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Figure S1. Efficacy of pemetrexed in Lewis Lung carcinoma model and metabolomic analysis of MC38 tumor bearing mice treated with 50 mg/kg of pemetrexed Figure S2. T cell inflamed phenotype induced by pemetrexed is modestly reduced in tumors upon combination with cisplatin Figure S3. Combination Effects of pemetrexed -/+ anti-PD-L1 in MC38, LLC and Colon26 tumor models Figure S4 (A) Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with pemetrexed (50 mg/kg, 5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse, weekly IP), and tumors were isolated after 14 days of treatment and single cell suspensions were subjected to flow cytometry analysis. FACS plots show representative data of intra-tumor CD45+ tumor-infiltrating lymphocyte (TIL) T cell (CD3+) and Myeloid cell (CD11b+) subsets frequencies; and percentage of Ki67+ CD4+ T effector cells based on the gating scheme indicated. (B) Spleens, TDLNs and tumors were isolated and antigen specific T cell responses were measured by IFNγ ELISpot, gp70 Tetramer staining (Tet) and Intracellular cytokine staining. (Left) CD8 T cells isolated from pooled spleens and TDLNs from individual mice were cultured alone (control) with irrelevant BALB/c tumor EMT6 or cognate Colon26 tumor cells overnight and number of IFNγ producing T cells was measured by ELISpot. (Middle) Freshly isolated CD8+ T cells from spleens and TDLNs were assessed for % of gp70 Tet+ cells. (Right) Freshly isolated tumors single cell suspensions were stimulated with PMA/ phorbol myristate acetate (PMA) and ionomycin with monensin for 4 hrs and the % of TNF-alpha positive and gp70 Tet+ CD8 T cells were measured from the Live CD45+, CD3+ T cells by FACS. Figure S5. Concordance between QuantiGene and nCounter gene expression data for in vitro activated T cells treated in the presence or absence of pemetrexed (related to Figure 6F). Supplementary Table 1. Ingenuity pathway analysis (IPA) of MC38 tumors treated with pemetrexed or paclitaxel with or without carboplatin Supplementary Table 2: IPA results for Colon26 experiment in Figure 4 sorted by functional class. Pemetrexed monotherapy in this experiment at this timepoint did not have enough differentially expressed genes to generate IPA results.

Purpose:Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non–small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown.Experimental Design:Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis.Results:Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell–intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1.Conclusions:Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell–intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890