Volume 22, Issue 1, Pages 206-218 (January 2014) Immunotherapy-induced CD8+ T Cells Instigate Immune Suppression in the Tumor  A J Robert McGray, Robin Hallett, Dannie Bernard, Stephanie L Swift, Ziqiang Zhu, Florentina Teoderascu, Heather VanSeggelen, John A Hassell, Arthur A Hurwitz, Yonghong Wan, Jonathan L Bramson  Molecular Therapy  Volume 22, Issue 1, Pages 206-218 (January 2014) DOI: 10.1038/mt.2013.255 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 CD8+ T cells and interferon (IFN)-γ mediate tumor growth suppression that is associated with the activity of CD8+ T cells that infiltrate the tumor early following vaccination. (a) DCT (square) and gp100 (triangle)-specific CD8+ peripheral blood lymphocytes (PBLs) were measured based on IFN-γ production following ex vivo peptide stimulation at different time points post-rHuAd5-hDCT or rHuAd5-hgp100 immunization (n = 5–12). (b) Tumor-bearing mice were immunized with either rHuAd5-hDCT (square), rHuAd5-hgp100 (triangle), or left untreated (circle). (c) Tumor-bearing mice were treated with rHuAd5-hDCT and depleted of CD4+ (filled square), CD8+ (inverted triangle), or both cell subsets (diamond) or left nondepleted (NT) (square). (d) Tumor-bearing mice were treated with rHuAd5-hDCT (square) or the vaccine in combination with IFN-γ neutralization (triangle). (e) Expression of IFN-γ in tumors from mice treated with either rHuAd5-hDCT (square), rHuAd5-hgp100 (triangle) or left untreated (circle) (n = 4). (f) Intratumoral tumor necrosis factor (TNF)-α expression as described in e. (g) DCT-specific CD8+ TIL were enumerated following rHuAd5-hDCT immunization (n = 8–19). Tumor volumes in b–d reflect individual representative experiments (n = 4–6). Data presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. DCT, dopachrome tautomerase. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Local immune activity instigates an adaptive immunosuppressive response in the tumor. (a) Heat Map displaying the relative expression of immunosuppressive genes in rHuAd5-hDCT or Untreated tumors (n = 4). (b) Expression of PD-L1, PD-L2, iNOS, and arginase 1 in tumors from mice treated with rHuAd5-hDCT and depleted of CD4+ (αCD4), CD8+ (αCD8), both cell subsets (αCD8/αCD4) or left nondepleted (NT) (n = 4). Data points correspond to peak expression of individual genes and grey areas correspond to the mean ± SEM for genes expressed in untreated tumors. (c) Expression of PD-L1, PD-L2, iNOS, and arginase 1 in tumors from wild type (WT) (day 5 or 9, corresponding to peak expression) or interferon (IFN)-γ−/− (day 5 and 8) mice treated with rHuAd5-hDCT (n = 4). (d) Expression of IFN-γ in tumors following treatment with rHuAd5-hDCT or rHuAd5-hDCT in combination with IFN-γ neutralization (n = 4–5). (e) Tumor-bearing mice were immunized with rHuAd5-hDCT and subsequently treated with αPD-1 or the combination of αPD-1/αTIM-3/αLAG3 beginning 3 days following immunization (n = 5). Data presented as mean ± SEM. *P < 0.05, **P < 0.01. DCT, dopachrome tautomerase; NS, not significant. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 rHuAd5-hDCT immunization instigates global transcriptional changes in the tumor. (a) Hierarchical clustering of differentially expressed genes in tumors following treatment with rHuAd5-hDCT, rHuAd5-hgp100, or untreated tumors (n = 3). (b) Graphical representation of gene ontology (GO) analysis of genes differentially expressed in rHuAd5-hDCT treated tumors. (c) Positively enriched pathways in the tumor following treatment with rHuAd5-hDCT. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Increasing the magnitude of the DCT-specific CD8+ T-cell response results in tumor regression and improved survival that correlates with the early CD8+ T-cell response. (a) Tumor-bearing mice were immunized with rHuAd5-hDCT + 104 (square), 105 (triangle), or 106 (circle) DCTT cells, rHuAd5-LCMV-GP + 106 DCTT cells (diamond), or rHuAd5-hDCT alone (filled square) (b) Survival data corresponding to the treatments outlined in a. (c) DCT-specific CD8+ peripheral blood lymphocytes (PBLs) were measured (based on interferon (IFN)-γ production in response to ex vivo peptide stimulation) following treatment with rHuAd5-hDCT, rHuAd5-hDCT + 104–106 DCTT cells, or rHuAd5-LCMV-GP + 106 DCTT cells (n = 7–14). (d) Analysis of the early (day 5) and late (day 10) frequencies of DCT-specific CD8+ PBL, clustered based on tumor regression versus tumor growth, for mice immunized with rHuAd5-hDCT ± transfer of DCTT cells (n = 14–35). Tumor volumes were calculated from a single representative experiment (n = 4–5) and survival data was compiled from independent experiments (n = 7–14). Data presented as mean ± SEM. *P < 0.05, ***P < 0.001. DCT, dopachrome tautomerase; NS, not significant. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 DCT-specific CD8+ TIL display a progressive decline in functionality despite increased tumor infiltration over time. Representative FACs plots showing interferon (IFN)-γ production by transferred (Thy1.1+) and endogenous (Thy1.1−) DCT-specific CD8+ T cells in response to peptide stimulation following rHuAd5-hDCT immunization + DCTT-cell transfer (n = 5–9). (b) Representative FACs plots showing frequencies of Thy1.1+ CD8+ TIL able to produce multiple cytokines (IFN-γ/TNF-α) and degranulate (IFN-γ/CD107a) over time as described in a (n = 5–9). (c) Transferred DCT-specific CD8+ TIL (Thy1.1+CD8+) were enumerated following vaccination with rHuAd5-hDCT (n = 5–9). Data presented as mean ± SEM. **P < 0.01. DCT, dopachrome tautomerase; TIL, tumor infiltrating lymphocyte; TNF, tumor necrosis factor. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Transfer of high dose DCTT cells in combination with rHuAd5-hDCT results in a transient increase in local intratumoral immune activity, but is accompanied by increased expression of immunosuppressive genes in the tumor. (a) Intratumoral expression of interferon (IFN)-γ (left), TNF-α (middle), and TCR-α (right) in tumors following treatment with rHuAd5-hDCT (square), rHuAd5-hDCT + 106 DCTT cells (triangle), or rHuAd5-LCMV-GP + 106 DCTT cells (circle) (n = 4) (b) Relative IFN-γ expression normalized to relative TCR-α expression in tumors following treatment with rHuAd5-hDCT (square) or rHuAd5-hDCT + 106 DCTT cells (triangle) (n = 4). (c) Heat Map displaying the relative expression of immunosuppressive genes in tumors following treatment with rHuAd5-hDCT ± 106 DCTT cells and rHuAd5-LCMV-GP + 106 DCTT cells (n = 4). Data presented as mean ± SEM. DCT, dopachrome tautomerase; TCR, T-cell receptor; TNF, tumor necrosis factor. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Transfer of high dose DCTT cells in combination with rHuAd5-hDCT initiates similar global changes within the tumor as vaccination alone, but of greater magnitude. (a) Hierarchical clustering of differentially expressed genes in untreated tumors compared to tumors treated with rHuAd5-hDCT (left) or rHuAd5-hDCT + 106 DCTT cells (right) (n = 4). (b,c) Comparison of differentially expressed genes shown in a using Venn diagrams. (d) Comparison of average relative expression for all codifferentially expressed genes following treatment with rHuAd5-hDCT ± 106 DCTT cells (n = 4). Data presented as mean ± SEM. DCT, dopachrome tautomerase. Molecular Therapy 2014 22, 206-218DOI: (10.1038/mt.2013.255) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions