Recombinant mouse il 2

Lymphocytes from the lymph nodes and spleens of LLO118 TCR transgenic mice were primed with 5 μM LLO_190-205 peptide under various T_H-skewing conditions for 4 days. Cytokine production by CD4+ T cells was determined by intracellular staining after 4 hours of stimulation with 0.9 nM PMA and ionomycin (0.5 μg/ml, Sigma) in the presence of brefeldin A (5 μg/ml, Sigma) and 2 μM monensin (eBioscience). CD4+ T cells were sorted on a flow cytometer based on surface CD4 expression and relative mRNA abundances were measured by quantitative PCR analysis. T_H cell skewing conditions were as follows: Th1-skewing condition: recombinant mouse IL-12 (50 ng/ml, Peprotech), purified anti-IL-4 antibody (10 μg/ml, 11B11), and recombinant mouse IL-2 (50 U/ml, Peprotech); Th2-skewing condition: recombinant mouse IL-2 (50 U/ml), recombinant mouse IL-4 (50 ng/ml, Peprotech), purified anti-IFN-γ antibody (10 μg/ml, XNG1.2) and anti-IL-12 antibody (5 μg/ml, BD Biosciences); Th17-skewing condition: recombinant mouse IL-6 (20 ng/ml, Peprotech), recombinant TGF-β (4 ng/ml, Peprotech), purified anti-IFN-γ antibody (10 μg/ml, XNG1.2) and purified anti-IL-4 antibody (10 μg/ml, 11B11). Staining antibodies were purchased from Biolegend.

Mouse spleens were mechanically disrupted over a 100-μm filter and subjected to magnetic bead negative enrichment for CD8⁺ T cells, using a CD8⁺ T-cell isolation kit (STEMCELL Technologies). Isolated CD8⁺ T cells were cultured in RPMI (Gibco) containing 10% FBS, L-glutamine, penicillin/streptomycin, and β-mercaptoethanol (50 μM). All cultures were maintained at a concentration of 1 × 10⁶ cells/mL. T cells were stimulated with 1 μg/mL anti-CD28 (clone 37.51, ThermoFisher, Cat# 14–0281-82) or anti-CD3/anti-CD28 microbeads according to manufacturer’s recommendation (Dynabeads Mouse T-Activator CD3/CD28; Invitrogen). Stimulated cells were incubated in media supplemented with 10 ng/mL of recombinant mouse IL-2 (ThermoFisher).

Retroviruses encoding Foxo1-GFP variants were packaged in 293T cells. Whole splenocytes from OT1 TCR transgenic mice were cultured in complete RPMI-1640 media with 0.4 mg/mL chicken ovalbumin (MilliporeSigma, #A5503) and 50 ng/mL recombinant mouse IL-2 (BioLegend, #575406). At 24-32 hours post activation, OT1 T cells were transduced with Foxo1 retroviral supernatants containing 8 μg/mL polybrene and centrifuged at 1,500g for 1.5 hours. After centrifugation, OT1 T cells were rested at 37°C for 1 hour before replacing the viral supernatant with fresh complete RPMI-1640 media containing 50 ng/mL recombinant mouse IL-2. At 48 hours post-transduction, Foxo1-GFP positive OT1 T cells were sorted by fluorescence-activated cell sorting (FACS) and seeded on 8-well chambered coverglass (ThermoFisher, #155409) at a density of 2.1 x 10⁵ cells per well in complete RPMI-1640 media with 50 ng/mL recombinant mouse IL-2. At 24 hours post-seeding, OT1 T cells were treated with 2 μM MK2206 or vehicle control (sterile H₂O) for 45 minutes and visualized on a Zeiss Axio Observer Z1 microscope at 63X magnification. Foxo1-GFP fluorescence intensity in the nuclear and cytoplasmic compartments was measured using FIJI and the nuclear-to-cytoplasmic ratio (N:C) of Foxo1-GFP was calculated on a per cell basis.