Regulatory strategies limiting endosomal Toll-like receptor activation in B cells.

The recognition of pathogen-associated nucleic acid (NA) promotes effective immunity against invading pathogens. However, endosomal Toll-like receptor (TLR) activation by self-NA also underlies the pathogenesis of systemic autoimmune diseases, such as systemic lupus erythematosus (SLE). For this reason, the activation thresholds of NA-sensing TLRs must be tightly regulated to balance protective and pathogenic immune responses. In this study, we will provide an overview of the evolutionary mechanisms designed to limit the aberrant activation of endosomal TLRs by self-ligands, focusing on four broad strategies. These include the following: 1) the production of nucleases able to degrade self-DNA and RNA; 2) the cell-specific regulation of endosomal TLR expression; 3) the spatial and temporal control of TLR positioning at a sub-cellular level; and 4) the modulation of downstream TLR signaling cascades. Given the critical role of B cells in lupus pathogenesis, where possible, we will describe evidence for B cell-specific induction of these regulatory mechanisms. We will also highlight our own work showing how modulation of B cell endolysosomal flux tunes NA-sensing TLR activation signals. In the face of inevitable generation of self-NA during normal cellular turnover, these parallel mechanisms are vital to protect against pathogenic inflammation.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells.

Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17(+) B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17(+) B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

Cutting Edge: Systemic Autoimmunity in Murine STAT3 Gain-of-Function Syndrome Is Characterized by Effector T Cell Expansion in the Absence of Overt Regulatory T Cell Dysfunction.

Germline gain-of-function mutations in the transcriptional factor STAT3 promote early-onset multisystemic autoimmunity. To investigate how increased STAT3 promotes systemic inflammation, we generated a transgenic knock-in strain expressing a pathogenic human mutation STAT3K392R within the endogenous murine locus. As predicted, STAT3K392R mice develop progressive lymphoid hyperplasia and systemic inflammation, mirroring the human disease. However, whereas the prevailing model holds that increased STAT3 activity drives human autoimmunity by dysregulating the balance between regulatory T cells and Th17 cell differentiation, we observed increased Th17 cells in the absence of major defects in regulatory T cell differentiation or function. In addition, STAT3K392R animals exhibited a prominent accumulation of IFN-γ-producing CD4+ and CD8+ T cells. Together, these data provide new insights into this complex human genetic syndrome and highlight the diverse cellular mechanisms by which dysregulated STAT3 activity promotes breaks in immune tolerance.

Anti-HLA antibodies in recipients of CD19 versus BCMA-targeted CAR T-cell therapy.

Antibodies against foreign human leukocyte antigen (HLA) molecules are barriers to successful organ transplantation. B cell-depleting treatments are used to reduce anti-HLA antibodies but have limited efficacy. We hypothesized that the primary source for anti-HLA antibodies is long-lived plasma cells, which are ineffectively targeted by B cell depletion. To study this, we screened for anti-HLA antibodies in a prospectively enrolled cohort of 49 patients who received chimeric antigen receptor T-cell therapy (CARTx), targeting naïve and memory B cells (CD19-targeted, n = 21) or plasma cells (BCMA-targeted, n = 28) for hematologic malignancies. Longitudinal samples were collected before and up to 1 year after CARTx. All individuals were in sustained remission. We identified 4 participants with anti-HLA antibodies before CD19-CARTx. Despite B cell depletion, anti-HLA antibodies and calculated panel reactive antibody scores were stable for 1 year after CD19-CARTx. Only 1 BCMA-CARTx recipient had pre-CARTx low-level anti-HLA antibodies, with no follow-up samples available. These data implicate CD19neg long-lived plasma cells as an important source for anti-HLA antibodies, a model supported by infrequent HLA sensitization in BCMA-CARTx subjects receiving previous plasma cell-targeted therapies. Thus, plasma cell-targeted therapies may be more effective against HLA antibodies, thereby enabling improved access to organ transplantation and rejection management.

Cutting Edge: A Threshold of B Cell Costimulatory Signals Is Required for Spontaneous Germinal Center Formation in Autoimmunity.

