Volume 44, Issue 3, Pages 609-621 (March 2016) MAP Kinase Inhibition Promotes T Cell and Anti-tumor Activity in Combination with PD- L1 Checkpoint Blockade  Peter J.R. Ebert, Jeanne Cheung, Yagai Yang, Erin McNamara, Rebecca Hong, Marina Moskalenko, Stephen E. Gould, Heather Maecker, Bryan A. Irving, Jeong M. Kim, Marcia Belvin, Ira Mellman  Immunity  Volume 44, Issue 3, Pages 609-621 (March 2016) DOI: 10.1016/j.immuni.2016.01.024 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Tumor and TIL Response to MEKi Treatment (A) BALB/c mice were inoculated with CT26 tumor cells, then enrolled when tumor volume reached ∼200 mm3, and treated daily with either vehicle alone (“no drug”) or the Mek inhibitor G-38963 at 75 mg/kg (“MEKi”). (B– F) Tumors were dissected and dissociated at day 12 after the initiation of treatment, fixed, and stained for phospho-Erk: (B) amounts of pErk in gated tumor cells; (C) mean MFI of pErk staining among five tumors per group (±SEM). (D) The percentage of CD8+ cells among all tumor-resident cells; (E) the percentage of CD8+ cells per mm3 of tumor volume; and (F) the percentage of CD8+ TILs that expressed low amounts of PD-1. All graphs show the mean among five tumors per group (±SEM). (G and H) Representative PD-1 expression on CD8+ TILs from (G) a control mouse and (H) a MEKi-treated mouse. Data are representative of three independent experiments. Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 CD8+ T Cell Phenotypic Changes in MEKi-Treated Mice (A and B) T-bet and Eomes expression of gated CD8+ TILs from either representative control (A) or MEKi-treated (B) CT26-tumor-bearing BALB/c mice, at day 12 after the initiation of treatment. (C) The mean total numbers of T-bethiCD8+ TILs from CT26-tumor-bearing mice (n = 5, ±SEM). (D) Total TILs from tumor-bearing mice were stimulated 6 hr in vitro with PMA and ionomycin in the presence of Brefeldin A, then fixed and stained for IFN-γ. The percentage of gated CD8+ cells that were also IFN-γ+ is shown (n = 5, ±SEM). (E and F) gp70-H-2Ld dextramer staining for gated CD8+ TILs from representative control (E) or MEKi-treated mice (F). (G) Average percentage of TILs that were also positive for gp70-H-2Ld-binding: gp70-H-2Ld-dextramer+ as a percentage of all CD8+ TILs (in red); gp70-H-2Ld-positive as a percentage of T-bethiEomesloCD8+ TILs (in blue); and gp70-H-2Ld-positive as a percentage of PD-1loCD8+ TILs (in orange; n = 5, ±SEM). Data are representative of three independent experiments. Additional individual samples are shown in Figure S1. Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 MEKi Inhibits Naive CD8+ T Cell Expansion and Priming In Vitro (A–G) OT-I lymph node cells were loaded with CFSE, then left untreated or stimulated with anti-CD3- and anti-CD28-coated beads, in the presence or absence of 0.5 μM Cobimetinib (MEKi) for 3 days. Shown are representative flow cytometry plots of CFSE dilution versus T-bet upregulation for gated CD8+ cells, for (A) unstimulated, untreated LN cells; (B) anti-CD3- plus anti-CD28-stimulated, untreated LN cells; (C) unstimulated, MEKi-treated LN cells; and (D) anti-CD3- plus anti-CD28-stimulated, MEKi-treated LN cells. (E) The total number of live CD8+ cells after 3 days; (F) the percentage of CD8+ cells that were T-bethi after 3 days; and (G) the percentage of CD8+ cells that had divided at least once, as indicated by dilution of CFSE signal. All graphs show mean ± SEM among duplicate samples and are representative of three independent experiments. (H and I) MEKi dose response curves of OT-I T cells’ 3H-thymidine incorporation, in response to (H) anti-CD3 stimulation over 72 hr or (I) splenocytes pulsed with SIINFEKL peptide, over 48 hr. Means of triplicate samples are shown ± SEM and represent two separate experiments. Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 MEKi Impairs Tumor-Specific CD8+ T Cell Expansion and Priming in Tumor-Bearing Mice (A and B) T-bet and Eomes expression of gated CD8+ cells from the tumor-draining lymph nodes of either representative control (A) or G-38963 (MEKi)-treated (B) CT26-tumor-bearing BALB/c mice, at day 12 after the initiation of treatment. (C) The total number of T-bethiEomesloCD8+ cells in the tumor-draining lymph nodes of control or MEKi-treated tumor-bearing mice (n = 5, ±SEM) and using the gating shown in (A) and (B). (D and E) gp70-H-2Ld dextramer staining of gated CD8+ cells from the tumor-draining lymph nodes of either representative control (D) or MEKi-treated (E) tumor-bearing mice, at day 12 after the initiation of treatment. (F) The total number of gp70-H-2Ld dextramer+ CD8+ cells in the tumor-draining lymph nodes of control or MEKi-treated tumor-bearing mice (n = 5, ±SEM), and using the gating shown in (D) and (E). (G and H) T-bet and Eomes expression of gated gp70-H-2Ld-dextramer+CD8+ cells from the tumor-draining lymph nodes of either representative control (G) or MEKi-treated (H) CT26-tumor-bearing BALB/c mice, at day 12 after the initiation of treatment. (I) The mean percentage of gp70-H-2Ld-dextramer+ cells in the tumor-draining lymph nodes of control or MEKi-treated tumor-bearing mice, either as a percentage of total CD8+ cells (blue) or as a percentage of T-bethiEomesloCD8+ cells (red; n = 5, ±SEM). (J–M) Representative PD-1 expression on gp70-H-2Ld-dextramer+CD8+ LN cells from (J) control mice and (K) MEKi-treated mice. PD-1 expression is quantified for (L) CD8+ LN cells and (M) gp70-H-2Ld-dextramer+CD8+ LN cells (n = 5, mean ± SEM). (N) Total LN T-bethiCD8+ cells in mice treated 10 days with vehicle (black circles) or MEKi, then continued on MEKi for an additional 2 days (12 days total, red squares) or with MEKi withdrawn for the final 2 days (blue triangles) (n = 5, mean ± SEM). Data are representative of three (A–M) or two (N) independent experiments. For (A) and (B), additional individual samples are shown in Figure S2. For (N), additional data are shown in Figure S3. Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 MEKi Impedes TCR-Triggered Death of Chronically Stimulated CD8+ T Cells OT-I LN cells were primed and stimulated weekly for 2 additional weeks using SIINFEKL-pulsed splenocytes. These cells were re-isolated and allowed to rest for 1 day prior to labeling with CellTrace Violet and then further stimulated with anti-CD3- or anti-CD3- plus anti-CD28-coated beads for 18 hr, with or without 0.5 μM Cobimetinib (MEKi), and in the presence of 5 ng/mL IL-2. Representative flow cytometry plots of these cells’ Nur77 expression and Fitc-zVAD-FMK incorporation are shown in (A); zVAD-FMK incorporation by gated Nur77− and Nur77+ cells under conditions of anti-CD3+anti-CD28 without drug are shown in (B); and aggregate Nur77 expression data are shown in (C) (mean ± SEM of duplicate samples, and representative of three separate experiments); representative flow plots of the cells’ zVAD-FMK incorporation are shown in (D); and aggregate zVAD-FMK incorporation data are shown in (E) (mean ± SEM of duplicate samples, and representative of three separate experiments). Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 MEKi Impedes TCR-Triggered Death of CT26 Tumor-Infiltrating CD8+ T Cells In Vitro CD8+ T cells were isolated from 150–250 mm3 CT26 tumors using MACS beads; (A) shows their purity in the left panel, and their T-bet and Eomes expression in the right panel, whereas (B) shows the concordance of caspase activity with Nur77 expression among total CD8+ TILs, and (C) shows the Nur77 and Fitc-zVAD-FMK profile of the three T-bet- and/or Eomes-expressing populations as gated in (A). These CD8+ TILs were then labeled with CellTrace Violet and stimulated 18 hr with anti-CD3- or anti-CD3- and anti-CD28-coated beads, with or without 0.5 μM Cobimetinib (MEKi), and in the presence of 5 ng/mL IL-2. Representative flow cytometry plots of Nur77 expression among T-bethi TILs after 18 hr’ treatment shown in (D), and (E–G) show aggregated Nur77 positivity and zVAD-FMK incorporation among T-bethiEomeshi and/or T-bethiEomeslo CD8+ TILs (mean ± SEM of duplicate samples, and representative of two independent experiments). Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 MEKi Does Not Impair Cytolysis and Combines with Anti-PD-L1 to Yield Anti-tumor Efficacy (A and B) pErk amounts in gated CD8+ LN cells pre-treated with vehicle or MEKi and stimulated with PMA plus ionomycin for 4 min, and quantified in (B) (as gated in A; mean ± SD for duplicate samples and representative of two separate experiments). (C and D) A 50:50 mix of SIINFEKL+CFSE− and SIINFEKL−CFSE+ LN cells were incubated 18 hr with or without MEKi, and with or without OT-I CTLs (at a ratio of 1:1 CTL:target). The CFSE profile of live target cells remaining in the cultures (C), which is quantified in (D) (mean ± SEM for duplicate samples and representative of two separate experiments). Figure S4 provides a more thorough presentation of these results. (E and F) CT26 target cells were incubated 18 hr with or without MEKi and with or without gp70-H-2Ld-specific CTLs. Percentage of CT26 target cells that were Annexin V+ after culture (E), and total live CT26 targets (F; mean ± SEM for duplicate samples and representative of two separate experiments). (G) BALB/c mice were inoculated subcutaneously with CT26 tumor cells, then enrolled when tumor volume reached ∼200 mm3, and treated daily with either vehicle alone (“control”) or the MEK inhibitor G-38963 at 75 mg/kg (“MEKi”). At day 7, CT26 tumors were excised and CD45− tumor cells were analyzed for PD-L1 expression by FACS: average expression among five tumors is shown (mean ± SEM). (H) BALB/c mice were inoculated and enrolled as in (G), then treated daily with either vehicle alone (“control”), the MEK inhibitor G-38963 at 75 mg/kg (“MEKi”), anti-PD-L1, or the combination of MEK inhibitor plus anti-PD-L1. Red slashes indicate where mice in the MEKi+anti-PD-L1 group were euthanized due to excessive tumor volume: six of ten mice in this group survived to day 92 with no sign of tumor burden. Data represent three independent experiments. Additional data are provided in Figures S4–S7 and Table S1. Immunity 2016 44, 609-621DOI: (10.1016/j.immuni.2016.01.024) Copyright © 2016 Elsevier Inc. Terms and Conditions