Phospho-specific Flow Cytometry Research Articles

B-cell receptor signaling activity identifies patients with mantle cell lymphoma at higher risk of progression

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.

Dynamic hydrogen peroxide levels reveal a rate-dependent sensitivity in B-cell lymphoma signaling

Although in vivo extracellular microenvironments are dynamic, most in vitro studies are conducted under static conditions. Here, we exposed diffuse large B-cell lymphoma (DLBCL) cells to gradient increases in the concentration of hydrogen peroxide (H2O2), thereby capturing some of the dynamics of the tumour microenvironment. Subsequently, we measured the phosphorylation response of B-cell receptor (BCR) signalling proteins CD79a, SYK and PLCγ2 at a high temporal resolution via single-cell phospho-specific flow cytometry. We demonstrated that the cells respond bimodally to static extracellular H2O2, where the percentage of cells that respond is mainly determined by the concentration. Computational analysis revealed that the bimodality results from a combination of a steep dose–response relationship and cell-to-cell variability in the response threshold. Dynamic gradient inputs of varying durations indicated that the H2O2 concentration is not the only determinant of the signalling response, as cells exposed to more shallow gradients respond at lower H2O2 levels. A minimal model of the proximal BCR network qualitatively reproduced the experimental findings and uncovered a rate-dependent sensitivity to H2O2, where a lower rate of increase correlates to a higher sensitivity. These findings will bring us closer to understanding how cells process information from their complex and dynamic in vivo environments.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=<0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=<0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Combined Single-Cell Transcriptomics and Mitochondrial Lineage Tracing Identify IL-6 Signaling Responses in TET2 Clonal Hematopoiesis of Indeterminate Potential

Clonal hematopoiesis of indeterminate potential (CHIP) arises from acquired mutations in hematopoietic stem cells, most frequently DNMT3A and TET2. Surprisingly, CHIP is associated with not only hematologic malignancy but also cardiovascular disease and an expanding array of inflammatory conditions with differential pathogenicity due to DNMT3A and TET2 mutations. In mouse models, CHIP increases risk of atherosclerosis in part due to signaling through the IL-6/IL-1β signaling axis. Here, we define the differential signaling response to IL-6 cytokine stimulation in human monocytes from patients with TET2 and DNMT3A CHIP. We stimulated peripheral blood mononuclear cells ex vivo and performed single-cell RNA sequencing (scRNA-seq) on ~150,000 single cells. At basal levels, we observed that genes involved in inflammation, monocyte activation, cell adhesion, and antigen presentation were enriched in monocytes from patients with TET2 CHIP (n=4) compared to control patients without CHIP (n=3). Following IL-6 stimulation, we found inflammatory genes significantly upregulated in TET2 patient monocytes but not in DNMT3A patient monocytes (n=3 patients). We used CellChat to further characterize cell-cell interactions in the IL-6 stimulated CHIP samples. We found that inflammatory ligands such as IL1B, CXCL3, and IL-8 were uniquely enriched in TET2-mutated monocytes after stimulation. We also identified inferred Macrophage Migration Inhibitory Factor (MIF) ligand signaling from TET2 monocytes was significantly higher than DNMT3A monocytes (Figure 1). Larger CHIP clones confer increased disease risk. To test the hypothesis that IL-6 responses are elevated in high TET2 VAF samples, we performed phospho-specific flow cytometry on TET2 mutant samples at baseline and following IL-6 stimulation. We measured pSTAT3 positivity in high VAF samples (TET2hi, >25% VAF, n=3), low VAF samples (TET2lo, <25% VAF, n=3) and controls (n=4). Despite all samples exhibiting response to stimulation, TET2hi monocytes displayed the highest proportion of pSTAT3+, significantly higher than both TET2lo and control samples (Figure 2, ANOVA p= 0.008, Tukey HSD p=0.008 (CTL vs. TET2hi) and p=0.02 (TET2lo vs. TET2hi)). Combining scRNA-seq with genotyping presents the opportunity to compare CHIP mutated and non-mutated cells from within a single patient. To assign clonality within the monocyte population, we performed MAESTER, a mitochondrial lineage tracing technique, in parallel with nuclear DNA genotyping. We then sought to isolate active transcription factor networks using pySCENIC, a tool that derives active transcription factors and their downstream targets from scRNA-seq data. For our analysis, we selected a TET2 mutant sample with high VAF (51%). In the stimulated monocyte condition, we discovered 237 active regulons within the mutant population. Notably, the XBP1 regulon, a known regulator of MHC-II expression, had the highest regulon specificity score (RSS), indicating activation in TET2-mutated cells. The SPI1 regulon, involved in myeloid lineage determination, the pro-inflammatory regulon, REL, and hypoxia-inducible factor HIF1A also had high RSS scores. We demonstrate that TET2 mutant monocytes exhibit unique signaling responses in response to IL-6 stimulation compared to both control samples and non-mutated cells from within the same patient. High VAF TET2 mutant samples displayed an elevated pSTAT3 response compared to lower VAF and control samples, suggesting that IL-6 signaling is a constitutively active, cell-intrinsic property. Our phospho-specific flow cytometry method has potential diagnostic and prognostic utility in stratifying individual risk for inflammation-related consequences of CHIP, such as cardiovascular disease and malignant transformation. Future prospective randomized clinical trials are required to test whether CHIP patients with TET2 mutation status and high VAF would benefit from IL-6/IL-1β interventions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

