File S1 - Pharmacological Inhibition of TPL2/MAP3K8 Blocks Human Cytotoxic T Lymphocyte Effector Functions

File S1 contains the following: Figure S1. Tpl2−/− and Tpl2+/+ mice display comparable CD4 and CD8 profiles in primary and secondary lymphoid organs. Thymus, peripheral blood, lymph nodes, and spleen were harvested from 4 month old Tpl2+/+, Tpl2+/−, and Tpl2−/− mice. Cells were stained for surface expression of CD4 and CD8 ex vivo and analyzed by flow cytometry. Figure S2. TCR-mediated induction of CD25 in human CD8+ T cells is not altered by specific MAP kinase inhibitors. Naïve CD8+ (CD8+CD45RA+) T cells were isolated by negative selection from healthy human PBMCs and stimulated with plate-bound anti-CD3+anti-CD28 and rhIL-12. Cells were split 1:10 with 100 U/mL IL-2 and cultured until day 7 when cells were counted and re-stimulated with plate-bound anti-CD3±indicated inhibitors or left unstimulated. CD25 expression was measured by staining for the surface marker and analysis with flow cytometry 24 h post 2° stimulation. Data shown are representative of 3 experiments from separate healthy donors. Percent of CD25+ cells within the live gate were determined and mean±SD plotted. Figure S3. Specific MAP kinase inhibitors do not alter human TEM CTL cell viability. CD8+CCR7lo T cells were isolated from healthy human PBMCs by FACS sorting. Cells were stimulated with plate-bound anti-CD3 in the presence of the highest concentration of the Tpl2 inhibitor (10 mM) used throughout the study. Cell viability was measured 24 h post stimulation by staining for AnnexinV and 7AAD. Data are expressed as dot plots of total events without live cell gating. (DOCX)