Figure 2. MAPK/ERK activation in glioma cell contributes to susceptibility and durable anti-tumoral immunity following anti-PD-1 therapy.

(A and B) Schematic illustration of cell line generation and in vivo survival studies for GL261 Mek1/2 overexpression stable cell line was established by lentiviral transduction and confirmed by Western blot to confer Erk1/2 phosphorylation. For treatment with ICB antibodies, treatment started 1 week after tumor implantation and delivered intravenously 4 times every 3 or 4 days. (C) Flow cytometry based immune cell infiltration analysis. After 20 days of tumor cell infusion, tumor infiltrating lymphocytes were analyzed comparing control GL261 tumor (n = 5) and GL261-Mek1/2 p-Erk^high tumor (n = 5). (D) Kaplan-Meier survival curve of GL261-CV (IC n = 10, aPD-1 n = 10) and GL261-Mek1/2 p-Erk^high (IC, n = 6 and aPD-1, n = 9) treated with IC or aPD-1 antibody, conducted in wildtype C57BL/6 mice. (E) Kaplan-Meier survival curve of control GL261-CV (IC, n = 4 and aPD-1, n = 5) and GL261-Mek1/2 p-Erk^high (IC, n = 5 and aPD-1, n = 5) treated with IC or aPD-1 antibody, conducted in Cd8 KO C57BL/6 mice. (F) Western blot analysis of Erk and Mek phosphorylations and expressions after several passages and freeze/thaw cycles. The cell line was referred as GL261-Mek1/2 p-Erk^low. (G) Kaplan-Meier survival curve of control GL261 tumor and GL261-Mek1/2 p-Erk^low treated with IC (n = 9) or aPD-1 (n = 10), conducted in wildtype C57BL/6 mice. (H) The mice cohort from GL261-Mek1/2 p-Erk^high, the long-term survivors received contralateral injection of wildtype GL261 cell infusion on day 120 post tumor cell injection (control, n = 10 and long-term survivors, n = 6). (I) Immune landscape after 120 days of the rechallenge. Comparing with brand-new tumor (CTL, n = 4), brains from long-term survivors (LTS, n = 4) were analyzed for immune phenotyping. Flow cytometry data showing percoll-enriched cells (left). Bar graph showing CD8⁺ and CD4⁺ T cell ratio (middle), Foxp3⁺ cells in CD4⁺ T cell population (right). (I) Flow cytometry data showing expression of CD44 and CD62L in CD8⁺ T cells (left). Bar graph showing central memory T cell (CD44⁺CD62L⁺) to effector memory T cell (CD44⁺CD62L⁻) ratio (middle). Flow cytometry data showing PD-1 expression in CD8⁺ T cells. The P values for survival studies were generated from low-rank test. Statistical analysis for group comparison was done using unpaired two-tailed T test.