STAT3 gain-of-function is not responsible for low total IgE levels in patients with autoimmune chronic spontaneous urticaria.

BackgroundThe pathogenesis of chronic spontaneous urticaria (CSU) has not been clarified entirely. Type IIb autoimmune chronic spontaneous urticaria (CSUaiTIIb) is a distinct subtype of CSU that is often difficult to treat and is connected to low levels of total IgE. Previous findings indicate that an enhanced signal transducer and activator of transcription 3 (STAT3) may be responsible for reduced IgE serum levels.ObjectiveOur aim was to investigate a possible underlying gain-of-function mutation or activating polymorphism in STAT3 that could be responsible for the low levels of IgE in patients with CSUaiTIIb.MethodsWe included 10 patients with CSUaiTIIb and low levels of IgE and sequenced selected single nucleotide polymorphisms (SNP) in STAT3 associated with common autoimmune diseases. Exon sequencing was performed for the most relevant exons of STAT3. To test for a gain-of-function of STAT3, we performed a phospho-specific flow cytometry analysis of STAT3 in peripheral blood mononuclear cells before and after stimulation with interleukin-6.ResultsNo differences were found in the prevalence of the tested SNPs between our patients and a control population. Moreover, we could not find any mutations or variants on the tested exons of STAT3. The function of STAT3 was also not altered in our patients.ConclusionIn total, we could not find any evidence for our hypothesis that low IgE in patients with CSUaiTIIb is linked to mutations in STAT3 or altered activity of STAT3. Thus, it remains to be discovered what causes the low serum levels of IgE in patients with CSUaiTIIb.

Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy.

Peripheral lymphocyte signaling pathway deficiencies predict treatment response in first-onset drug-naïve schizophrenia

Despite being a major cause of disability worldwide, the pathophysiology of schizophrenia and molecular basis of treatment response heterogeneity continue to be unresolved. Recent evidence suggests that multiple aspects of pathophysiology, including genetic risk factors, converge on key cell signaling pathways and that exploration of peripheral blood cells might represent a practical window into cell signaling alterations in the disease state. We employed multiplexed phospho-specific flow cytometry to examine cell signaling epitope expression in peripheral blood mononuclear cell (PBMC) subtypes in drug-naïve schizophrenia patients (n = 49) relative to controls (n = 61) and relate these changes to serum immune response proteins, schizophrenia polygenic risk scores and clinical effects of treatment, including drug response and side effects, over the longitudinal course of antipsychotic treatment. This revealed both previously characterized (Akt1) and novel cell signaling epitopes (IRF-7 (pS477/pS479), CrkL (pY207), Stat3 (pS727), Stat3 (pY705) and Stat5 (pY694)) across PBMC subtypes which were associated with schizophrenia at disease onset, and correlated with type I interferon-related serum molecules CD40 and CXCL11. Alterations in Akt1 and IRF-7 (pS477/pS479) were additionally associated with polygenic risk of schizophrenia. Finally, changes in Akt1, IRF-7 (pS477/pS479) and Stat3 (pS727) predicted development of metabolic and cardiovascular side effects following antipsychotic treatment, while IRF-7 (pS477/pS479) and Stat3 (pS727) predicted early improvements in general psychopathology scores measured using the Brief Psychiatric Rating Scale (BPRS). These findings suggest that peripheral blood cells can provide an accessible surrogate model for intracellular signaling alterations in schizophrenia and have the potential to stratify subgroups of patients with different clinical outcomes or a greater risk of developing metabolic and cardiovascular side effects following antipsychotic therapy.

