Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry

Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors controlling functions in immune responses and other cell types. Given their importance, we developed a flow cytometry panel to assess eight phosphorylated STAT residues in human T-cells, including six tyrosine residues across six STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT6) and additional serine residues on STAT1 and STAT3. We applied this protocol to test the in vitro induction of pSTATs in response to CD3/CD28 activation and a panel of recombinant cytokines. We also assessed the pSTAT expression profiles of naïve CD4+ T-cells polarized to Th1, Th2, Th17 or iTregs. pSTAT1(S727), pSTAT2(Y689) and pSTAT3(S727) were constitutively expressed in most T-cells, even in the absence of stimulation. For pSTAT1(S727) and pSTAT3(S727), we observed two positive states, high and low. Conversely, expression of pSTAT1(Y701), pSTAT3(Y705), pSTAT4(Y693) and pSTAT6(Y641) were absent in resting T-cells and only expressed with CD3/CD28 activation or with specific cytokines. Variable frequencies of pSTAT5a(Y694) expression were observed in resting T-cells, which increased with activation or specific cytokine stimulation (e.g. IL-2). IFNβ stimulation enhanced frequencies of expressing cells for all pSTATs. Correlations among several pSTATs, particularly pSTAT1(S727)high and pSTAT3(S727)high were observed. While polarization resulted in increases in canonically associated pSTATs, other non-canonical pSTAT changes were also observed. Collectively, we developed, optimized, and tested a sensitive and rapid approach for simultaneously assessing phosphorylation of six STAT proteins. Using this approach, we made several novel observations of T-cell pSTAT induction in response to stimuli. ### Competing Interest Statement Emily Monk: No conflicts of interest to declare. Melinda Vassallo: No conflicts of interest to declare. Paulo Burke: No conflicts of interest to declare. Jeffrey Weber: consults for and has received less than $10,000 dollars per annum from Merck, Genentech, Astra Zeneca, Pfizer, Regeneron, GSK, Alkermes, Novartis, Celldex, Incyte and EMD Serono and >$10,000 dollars annually from BMS for membership on advisory boards; holds equity in CytoMx, Biond, Neximmune and Immunimax; on scientific advisory boards for Celldex, CytoMx, Incyte, Biond, Neximmune and Sellas; named on patents for an ipilimumab biomarker (Moffitt Cancer Center), a TIL growth method (Moffitt Cancer Center) and a PD-1 biomarker (Biodesix). Pratip Chattopadhyay: owns Talon Biomarkers. David Woods: owns less than $10,000 stock in Seattle Genetics, Bristol Myers-Squibb, Merck, Glaxo-Kline Smith, Roche, Gilead, Moderna, Sorrento Therapeutics, Iovance, Lumos Pharma, Cue Biopharma, Atara Biotherapeutics, Lyra Therapeutics, Onconova Therapeutics, Fortress Biotech, Ziopharm Oncology, Arcus Biosciences, Surface Oncology, and Tiziana Life Sciences.