Cognate interactions between autoreactive B and T cells promote systemic lupus erythematosus pathogenesis by inter alia facilitating spontaneous germinal center (GC) formation. Whereas both myeloid and B cell APCs express B7 ligands (CD80 and CD86), the prevailing model holds that dendritic cell costimulation is sufficient for CD28-dependent T cell activation. In this study, we report that B cell-intrinsic CD80/CD86 deletion unexpectedly abrogates GCs in murine lupus. Interestingly, absent GCs differentially impacted serum autoantibodies. In keeping with distinct extrafollicular and GC activation pathways driving lupus autoantibodies, lack of GCs correlated with loss of RNA-associated autoantibodies but preserved anti-dsDNA and connective tissue autoantibody titers. Strikingly, even heterozygous B cell CD80/CD86 deletion was sufficient to prevent autoimmune GCs and RNA-associated autoantibodies. Together, these findings identify a key mechanism whereby B cells promote lupus pathogenesis by providing a threshold of costimulatory signals required for autoreactive T cell activation.

Recent advances in immunotherapies for lupus nephritis.

Childhood-onset systemic lupus erythematosus (SLE) is characterized by increased rates of kidney involvement, termed lupus nephritis. Despite the significant morbidity and mortality associated with this disease, lupus nephritis trials have been plagued by repeated failures to meet clinical endpoints. However, improvements in trial design and the development of targeted approaches have begun to yield promising results, including two new FDA-approved lupus nephritis treatments since 2020. These include belimumab, a monoclonal antibody targeting the B cell survival cytokine BAFF (B cell activating factor), and voclosporin, a cyclosporin analog with improved pharmacokinetic characteristics. In this review, we will summarize the data supporting regulatory approval for these agents in lupus nephritis and highlight ongoing clinical trials targeting the diverse immunologic drivers of renal inflammation in SLE. While pediatric patients remain underrepresented in lupus clinical trials, given the increased severity of childhood-onset SLE and need for long-term protection from kidney damage, we anticipate the need for off-label use of these targeted therapies in the pediatric population. Future studies are needed to define optimal patient selection, drug combinations, and treatment duration in pediatric lupus nephritis.

BAFF inhibition in SLE-Is tolerance restored?

The B cell activating factor (BAFF) inhibitor, belimumab, is the first biologic drug approved for the treatment of SLE, and exhibits modest, but durable, efficacy in decreasing disease flares and organ damage. BAFF and its homolog APRIL are TNF-like cytokines that support the survival and differentiation of B cells at distinct developmental stages. BAFF is a crucial survival factor for transitional and mature B cells that acts as rheostat for the maturation of low-affinity autoreactive cells. In addition, BAFF augments innate B cell responses via complex interactions with the B cell receptor (BCR) and Toll like receptor (TLR) pathways. In this manner, BAFF impacts autoreactive B cell activation via extrafollicular pathways and fine tunes affinity selection within germinal centers (GC). Finally, BAFF and APRIL support plasma cell survival, with differential impacts on IgM- and IgG-producing populations. Therapeutically, BAFF and combined BAFF/APRIL inhibition delays disease onset in diverse murine lupus strains, although responsiveness to BAFF inhibition is model dependent, in keeping with heterogeneity in clinical responses to belimumab treatment in humans. In this review, we discuss the mechanisms whereby BAFF/APRIL signals promote autoreactive B cell activation, discuss whether altered selection accounts for therapeutic benefits of BAFF inhibition, and address whether new insights into BAFF/APRIL family complexity can be exploited to improve human lupus treatments.

Lupus nephritis transcriptomics across space and time.

Current immunosuppression regimens for lupus nephritis are incompletely effective, placing patients at risk for poor long-term outcomes. This emphasizes the need to dissect pathogenic mechanisms in lupus nephritis, to inform the development of targeted therapies. In this issue of Kidney International, Parikh et al. performed transcriptomic analysis of pretreatment and posttreatment protocol kidney biopsies, segregated into glomerular and tubulointerstitial compartments, to identify candidate molecular pathways distinguishing treatment responders and nonresponders.

TACI haploinsufficiency protects against BAFF-driven humoral autoimmunity in mice.