Isocitrate dehydrogenase mutations are associated with altered IL-1β responses in acute myeloid leukemia.

Mutations in isocitrate dehydrogenase 2 (IDH2) have been noted to impact cellular differentiation in addition to DNA and histone methylation. However, little is known about the impact of IDH2 mutations on intracellular signaling. Using an isogenic cell line model, we investigated both differentiation and signaling responses in IDH2 mutant cells and show augmented responses to inflammatory immune ligands. Using phospho-specific flow and mass cytometry, we demonstrate IDH2mutant cells were significantly more sensitive to IL-1β at multiple downstream readouts. Further, bulk RNA sequencing confirmed increases in cytokine-related signaling pathways and NF-κB target genes. Single-cell RNA sequencing of unstimulated and stimulated cells confirmed altered IL-1β transcriptional responses in the IDH2 mutant cells. Targeted inhibition of the IKK complex reduced IL-1β responses and induced cell death in primary IDH-mutated leukemia samples. Together, these results confirm altered IL-1β signaling in IDH2 mutant cells and identify this pathway as a potential therapeutic target.

The STAT Signaling Biosignature at the Single Cell Level Reveals Novel Insights in the Association of FOXP3+ T Regulatory Cells with Recurrent Spontaneous Abortions (RSA)

BackgroundTregs play an important role in the establishment and maintenance of immune tolerance during pregnancy and they are expanded early after embryo implantation. Decreased frequency and function of Tregs in women suffering from recurrent spontaneous abortions (RSA) has been reported in literature. Signal transducer and activator of transcription (STAT) proteins play a prominent role in the epigenetic control of Treg differentiation. Deficiency of STAT5 leads to loss of Foxp3+ T regulatory cells (Tregs) and inability to induce Tregs in vitro whereas constitutive activation of STAT5β enforces Foxp3 positive Treg development. Aberrant immune signaling via STATs is involved in disease pathobiology in numerous autoimmune conditions, therefore explored the STAT signaling biosignature of Tregs by single-cell phospho-specific flow cytometry in women with RSA before and after treatment with lymphocyte immunotherapy (LIT).Materials and methodsPeripheral blood mononuclear cells from 31 women with either RSA (n=23) or with infertility and ≥2 unsuccessful IVF (n=8) and 10 age-matched females without RSA who had at least one successful pregnancy, were obtained before and after LIT. Basal levels of phospho-STAT1, STAT3 and STAT5 and potentiated, i.e. target/stimuli, nodes (IFNα/pSTAT1, IFNα/pSTAT5, IL-6/pSTAT1, IL-6/pSTAT3 and IL-2/pSTAT5) were measured by phospho-specific flow cytometry. Treg signaling profiles were grouped by using unsupervised hierarchical clustering and correlated with clinical parameters by using χ2 or Fisher Exact tests. Associations of single signaling nodes with the aforementioned parameters were performed by Mann-Whitney and Wilcoxon signed-rank tests as appropriate.ResultsNo differences were observed between women with RSA and age matched women without RSA concerning the pretreatment and post treatment levels of Tregs. By contrast, clustering of pretreatment signaling nodes identified three signaling clusters (SC, figure 1a). SC#3 was enriched in women without RSA (p<0.001) and characterized by high potentiated responses of STATs, whereas women with RSA segregated in SC#1 and SC#2 which were characterized by blunted STAT responses in cytokine stimulation. Of note, all women with successful pregnancy after LIT (n=7) were clustered in SC#1 (p=0.028) which was characterized by high basal levels of pSTAT1 and pSTAT5 but weak potentiated responses of all nodes. Also, Tregs in women with a successful pregnancy after LIT displayed a significantly decreased response of pSTAT5 to IFNα (p= 0.05) and increased basal pSTAT5 levels (p=0.047), compared to women who failed LIT. Τhe post-treatment STAT signaling biosignature remained abnormal in women with RSA (p<0.001, figure 1b). However, compared to women with at least one miscarriage (n=23), women with infertility and unsuccessful IVFs displayed decreased post treatment basal levels of pSTAT1 (p=0.004) and pSTAT3 (p=0.01) and increased responses of pSTAT1 (p=0.047) and pSTAT3 (p=0.047) to IL6. Also, clustering the fold change of pre versus post-treatment STAT signaling profiles in Tregs revealed two signaling clusters. The #SC2 was marginally enriched in women with successful pregnancy after LIT (p=0.1) and was characterized by upregulation of all potentiated nodes after treatment (figure 1c).Collectively, we show for the first time that Tregs in women with RSA bear a pathological STAT biosignature which correlates with LIT outcome. Moreover, LIT can potentially induce a beneficial modulation of STAT signaling by upregulating Treg responsiveness to cytokines. Our work provides novel biological data on the mechanisms of immune tolerance in pregnancy and may point to therapies that can overcome pregnancy loss by modifying cell and cytokine responses. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The delta isoform of phosphatidylinositol-3-kinase predominates in chronic myelomonocytic leukemia and can be targeted effectively with umbralisib and ruxolitinib

