Aberrant B cell receptor signaling in circulating naïve and IgA+ memory B cells from newly-diagnosed autoantibody-positive rheumatoid arthritis patients.

OBJECTIVE: Altered B cell receptor (BCR) signaling has been implicated in the pathogenesis of rheumatoid arthritis (RA). Here we aimed to identify signaling aberrations in autoantibody-positive and autoantibody-negative RA patients by performing a comprehensive analysis of the BCR signaling cascade in different B cell subsets. METHODS: We first optimized phosphoflow cytometry for an in-depth analysis of BCR signaling across immunoglobulin isotypes in healthy donors. Subsequently, we compared BCR signaling in circulating B cell subsets from treatment-naïve, newly-diagnosed autoantibody-positive RA and autoantibody-negative RA patients and healthy controls (HCs). RESULTS: We observed subset-specific phosphorylation patterns of the BCR signalosome in circulating B cells from healthy donors. Compared with HCs, autoantibody-positive RA patients displayed enhanced responses to BCR stimulation for multiple signaling proteins, specifically in naïve and IgA+ memory B cells. Whereas in unstimulated healthy donor B cells, the phosphorylation status of individual signaling proteins showed only limited correlation, BCR stimulation enhanced the interconnectivity in phosphorylation within the BCR signalosome. However, this strong interconnectivity within the BCR signalosome in stimulated B cells from HCs was lost in RA, especially in autoantibody-positive RA patients. Finally, we observed strong correlations between SYK and BTK protein expression, and IgA and IgG anti-citrullinated protein antibody concentrations in serum from autoantibody-positive RA patients. CONCLUSION: Collectively, the isotype-specific analysis of multiple key components of the BCR signalosome identified aberrant BCR signaling responses in treatment-naïve autoantibody-positive RA patients, particularly in naïve B cells and IgA+ memory B cells. Our findings support differential involvement of dysregulated BCR signaling in the pathogenesis of autoantibody-positive and autoantibody-negative RA.

Increased proportions of circulating PD-1+ CD4+ memory T cells and PD-1+ regulatory T cells associate with good response to prednisone in pulmonary sarcoidosis.

BACKGROUND: The treatment response to corticosteroids in patients with sarcoidosis is highly variable. CD4+ T cells are central in sarcoid pathogenesis and their phenotype in peripheral blood (PB) associates with disease course. We hypothesized that the phenotype of circulating T cells in patients with sarcoidosis may correlate with the response to prednisone treatment. Therefore, we aimed to correlate frequencies and phenotypes of circulating T cells at baseline with the pulmonary function response at 3 and 12 months during prednisone treatment in patients with pulmonary sarcoidosis. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell populations in 22 treatment-naïve patients and 21 healthy controls (HCs). Pulmonary function tests at baseline, 3 and 12 months were used to measure treatment effect. RESULTS: Patients with sarcoidosis showed an absolute forced vital capacity (FVC) increase of 14.2% predicted (± 10.6, p < 0.0001) between baseline and 3 months. Good response to prednisone (defined as absolute FVC increase of ≥ 10% predicted) was observed in 12 patients. CD4+ memory T cells and regulatory T cells from patients with sarcoidosis displayed an aberrant phenotype at baseline, compared to HCs. Good responders at 3 months had significantly increased baseline proportions of PD-1+CD4+ memory T cells and PD-1+ regulatory T cells, compared to poor responders and HCs. Moreover, decreased fractions of CD25+ cells and increased fractions of PD-1+ cells within the CD4+ memory T cell population correlated with ≥ 10% FVC increase at 12 months. During treatment, the aberrantly activated phenotype of memory and regulatory T cells reversed. CONCLUSIONS: Increased proportions of circulating PD-1+CD4+ memory T cells and PD-1+ regulatory T cells and decreased proportions of CD25+CD4+ memory T cells associate with good FVC response to prednisone in pulmonary sarcoidosis, representing promising new blood biomarkers for prednisone efficacy. TRIAL REGISTRATION: NL44805.078.13.

Circulating T cells in sarcoidosis have an aberrantly activated phenotype that correlates with disease outcome.

RATIONALE: Disease course in sarcoidosis is highly variable. Bronchoalveolar lavage fluid and mediastinal lymph nodes show accumulation of activated T cells with a T-helper (Th)17.1 signature, which correlates with non-resolving sarcoidosis. We hypothesize that the peripheral blood (PB) T cell phenotype may correlate with outcome. OBJECTIVES: To compare frequencies, phenotypes and function of circulating T cell populations in sarcoidosis patients with healthy controls (HCs) and correlate these parameters with outcome. METHODS: We used multi-color flow cytometry to quantify activation marker expression on PB T cell subsets in treatment-naïve patients and HCs. The disease course was determined after 2-year follow-up. Cytokine production was measured after T cell stimulation in vitro. MEASUREMENTS AND MAIN RESULTS: We observed significant differences between patients and HCs in several T cell populations, including CD8+ and CD4+ T cells, Th1/Th17 subsets, CD4+ T memory stem cells, regulatory T cells (Tregs) and γδ T cells. Decreased frequencies of CD4+ T cells and increased frequencies of Tregs and CD8+ γδ T cells correlated with worse outcome. Naïve CD4+ T cells displayed an activated phenotype with increased CD25 expression in patients with active chronic disease at 2-year follow-up. A distinctive Treg phenotype with increased expression of CD25, CTLA4, CD69, PD-1 and CD95 correlated with chronic sarcoidosis. Upon stimulation, both naïve and memory T cells displayed a different cytokine profile in sarcoidosis compared to HCs. CONCLUSIONS: Circulating T cell subpopulations of sarcoidosis patients display phenotypic abnormalities that correlate with disease outcome, supporting a critical role of aberrant T cell activation in sarcoidosis pathogenesis.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Bruton's tyrosine kinase inhibition induces rewiring of proximal and distal B-cell receptor signaling in mice.

Bruton's tyrosine kinase (Btk) is a crucial signaling molecule in BCR signaling and a key regulator of B- cell differentiation and function. Btk inhibition has shown impressive clinical efficacy in various B-cell malignancies. However, it remains unknown whether inhibition additionally induces changes in BCR signaling due to feedback mechanisms, a phenomenon referred to as BCR rewiring. In this report, we studied the impact of Btk activity on major components of the BCR signaling pathway in mice. As expected, NF-κB and Akt/S6 signaling was decreased in Btk-deficient B cells. Unexpectedly, phosphorylation of several proximal signaling molecules, including CD79a, Syk, and PI3K, as well as the key Btk-effector PLCγ2 and the more downstream kinase Erk, were significantly increased. This pattern of BCR rewiring was essentially opposite in B cells from transgenic mice overexpressing Btk. Importantly, prolonged Btk inhibitor treatment of WT mice or mice engrafted with leukemic B cells also resulted in increased phosho-CD79a and phospho-PLCγ2 in B cells. Our findings show that Btk enzymatic function determines phosphorylation of proximal and distal BCR signaling molecules in B cells. We conclude that Btk inhibitor treatment results in rewiring of BCR signaling, which may affect both malignant and healthy B cells.

Phosphoflow Protocol for Signaling Studies in Human and Murine B Cell Subpopulations.

BCR signaling, involving phosphorylation of various downstream molecules, including kinases, lipases, and linkers, is crucial for B cell selection, survival, proliferation, and differentiation. Phosphoflow cytometry (phosphoflow) is a single-cell-based technique to measure phosphorylated intracellular proteins, providing a more quantitative read-out than Western blotting. Recent advances in phosphoflow basically allow simultaneous analysis of protein phosphorylation in B cell (sub)populations, without prior cell sorting. However, fixation and permeabilization procedures required for phosphoflow often affect cell surface epitopes or mAb conjugates, precluding the evaluation of the phosphorylation status of signaling proteins across different B cell subpopulations present in a single sample. In this study, we report a versatile phosphoflow protocol allowing extensive staining of B cell subpopulations in human peripheral blood or various anatomical compartments in the mouse, starting from freshly isolated or frozen cell suspensions. Both human and mouse B cell subpopulations showed different basal and BCR stimulation-induced phosphorylation levels of downstream signaling proteins. For example, peritoneal B-1 cells and splenic marginal zone B cells exhibited significantly increased basal (ex vivo) signaling and increased responsiveness to in vitro BCR stimulation compared with peritoneal B-2 cells and splenic follicular B cells, respectively. In addition, whereas stimulation with anti-IgM or anti-Igκ L chain Abs resulted in strong pCD79a and pPLCγ2 signals, IgD stimulation only induced CD79a but not pPLCγ2 phosphorylation. In summary, the protocol is user friendly and quantifies BCR-mediated phosphorylation with high sensitivity at the single-cell level, in combination with extensive staining to identify individual B cell development and differentiation stages.

Overexpression of SH2-Containing Inositol Phosphatase Contributes to Chronic Lymphocytic Leukemia Survival.

Balanced activity of kinases and phosphatases downstream of the BCR is essential for B cell differentiation and function and is disturbed in chronic lymphocytic leukemia (CLL). In this study, we employed IgH.TEµ mice, which spontaneously develop CLL, and stable EMC CLL cell lines derived from these mice to explore the role of phosphatases in CLL. Genome-wide expression profiling comparing IgH.TEµ CLL cells with wild-type splenic B cells identified 96 differentially expressed phosphatase genes, including SH2-containing inositol phosphatase (Ship2). We found that B cell-specific deletion of Ship2, but not of its close homolog Ship1, significantly reduced CLL formation in IgH.TEµ mice. Treatment of EMC cell lines with Ship1/2 small molecule inhibitors resulted in the induction of caspase-dependent apoptosis. Using flow cytometry and Western blot analysis, we observed that blocking Ship1/2 abrogated EMC cell survival by exerting dual effects on the BCR signaling cascade. On one hand, specific Ship1 inhibition enhanced calcium signaling and thereby abrogated an anergic response to BCR stimulation in CLL cells. On the other hand, concomitant Ship1/Ship2 inhibition or specific Ship2 inhibition reduced constitutive activation of the mTORC1/ribosomal protein S6 pathway and downregulated constitutive expression of the antiapoptotic protein Mcl-1, in both EMC cell lines and primary IgH.TEµ CLL cells. Importantly, also in human CLL, we found overexpression of many phosphatases including SHIP2. Inhibition of SHIP1/SHIP2 reduced cellular survival and S6 phosphorylation and enhanced basal calcium levels in human CLL cells. Taken together, we provide evidence that SHIP2 contributes to CLL pathogenesis in mouse and human CLL.

Decreased BAFF Receptor Expression and Unaltered B Cell Receptor Signaling in Circulating B Cells from Primary Sjögren's Syndrome Patients at Diagnosis.

Animal models of autoimmunity and human genetic association studies indicate that the dysregulation of B-cell receptor (BCR) signaling is an important driver of autoimmunity. We previously showed that in circulating B cells from primary Sjögren's syndrome (pSS) patients with high systemic disease activity, protein expression of the BCR signaling molecule Bruton's tyrosine kinase (BTK) was increased and correlated with T-cell infiltration in the target organ. We hypothesized that these alterations could be driven by increased B-cell activating factor (BAFF) levels in pSS. Here, we investigated whether altered BCR signaling was already present at diagnosis and distinguished pSS from non-SS sicca patients. Using (phospho-)flow cytometry, we quantified the phosphorylation of BCR signaling molecules, and investigated BTK and BAFF receptor (BAFFR) expression in circulating B cell subsets in an inception cohort of non-SS sicca and pSS patients, as well as healthy controls (HCs). We found that both BTK protein levels and BCR signaling activity were comparable among groups. Interestingly, BAFFR expression was significantly downregulated in pSS, but not in non-SS sicca patients, compared with HCs, and correlated with pSS-associated alterations in B cell subsets. These data indicate reduced BAFFR expression as a possible sign of early B cell involvement and a diagnostic marker for pSS.

Splenic Architecture and Function Requires Tight Control of Transmembrane TNF Expression.

Soluble tumor necrosis factor (sTNF) is an important inflammatory mediator and essential for secondary lymphoid organ (SLO) development and function. However, the role of its transmembrane counterpart (tmTNF) in these processes is less well established. Here, the effects of tmTNF overxpression on SLO architecture and function were investigated using tmTNF-transgenic (tmTNF-tg) mice. tmTNF overexpression resulted in enlarged peripheral lymph nodes (PLNs) and spleen, accompanied by an increase in small splenic lymphoid follicles, with less well-defined primary B cell follicles and T cell zones. In tmTNF-tg mice, the spleen, but not PLNs, contained reduced germinal center (GC) B cell fractions, with low Ki67 expression and reduced dark zone characteristics. In line with this, smaller fractions of T follicular helper (Tfh) and T follicular regulatory (Tfr) cells were observed with a decreased Tfh:Tfr ratio. Moreover, plasma cell (PC) formation in the spleen of tmTNF-tg mice decreased and skewed towards IgA and IgM expression. Genetic deletion of TNFRI or -II resulted in a normalization of follicle morphology in the spleen of tmTNF-tg mice, but GC B cell and PC fractions remained abnormal. These findings demonstrate that tightly regulated tmTNF is important for proper SLO development and function, and that aberrations induced by tmTNF overexpression are site-specific and mediated via TNFRI and/or TNFRII signaling.