Polymorphisms in TACI, a BAFF family cytokine receptor, are linked to diverse human immune disorders including common variable immunodeficiency (CVID) and systemic lupus erythematosus (SLE). Functional studies of individual variants show modest impacts on surface TACI expression and/or downstream signal transduction, indicating that relatively subtle variation in TACI activity can impact human B-cell biology. However, significant complexity underlies TACI biology, including both positive and negative regulation of physiologic and pathogenic B-cell responses. To model these contradictory events, we compared the functional impact of TACI deletion on separate models of murine SLE driven by T cell-independent and -dependent breaks in B-cell tolerance. First, we studied whether reduced surface TACI expression was sufficient to protect against progressive BAFF-mediated systemic autoimmunity. Strikingly, despite a relatively modest impact on surface TACI levels, TACI haploinsufficiency markedly reduced pathogenic RNA-associated autoantibody titers and conferred long-term protection from BAFF-driven lupus nephritis. In contrast, B cell-intrinsic TACI deletion exerted a limited impact of autoantibody generation in murine lupus characterized by spontaneous germinal center formation and T cell-dependent humoral autoimmunity. Together, these combined data provide new insights into TACI biology and highlight how TACI signals must be tightly regulated during protective and pathogenic B-cell responses.

Functional Characterization of CD11c+ Age-Associated B Cells as Memory B Cells.

Age-associated B cells (ABCs) are a unique subset of B cells defined by surface CD11b and CD11c expression. Although ABC expansion has been observed in both human and animal studies in the setting of advanced age, during humoral autoimmunity and following viral infection, the functional properties of this cellular subset remain incompletely defined. In the current study, we demonstrate that ABCs fulfill the criteria for memory B cells (MBCs), based on evidence of Ag-dependent expansion and persistence in a state poised for rapid differentiation into Ab-secreting plasma cells during secondary responses. First, we show that a majority of ABCs are not actively cycling but exhibit an extensive replication history consistent with prior Ag engagement. Second, despite unswitched surface IgM expression, ABCs show evidence of activation-induced cytidine deaminase (AID)-dependent somatic hypermutation. Third, BCRs cloned from sorted ABCs exhibit broad autoreactivity and polyreactivity. Although the overall level of ABC self-reactivity was not increased relative to naive B cells, ABCs lacked features of functional anergy characteristic of autoreactive B cells. Fourth, ABCs express MBC surface markers consistent with being poised for rapid plasma cell differentiation during recall responses. Finally, in a murine model of viral infection, adoptively transferred CD11c+ B cells rapidly differentiated into class-switched Ab-secreting cells upon Ag rechallenge. In summary, we phenotypically and functionally characterize ABCs as IgM-expressing MBCs, findings that together implicate ABCs in the pathogenesis of systemic autoimmunity.

Generation of functional murine CD11c+ age-associated B cells in the absence of B cell T-bet expression.

Age-associated B cells (ABC), a novel subset of activated B cells defined by CD11b and CD11c expression, have been linked with both protective anti-viral responses and the pathogenesis of systemic autoimmunity. Expression of the TH 1 lineage transcription factor T-bet has been identified as a defining feature of ABC biology, with B cell-intrinsic expression of this transcription factor proposed to be required for ABC formation. In contrast to this model, we report that Tbx21 (encoding T-bet)-deficient B cells upregulate CD11b and CD11c surface expression in vitro in response to integrated TLR and cytokine signals. Moreover, B cell-intrinsic T-bet deletion in a murine lupus model exerted no impact of ABC generation in vivo, with Tbx21-/- ABCs exhibiting an identical surface phenotype to wild-type (WT) ABCs. Importantly, WT and Tbx21-/- ABCs sorted from autoimmune mice produced equivalent amounts of IgM and IgG ex vivo following TLR stimulation, indicating that T-bet-deficient ABCs are likely functional in vivo. In summary, our data contradict the established literature by demonstrating that T-bet expression is not uniformly required for ABC generation.

Integrated B Cell, Toll-like, and BAFF Receptor Signals Promote Autoantibody Production by Transitional B Cells.

