Valeria Estrada Nevarro

Supplementary Table 1. Flow cytometry antibody details:

Supplementary Figure 14. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in CT26 tumor. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Tumor weights, numbers of cells/mg, and CD8:Treg ratio of TILs; n=4-5. (C) Phenotypes of TILs; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 12. The triple combination increases activation of CD8+ T cells, CD4+ Teffs, and Tregs in CT26 TDLN. (A) Schema of CT26 tumor bearing mice treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. All timepoints were harvested on day 21 (7 days post treatment). (B) Absolute number of immune cell populations in the TDLN; n=4-5. (C) Phenotypes of T cells from the TDLN; n=4-5. Data are shown as mean±SEM. One-way ANOVA test with Bonferroni’s correction for multiple comparisons was used in all panels. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 4. Intermittent CKI27 treatment partially relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr) or washout CKI27 (24hr on, 72hr off); n=2-3. (A) Proliferation fold change of CTVlow CD8+ and CD4+ T cells was calculated by normalizing to DMSO. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 9. Intermittent CKI27 treatment and GITR engagement relieves suppressive effects of MEK inhibition on T cell proliferation, cytokine production, and effector function. (A-C) Human PBMCs were labelled with CTV, sub-optimally stimulated with 1:25 or 1:100 CD3/CD28 Dynabeads, and treated with DMSO, continuous CKI27 (96hr), washout CKI27 (24hr on, 72hr off), and/or GITR-L; n=2-3. (A) % proliferation of CTVlow CD8+ and CD4+ T cells. (B) FACS analysis of co-inhibitory, co-stimulatory, and activation markers on CD8+ and CD4+ T cells. Heatmaps represent fold changes of positive percentages of each marker normalized to DMSO. (C) Cytokine analysis of supernatants collected from all PBMCs. Heatmap represents fold changes of concentrations (pg/mL) of proteins normalized to DMSO. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

Supplementary Figure 3. MEK inhibition with CKI27 increases HLA and checkpoint ligand expression. (A-B) Human tumor cell lines were treated with DMSO or CKI27 for 72hr and either with or without IFNγ (10ng/mL) for the last 24hr; n=3. FACS analysis of (A) HLA-ABC and HLA-DR and (B) PD-L1, CD80 and CD86 surface expression. MFI values are shown as mean±SEM.

Supplementary Figure 1. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. (A-B) Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of (A) MHC-I (H2Kb/Kd and H2Db/Dd) and MHC-II and (B) PD-L1, CD80 and CD86 surface expression. Median fluorescence intensity (MFI) values were normalized to DMSO and log transformed. Data are shown as mean±SEM.

Supplementary Figure 8. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of activation markers. Representative dot plot data for FACS analysis of activation markers expressed by CD8+ T cells.

Supplementary Figure 10. The triple combination reduces tumor growth, is T cell dependent, and protects from re-challenge in LLC and CT26. (A-D) LLC tumor bearing mice were treated with vehicle, isotypes, GITR, CTLA-4, 5mg/kg 4on/3off CKI27, and/or CD8 for 4 weeks and tumor growth was monitored over time. (A) Average tumor growth (volume, mm3) of immunocompetent mice. (B) Average tumor growth (volume, mm3) of immunodeficient mice. (C) Average tumor growth (volume, mm3) of CD8 depleted mice. (D) Average tumor growth (volume, mm3) of mice from (A) that were re-challenged. (E-H) CT26 tumor bearing mice were treated with vehicle, isotypes, GITR, αCTLA-4, 2mg/kg 4on/3off CKI27, and/or αCD8 for 4 weeks and tumor growth was monitored over time. (E) Average tumor growth (volume, mm3) of immunocompetent mice. (F) Average tumor growth (volume, mm3) of immunodeficient mice. (G) Average tumor growth (volume, mm3) of CD8 depleted mice. (H) Average tumor growth (volume, mm3) of mice from (E) that were re-challenged. Two-way ANOVA test with Bonferroni’s correction for multiple comparisons was used. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).

Supplementary Figure 2. MEK inhibition with CKI27 increases MHC and checkpoint ligand expression. Murine tumor cell lines were treated with DMSO or CKI27 for 72 hr and either with or without IFNγ (5ng/mL) for the last 24hr; n=3. FACS analysis of representative histograms for the MFI of MHC-I (H2Kb/Kd and H2Db/Dd), MHC-II, PD-L1, CD80 and CD86 are shown.