Loss of immune homeostasis in patients with idiopathic pulmonary arterial hypertension.

INTRODUCTION: Autoreactivity against pulmonary vascular structures is thought to be involved in idiopathic pulmonary arterial hypertension (IPAH), but the underlying mechanisms remain poorly understood. We hypothesised that aberrant B-cell activation contributes to IPAH aetiology. METHODS: Mice with enhanced B-cell activation due to B-cell-specific overexpression of the B-cell receptor (BCR) signalling molecule Bruton's tyrosine kinase (BTK) were subjected to lung injury and examined for several pulmonary hypertension (PH) indices. Peripheral blood lymphocytes from patients with IPAH (n=13), connective tissue disease-associated PAH (CTD-PAH, n=9), congenital heart disease PAH (n=7), interstitial lung disease associated PH (n=17) and healthy controls (n=19) were characterised by 14-colour flow cytometry. RESULTS: Following pulmonary injury, BTK-overexpressing mice showed prolonged activation of B cells and CXCR5+ follicular T-helper (Tfh) cells, as well as features of PH development. Patients with CTD-PAH and CHD-PAH displayed reduced proportions of circulating non-switched-memory B cells (p=0.03, p=0.02, respectively). Interestingly, we observed increased BTK protein expression in naive (p=0.007) and memory B-cell subsets of patients with IPAH and CTD-PAH. BTK was particularly high in patients with IPAH with circulating autoantibodies (p=0.045). IPAH patients had low frequencies of circulating CXCR5+ Tfh cells (p=0.005). Hereby, the increased BTK protein expression in B cells was associated with high proportions of Tfh17 (p=0.018) and Tfh17.1 (p=0.007) cells within the circulating Tfh population. CONCLUSIONS: Our study shows that pulmonary injury in combination with enhanced B-cell activation is sufficient to induce PH symptoms in mice. In parallel, immune homeostasis in patients with IPAH is compromised, as evidenced by increased BCR signalling and cTfh17 polarisation, indicating that adaptive immune activation contributes to IPAH disease induction or progression.

Evidence for enhanced Bruton's tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis.

OBJECTIVES: To determine Bruton's tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production. METHODS: BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA- and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures. RESULTS: BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells. CONCLUSION: We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.

Enhanced Bruton's tyrosine kinase in B-cells and autoreactive IgA in patients with idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic Pulmonary Fibrosis (IPF) is thought to be triggered by repeated alveolar epithelial cell injury. Current evidence suggests that aberrant immune activation may contribute. However, the role of B-cell activation remains unclear. We determined the phenotype and activation status of B-cell subsets and evaluated the contribution of activated B-cells to the development of lung fibrosis both in humans and in mice. METHODS: B-cells in blood, mediastinal lymph node, and lung single-cell suspensions of IPF patients and healthy controls (HC) were characterized using 14-color flow cytometry. Mice were exposed to bleomycin to provoke pulmonary fibrosis. RESULTS: More IgA+ memory B-cells and plasmablasts were found in blood (n = 27) and lungs (n = 11) of IPF patients compared to HC (n = 21) and control lungs (n = 9). IPF patients had higher levels of autoreactive IgA in plasma, which correlated with an enhanced decline of forced vital capacity (p = 0.002, r = - 0.50). Bruton's tyrosine kinase expression was higher in circulating IPF B-cells compared to HC, indicating enhanced B-cell activation. Bleomycin-exposed mice had increased pulmonary IgA+ germinal center and plasma cell proportions compared to control mice. The degree of lung fibrosis correlated with pulmonary germinal center B-cell proportions (p = 0.010, r = 0.88). CONCLUSION: Our study demonstrates that IPF patients have more circulating activated B-cells and autoreactive IgA, which correlate with disease progression. These B-cell alterations were also observed in the widely used mouse model of experimental pulmonary fibrosis. Autoreactive IgA could be useful as a biomarker for disease progression in IPF.

