Anti mouse cd28 antibody clone 37

For proliferation analysis, cells isolated from spleens were cultured in T cell culture medium with 0.5 μg/ml Ultra-LEAF purified anti-mouse CD3ε antibody (clone 145-2C11, Biolegend), and 0.5 μg/ml Ultra-LEAF purified anti-mouse CD28 antibody (clone 37.51, Biolegend). 20 ng/ml interleukin 2 (IL-2, Proleukin, Norvatis) was added on days 2 and 3. CD4 or CD8 T cell numbers and forward scatter (FSC-A) were assessed using a Novocyte Flow Cytometer (Aceabio) or Attune NxT Flow Cytometer (Thermo Fisher Scientific). CD4 T cells were MACS magnet sorted using a mouse CD4 + T cell Kit (130-104-454; Miltenyi Biotec). Live and T cell numbers and purity (resulting in a minimum of 90%) were assessed using a Novocyte flow cytometer (Aceabio). Gating strategies for FACS are presented in Supplemental Figs. 1–4.

To measure proliferation, apoptosis, puromycin incorporation and forward scatter, lymph node cells were cultured in T cell culture medium (RPMI + 10% heat inactivated FCS + pen/strep + 50 μM 2ME) with 0.5 ug/ml Ultra-LEAF purified anti-mouse CD3ϵ antibody (clone 145-2C11, Biolegend), and 0.5 ug/ml Ultra-LEAF purified anti-mouse CD28 antibody (clone 37.51, Biolegend). 20ng/ml IL-2 (proleukin, Norvatis) was added on day 2. For analysis of protein or RNA in CD4 T cells, magnet sorted CD4 T cells were cultured in T cell culture medium on anti-CD3 (5 μg/ml)/anti-CD28 (1 μg/ml) coated plates.

Naive WT and eEF-2K KO CD8⁺ T cells isolated from B6 Thy1.2 mice were activated by anti-mouse CD3 antibody (clone 2C11; BioLegend, San Diego, CA)/anti-mouse CD28 antibody (clone 37.51; BioLegend, San Diego, CA) and monitored for their survival by trypan blue cell exclusion method using a TC20 automated cell counter (Bio-Rad, USA). The live CD8⁺ T cells were counted and plotted graphically with GraphPad Prism 9. CD8⁺ T cell proliferation was measured by CFSE (Invitrogen, Carlsbad, CA) assay as described previously (2 (link), 30 (link)). WT and eEF-2K KO CD8⁺ T cell IL-2 secretion was assessed from day 1 to 3 cell culture supernatants using enzyme-linked immunosorbent assay (ELISA) kits (BioLegend, San Diego, CA) as per the manufacturer’s instructions.