Intracellular Phospho‐Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot

Abstract

Phospho-site specific antibodies become increasingly available, enabling the study of signaling events by Western blotting (WB) or intracellular flow cytometry (PhosphoFlow). Here we compared data generated by WB or Phospho-Flow regarding the kinetics and degree of phosphorylation of membrane proximal TCR signaling molecules. Phosphorylation events in Jurkat T cells were triggered by anti-CD3 stimulation (OKT3) or by oxidative stress (H 2 O 2 ) and were analyzed by Phospho-Flow or WB. Both techniques showed that OKT3-or H 2 O 2 -induced, transient phosphorylation of ZAP70 or LAT was dependent on functional Lck. Phospho-Flow data revealed differences in the kinetics and the degree of H 2 O 2 -or OKT3-mediated protein phosphorylation compared with WB data. In addition, using Phospho-Flow we discovered that H 2 O 2 -induced phosphorylation of TCR signaling proteins was inhibited by small molecular weight kinase inhibitors far more potently than OKT3-triggered protein phosphorylation, despite a superior induction of phosphorylation by H 2 O 2 .This finding was confirmed by WB. Interestingly, we identified by Phospho-Flow that, in P116 Jurkat cells lacking ZAP70 protein expression, H 2 O 2 potently triggered the phosphorylation of ZAP70 residues Y493 and Y292 but not Y319. The phosphorylation of these ZAP70 tyrosine residues cells was blocked by an Lck inhibitor, suggesting the existence of an Lck-coupled truncated ZAP70 protein or a novel isoform of ZAP70 in P116 cells. Phospho-Flow is a largely quantitative technology with excellent throughput, highly suited in studying the function or inhibition of TCR signaling pathways and allowing the detection of novel pathway insights. It can serve as a good complement to Western blot analysis. '

Regulatory T cells Suppress Zap70 Phosphorylation in Responding T cells

“…Phospho-flow has been shown to be an excellent tool to detect intracellular signaling in complex populations of cells [15] including Zap70 phosphorylation in CD4 + T cells [16]. We show that Zap70 phosphorylation occurs within few minutes of Tresp stimulation and is detected for only 10 minutes, similar to the short period of Zap70 phosphorylation previously reported [11]. Treg were reported to require several hours for exerting their effect on Tresp [9], therefore we employed a "2-stages approach" for activating Treg and then measuring Zap70 phosphorylation in Tresp using anti-CD3 and anti-CD28 antibodies for both stages.…”

“…These events induce transcription of diverse gene products, culminating in cytokine secretion by T cells as well as proliferation and enhancement of other T cell functions [10]. Zap70 phosphorylation occurs within minutes after activation of T cells [11]. Accordingly, we hypothesized that activated Treg can suppress Zap70 phosphorylation in activated Tresp.…”

STAT5 in human basophils: IL‐3 is required for its FcεRI‐mediated phosphorylation

“…Recently, it has been demonstrated that flow cytometry can generate qualitative results concerning dose responses and kinetics of signaling molecules similar and complementary to the data obtained with blotting techniques (19,(26)(27)(28). Flow cytometry does not require a purification of the basophils.…”

Comparison of Acalabrutinib, A Selective Bruton Tyrosine Kinase Inhibitor, with Ibrutinib in Chronic Lymphocytic Leukemia Cells

“…Cells were stained with antibodies against phospho-LCK (#558552; BD Biosciences, Franklin Lakes, NJ), phospho-SRC (#560096; BD Biosciences), cleaved Poly(ADP-ribose) polymerase (PARP) (#560640; BD Biosciences) and CD3 (#562426; BD Biosciences). A similar assay was previously described for T-cell phosphoflow analysis using CD3/CD28 and H 2 O 2 stimuli (38,39). Data were acquired on a FACSVerse flow cytometer and analyzed with FCS Express (version 4; BD Biosciences).…”