Rmil 7

T-ALL cell lines CEM, MOLT-4, DND4.1, Jurkat, Loucy, and TALL-1 were cultured in RPMI 1640 (Gibco, Thermo Fisher Scientific, Gaithersburg, MD, USA) supplemented with 10% FBS (Biowest, Nuaillé, France), 1% penicillin/streptomycin (Gibco) and 1% HEPES (Gibco), at 37 °C in a 5% CO₂ environment. Cells were kept at an optimal concentration of 0.5 × 10⁶ cells/mL. D1 cells were cultured in the same conditions plus 25 ng/mL rmIL-7 (PeproTech EC, London, UK). HEK-293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin. All cell lines were periodically checked for mycoplasma by PCR and were found to be negative.

Lipopolysaccharide (LPS)-matured BMDCs were generated as previously described (Lutz et al., 1999 (link)), then irradiated (21 Gy). Naive T cells and memory T cells were fluorescence-activated cell sorted, labeled with 5 μM of carboxyfluorescein succinimidyl ester (CFSE, Life Technologies), and cocultured with BMDCs (1:1 ratio) for 5 days in the presence of DMSO (Sigma-Aldrich), the p110δ-specific inhibitors IC87114 (UCB Cell Tech) and idelalisib (Selleckchem), or the pan-PI3K inhibitor LY294002 at the indicated doses. Then, T cell proliferation (CFSE dilution) was assessed by flow cytometry. Generation of alloreactive T cells was based on a previously described protocol (Sauer et al., 2004 (link)). In brief, naive and memory T cells were primed with irradiated (21 Gy) BALB/c BMDCs pulsed with A20 lysate, obtained by four freeze-thaw cycles, at a 20:1 ratio for 5 days. T cells were expanded 2 days further with plate-bound anti-CD3 antibody (clone 145-2C11) at 10 μg/ml, soluble anti-CD28 (37.51) at 2 μg/ml, rhIL-2 (Proleukin, Novartis) at 20 IU/ml, and rmIL-7 (Peprotech) at 4 ng/ml, then maintained in culture with cytokines only.

CD8⁺ T cells were isolated from spleens and inguinal lymph nodes of C57BL/6 mice using CD8a⁺ T cell Isolation Kit II (Miltenyi Biotec). The purity obtained was assessed by staining for anti-mouse CD8-PE (eBioscience) and analyzing on FACS (BD) and was always higher than 90%. CD8⁺ T cells were plated overnight at 1.5 × 10⁶ cells/well in IMDM containing 10 ng/ml rmIL-2, 10 ng /ml rmIL-7, 10 ng/ml rhIL-15 (all PeproTech) in 24 well plates previously coated with 2 μg/ml NA/LE anti-mouse CD3ε (BD) and 1 μg/ml NA/LE anti-mouse CD28 (BD Pharmingen).

For all signaling assays, non-sorted splenocytes or lymph node cells were serum-starved for 1 h at 37 °C before stimulation in complete media without serum. For evaluation of TCR signaling, splenocytes and lymphocytes were stimulated with 20 μg/mL anti-CD3ε (145-2C11; Biolegend) and 50 µg/mL anti-IgG (polyclonal, α-Armenian hamster, Jackson ImmunoResearch) for the indicated times^{69 (link),70 (link)}. For evaluation of pSTAT5 signaling, cells were stimulated for 15 min with recombinant human IL-2 (rhIL-2; NIH AIDS Reagent Program) at indicated concentrations, recombinant mouse IL-15 (rmIL-15; Peprotech) at indicated concentrations, or 10 ng/mL recombinant IL-7 (rmIL-7; Peprotech). Immediately following stimulation cells were fixed with a final concentration of 1.5% methanol-free formaldehyde (Fisher Scientific; Cat. PI28906) for 15 min, and permeabilized with 500 µL of ice-cold methanol per 1 × 10⁶ cells for 30 min^{69 (link),71 (link)}. Cells were washed twice with PBS/2%FBS (Omega Scientific) then stained for 60 min in anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-FoxP3 (FJK-16s), anti-pSTAT5 (Y694; C71E5; Cell Signaling Technologies) or anti-pS6 (S235/236; D57.2.2E; Cell Signaling Technologies), and, for exclusion of non-relevant cells, anti-CD45R (B220; RA3-6B2), anti-CD11b (MI/70), anti-CD11c (N418), and anti-Ly-6G (Gr-1; RB6-8C5).

M-ATOs were generated as previously described (27 (link)). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free M-ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in 1X PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (of note SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522). D/F12-B27 was made fresh weekly. 1.5x10⁵ MS5-mDLL4 cells were combined with purified murine HSPCs (100-4000 cells/ATO). M-ATOs were plated on a 0.4 µm Millicell transwell insert (EMD Millipore, Billerica, MA; Cat. PICM0RG50) placed in a 6-well plate containing 1 ml D/F12-B27 per well. Medium was changed completely every 3-4 days by aspiration from around the cell insert followed by replacement with 1 ml with fresh D/F12 and cytokines.