Extended Data Fig. 5: Frequency of CD8+ T cells in various tissues following ACT, the expression levels of mTORC1 in MEKi-treated CD8+ T cells, and a proposed model for MEKi-mediated TSCM generation.
From: MEK inhibition reprograms CD8+ T lymphocytes into memory stem cells with potent antitumor effects
Related to Fig. 8. a, The frequency of CD8+ T cell engrafted in spleen and tumors of mice that received variously treated pMel-1 CD8+ T cells (48 h post-T cell infusion). FACS analysis of the tumor and spleen samples was performed by gating upon Thy1.1 cell population. A representative of two experiments is shown. Each symbol corresponds to one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. Statistical analysis was performed by unpaired, one-tailed Student’s t-test. Significant differences in engraftment were not observed between MEKi-treated and untreated spleen and tumor samples. b, Loci-specific bisulfite sequencing analysis of the Ifng and Prf1 in TCM CD8+ T cells generated after activation of human CD8+ T cells with anti-CD3/28 with or without MEKi treatment. Horizontal lines represent individual sequenced clones from the pool of FACS-purified CD8+ T cells. Bar graphs represent the frequencies of methylated CpGs in respective sample as shown in the figure. Representative data from one of two experiments are shown. c, Estimation of numbers of adoptively transferred cells in tumors of variously treated mice. The mice were sacrificed at 22 days after ACT and tumors were harvested. Samples were stained and processed for FACS analysis. A representative of two experiments is shown. Each symbol represents one mouse with the indicated number of mice per group given in parentheses. The error bars indicate the s.e.m. Statistical analysis was performed by unpaired, one-tailed Student’s t-test (*P≤0.05). d, Levels of phosphorylated-(p)-mTORC1 and total mTORC1 in MEKi-treated mouse CD8+ T cells during initial cell activation and following antigenic re-challenge as detailed in the figure. Expression of β-actin is shown as a control. Number on the bands show band intensity. Experiments were repeated twice with similar results. e, Proposed model for MEKi-mediated TSCM generation. Inhibition of MEK1/2 during antigen-activation of naive cells: 1) results in a decrease in the levels of ERK1/2 and cyclin D1, delaying cell division and accumulating these cells in early phases of differentiation; 2) results in an increase in PGC1α and its downstream SIRT3, enhancing FAO-mediated metabolic fitness that drives memory generation; and 3) does not affect PI3K-Akt-mediated T cell activation. This crosstalk between MAPK pathway, cellular metabolism and TCR-mediated signaling after MEK-inhibition leads to induction of memory characteristics in naive CD8+ T cells, generating TSCM. These TSCM produce highly activated effector cells following re-stimulation with the cognate antigen resulting in robust recall responses.