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome characterized by monocytic proliferation in the presence of dysplastic bone marrow changes, inflammatory symptoms, and propensity for transformation to acute myeloid leukemia (AML), with a poor prognosis and limited treatment options. Unlike the α and β isoforms, the phosphatidylinositol-3-kinase (PI3K)-δ signaling protein is predominantly expressed by hematopoietic cells and therefore has garnered interest as a potential target for the treatment of lymphomas and leukemias. We revealed a pattern of increased PIK3CD:PIK3CA ratio in monocytic M5 AML patients and cell lines, and this ratio correlated with responsiveness to pharmacological PI3K-δ inhibition in vitro. Because CMML is a disease defined by monocytic clonal proliferation, we tested the PI3K-δ inhibitor umbralisib as a single agent and in combination with the JAK1/2 inhibitor ruxolitinib, in CMML. Our ex vivo experiments with primary CMML patient samples revealed synergistic inhibition of viability and clonogenicity with this combination. Phospho-specific flow cytometry revealed that dual inhibition had the unique ability to decrease STAT5, ERK, AKT, and S6 phosphorylation simultaneously, which offers a mechanistic hypothesis for the enhanced efficacy of the combination treatment. These preclinical data indicate promising activity by co-inhibition of PI3K-δ and JAK1/2 and support the use of ruxolitinib + umbralisib combination therapy in CMML under active clinical investigation.

Effects of CD20 antibodies and kinase inhibitors on B-cell receptor signalling and survival of chronic lymphocytic leukaemia cells.

Recently, clinical trial results have established inhibitors of B-cell receptor (BCR)-associated kinase (BAKi), with or without CD20 moniclonal antibodies (mAbs), as the preferred first-line treatment for most chronic lymphocytic leukaemia (CLL) patients. Using phosphospecific flow cytometry, we showed that in leukaemic cells from CLL patients the CD20 therapeutic antibodies - rituximab, ofatumumab, and obinutuzumab - inhibited BCR signalling pathways targeting preferentially pBTKY551 - but not BTKY223 - and pAKT. On the contrary, ibrutinib and idelalisib reduced pBTKY223 to a higher extent than pBTKY551 . The strong reduction of pAKT induced by idelalisib was enhanced by its combination with rituximab or ofatumumab. Moreover, CD20 mAbs and BAKi induced the death of leukaemia cells that was significantly potentiated by their combination. Analysis of the enhancement of cell death in these combinations revealed an approximately additive enhancement induced by rituximab or obinutuzumab combined with ibrutinib or idelalisib. Taken together, our data identified negative regulatory effects of CD20 mAbs and their combinations with BAKi on BCR signalling and cell survival in CLL. In conclusion, this study advances our understanding of mechanisms of action of CD20 mAbs as single agents or in combination with BAKi and could inform on the potential of combined therapies in ongoing and future clinical trials in patients with CLL.