The Role of Bruton's Tyrosine Kinase in Immune Cell Signaling and Systemic Autoimmunity.

Bruton's tyrosine kinase (BTK) is an intracellular signaling molecule first identified as the molecule affected in X-linked agammaglobulinemia (XLA) patients, who almost completely lack peripheral B cells and serum immunoglobulins. BTK is crucial for B cell development and various B cell functions, including cytokine and natural antibody production. Importantly, it is also expressed in numerous other cells, including monocytes, macrophages, granulocytes, dendritic cells, and osteoclasts. A few rare cases of autoimmune disease in XLA patients have been described. Interestingly, increased BTK protein expression in patients with systemic autoimmune disease appears to be correlated with autoantibody production. In addition, BTK may promote autoimmunity as an important driver of an imbalance in B-T cell interaction. Because of this overwhelming evidence of a pathogenic role of BTK in autoimmunity, several clinical trials in rheumatoid arthritis and systemic lupus erythematosus patients with BTK inhibitors are currently running. Here, we review BTK function in different signaling pathways and in different cell lineages, focusing on the growing body of literature indicating a critical role for BTK in autoimmunity. We also discuss BTK and the promising results of BTK inhibition in animal models of autoimmune disease.

Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase.

Th17 cells in primary Sjögren's syndrome: Pathogenicity and plasticity.

Th17 cells play an important physiological role at mucosal barriers, and are involved in inflammatory responses to pathogens. Th17 cells and their signature cytokine IL-17 are also present in salivary gland lesions of primary Sjögren's syndrome (pSS) patients and can be elevated in their peripheral blood. In pSS patients, clear correlations between increased Th17 cell activity and symptoms of the disease have not been found, but Th17 cells may contribute to disease progression, for example by supporting autoreactive B cell responses. In mouse models of pSS, Th17 cells play an important role in pathogenesis, particularly at disease onset, when there is a disturbed balance between T effector and T regulatory cells. Studying the pathogenicity of Th17 cells in humans is complicated due to the plasticity of this cell subset, allowing them to obtain different effector functions depending on the local environment. Th17 cells can develop towards Th17.1 cells, producing both IL-17 and IFN-γ, or even towards Th1-like cells producing IFN-γ in the absence of IL-17. These effector subsets may be more pathogenic than bona fide Th17 cells. Co-expression of IFN-γ by Th17 cells has been shown to promote chronic inflammation in several autoimmune diseases and may also contribute to pSS pathogenesis. In line with the noticeable role of IL-17 in pSS mouse models, interference with Th17 cell generation, recruitment or effector functions (e.g. IL-17 inhibition) can prevent or ameliorate disease in these models. Therapies targeting Th17 cells or IL-17 have not been tested so far in pSS patients, although treatment with rituximab seems to lower local and systemic IL-17 protein levels, and to a lesser extent also chemokine receptor-defined Th17 cells. In this review we discuss current knowledge of pathogenicity and plasticity of Th17 cells in human pSS and murine models of pSS. We postulate that plasticity towards Th17.1 cells in pSS may enhance pathogenicity of Th17 cells at the main target sites of the disease, i.e. salivary and lacrimal glands.

Enhanced Expression of Bruton's Tyrosine Kinase in B Cells Drives Systemic Autoimmunity by Disrupting T Cell Homeostasis.

Upon BCR stimulation, naive B cells increase protein levels of the key downstream signaling molecule Bruton's tyrosine kinase (BTK). Transgenic CD19-hBtk mice with B cell-specific BTK overexpression show spontaneous germinal center formation, anti-nuclear autoantibodies, and systemic autoimmunity resembling lupus and Sjögren syndrome. However, it remains unknown how T cells are engaged in this pathology. In this study, we found that CD19-hBtk B cells were high in IL-6 and IL-10 and disrupted T cell homeostasis in vivo. CD19-hBtk B cells promoted IFN-γ production by T cells and expression of the immune-checkpoint protein ICOS on T cells and induced follicular Th cell differentiation. Crosses with CD40L-deficient mice revealed that increased IL-6 production and autoimmune pathology in CD19-hBtk mice was dependent on B-T cell interaction, whereas IL-10 production and IgM autoantibody formation were CD40L independent. Surprisingly, in Btk-overexpressing mice, naive B cells manifested increased CD86 expression, which was dependent on CD40L, suggesting that T cells interact with B cells in a very early stage of immune pathology. These findings indicate that increased BTK-mediated signaling in B cells involves a positive-feedback loop that establishes T cell-propagated autoimmune pathology, making BTK an attractive therapeutic target in autoimmune disease.

