Supplementary Table 1. Flow cytometry antibody details:

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 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.

Supplementary Figure 13. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while destabilizing Tregs in LLC tumor. (A) Schema of LLC 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) Numbers of cells/mg 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).

Supplemental Figure 11. The triple combination increases activation of CD8+ T cells and CD4+ Teffs while Tregs remain unaffected in LLC TDLN. (A) Schema of LLC 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) Image of TDLNs from LLC tumor bearing mice. (C) Gating strategy for all in vivo flow experiments. (D) Absolute number of immune cell populations in the TDLN; n=4-5. (E) Phenotypes of T cells from the TDLN; n=9-10. 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 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 5. Intermittent CKI27 allows for immune cell recovery in the spleen, increases frequencies in the TDLN, and inhibits TILs similarly to continuous treatment. (A) Schema of LLC tumor bearing mice treated with vehicle, daily 2mg/kg CKI27, or intermittent 5mg/kg 4on/3off CKI27. Mice were treated in a staggered schedule and all timepoints were harvested on day 23. (B-D) All fold changes were calculated by normalizing to DMSO. (B) Fold changes of absolute number (cells/uL) of spleen immune cell populations. (C) Fold changes of absolute number (cells/uL) of TDLN immune cell populations. (D) Fold changes of tumor weights (mg) and TILs (cells/mg); n=4-5. Data are shown as mean±SEM. Unpaired, nonparametric Mann-Whitney test was used to compare each time point. Significance levels are indicated by asterisks (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001)

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

Supplementary Figure 15. The triple combination favorably alters the genetic profile of immune cells in the TDLN. LLC tumor bearing mice were treated with vehicle, CKI27, isotypes, GITR, and/or CTLA-4. TLDNs were harvested on day 21 (7 days post treatment). Live CD45+ cells were FACS sorted and processed for sc-RNA sequencing. (A-B) Heatmap showing top genes expressed by each cluster for (A) CD8+ and (B) CD4+ T cells. (C) UMAPs of each treatment groups showing different clusters and annotations. (D) UMAPs of CD8+ and CD4+ T cells from each treatment group showing specific activation genes and proteins.

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 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 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 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 6. Intermittent CKI27 treatment and GITR co-stimulation relieves expression of co-inhibitory markers. (A) Gating strategy for all T cell activation assays. (B) Representative dot plot data for FACS analysis of co-inhibitory markers expressed by CD8+ T cells.

Supplementary Table 2. Gene list associated with each cluster.

Abstract T cell immune checkpoint blockade (ICB) has shown remarkable promise in melanoma and other cancers. However, most patients do not show clinical benefit. This is because tumors can activate multiple checkpoints and immunosuppressive pathways to evade anti-tumor immune responses. Inhibition of these immune suppressive mechanisms and immune checkpoints or repolarizing the tumor microenvironment (TME) to become more accessible to the immune system may be necessary for maximal therapeutic efficacy of immunotherapies. There is increasing evidence that tumor hypoxia can attenuate the antitumor immune response by promoting immune suppression and inhibiting direct killing by cytotoxic immune cells. We propose that blocking tumor oxygen consumption using drugs that target the mitochondrial complex I (phenformin and metformin) will enhance the efficacy of immunotherapies such as ICB in preclinical models such as B16 melanoma. In this study, we examined how low oxygen levels (hypoxia) influence T cell effector function such as cytotoxic activity and cytokine production in vitro. We co-cultured B16 melanoma cells in the presence of tumor specific T cells (Pmels) and incubated at 37ºC in either normoxia (~21% O2) or hypoxia (3% O2) and assessed killing and intracellular cytokine production 24 - 48 hours later. We show that the ability of tumor antigen-specific T cells to kill tumor cells or make effector cytokine such as IFNγ and TNFα is significantly reduced in hypoxia settings. We also examined the effects of hypoxia targeting drugs (metformin and phenformin) on T cell priming and activation. When T cells were activated in the presence of each drug in vitro, there was enhanced expression of T cell activation markers (CD25, Granzyme B) as well as an increase in their differentiation into central memory T cells. We next determined whether these drugs could alter the TME in vivo by treating C56BL/6J mice bearing established B16 tumors with phenformin and metformin and then examine the effects on the immune system in the tumor, tumor draining lymph nodes and spleen by flow cytometry. We found that these three drugs have differential effects on both the innate and adaptive the immune system in the tumors and periphery. Finally, we tested whether these drugs could delay B16 melanoma growth in vivo as a monotherapy or in combination with anti-PD1. We found that while both drugs can significantly delay tumor growth as monotherapies, only metformin showed improved anti-tumor efficacy when combined with anti-PD-1 in vivo. These studies show that while both drugs target tumor oxygen consumption (via inhibition of complex I), and can delay B16 tumor growth in vivo, they have differential effects on the TME and will need further considerations when combining with immune based therapies such as ICBs. Citation Format: Sadna Budhu, Anais Assouvie, Mamadou Bah, Svena Verma, Inna Serganova, Mayuresh Mane, Juan Zurita, Jason Koutcher, Vladimir Ponomarev, Jedd D. Wolchok, Taha Merghoub. Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4053.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults presenting with a 95% five-year mortality rate. Due to high relapse rates, GBM remains a major unmet clinical need for new treatment approaches. An innovative approach to target GBM is the administration of chimeric antigen receptor (CAR) T cells to treat relapsed/refractory disease. In this study we identified B7-H3 as an optimal target antigen for CAR T cell therapy of GBM, and developed a novel B7-H3 targeting CAR T cell. These CAR T cells demonstrated robust, antigen-specific cytolysis and cytokine production, while demonstrating low tonic signaling and preservation of a stem/naïve cell phenotype. Using multiple orthotopic xenograft models of GBM we show that a single intravenous administration of B7-H3 targeted CAR T cells confers tumor control, and long-term survival. We identified upregulation of T cell inhibitory ligands PD-L1 and PD-L2 as a mechanism of resistance to CAR T cell therapy. Accordingly, we modified our B7-H3 targeting CAR T cells to secrete a PD-1 blocking scFv downstream of the CAR. Using this armored approach, we further demonstrated that concomitant PD-1 blockade through secretion of a PD-1 blocking scFv by CAR T cells augments the antitumor capabilities of B7-H3-targeting CAR T cells. This was demonstrated through increased infiltration of the adoptively transferred T cells into tumors and improved effector function which conferred durable, long-term remissions in a disease-relevant model of GBM. Together, these data demonstrate B7-H3 targeting CAR T cells armored with concomitant PD-1 blockade are a safe and efficient method for improving the antitumor capacity of B7-H3 targeting CAR T cells in the context of GBM.