The B cell survival cytokine BAFF has been linked with the pathogenesis of systemic lupus erythematosus (SLE). BAFF binds distinct BAFF-family surface receptors, including the BAFF-R and transmembrane activator and CAML interactor (TACI). Although originally characterized as a negative regulator of B cell activation, TACI signals are critical for class-switched autoantibody (autoAb) production in BAFF transgenic mice. Consistent with this finding, a subset of transitional splenic B cells upregulate surface TACI expression and contribute to BAFF-driven autoAb. In the current study, we interrogated the B cell signals required for transitional B cell TACI expression and Ab production. Surprisingly, despite established roles for dual BCR and TLR signals in autoAb production in SLE, signals downstream of these receptors exerted distinct impacts on transitional B cell TACI expression and autoAb titers. Whereas loss of BCR signals prevented transitional B cell TACI expression and resulted in loss of serum autoAb across all Ig isotypes, lack of TLR signals exerted a more limited impact restricted to autoAb class-switch recombination without altering transitional B cell TACI expression. Finally, in parallel with the protective effect of TACI deletion, loss of BAFF-R signaling also protected against BAFF-driven autoimmunity. Together, these findings highlight how multiple signaling pathways integrate to promote class-switched autoAb production by transitional B cells, events that likely impact the pathogenesis of SLE and other BAFF-dependent autoimmune diseases.

Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells.

The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.

TACI deletion protects against progressive murine lupus nephritis induced by BAFF overexpression.

B cells are known to promote the pathogenesis of systemic lupus erythematosus (SLE) via the production of pathogenic anti-nuclear antibodies. However, the signals required for autoreactive B cell activation and the immune mechanisms whereby B cells impact lupus nephritis pathology remain poorly understood. The B cell survival cytokine B cell activating factor of the TNF Family (BAFF) has been implicated in the pathogenesis of SLE and lupus nephritis in both animal models and human clinical studies. Although the BAFF receptor has been predicted to be the primary BAFF family receptor responsible for BAFF-driven humoral autoimmunity, in the current study we identify a critical role for signals downstream of Transmembrane Activator and CAML Interactor (TACI) in BAFF-dependent lupus nephritis. Whereas transgenic mice overexpressing BAFF develop progressive membranoproliferative glomerulonephritis, albuminuria and renal dysfunction, TACI deletion in BAFF-transgenic mice provided long-term (about 1 year) protection from renal disease. Surprisingly, disease protection in this context was not explained by complete loss of glomerular immune complex deposits. Rather, TACI deletion specifically reduced endocapillary, but not mesangial, immune deposits. Notably, although excess BAFF promoted widespread breaks in B cell tolerance, BAFF-transgenic antibodies were enriched for RNA- relative to DNA-associated autoantigen reactivity. These RNA-associated autoantibody specificities were specifically reduced by TACI or Toll-like receptor 7 deletion. Thus, our study provides important insights into the autoantibody specificities driving proliferative lupus nephritis, and suggests that TACI inhibition may be novel and effective treatment strategy in lupus nephritis.

Cutting Edge: BAFF Overexpression Reduces Atherosclerosis via TACI-Dependent B Cell Activation.

Patients with systemic lupus erythematosus exhibit accelerated atherosclerosis, a chronic inflammatory disease of the arterial wall. The impact of B cells in atherosclerosis is controversial, with both protective and pathogenic roles described. For example, natural IgM binding conserved oxidized lipid epitopes protect against atherosclerosis, whereas anti-oxidized low-density lipoprotein (oxLDL) IgG likely promotes disease. Because BAFF promotes B cell class-switch recombination and humoral autoimmunity, we hypothesized that excess BAFF would accelerate atherosclerosis. In contrast, BAFF overexpression markedly reduced hypercholesterolemia and atherosclerosis in hyperlipidemic mice. BAFF-mediated atheroprotection required B cells and was associated with increased protective anti-oxLDL IgM. Surprisingly, high-titer anti-oxLDL IgM production and reduced atherosclerosis was dependent on the BAFF family receptor transmembrane activator and CAML interactor. In summary, we identified a novel role for B cell-specific, BAFF-dependent transmembrane activator and CAML interactor signals in atherosclerosis pathogenesis, of particular relevance to the use of BAFF-targeted therapies in systemic lupus erythematosus.

Cutting Edge: BAFF Promotes Autoantibody Production via TACI-Dependent Activation of Transitional B Cells.

