Holly M. Jacobs

B cell IFN-γ receptor signaling promotes autoimmune germinal centers via cell-intrinsic induction of BCL-6.

Jackson et al. propose a role for B cell–intrinsic IFN-γ receptor signaling in spontaneous germinal center activation and autoantibody production.

Altered BCR and TLR signals promote enhanced positive selection of autoreactive transitional B cells in Wiskott-Aldrich syndrome.

Kolhatkar et al. report that altered BCR and TLR signaling orchestrates increased positive selection of transitional B cells expressing low-affinity self-reactive BCRs, leading to their enrichment within the naive B cell compartment. These findings have important implications to understand events that promote altered B cell selection in both Wiskott-Aldrich syndrome patients and in other autoimmune-prone individuals.

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–derived IL-6 initiates spontaneous germinal center formation during systemic autoimmunity

Recent studies have identified critical roles for B cells in triggering autoimmune germinal centers (GCs) in systemic lupus erythematosus (SLE) and other disorders. The mechanisms whereby B cells facilitate loss of T cell tolerance, however, remain incompletely defined. Activated B cells produce interleukin 6 (IL-6), a proinflammatory cytokine that promotes T follicular helper (TFH) cell differentiation. Although B cell IL-6 production correlates with disease severity in humoral autoimmunity, whether B cell–derived IL-6 is required to trigger autoimmune GCs has not, to our knowledge, been addressed. Here, we report the unexpected finding that a lack of B cell–derived IL-6 abrogates spontaneous GC formation in mouse SLE, resulting in loss of class-switched autoantibodies and protection from systemic autoimmunity. Mechanistically, B cell IL-6 production was enhanced by IFN-&ggr;, consistent with the critical roles for B cell–intrinsic IFN-&ggr; receptor signals in driving autoimmune GC formation. Together, these findings identify a key mechanism whereby B cells drive autoimmunity via local IL-6 production required for TFH differentiation and autoimmune GC formation.

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

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.

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.

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.

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 TH1 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.

AI-18 B cell IFN-γ receptor signalling promotes autoimmune germinal centresvia cell-intrinsic induction of BCL-6

Background Dysregulated germinal centrecenter (GC) responses are implicated in the pathogenesis of human autoimmune diseases, including systemic lupus erythematosus (SLE). Although type 1 interferons (IFNs) are most frequently associated with lupus pathogenesis, type 2 interferon (IFN-γ) has also been shown to promote SLE. However, the respective impacts of these cytokines in promoting B cell activation during humoral autoimmunity have not been addressed. Materials and methods We recently developed a chimeric murine lupus model in which Wiskott-Aldrich syndrome protein (WAS)-deficient B cells promote spontaneous humoral autoimmunity (Jackson, et al. J Immunol 2014). An important advantage of the WAS chimaera model is that dysregulated immune responses are limited to the B cell compartment, allowing genetic manipulation in a B cell-intrinsic fashion. In the current study, we contrast the impact B cell-intrinsic type 1 IFN vs. IFN-γ signals on autoimmune GC formation and the pathogenesis of SLE. Results Although type 1 IFN prominently enhanced B cell responses in vitro, B cell-intrinsic IFNAR deletion exerted surprisingly minimal impacts on class-switched autoantibody titers and spontaneous GC formation in vivo. This finding suggested that other cytokines promote B cell activation in the WAS chimaera model. Notably, B cells directly initiated CD4+ T cell activation and T follicular helper cell formation via MHC Class II (MHC-II)-dependent antigen presentation. In addition, activated T cells exhibited prominent IFN-γ production that was lost following B cell-intrinsic MHC-II deletion, suggesting a direct role for IFN-γ in promoting autoimmune GC formation. Strikingly, B cell-intrinsic deletion of the IFN-γ receptor was sufficient to abrogate spontaneous GCs, class-switched autoantibodies and systemic autoimmunity. Mechanistically, although IFN-γ receptor signals increased B cell T-bet expression, B cell-intrinsic deletion of T-bet exerted an isolated impact on class-switch recombination to pathogenic IgG2c autoantibody subclasses without impacting GC development. Rather, in both murine and human B cells, IFN-γ synergized with BCR, TLR and/or CD40 activation signals to promote cell-intrinsic BCL-6 expression. Finally, IFN-γ driven BCL-6 expression in B cells was blocked using clinically-relevant Janus kinase inhibitors, ruxolitinib and tofacitinib. Conclusions Our study demonstrates that B cell-intrinsic IFN-γ receptor signals promote lupus pathogenesis via formation of spontaneous, autoimmune GCs. In addition, we have uncovered a novel cell-intrinsic program whereby IFN-γ, together with BCR-, TLR- and/or CD40 signals, orchestrates B cell expression of the GC master transcription regulator BCL-6. Our combined findings suggest that this IFN-γ signalling program may be a potential therapeutic target in SLE. Acknowledgements This work was supported by the National Institutes of Health under award numbers: R01HL075453 (DJR), R01AI084457 (DJR), R01AI071163 (DJR), DP3DK097672 (DJR) and K08AI112993 (SWJ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional support provided by the Benaroya Family Gift Fund (DJR); by the ACR REF Rheumatology Scientist Development Award (SWJ); and by the Arnold Lee Smith Endowed Professorship for Research Faculty Development (SWJ).