of pathogenic cytokines when cocultured with RASFib despite the presence of proliferating Treg with superior regulatory potency. In summary, RASFib IL-15 exerts a dual action on the equilibrium between Treg and Tresp by potentiating the suppressive effect of Treg while augmenting the proinflammatory action of Tresp; the result is a shift of the Treg/Tresp balance toward a proinflammatory state. This alteration of the Treg/Tresp equilibrium is not observed in the presence of osteoarthritis synovial fibroblasts or dermal fibroblasts, which do not constitutively express surface IL-15. Additionally, Treg with superior suppressive potency were present in the peripheral blood and the synovial fluid of RA patients, but this enhanced immunoregulatory activity was not able to overcome the increased secretion of pathogenic cytokines by RA-Tresp, indicating that rheumatoid arthritis patients demonstrate an altered Treg/Tresp equilibrium in vivo.