Loss of IL-22 inhibits autoantibody formation in collagen-induced arthritis in mice.

Interleukin 22 (IL-22) expression is associated with increased joint destruction and disease progression in rheumatoid arthritis (RA). Although IL-22 is considered a pro-inflammatory cytokine, its mechanism of action in RA remains incompletely understood. Here, we used the collagen-induced arthritis model in IL-22 deficient (IL-22(-/-) ) mice to study the role of IL-22 in RA. In spite of normal disease incidence, disease severity is significantly diminished in IL-22(-/-) mice. Moreover, pathogenicity of Th17 cells and development and function of B cells are unaffected. In contrast, splenic plasma cells, as well as serum autoantibody titers, are reduced in the absence of IL-22. At the peak of disease, germinal centers (GCs) are severely reduced in the spleens of IL-22(-/-) mice, correlating with a decline in GC B-cell numbers. Within the GC, we identified IL-22R1 expressing follicular dendritic cell-like stromal cells. Human lymphoid stromal cells respond to IL-22 ex vivo by inducing transcription of CXCL12 and CXCL13. We therefore postulate IL-22 as an important enhancer of the GC reaction, maintaining chemokine levels for the persistence of GC reactions, essential for the production of autoantibody-secreting plasma cells. Blocking IL-22 might therefore prevent immune-complex deposition and destruction of joints in RA patients.

BTK Signaling in B Cell Differentiation and Autoimmunity.

Since the original identification of Bruton's tyrosine kinase (BTK) as the gene defective in the primary immunodeficiency X-linked agammaglobulinemia (XLA) in 1993, our knowledge on the physiological function of BTK has expanded impressively. In this review, we focus on the role of BTK during B cell differentiation in vivo, both in the regulation of expansion and in the developmental progression of pre-B cells in the bone marrow and as a crucial signal transducer of signals downstream of the IgM or IgG B cell antigen receptor (BCR) in mature B cells governing proliferation, survival, and differentiation. In particular, we highlight BTK function in B cells in the context of host defense and autoimmunity. Small-molecule inhibitors of BTK have very recently shown impressive anti-tumor activity in clinical studies in patients with various B cell malignancies. Since promising effects of BTK inhibition were also seen in experimental animal models for lupus and rheumatoid arthritis, BTK may be a good target for controlling autoreactive B cells in patients with systemic autoimmune disease.

Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion.

While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.

Btk levels set the threshold for B-cell activation and negative selection of autoreactive B cells in mice.

On antigen binding by the B-cell receptor (BCR), B cells up-regulate protein expression of the key downstream signaling molecule Bruton tyrosine kinase (Btk), but the effects of Btk up-regulation on B-cell function are unknown. Here, we show that transgenic mice overexpressing Btk specifically in B cells spontaneously formed germinal centers and manifested increased plasma cell numbers, leading to antinuclear autoantibody production and systemic lupus erythematosus (SLE)-like autoimmune pathology affecting kidneys, lungs, and salivary glands. Autoimmunity was fully dependent on Btk kinase activity, because Btk inhibitor treatment (PCI-32765) could normalize B-cell activation and differentiation, and because autoantibodies were absent in Btk transgenic mice overexpressing a kinase inactive Btk mutant. B cells overexpressing wild-type Btk were selectively hyperresponsive to BCR stimulation and showed enhanced Ca(2+) influx, nuclear factor (NF)-κB activation, resistance to Fas-mediated apoptosis, and defective elimination of selfreactive B cells in vivo. These findings unravel a crucial role for Btk in setting the threshold for B-cell activation and counterselection of autoreactive B cells, making Btk an attractive therapeutic target in systemic autoimmune disease such as SLE. The finding of in vivo pathology associated with Btk overexpression may have important implications for the development of gene therapy strategies for X-linked agammaglobulinemia, the immunodeficiency associated with mutations in BTK.