Perfix expose kit

CD3, CD25 (SK7 and M-A251 respectively; BD Biosciences) and CD127 (eBioRDR5; eBiosciences Hatfield, UK) antibodies were used for cell surface phenotyping. Cells were then further stained for intranuclear Foxp3 (PCH101; eBiosciences) using the Foxp3 staining kit as per the manufacturer's instructions (eBiosciences).
For intracellular cytokine staining on day 7, cells were restimulated for 4 hr with 5 ng/ml PMA and 500 ng/ml ionomycin, with 2 μm monensin (Sigma-Aldrich, St Louis, MO) added for the final 2 hr. Cells were washed, fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences) and then stained with fluorescently labelled monoclonal antibodies to IL-2 (MQ1-17H12; eBiosciences). Dead cells (7-aminoactinomycin D-positive; Sigma-Aldrich, Gillingham, UK) were gated out.
For phospho-STAT5 staining two different buffer kits were used with the same antibody clone: 47/Stat5[pY694] obtained from BD Biosciences. The buffer set used for phospho-STAT5 staining on total CD4⁺ cell populations was PerFix EXPOSE Kit (Beckman Coulter, High Wycombe, UK). The buffer set used for phopspho-STAT5 staining on sorted Treg cell and effector T cell populations was CytoFix/CytoPerm Buffer and Perm Buffer III (BD Biosciences). Both buffer sets were used as per the manufacturer's instructions.

Splenocytes recovered from QM X C57Bl/6 F1 mice were used for analysis of Syk and Blnk phosphorylation following in vitro stimulation of NP-specific B cells with NP-dextran or NP-KLH. Enriched BM plasma cells populations prepared by positive CD138 selection from immunized Blimp^gfp/+ mice were used for analysis of Syk and Blnk phosphorylation in TI or TD BM plasma cells (Fig. 4). Cells were stimulated ex vivo with antigen (NP-dextran, NP-KLH) or surrogate antigen (anti-Ig Abs) for 3 min then fixed using the PerFix EXPOSE kit (Beckman Coulter) before staining with anti-p-Syk and anti-p-Blnk mAbs, together with NP-PE, anti-B220 and anti-CD19 mAbs (for B cell samples). NP-binding (NP⁺) and non NP-binding (NP⁻) B cells were gated as B220⁺/CD19⁺/NP-PE⁺ and B220⁺/CD19⁺/NP-PE⁻ cells, respectively. NP-binding and non NP-binding plasma cells were gated as GFP⁺/CD138⁺/NP-PE⁺ and GFP⁺/CD138⁺/NP-PE⁻ cells respectively. DAPI (Molecular Probes) was used to exclude dead cells. The delta MFI values used to quantitate the level of Syk and Blnk phosphorylation in Figs 4e and 5c,e,h were calculated by subtracting the MFI values of the staining histograms for unstimulated cells from those of stimulated cells.

For phosphoflow experiments, T cells were isolated by magnetic-activated cell sorting (MACS) using the Pan T cell isolation kit according to the manufacturer’s protocol (Miltenyi Biotech, Köln, Germany). T cells were then cultured in RPMI 1640 media and different TKI at peak concentrations overnight. 96-well flat bottom high protein binding plates (Nunc Maxisorp, ThermoFisher) were coated overnight with blinatumomab at 100 ng/ml in PBS. T cells were transferred to FCS with different TKI present at peak concentrations and stimulated on 96-well plates. After 0, 5, and 15 min, signalling was stopped by buffer 1 of the Perfix Expose Kit (Beckman Coulter) and T cells were further treated according to the manufacturer’s protocol. Intracellular staining was performed with an anti-LCK(pTyr394) antibody (Genetex, 1:500) and anti-rabbit-PE secondary antibody (Cell Signaling Technologies, 1:200). Data acquisition was performed using a FACSCanto II (BD Biosciences).

Splenic B cells were stained with CD19-BV510 and CD5-APC mAb for 15 min, washed and diluted in FCS. Cells were stimulated with 10 μg/mL α-IgM (Southern Biotech) for the indicated time points at 37°C. Cells were immediately fixed and permeabilized with the PerFix EXPOSE Kit (Beckman Coulter) according to the manufacturer's instructions. Intracellular staining of ERK1/2/P-ERK1/2(T202/Y204) (1:400/1:800) and SYK/P-SYK(Y525/526) (both 1:200) and respective isotype controls was performed, followed by detection with an α-rabbit F(ab′)₂-PE conjugate (1:250) (all from Cell Signaling Technology) and measured with the LSRFortessa cytometer (BD Bioscience).

Ex vivo assessment of S6 Ser235/236 phosphorylation was performed on whole blood using an antibody against phosphorylated S6 Ser235/236 (clone D57.2.2E, catalog 8520S, Cell Signaling Technology) and the PerFix EXPOSE kit, according to the manufacturer’s recommendations (Beckman Coulter). Briefly, 100 μl of fresh blood was incubated in the presence of surface antibody for 10 min at 37 °C in a water bath. B- and T-cell surface markers were: CD19 (clone HIB19, catalog 2,111,030, Sony), CD3 (clone UCHT1, catalog 25-0038-42, BD), CD4 (clone VIT4, catalog 130-092- 373, MiltenyiBiotec), CD8 (clone BW135/80, catalog 130-096-902, MiltenyiBiotec). The reaction was stopped using buffer 1 and red blood cells were lysed with buffer 2 for 5 min at 37 °C. The samples were centrifuged, and the pellets underwent intracellular staining with buffer 3 for 1 h. The cells were then washed with the dedicated buffer, re-suspended, and analyzed with a FacsAriaIII flow cytometer (Becton–Dickinson).