Soluble αcd28 37

CD8⁺ T cells were purified from mouse spleen by MACS with the CD8a⁺ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and stained with cell trace violet (CTV) proliferation dye eBioscience (Thermo Fisher Scientific, Eugene, OR, USA). 0.2 x10⁶ cells were stimulated with plate bound anti-CD3ε (145-2C11, 2 μg/mL) and soluble αCD28 (37.51, 1 μg/mL) (both from BioXcell, West Lebanon, NH, USA) in T cell media supplemented with murine IL-2 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA). Proliferation was measured by dilution of the proliferation dye and apoptosis was assessed with an Annexin V apoptosis detection kit with 7-AAD from BioLegend (San Diego, CA, USA). For in vivo proliferation comparison, CD8⁺ T cells were purified from WT and Bcl3 KO P14 mouse spleen by MACS with the CD8a⁺ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and stained with cell trace violet (CTV) proliferation dye eBioscience (Thermo Fisher Scientific, Eugene, OR, USA). 1 x10⁶ cells were adoptively transferred to congenic recipient mice. The next day, mice were infected with LCMV Armstrong and mesenteric lymph nodes were analyzed 4 days after infection.

CD8⁺ T cells were purified from WT mice spleens by MACS with the CD8a⁺ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). Purified cells were kept at rest for 12 hours in T cell media (RPMI with 10% FBS, 10 mM HEPES, 1% non-essential amino acids, 1 mM sodium pyruvate, 100 units/ mL penicillin, 100 units/ mL streptomycin and 55 nM β-ME). After 12 h, cells were stimulated with plate bound CD3ε (145-2C11, 2 μg/mL) and soluble αCD28 (37.51, 1 μg/mL) (both from BioXcell, West Lebanon, NH, USA) in T cell media supplemented with murine IL-2 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA). Cells were collected at various time points. For in vivo Bcl3 kinetic analysis, CD8⁺ T cells were purified from LCMV infected or naïve mice spleen by CD8a (Ly-2) microbeads (Miltenyi Biotec, Auburn, CA, USA) at various time points. RNA was prepared by RNeasy mini kit and cDNAs were synthesized using QuantiTect reverse transcription kit (both from Qiagen, Hilden, Germany). Bcl3 levels were quantified relative to Gapdh by TaqMan assays on Quantstudio3 real time system (Applied Biosystems by Thermo Fisher Scientific, Carlsbad, CA, USA).