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ObjectivePancreatic ductal adenocarcinoma (PDAC) has the characteristics of high-density desmoplastic stroma, a distinctive immunosuppressive microenvironment and is profoundly resistant to all forms of chemotherapy and immunotherapy, leading to a 5-year survival rate of 9%. Our study aims to add novel small molecule therapeutics for the treatment of PDAC.DesignWe have studied whether TAK-981, a novel highly selective and potent small molecule inhibitor of the small ubiquitin like modifier (SUMO) activating enzyme E1 could be used to treat a preclinical syngeneic PDAC mouse model and we have studied the mode of action of TAK-981.ResultsWe found that SUMOylation, a reversible post-translational modification required for cell cycle progression, is increased in PDAC patient samples compared with normal pancreatic tissue. TAK-981 decreased SUMOylation in PDAC cells at the nanomolar range, thereby causing a G2/M cell cycle arrest, mitotic failure and chromosomal segregation defects. TAK-981 efficiently limited tumour burden in the KPC3 syngeneic mouse model without evidence of systemic toxicity. In vivo treatment with TAK-981 enhanced the proportions of activated CD8 T cells and natural killer (NK) cells but transiently decreased B cell numbers in tumour, peripheral blood, spleen and lymph nodes. Single cell RNA sequencing revealed activation of the interferon response on TAK-981 treatment in lymphocytes including T, B and NK cells. TAK-981 treatment of CD8 T cells ex vivo induced activation of STAT1 and interferon target genes.ConclusionOur findings indicate that pharmacological inhibition of the SUMO pathway represents a potential strategy to target PDAC via a dual mechanism: inhibiting cancer cell cycle progression and activating anti-tumour immunity by inducing interferon signalling.

Systemic autoimmune disorders are complex heterogeneous chronic diseases involving many different immune cells. A significant proportion of patients respond poorly to therapy. In addition, the high burden of adverse effects caused by “classical” anti-rheumatic or immune modulatory drugs provides a need to develop more specific therapies that are better tolerated. Bruton’s tyrosine kinase (BTK) is a crucial signaling protein that directly links B-cell receptor (BCR) signals to B-cell activation, proliferation, and survival. BTK is not only expressed in B cells but also in myeloid cells, and is involved in many different signaling pathways that drive autoimmunity. This makes BTK an interesting therapeutic target in the treatment of autoimmune diseases. The past decade has seen the emergence of first-line BTK small-molecule inhibitors with great efficacy in the treatment of B-cell malignancies, but with unfavorable safety profiles for use in autoimmunity due to off-target effects. The development of second-generation BTK inhibitors with superior BTK specificity has facilitated the investigation of their efficacy in clinical trials with autoimmune patients. In this review, we discuss the role of BTK in key signaling pathways involved in autoimmunity and provide an overview of the different inhibitors that are currently being investigated in clinical trials of systemic autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus, as well as available results from completed trials.

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.

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.

Understanding the mechanisms and impact of booster vaccinations can facilitate decisions on vaccination programmes. This study shows that three doses of the same synthetic peptide vaccine eliciting an exclusive CD8+ T cell response against one SARS-CoV-2 Spike epitope protected all mice against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, while only a second vaccination with this T cell vaccine was insufficient to provide protection. The third vaccine dose of the single T cell epitope peptide resulted in superior generation of effector-memory T cells in the circulation and tissue-resident memory T (TRM) cells, and these tertiary vaccine-specific CD8+ T cells were characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping showed that a substantial fraction of the tertiary effector-memory CD8+ T cells developed from remigrated TRM cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.SummaryA third dose with a single T cell epitope-vaccine promotes a strong increase in tissue-resident memory CD8+ T cells and fully protects against SARS-CoV-2 infection, while single B cell epitope-eliciting vaccines are unable to provide protection.

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.

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease in which an impaired healing response to recurrent micro-injuries is thought to lead to fibrosis. Recent findings hint at a role for B cells and autoimmunity in IPF pathogenesis. We previously reported that circulating B cells from a fraction of patients, compared with healthy controls, express increased levels of the signaling molecule Bruton’s tyrosine kinase (BTK). However, it remains unclear whether B cell receptor (BCR) signaling is altered in IPF. Here, we show that the response to BCR stimulation is enhanced in peripheral blood B cells from treatment-naïve IPF patients. We observed increased anti-immunoglobulin-induced phosphorylation of BTK and its substrate phospholipase Cγ2 (PLCγ2) in naïve but not in memory B cells of patients with IPF. In naïve B cells of IPF patients enhanced BCR signaling correlated with surface expression of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) but not B cell activating factor receptor (BAFFR), both of which provide pro-survival signals. Interestingly, treatment of IPF patients with nintedanib, a tyrosine kinase inhibitor with anti-fibrotic and anti-inflammatory activity, induced substantial changes in BCR signaling. These findings support the involvement of B cells in IPF pathogenesis and suggest that targeting BCR signaling has potential value as a treatment option.

Bruton's tyrosine kinase (Btk) is a crucial signaling molecule in B-cell receptor (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 wild-type 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. This article is protected by copyright. All rights reserved.

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.

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.

Background Tumour necrosis factor (TNF) is important in immune-mediated inflammatory diseases such as spondyloarthritis (SpA). SpA patients have extensive angiogenesis in inflammatory and bone forming regions. Transmembrane (tm)TNF-transgenic (tg) mice (1) that overexpress tmTNF develop SpA symptoms, including inflammation, bone destruction and bone formation. Interestingly, these mice also develop lymphoid aggregates in the bone marrow (BM) of the axial and peripheral skeleton. Objectives Characterization of lymphoid aggregates in tmTNF tg mouse BM in the context of angiogenesis and bone formation. Methods Ankles, femora, tibiae, vertebrae and spleens from tmTNF tg mice and wild-type (WT) littermates (6 weeks, 12 weeks, and 8 months old; n=5 per age per group) were dissected and analyzed by confocal microscopy. In addition, 12 week old mice (n=5 per group) were analyzed by flow cytometry. To study the importance of TNF-R signaling in these processes, tmTNF tg mice lacking TNF-RI (tmTNF tgxTNF-RI-/-) or TNF-RII (tmTNF tgxTNF-RII-/-) (n=4 per group) were investigated. Results Immunofluorescent (IF) evaluation demonstrated that BM of tmTNF tg mice contained extensive lymphoid aggregates, both in the vertebrae and the ankles, but not in the femurs or spleen. IF microscopy demonstrated that the aggregates in the BM contain characteristics of ectopic lymphoid structures (ELS) and consisted of B220+ B cells and FDC-M1+ follicular dendritic cells that are in close proximity of MECA79+high endothelial venules (HEVs). Flow cytometric analysis revealed that most B220+ B cells are IgD+ naive B cells and that tmTNF tg vertebrae contain significantly more IgD-CD95+ germinal center B cells (P Conclusion: tmTNF overexpression in mice results in extensive lymphoid aggregates in the BM that are often organized in ELS, which may be mediated via TNF-RI signaling. These ELS might result in an increase of IgA+ plasma cells in tmTNF tg mice. Ongoing studies will look further into this and may indicate whether these findings contribute to the pathology observed in these mice. Reference: [1] Alexopoulou L, et al. Eur J Immunol1997; 27(10):2588-92 Disclosure of Interests Merlijn Kaaij: None declared, Jan Piet van Hamburg: None declared, Jasper Rip: None declared, George Kollias: None declared, Dominique Baeten Employee of: UCB Pharma, Martijn Nolte: None declared, Leonie van Duivenvoorde: None declared, Sander W. Tas: None declared

Career situation of first and presenting author Student for a master or a PhD. Introduction Tumour necrosis factor (TNF) is important in immune-mediated inflammatory diseases such as spondyloarthritis (SpA). Transmembrane (tm)TNF-transgenic (tg) mice1 that overexpress tmTNF develop SpA symptoms, including inflammation, ectopic lymphoid structures (ELS) in bone marrow (BM), bone destruction and bone formation. SpA patients have extensive angiogenesis in inflammatory and bone forming regions. Objectives To investigate whether there is a link between pathological angiogenesis and ELS in tmTNF tg mice in the BM. Methods Ankles, femora, tibiae, vertebrae and spleens from 6 and 12 weeks and 8 months old tmTNF tg mice and wild-type (WT) littermates (n=5 per age per group) were dissected and analyzed with confocal microscopy and analyzed 12 week old tmTNF tg and WT mice (n=4) with flow cytometry. To study the importance of TNF-R signaling, tmTNF tg mice lacking TNF-RI (tmTNF tgxTNF-RI-/-) or TNF-RII (tmTNF tgxTNF-RII-/-) (n=4 per group) were used. Results Immunofluorescent evaluation demonstrated that BM of tmTNF tg mice contained significantly more and extensive ELS. These ELS are limited to BM of the vertebrae and ankles, and are in close proximity of MECA79+high endothelial venules (HEVs). ELS predominantly consisted of B220+ B cells, of which most are IgD+ naive B cells. Preliminary flow cytometric analysis revealed a trend towards an increase in IgD-CD95+ germinal center B cells and CXCR5+PD-1+FoxP3-CTLA4- T follicular helper cells and CXCR5+PD-1+FoxP3+CTLA4+ in the vertebrae of tmTNF tg mice compared to WT littermates. Meanwhile, B cell lineages in the BM of tmTNF tg hind limbs were not altered. Furthermore, preliminary data indicates that BM and spleen from tmTNF tg mice contain more IgA+ plasma cells compared to WT littermates. tmTNF tgxTNF-RI-/- mice did not display lymphoid aggregates or HEVs in the BM, while tmTNF tgxTNF-RII-/- mice did, although to a lesser extent than tmTNF tg mice. Conclusions tmTNF overexpression in mice results in extensive ELS associated with HEVs in the BM, which is likely to be mediated through TNF-RI signaling. HEV formation may lead to persistence of inflammation in the BM which contributes to pathology. Reference Alexopoulou L, et al. Eur J Immunol 1997;27(10):2588–92 Disclosure of Interest None declared

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.

CLL is the most common leukemia in the western world and is characterized by the accumulation of monoclonal mature circulating CD5+ B cells that generally express low levels of surface immunoglobulin (Ig). Many lines of evidence indicate that chronic signaling through the B-cell receptor (BCR) plays a key role in CLL pathogenesis. CLL prognosis is correlated with the BCR somatic hypermutation status and the CLL BCR repertoire is highly restricted. Often, CLL cells show constitutive activation of several kinases that are activated immediately downstream of the BCR. Thus, the BCR signaling pathway is aberrantly active in CLL and may play a role in disease development. One of the signal transduction molecules downstream of the BCR is BTK, a Tec family non-receptor kinase that is primarily expressed in most hematopoietic lineages, but not in T cells. BTK has been shown to be essential for several constitutively active pathways implicated in CLL cell survival, including the AKT, ERK and NF-κB pathway. Importantly, small molecule inhibitors of BTK have shown impressive anti-tumor activity in clinical studies, with high response rates in various B cell malignancies, in particular CLL. [...]

ObjectiveBruton's tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease.MethodsUsing intracellular flow cytometry, we quantified BTK expression and phosphorylation in subsets of peripheral blood B cells from 30 patients with rheumatoid arthritis (RA), 26 patients with primary Sjogren's syndrome (SS), and matched healthy controls.ResultsIn circulating B cells, BTK protein expression levels correlated with BTK phosphorylation. BTK expression was up-regulated upon BCR stimulation in vitro and was significantly higher in CD27+ memory B cells than in CD27-IgD+ naive B cells. Importantly, BTK protein and phospho-BTK were significantly increased in B cells from anti-citrullinated protein antibody (ACPA)-positive RA patients but not in B cells from ACPA-negative RA patients. BTK was increased both in naive B cells and in memory B cells and correlated with frequencies of circulating CCR6+ Th17 cells. Likewise, BTK protein was increased in B cells from a major fraction of patients with primary SS and correlated with serum rheumatoid factor levels and parotid gland T cell infiltration. Interestingly, targeting T cell activation in patients with primary SS using the CTLA-4Ig fusion protein abatacept restored BTK protein expression in B cells to normal levels.ConclusionThese data indicate that autoimmune disease in humans is characterized by enhanced BTK activity, which is linked not only to autoantibody formation but also to T cell activity.