Mice overexpressing B cell activating factor of the TNF family (BAFF) develop systemic autoimmunity characterized by class-switched anti-nuclear Abs. Transmembrane activator and CAML interactor (TACI) signals are critical for BAFF-mediated autoimmunity, but the B cell developmental subsets undergoing TACI-dependent activation in settings of excess BAFF remain unclear. We report that, although surface TACI expression is usually limited to mature B cells, excess BAFF promotes the expansion of TACI-expressing transitional B cells. TACI(+) transitional cells from BAFF-transgenic mice are characterized by an activated, cycling phenotype, and the TACI(+) cell subset is specifically enriched for autoreactivity, expresses activation-induced cytidine deaminase and T-bet, and exhibits evidence of somatic hypermutation. Consistent with a potential contribution to BAFF-mediated humoral autoimmunity, TACI(+) transitional B cells from BAFF-transgenic mice spontaneously produce class-switched autoantibodies ex vivo. These combined findings highlight a novel mechanism through which BAFF promotes humoral autoimmunity via direct, TACI-dependent activation of transitional B cells.

B-cell intrinsic TLR7 signals promote depletion of the marginal zone in a murine model of Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) exhibit prominent defects in splenic marginal zone (MZ), resulting in abnormal T-cell-independent antibody responses and increased bacterial infections. B-cell-intrinsic deletion of the affected gene WAS protein (WASp) markedly reduces splenic MZ B cells, without impacting the rate of MZ B-cell development, suggesting that abnormal B-cell retention within the MZ accounts for MZ defects in WAS. Since WASp regulates integrin-dependent actin cytoskeletal rearrangement, we previously hypothesized that defective B-cell integrin function promotes MZ depletion. In contrast, we now report that B-cell-intrinsic deletion of the TLR signaling adaptor MyD88 is sufficient to restore the MZ in WAS. We further identify TLR7, an endosomal single-stranded RNA (ssRNA) receptor, as the MyD88-dependent receptor responsible for WAS MZ depletion. These findings implicate spontaneous activation of MZ B cells by ssRNA-containing self-ligands (likely derived from circulating apoptotic material) as the mechanism underlying MZ depletion in WAS. Together, these data suggest a previously unappreciated role for B-cell intrinsic TLR signals in MZ homeostasis, of relevance to both pathogen responses and to the development of systemic autoimmunity.

Opposing impact of B cell-intrinsic TLR7 and TLR9 signals on autoantibody repertoire and systemic inflammation.

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by autoantibodies targeting nucleic acid-associated Ags. The endosomal TLRs TLR7 and TLR9 are critical for generation of Abs targeting RNA- or DNA-associated Ags, respectively. In murine lupus models, deletion of TLR7 limits autoimmune inflammation, whereas deletion of TLR9 exacerbates disease. Whether B cell or myeloid TLR7/TLR9 signaling is responsible for these effects has not been fully addressed. In this study, we use a chimeric strategy to evaluate the effect of B cell-intrinsic deletion of TLR7 versus TLR9 in parallel lupus models. We demonstrate that B cell-intrinsic TLR7 deletion prevents RNA-associated Ab formation, decreases production of class-switched Abs targeting nonnuclear Ags, and limits systemic autoimmunity. In contrast, B cell-intrinsic TLR9 deletion results in decreased DNA-reactive Ab, but increased Abs targeting a broad range of systemic autoantigens. Further, we demonstrate that B cell-intrinsic TLR9 deletion results in increased systemic inflammation and immune complex glomerulonephritis, despite intact TLR signaling within the myeloid compartment. These data stress the critical importance of dysregulated B cell-intrinsic TLR signaling in the pathogenesis of systemic lupus erythematosus.

Cutting edge: regulation of TLR4-driven B cell proliferation by RP105 is not B cell autonomous.

Mechanistic understanding of RP105 has been confounded by the fact that this TLR homolog has appeared to have opposing, cell type-specific effects on TLR4 signaling. Although RP105 inhibits TLR4-driven signaling in cell lines and myeloid cells, impaired LPS-driven proliferation by B cells from RP105(-/-) mice has suggested that RP105 facilitates TLR4 signaling in B cells. In this article, we show that modulation of B cell proliferation by RP105 is not a function of B cell-intrinsic expression of RP105, and identify a mechanistic role for dysregulated BAFF expression in the proliferative abnormalities of B cells from RP105(-/-) mice: serum BAFF levels are elevated in RP105(-/-) mice, and partial BAFF neutralization rescues aberrant B cell proliferative responses in such mice. These data indicate that RP105 does not have dichotomous effects on TLR4 signaling and emphasize the need for caution in interpreting the results of global genetic deletion.