Supplemental Information. Checkpoint Blockade Immunotherapy. Induces Dynamic Changes. in PD-1 CD8 + Tumor-Infiltrating T Cells

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1 Immunity, Volume 50 Supplemental Information Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1 CD8 + Tumor-Infiltrating T Cells Sema Kurtulus, Asaf Madi, Giulia Escobar, Max Klapholz, Jackson Nyman, Elena Christian, Mathias Pawlak, Danielle Dionne, Junrong Xia, Orit Rozenblatt-Rosen, Vijay K. Kuchroo, Aviv Regev, and Ana C. Anderson

2 Figure S1 (Related to Figure 4). Marker expression and poly-functionality in PD-1 CD8 + TIL subsets. A) Representative flow cytometry data showing expression of CD62L, CX3CR1, and KLRG1 within PD-1 CD8 + TILs. (B C) Frequency (mean ± SEM) of IL-2 + IFN-γ + (B) and TNF-α + IFN-γ + (C) cells among the indicated PD-1 CD8 + TIL populations after ex vivo stimulation with 5 µg/ml OVA peptide. ***p < and ****p < , One-way ANOVA, Tukey s multiple comparison test.

3 Figure S2 (Related to Figure 5). Changes in PD-1 CD8 + TIL subsets after Tim3 + PD-1 blockade. A) Numbers (mean ± SEM) of the indicated PD1 CD8 + TIL subsets in tumors from Tim3 + PD1 blockade- or isotype-treated MC38OVA-bearing mice overtime. **p < 0.01, ***p < 0.001, Mann Whitney U test. B) Frequency (mean ± SEM) of the indicated PD-1 CD8 + TIL subsets from Tim3 + PD-1 blockade- or isotype-treated B16F10-bearing mice. *p < 0.05, ****p < Mann Whitney U test. Data are from two independent experiments.

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5 Figure S3 (Related to Figure 6). Analysis of single-cell RNA profiles from Tim-3 + PD-1 blockade- versus isotype-treated mice. A) Bar graphs show the frequency of cells present in each cluster from isotype (blue) or anti-tim- 3 + PD-1 (red)-treated groups, *p < 0.001, Fisher s exact test. B) I, tsne plot showing projection of an effector CD8 + T cell signature (Kaech et al., 2002), II) projection of the CD62L hi Slamf7 naïve-like signature, III) projection of the Slamf7 hi CX3CR1 memory-precursor-like signature, and IV) projection of the Slamf7 hi CX3CR1 + effector-like signature onto the PD-1 - CD8 + TILs single-cell data. Color scale indicates the signature score. The contour marks the region of highly scored cells by taking into account only those cells that have a signature score above the 10th percentile. Cells with a statistically significant score are marked with a + (Methods). C) I) tsne plot showing projection of IFNβ (Iwata et al., 2017), II) IFNγ (Iwata et al., 2017), III) IL-6 (Hirahara et al., 2015), and IV) IL-12 (Agarwal et al., 2009) signatures onto the PD-1 CD8 + TILs single-cell data. Color scale indicates the signature score. The contour marks the region of highly scored cells by taking into account only those cells that have a signature score above the 10th percentile (Methods). Cells with a statistically significant score are marked with a +. Violin plots show the cytokine signature score from isotype- vs Tim-3 + PD-1 blockade-treated mice. ***p < , t-test. D) Dot plot showing expression of the indicated genes in each of the single-cell clusters. Color scale indicates the expression score of each gene in the indicated cluster. Circle size indicates the percentage of cells that expresses the gene within the indicated cluster.

6 Figure S4 (Related to Figure 6). Naïve, effector, and memory-precursor-like cells in patients. A) GSEA of signatures from human CD8 + TILs (Methods) in the memory-precursor-like CD62L Slamf7 hi CX3CR1 PD-1 (green) and effector-like CD62L Slamf7 hi CX3CR1 + PD-1 (blue) CD8 + TIL subsets. B) Projection of several human signatures (Methods) onto our single-cell clusters (Figure 6A, panel II). The color scale shows the average expression signature score of all the cells that compose the cluster. Circle size indicates the percentage of the cells in each cluster that expresses a signature above the median and the dark borders indicate clusters that were either significantly concentrated or depleted of high-scoring cells (FDR- adjusted P value < 0.05, t-test). A + sign indicates clusters that had a statistically significant score (FDR- adjusted P value <0.05) compared to randomly generated signatures (Methods). Clusters that are naïve-like, effector-like, and memory-precursor-like (Fig. 6E) are indicated by the colored bars.

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8 Figure S5 (Related to Figure 7). Analysis of Tcf7/Tcf1 expression in PD-1 CD8 + TIL subsets and thymic development and peripheral homeostasis in E8i-Cre + Tcf7 fl/fl mice. A) Mean fluorescence intensity (MFI; mean ± SEM) of Tcf1 protein (encoded by Tcf7) in the indicated populations of PD-1 CD8 + TILs. *p < 0.05, **p < 0.01, ****p < , One-way ANOVA, Tukey s multiple comparison test. B) Representative FACS plots showing CD4 and CD8 expression in the thymus (top) and spleen (bottom) of E8i-Cre Tcf7 fl/fl vs E8i-Cre + Tcf7 fl/fl mice (n = 3 per group).

9 Figure S6 (Related to Figure 7). Tumor-antigen specific CD8 + TILs in the absence of Tcf7 and tumor regression in TLR9 agonist treated mice A) E8i-Cre Tcf7 fl/fl and E8i-Cre + Tcf7 fl/fl were implanted with MC38-OVA and TILs analyzed days post implant. Frequency (mean ± SEM) of Ova-specific cells in PD1 + and PD1 CD8 + TILs in E8i-Cre Tcf7 fl/fl and E8i-Cre + Tcf7 fl/fl mice is shown. *p < 0.05, ***p < 0.001, t-test. B) WT mice were implanted with MC38-OVA and treated with PBS (blue) or TLR9 agonist (red; IMO-2125) on days 4 and 7. Tumor size (mean ± SEM, top) and individual tumor size (bottom) in each group is shown. ****p < , linear regression. Data are representative of at least three independent experiments.