Time course of signaling profiles of blood leukocytes in acute pancreatitis and sepsis

Activation of intracellular signaling pathways in circulating leukocytes represents an early step in systemic immune-inflammatory response occurring e.g. in acute pancreatitis (AP) and sepsis. Previously, we found aberrations in the phosphorylation of leukocyte signaling proteins in patients with sepsis or AP (measured <48 h from hospital admission) resembling each other and associating with AP severity. Of these patients, those with sepsis or severe AP complicated by persistent organ dysfunction (OD+, n = 17) and patients with moderately severe AP (OD-, n = 6) were followed up in this study by measuring the phosphorylations at two additional time points (2–4 and 5–8 days after the initial sample) using phosphospecific whole blood flow cytometry. Twenty-eighty healthy subjects served as controls (HC). Constitutive STAT3 phosphorylation (pSTAT3) declined in monocytes and neutrophils of OD-/OD + and in lymphocytes of OD + and remained higher in OD- than HC. Monocytes of OD-/OD + showed low pSTAT3 and pSTAT1 levels in response to IL-6 through follow-up. Monocyte pNF-κB levels in response to TNF, LPS and E. coli in OD+, to E. coli in OD-, and lymphocyte pNF-κB levels in response to TNF in OD- increased during follow-up but remained lower than in HC, and neutrophil pNF-κB levels in response to TNF declined in OD-. Phorbol myristate acetate + Ca2+ ionophore-stimulated pERK1/2 decreased in neutrophils of OD−/OD+. To conclude, in patients with moderately severe or severe AP or sepsis, improvement and molecular events contributing to OD can be assessed at the level of blood leukocyte signaling.

Modelling Single Cell B-Cell Receptor Signaling Reveals Enhanced Activity in Primary CLL Cells Compared to Non-Malignant Cells While Fundamental Network Circuit Topology Remains Stable Even with Novel Therapeutic Inhibitors

B-cell receptor (BCR) signalling is central for the pathomechanism of chronic lymphocytic leukemia (CLL). Novel inhibitors of BCR signalling have recently substantially improved treatment of CLL, and a better characterization of the molecular circuitry of leukemic BCR signalling will allow a more refined targeting of this Achilles heel. In order to model malignant and non-malignant BCR signalling, we quantified after stimulation 5 components of BCR signaling (ZAP70/SYK, BTK, PLCy2, AKT, ERK1/2) in single cells from primary human leukemic and non-malignant tissue via phospho-specific flow cytometry over 6 time points. We stimulated cells from 11 patients and non-malignant CD19 negative enriched B-cells from 5 healthy donors by crosslinking the BCR with anti-IgM and/or anti-CD19 and synchronous inhibition of phosphatases with H2O2. As expected, we found more phosphorylation of all BCR signalling components after stimulation in malignant vs non-malignant cells and in IGHV non-mutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2O2 led to higher phosphorylation of BCR components in CLL cells with mutated IGHV genes compared to CLL cells with non-mutated IGHV genes, suggesting a stronger dampening of signalling activity in mutated IGHV CLL by phosphatases. In order to characterize the signalling circuitry, we modelled the connectivity of the cascade components by correlating signal intensities across single cells of the cell populations of single samples (Figure 1). Surprisingly, upon stimulation no substantial differences in network topology were observed between malignant and non-malignant cells. To additionally test for changes in network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib). Ibrutinib and idelalisib acted complementary, but not synergistic, and were similarly effective in IGHV mutated and non-mutated CLL. Effects of idelalisib were the same on malignant and non-malignant cells, whereas ibrutinib was mostly active on CLL cells, not on non-malignant B-cells. Upon stimulation with combinations of IgM and CD19 crosslinking augmented with H2O2, phosphorylation of PLCy2 could not be significantly inhibited by idelalisib or ibrutinib on a timescale of 28mins. We therefore aimed to identify central activating nodes of the BCR signalling cascade using targeted inhibitors. In fact, we found that inhibition of LYN with dasatinib and inhibition of SYK with entospletinib could substantially reduce phosphorylation of PLCy2, BTK and ERK but not AKT after all combinations of BCR stimulation. This suggests additional signalling cascades modulating AKT and a strong impact of SYK/LYN activity on the regulation of PLCy2. In summary, our findings underline the importance of single cell analysis of the dynamic circuitry of B-cell receptor signalling to understand development of resistance mechanisms and potential vulnerabilities. Figure 1: Workflow scheme of the Bayesian network learning and averaging approach. After discretizing the continuous single cell data, an optimal network is derived from each of R bootstrap samples. The Bayesian network learning strategy uses the BDe scoring function and a greedy hill-climbing algorithm to find the network model that represents the resampled data best. An average arc strength for each connection between nodes is derived from the number of occurrences of the respective connection in the set of R best scoring networks. Further averaging among networks derived from different data sets was applied for identifying conditional, temporal, and group-specific differences. Figure 1 Disclosures Döhner: Novartis: Consultancy, Honoraria, Research Funding; Astex: Consultancy, Honoraria; Bristol Myers Swuibb: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Arog: Research Funding; Seattle Genetics: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria. Stilgenbauer:GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Modulation of the IL-6/STAT3 Signaling Axis in CD4+ T Cells As a Potential Immune Mechanism of Action of Azacytidine in High-Risk Myelodysplastic Syndromes

Modulation of the IL-6/STAT3 Signaling Axis in CD4+ T Cells As a Potential Immune Mechanism of Action of Azacytidine in High-Risk Myelodysplastic Syndromes

The STAT signaling profile at the single cell level reveals novel insights in the association of FOXP3+ T regulatory cells with recurrent spontaneous abortions before and after lymphocyte immunotherapy

Foxp3+ T regulatory cell (Tregs) are central in the pathobiology of recurrent spontaneous abortions (RSA). Signal transducer and activator of transcription (STAT) proteins instruct Treg differentiation and polarization, but the STAT signaling architecture of Tregs in RSA and its modifications by lymphocyte immunotherapy (LIT) are yet unknown. By using single-cell phospho-specific flow cytometry we show that the STAT signaling biosignature of Tregs in women with RSA was characterized by marked downregulation of the IFNα/pSTAT1&5, IL-6/pSTAT1&3 and IL-2/pSTAT5 signaling nodes compared to age-matched fertile females. LIT partially restored all of these signaling axes in Tregs only in women who achieved pregnancy after treatment. Both the pretreatment biosignature of Tregs and its modulations by LIT were associated with therapeutic success. We conclude that STAT signaling pathways in Tregs are actively involved in the pathophysiology of RSA and may serve as a predictive tool for selecting patients who may benefit from LIT.

P38 mitogen-activated protein kinase impairment of innate immune cells is a characteristic feature of HBeAg-negative chronic hepatitis B.

The mitogen-activated protein kinase p38 (MAPK) is implicated in the induction of immune responses by regulating the differentiation of T lymphocytes and production of cytokines. Our aim was to investigate p38MAPK phosphorylation in different stages of the natural history of hepatitis B virus (HBV) infection. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density-based centrifugation from 10 patients with HBeAg-negative chronic hepatitis B [HBeAg(-) CHB;HBV-DNA>2000IU/mL], eight patients with HBeAg-negative chronic HBV infection [HBeAg(-) CI;undetectable HBV-DNA] and 8 healthy controls (HCs). p38MAPK phosphorylation was assessed by phospho-specific flow cytometry in PBMCs and cell subsets (CD3+,CD3-,CD56+,CD56-) after stimulation with cytokines (IL-12+IL-2 and IL-12+IL-18) or nonspecific stimuli [arsenite, phorbol 12-myristate 13-acetate (PMA) and ionomycin] at 0,30,60,120 and 240minutes using p38 phospho-specific conjugated antibodies. ΙFN-γ was determined by ELISA in PBMCs culture supernatants after stimulation with rhIL-2, rhIL-12 and rhIL-18, with and without pre-treatment with the p38 MAPK inhibitor, SB203580. HBeAg(-) CI patients showed the highest expression of phosphor-p38 MAPK in total PBMCs and subpopulations compared to HBeAg(-) CHB and HCs. A striking impairment in p38 phosphorylation was noted in CD56+ cells and in especially in NK cells (CD3-CD56+). SB203580-induced inhibition of p38MAPK phosphorylation was associated with suppression of IFN-γ production in all groups. The universal lack of p38 MAPK activation in CD56+ and in particular in NK cells from HBeAg(-) CHB patients during viremia suggests a potential cell-dependent implication of this pathway in the natural history of HBV infection.

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation

As antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets.

PF537 THE STAT SIGNALING BIOSIGNATURE OF CD4+ T CELL SUBSETS REFLECT THE IMMUNOGENICITY OF HIGH‐RISK MDS AND PREDICT AZACYTIDINE TREATMENT OUTCOME

Background:Azacytidine (AZA) is the mainstream treatment in high‐risk Myelodysplastic Syndromes (MDS), but the exact mechanism of action and resistance is largely unknown. AZA potentially acts, at least partially, by upregulating the antitumor immune response, but literature reports are still contradictory. Similarly, CD4+ T cells appear to play a critical role in the antitumor immune response in MDS, even though the exact mechanism remains unclear.Aims:As STAT proteins are key regulators of both physiological and pathological polarization and differentiation of CD4+cells, we investigated the STAT signaling profile of high‐risk MDS patients during AZA treatment and its correlations with clinical and biological parameters.Methods:Peripheral blood mononuclear cells were isolated before the initiation of AZA from 67 high‐risk MDS patients and 13 age‐matched healthy donors. We applied phosphospecific flow cytometry to measure both basal and potentiated levels of pSTAT1, pSTAT3 and pSTAT5. A signaling profile (SP) was constracted for T conventional (Tconv) and T regulatory cells (Tregs) and their functional subsets: PD1+ cells, central memory (TCM), naïve (TN), effector memory (TEM) and terminal effector memory (TEMRA) cells. We further characterized the functional and polarization state of T cells by staining with Perforin, IFNg, IL4, IL17 and Helios.Results:pSTAT signaling profile of Tconv cells revealed three signaling clusters (SC). SC#2 was characterized by higher potentiated responses of pSTAT3 after IL‐6 stimulation (IL‐6/pSTAT3) and was enriched in patients with longer median‐overall survival (mOS, p = 0.047, figure 1A). The SP of both PD1+ Tconv and TEM revealed three SCs. In both cases patients with shorter median event free survival (mEFS) segregated in SCs with downregulated potentiated responses (p = 0.055 and p = 0.04 respectively). The pretreatment SP of Tregs was also associated with mEFS though without reaching significance (p = 0.068), whereas the SP of PD1+ Tregs separated patients with intermediate potentiated responses which showed longer OS (p = 0.023).Summary/Conclusion:Collectively, are results reveal for the first time the clinically significant heterogeneity of CD4+ T cell subsets and their diverse role in the antitumor immunity in MDS. Moreover, the beneficial effect of the activation of IL‐6/pSTAT3 pathway in CD4conv cells comes in line with current data in CAR‐T cells and further suggests that the STAT signaling biosignature on CD4+ cells may represent a response biomarker to AZA and a therapeutic target for high‐risk MDS.image

Human Germinal Center B Cells Differ from Naïve and Memory B Cells in CD40 Expression and CD40L-Induced Signaling Response.

CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. © 2019 International Society for Advancement of Cytometry.

Single Cell Based Phosphorylation Profiling Identifies Alterations in Toll-Like Receptor 7 and 9 Signaling in Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and −9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and −9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways.