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.

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.

Activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase pathway in childhood B-cell acute lymphoblastic leukemia.

RAS mutations are frequently observed in childhood B-cell acute lymphoblastic leukemia (B-ALL) and previous studies have yielded conflicting results as to whether they are associated with a poor outcome. We and others have demonstrated that the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK) pathway can be activated through epigenetic mechanisms in the absence of RAS pathway mutations. Herein, we examined whether MAPK activation, as determined by measuring phosphorylated extracellular signal-regulated kinase (pERK) levels in 80 diagnostic patient samples using phosphoflow cytometry, could be used as a prognostic biomarker for pediatric B-ALL. The mean fluorescence intensity of pERK (MFI) was measured at baseline and after exogenous stimulation with or without pretreatment with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Activation levels (MFI stimulated/MFI baseline) ranged from 0.76 to 4.40 (median = 1.26), and inhibition indexes (MFI stimulated/MFI trametinib stimulated) ranged from 0.439 to 5.640 (median = 1.30), with no significant difference between patients with wildtype versus mutant RAS for either. Logistic regression demonstrated that neither MAPK activation levels nor RAS mutation status at diagnosis alone or in combination was prognostic of outcome. However, 35% of RAS wildtype samples showed MAPK inhibition indexes greater than the median, thus raising the possibility that therapeutic strategies to inhibit MAPK activation may not be restricted to patients whose blasts display Ras pathway defects.

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.

Phosflow assessment of PDGFRA phosphorylation state: A guide for tyrosine kinase inhibitor targeted therapy in hypereosinophilia patients.

Clonal eosinophilia is a hematologic disorder caused by translocation in growth factor receptor (GFR) genes. Despite the identified molecular mechanisms underlying clonal hypereosinophilia, the distinction between clonal and reactive eosinophilia has remained challenging due to the diversity of partner genes for translocated GFRs. This study aimed to examine the feasibility of phosphoflow cytometry in the diagnosis of clonal hypereosinophilia through evaluating the level of platelet-derived growth factor receptor alpha (PDGFRA) phosphorylation and its correlation with PDGFRA genetic aberration. Blood samples were collected from 45 hypereosinophilia patients and 10 healthy controls. Using phosphoflow cytometry method, the phosphorylation state of PDGFRA was assessed. The specificity of phosflow results was confirmed by western blotting and eventually compared with qRT-PCR expression analysis of 3'-region of PDGFRA. To detect the genetic aberration of PDGFRA, 5'-rapid amplification of cDNA ends (5'-RACE) was performed. Phosflow analysis illustrated that 9 of 45 hypereosinophilic patients had higher level of PDGFRA phosphorylation while sequence analysis of 5'-RACE-PCR fragments confirmed that in seven cases of them, there was a PDGFRA-FIP1L1 fusion. We also verified that two of nine patients with hyperposphorylated PDGFRA hold ETV6-PDGFRA and STRN-PDGFRA rearrangements. Importantly, nine cases also had significantly higher levels of PDGFRA mRNA expression when compared with healthy controls, and cases with no PDGFRA rearrangement. These findings highlight a robust correlation between hyperphosphorylation state of PDGFRA and aberrant PDGFRA gene fusions. This implicates phosflow as an efficient and reliable technique raising an intriguing possibility that it could replace other genomic and cDNA-amplification-based diagnostic approaches with limited effectiveness.

Aberrant STAT phosphorylation signaling in peripheral blood mononuclear cells from multiple sclerosis patients.

BACKGROUND: Multiple sclerosis (MS) is characterized by increased activation of peripheral blood mononuclear cells (PBMCs), linked to perturbations in the phosphorylation of signaling proteins. METHODS: We developed a phosphoflow cytometry protocol to assess the levels of 11 phosphorylated nuclear proteins at baseline conditions and after cell activation in distinct PBMC populations from 41 treatment-naïve relapsing-remitting (RR) MS subjects and 37 healthy controls, and in a second cohort of 9 untreated RRMS patients and 10 secondary progressive (SP) MS patients. Levels of HLA-ABC, HLA-E, and HLA-DR were also assessed. Phosphorylation levels of selected proteins were also assessed in mouse splenocytes isolated from myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE). RESULTS: Modest differences were observed at baseline between patients and controls, with general lower phosphorylation levels in cells from affected individuals. Conversely, a dramatic increase in phosphorylated p38MAPK and STAT proteins was observed across all cell types in MS patients compared to controls after in vitro activation. A similar phosphorylation profile was observed in mouse lymphocytes primed in vivo with MOG. Furthermore, levels of all p-STAT proteins were found directly correlated with HLA expression in monocytes. Levels of phosphorylated proteins did not differ between relapsing-remitting and secondary progressive MS patients either in baseline conditions or after stimulation. Lastly, phosphorylation levels appear to be independent of the genotype. CONCLUSION: The response to IFN-α through STAT proteins signaling is strongly dysregulated in MS patients irrespective of disease stage. These findings suggest that the aberrant activation of this pathway could lead to changes in the expression of HLA molecules in antigen presenting cells, which are known to play important roles in the regulation of the immune response in health and disease.

Wnt inhibition leads to improved chemosensitivity in paediatric acute lymphoblastic leukaemia.

While childhood acute lymphoblastic leukaemia (ALL) is now highly curable, the dismal prognosis for children who relapse warrants novel therapeutic approaches. Previously, using an integrated genomic analysis of matched diagnosis-relapse paired samples, we identified overactivation of the Wnt pathway as a possible mechanism of recurrence. To validate these findings and document whether Wnt inhibition may sensitize cells to chemotherapy, we analysed the expression of activated ß-catenin (and its downstream target BIRC5) using multiparameter phosphoflow cytometry and tested the efficacy of a recently developed Wnt inhibitor, iCRT14, in ALL cell lines and patient samples. We observed increased activation of ß-catenin at relapse in 6/10 patients. Furthermore, treatment of leukaemic cell lines with iCRT14 led to significant downregulation of Wnt target genes and combination with traditional chemotherapeutic drugs resulted in a synergistic decrease in viability as well as a significant increase in apoptotic cell death. Finally, pre-treatment of purified blasts from patients with relapsed leukaemia with the Wnt inhibitor followed by exposure to prednisolone, restored chemosensitivity in these cells. Our results demonstrate that overactivation of the Wnt pathway may contribute to chemoresistance in relapsed childhood ALL and that Wnt-inhibition may be a promising therapeutic approach.

Phosphoflow cytometry to assess cytokine signaling pathways in peripheral immune cells: potential for inferring immune cell function and treatment response in patients with solid tumors.

Tumor biopsy is often not available or difficult to obtain in patients with solid tumors. Investigation of the peripheral immune system allows for in-depth and dynamic profiling of patient immune response prior to and over the course of treatment and disease. Phosphoflow cytometry is a flow cytometry‒based method to detect levels of phosphorylated proteins in single cells. This method can be applied to peripheral immune cells to determine responsiveness of signaling pathways in specific immune subsets to cytokine stimulation, improving on simply defining numbers of populations of cells based on cell surface markers. Here, we review studies using phosphoflow cytometry to (a) investigate signaling pathways in cancer patients' peripheral immune cells compared with healthy donors, (b) compare immune cell function in peripheral immune cells with the tumor microenvironment, (c) determine the effects of agents on the immune system, and (d) predict cancer patient response to treatment and outcome. In addition, we explore the use and potential of phosphoflow cytometry in preclinical cancer models. We believe this review is the first to provide a comprehensive summary of how phosphoflow cytometry can be applied in the field of cancer immunology, and demonstrates that this approach holds promise in exploring the mechanisms of response or resistance to immunotherapy both prior to and during the course of treatment. Additionally, it can help identify potential therapeutic avenues that can restore normal immune cell function and improve cancer patient outcome.

A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations.

Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability - together with the relatively easy quantification of phosphorylation - phosphoflow is increasingly and more commonly used, compared to classical western blot analysis. It can however be challenging to set-up a method that works for all targets of interest. Here, we present a step-by-step phosphoflow protocol allowing the evaluation of the phosphorylation status of signaling molecules in conjunction with extensive staining to identify various human and murine B cell subpopulations, as was previously published in the original paper by Rip et al. (2020). Next to a description of phosphoflow targets from the original paper, we provide directions on additional targets that play a pivotal role in BCR signaling. The step-by-step phosphoflow protocol is user-friendly and provides sensitive detection of phosphorylation of various BCR signaling molecules in human and murine B cell subpopulations.

Toll-Like Receptor Signaling Drives Btk-Mediated Autoimmune Disease.

Bruton's tyrosine kinase (Btk) is a signaling molecule involved in development and activation of B cells through B-cell receptor (BCR) and Toll-like receptor (TLR) signaling. We have previously shown that transgenic mice that overexpress human Btk under the control of the CD19 promoter (CD19-hBtk) display spontaneous germinal center formation, increased cytokine production, anti-nuclear autoantibodies (ANAs), and systemic autoimsmune disease upon aging. As TLR and BCR signaling are both implicated in autoimmunity, we studied their impact on splenic B cells. Using phosphoflow cytometry, we observed that phosphorylation of ribosomal protein S6, a downstream Akt target, was increased in CD19-hBtk B cells following BCR stimulation or combined BCR/TLR stimulation, when compared with wild-type (WT) B cells. The CD19-hBtk transgene enhanced BCR-induced B cell survival and proliferation, but had an opposite effect following TLR9 or combined BCR/TLR9 stimulation. Although the expression of TLR9 was reduced in CD19-hBtk B cells compared to WT B cells, a synergistic effect of TLR9 and BCR stimulation on the induction of CD25 and CD80 was observed in CD19-hBtk B cells. In splenic follicular (Fol) and marginal zone (MZ) B cells from aging CD19-hBtk mice BCR signaling stimulated in vitro IL-10 production in synergy with TLR4 and particularly TLR9 stimulation, but not with TLR3 and TLR7. The enhanced capacity of CD19-hBtk Fol B cells to produce the pro-inflammatory cytokines IFNγ and IL-6 compared with WT B cells was however not further increased following in vitro BCR or TLR9 stimulation. Finally, we used crosses with mice deficient for the TLR-associated molecule myeloid differentiation primary response 88 (MyD88) to show that TLR signaling was crucial for spontaneous formation of germinal centers, increased IFNγ, and IL-6 production by B cells and anti-nuclear autoantibody induction in CD19-hBtk mice. Taken together, we conclude that high Btk expression does not only increase B cell survival following BCR stimulation, but also renders B cells more sensitive to TLR stimulation, resulting in increased expression of CD80, and IL-10 in activated B cells. Although BCR-TLR interplay is complex, our findings show that both signaling pathways are crucial for the development of pathology in a Btk-dependent model for systemic autoimmune disease.

Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks.

Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

Phosphoantigens and butyrophilin 3A1 induce similar intracellular activation signaling in human TCRVγ9+ γδ T lymphocytes.

Human γδ cells expressing TCRVγ9 are T lymphocytes with great potential for cancer immunotherapy and unconventional pattern of antigen specificity. These HLA-unrestricted lymphocytes are specifically reactive to non-peptide metabolites (phosphoantigens) and to the butyrophilin 3A (BTN3A/CD277) protein. Whether recognition of such highly different structures trigger the same activation signaling pathway remains unclear, however. Here we combined fluorescent cell barcoding and phosphoflow analysis of TCRVγ9(+) T lymphocytes to compare simultaneously the level of several signaling phosphoproteins after activation by phosphoantigen (BrHPP) or by anti-BTN3A (monoclonal antibody 20.1). This approach shows that the same pathways involving ZAP70, PLCγ2, Akt, NFκB p65, MAPK p38 and Erk1, were induced by either of these stimuli. These data strongly suggest the TCRVγ9(+) T lymphocytes detect phosphoantigens and butyrophilin A3 by the same recognition process.