Article Figures & Data
Figures
- Figure 1.
Expression of PD-1 and PD-L1 in murine and human PDA. A, representative histograms and quantification of PD-1 expression on tumor-infiltrating CD8+ (gated on live, CD45+, CD3+, CD8+), CD4+ (gated on live, CD45+, CD3+, CD4+), or regulatory (Tregs; gated on live, CD45+, CD3+, CD4+, FoxP3+) T cells in tumors (n = 6–11) or spleens (n = 4–17) from tumor-bearing KPC mice. **, P ≤ 0.01; ****, P ≤ 0.0001. B, representative histograms and quantification of PD-L1 expression on tumor cells, normal pancreatic epithelial cells (gated on live, CD45neg, CD31neg, CD90neg), DCs (gated on live, CD45+, F4/80neg, CD19neg, CD11c+), and macrophages (Macs; gated on live, CD45+, F4/80+) in tumors (n = 4–11) or spleens (n = 25) from tumor-bearing KPC mice; and normal pancreata (n = 5) from healthy C57BL/6 mice. **, P ≤ 0.01; ***, P ≤ 0.001. C, histology and quantification of PD-L1 expression and CD8+ T-cell infiltration in human pancreatic cancer sections. Left two, PD-L1 expression on malignant cells of a PDA tumor (PD-L1 expression score of 4+ (intense). See Materials and Methods and Supplementary Fig. S1. ×40 and ×400 magnification for top and bottom, respectively). Right top, CD8 expression in serial section of the tumor on left, showing few tumor-infiltrating CD8+ T cells (×40 magnification). Right bottom, plot describing correlation between intratumoral CD8 count and tumor PD-L1 score (n = 8). P = 0.69.
- Figure 2.
PD-1–PD-L1 axis is highly expressed and is not IFNγ-dependent in a subcutaneous murine model of PDA. A, experimental design for establishment of subcutaneous PDA tumors or chronic LCMV clone 13 (Cl-13) infection simultaneously in two cohorts of C57BL/6 mice. B, representative flow plots and quantification of coexpression of PD-1 and Lag-3 on CD8+ (gated on live, lymphocytes, B220neg, NK1.1neg, CD8+), CD4+ (gated on live, lymphocytes, B220neg, NK1.1neg, CD4+) and regulatory (Tregs; gated on live, lymphocytes, B220neg, NK1.1neg, CD4+, FoxP3+) T cells from spleens of mice infected with LCMV Cl-13 (day 30) or the tumors and spleens of mice bearing PDA tumors (day 14). C, representative histograms and quantification of PD-L1 expression on tumor cells, DCs, and macrophages (Mac) in subcutaneous PDA tumors or spleens from the same mice (day 14), gated as in Fig. 1B. ****, P ≤ 0.0001. See also Supplementary Fig. S2. D, histogram of KPC-derived PDA cell line interrogated for PD-L1 expression in vitro with or without IFNγ in the culture, representative of three experiments. E, quantification and MFI of PD-L1 expression on tumor cells from subcutaneous PDA tumors established in either C57BL/6 (B6) or IFNγ−/− (IFNγ ko) mice with or without CD4+ and CD8+ T-cell depletion (TCD; day 16; n = 6-8 mice per cohort). F, quantification and MFI of PD-L1 expression on DCs and macrophages in subcutaneous PDA tumors grown in either B6 or IFN-γ ko mice with or without TCD (day 16; n = 6–8 mice per cohort). One-way ANOVA:%DCs PD-L1+, P = 0.015; DC PD-L1 MFI, P = 0.0039;%Macs PD-L1+, P = 0.58; Macs PD-L1 MFI, P = 0.0007. Post hoc test P values are indicated where statistically significant: *, P ≤ 0.05; **, P ≤ 0.01.
- Figure 3.
T-cell stimulation with αCD40/gemcitabine/nab-paclitaxel potentiates efficacy of checkpoint blockade in murine model of PDA. A, experimental design for study of subcutaneous PDA tumors treated with checkpoint inhibitors and αCD40/chemotherapy, as further described in Materials and Methods (G, gemcitabine; nP, nab-paclitaxel; q3d, antibody administered every 3 days). B, tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as indicated (n = 9–10 per cohort; results for control and αPD-1 + αCTLA-4 cohorts representative of three independent experiments). See also Supplementary Fig. S7. C, tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as indicated (n = 9–10 per cohort; findings representative of three independent experiments). Two-way ANOVA: P ≤ 0.0001. Post hoc test P values are indicated where statistically significant: *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001. See also Supplementary Fig. S7. D, percentage of mice bearing subcutaneous PDA tumors treated with indicated regimens that rejected their tumors and survived tumor-free long-term (median follow-up of 42 days; range, 23–222 days). Data were compiled from five independent experiments.
- Figure 4.
Rejection of PDA tumors after αCD40/chemotherapy and checkpoint blockade is T cell–mediated. A, tumor growth and survival analyses of mice bearing subcutaneous PDA tumors treated as indicated (n = 9–10 per cohort; G, gemcitabine; nP, nab-paclitaxel; TCD, CD4/CD8 depletion). B, flow cytometric analysis of subcutaneous PDA tumors treated as indicated (day 18 after tumor injection, day 7 after αCD40 treatment; P, αPD-1; C, αCTLA-4). One-way ANOVA%CD8s of live cells: P = 0.17; one-way ANOVA%Tregs of CD4+ T cells: P = 0.0004; one-way ANOVA CD8:Treg ratio: P = 0.0005. Post hoc test P values are indicated where statistically significant: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. C, experimental design for first tumor rechallenge experiments. The table quantifies fraction and percentage of mice that rejected tumor rechallenge in mice that had rejected the initial tumor implantation and were tumor free for at least 43 days. Data were compiled from three independent experiments. D, experimental design for second tumor rechallenge experiment. The second rechallenge occurred on days 31 to 49 after the first rechallenge. The table quantifies fraction and percentage of mice that rejected the second tumor rechallenge in mice that had rejected a first tumor rechallenge. Host mice in this experiment were either treated with αCD8 (n = 11) or isotype (Iso; n = 6) antibodies. Survival analysis of mice after the second rechallenge with or without CD8 depletion is shown. Data were compiled from two independent experiments.
- Figure 5.
Combination of αCD40/chemotherapy and PD-1 blockade improves survival in KPC genetic model of PDA. A, experimental design for randomized, controlled study in tumor-bearing KPC mice, treated with αCD40/chemotherapy and αPD-1, as described in Materials and Methods. G, gemcitabine; nP, nab-paclitaxel; q3d, antibody administered every 3 days. B, overall survival analysis of tumor-bearing KPC mice treated as indicated (n = 6–8 per cohort). αPD-1 alone versus isotype alone P = 0.39; CD40/G/nP vs. isotype alone P = 0.76; CD40/G/nP + αPD-1 vs. isotype alone P = 0.015. C, median overall survival of tumor-bearing KPC mice treated as indicated.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - Supplementary Methods
- Supplemenatry Figure Legends - Supplementary Figure Legends
- Supplementary Figure S1 - Supplementary Figure S1. PD-L1 expression in human PDA.
- Supplementary Figure S2 - Supplementary Figure S2. PD-L1 expression on lineage labeled PDA cells in vivo.
- Supplementary Figure S3 - Supplementary Figure S3. CTLA-4 expression on T cell subsets in mice bearing subcutaneous PDA tumors.
- Supplementary Figure S4 - Supplementary Figure S4. CD40 expression on APCs in subcutaneous PDA tumors.
- Supplementary Figure S5 - Supplementary Figure S5. T cell changes following αCD40/chemotherapy treatment.
- Supplementary Figure S6 - Supplementary Figure S6. Antitumor effect of αCD40/chemotherapy is macrophage independent.
- Supplementary Figure S7 - Supplementary Figure S7. Tumor growth curves of individual mice with subcutaneous PDA treated as indicated.
- Supplementary Figure S8 - Supplementary Figure S8. Non-tumor related events and deaths in tumor bearing C57BL/6 mice after immunotherapy.
Volume 3, Issue 4
