Article Figures & Data
Figures
- Figure 1.
Anti-PD1 sensitivity correlates with reduced frequency of PD1-expressing CD8+ T cells. Groups of B6 WT mice (n = 5–8) were s.c. injected with MC38 tumor (1 × 106 cells; A, C, and E) or AT3 tumor (1 × 106 cells; B, D, and F) on day 0. A and B, tumor-bearing mice were treated with 250 μg of cIg or anti-PD1 on days 6, 10, and 14 (A) or days 15, 19, 23, and 27 (B). Tumor growth was measured using a digital caliper, and tumor sizes are presented as mean ± SEM. Statistical differences in tumor sizes between cIg- and anti-PD1–treated mice were determined by an unpaired t test (**, P < 0.01; ***, P < 0.001). Data are representative of two or more independent experiments. C–F, tumor-bearing mice were treated with 250 μg of cIg or anti-PD1 on day 10 (C and E) or day 14 (D and F), and tumors were harvested 2 or 3 days after antibody treatments for flow cytometric analyses. C and D, representative FACS plots for gating strategy to define frequencies of PD1-expressing CD4+ T cells (left) and CD8+ T cells (right) between cIg- and anti-PD1–treated mice are shown. E and F, frequencies of PD1-expressing CD4+ T cells and CD8+ T cells are shown. Statistical differences in frequencies of PD1-expressing CD4+ T cells and CD8+ T cells between cIg- and anti-PD1–treated mice were determined by an unpaired t test (****, P < 0.0001). Data shown are pooled from 12 (E) and 7 (F) independent experiments and are presented as the mean ± SD, with individual symbols representing individual mice. Data shown for frequencies of PD1-expressing CD4+ T cells, and PD1-expressing CD8+ T cells between cIg- and anti-PD1–treated mice (E and F) are the same data set as shown in Supplementary Fig. S1B and S1D.
- Figure 2.
Anti-PD1 mAb sensitivity correlates with intratumor T-cell inflammatory cytokines. Groups of B6 WT mice (n = 5–8) were s.c. injected with MC38 tumor (1 × 106 cells; A, B, E, and F) or AT3 tumor (1 × 106 cells; C, D, G, and H) on day 0. Tumor-bearing mice were treated with 250 μg of cIg or anti-PD1 on day 10 (A, B, E, and F) or day 14 (C, D, G, and H), and tumors were harvested 2 or 3 days after antibody treatments for flow cytometric analyses. A–D, frequencies of IFNγ- and TNF-expressing CD4+ T cells (A and C) and CD8+ T cells (B and D) between cIg- and anti-PD1–treated mice are shown. Data are presented as the mean ± SD, with individual symbols representing individual mice. Statistical differences in frequencies of IFNγ- and TNF-producing cells of CD4+; and CD8+ T-cell subsets between cIg- and anti-PD1–treated mice were determined by an unpaired t test (*, P < 0.05). Data shown are representative of three (A and B) and two (C and D) independent experiments. Tbet MFI (E and G) and Eomes MFI (F and H) of CD4+ T cells and CD8+ T cells between cIg- and anti-PD1–treated mice are shown. Data are presented as the mean ± SD, with individual symbols representing individual mice. Statistical differences in Tbet MFI (E and G) and Eomes MFI (F and H) of CD4+ and CD8+ T-cell subsets between cIg- and anti-PD1–treated mice were determined by an unpaired t test (* P < 0.05). Data shown are representative of four or more (E and F) and three (G and H) independent experiments.
- Figure 3.
T-cell PD1 level and frequency is independent of T-cell differentiation. Groups of B6 WT mice (n = 5–8) were s.c. injected with MC38 tumor (1 × 106 cells; A and B) or AT3 tumor (1 × 106 cells; C and D) on day 0. Tumor-bearing mice were treated with 250 μg of cIg or anti-PD1 on day 10 (A and B) or day 14 (C and D), and tumors were harvested 2 or 3 days after antibody treatments for flow cytometric analyses. Frequencies of CD44+CD62L− and CD44+CD62L+ in CD4+ T cells (A and C) and CD8+ T cells (B and D) between cIg- and anti-PD1–treated mice are shown. Data are presented as the mean ± SD of 5 mice, with individual symbols representing individual mice. Statistical differences in the frequencies of CD44+CD62L− and CD44+CD62L+ in CD4+ and CD8+ T-cell subsets between cIg- and anti-PD1–treated mice were determined by an unpaired t test (**, P < 0.01). Data shown are representative of four (A and B) and two (C and D) independent experiments.
- Figure 4.
Treg promotes anti-PD1–resistant PD1hi T cells. Groups of B6 WT (A and B) and Foxp3-DTR (C–F) mice (n = 5–10) were s.c. injected with MC38 tumor (1 × 106 cells; A, C, and D) or AT3 tumor (1 × 106 cells; B, E, and F) on day 0. A and B, tumor-bearing mice were treated with 250 μg of cIg or anti-PD1 on day 10 (A) or day 14 (B), and tumors were harvested 2 or 3 days after antibody treatments for flow cytometric analyses. Frequencies, numbers of Treg (CD4+Foxp3+), and CD8/Treg ratio between cIg- and anti-PD1–treated mice are shown. Data are presented as the mean ± SD, with individual symbols representing individual mice. Statistical differences in the frequencies, cell numbers, and CD8/Treg ratio between cIg- and anti-PD1–treated mice were determined by an unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data shown are pooled from 7 (A) and 5 (B) independent experiments. C–F, tumor-bearing Foxp3-DTR mice were treated with PBS or 500 ng of DT on day 10 (C and D) or day 14 (E and F), and tumors were harvested 3 or 4 days after PBS/DT treatment for flow cytometric analyses. C and E, frequencies of PD1-expressing CD4+ and CD8+ T cells with their representative histogram plots (shaded histogram, PBS-treated; open histogram, DT-treated) from PBS- and DT-treated mice are shown. Data are presented as the mean ± SD with individual symbols representing individual mice. D and F, Tbet MFI of CD4+ and CD8+ T cells of PBS- and DT-treated mice are shown. Data are presented as the mean ± SD, with individual symbols representing individual mice. Statistical differences in frequencies of PD1-expressing T cells (C and E) and Tbet MFI (D and F) of respective cell subsets between PBS- and DT-treated mice were determined by an unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). G and H, groups of Balb/c or B6 Foxp3-DTR mice (n = 5) were s.c. injected with CT26 tumor (1 × 105 cells; G) or AT3 tumor (5 × 105 cells; H) on day 0. G, CT26-bearing mice were treated PBS or 250 ng DT on day 14 as indicated. Mice then received 250 μg of cIg or anti-PD1 on days 17, 20, and 22. H, AT3-bearing mice were treated with PBS or 100 ng DT on day 16 as indicated. Mice then received 250 μg of cIg or anti-PD1 on days 20, 24, and 28. Tumor growth was measured using a digital caliper, and tumor sizes are presented as mean ± SEM. Statistical differences in tumor growth between DT- and DT and anti-PD1–treated mice were determined by a Mann–Whitney test (*, P < 0.05).
- Figure 5.
Anti-PDL1 mAb suppresses anti-PD1–resistant tumor. Groups of B6 WT mice (n = 5-8) were subcutaneously injected with MC38 tumor (1 × 106 cells; A and C) or AT3 tumor (1 × 106 cells; B and D) on day 0. A and B, tumor-bearing mice were treated with 250 μg of cIg or anti-PD1 on day 10 (A) or day 14 (B), and tumors were harvested 2 or 3 days after antibody treatments for flow cytometric analyses. PDL1 MFI of CD4+ T cells, CD8+ T cells, CD11b Gr1hi cells, CD11b Gr1int cells, CD11b Gr1lo cells, CD45.2− cells (7AAD− CD45.2−) between cIg- and anti-PD1–treated mice are shown. Statistical differences in PDL1 MFI of indicated cell subsets between cIg- and anti-PD1–treated mice were determined by an unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Data shown are pooled from 5 or more (A) and three or more (B) independent experiments. C and D, tumor-bearing mice were treated with 250 μg of cIg, anti-PD1, and/or anti-PDL1 on days 8, 12, and 16 (C) or days 14, 18, 22, and 26 (D). Tumor growth was measured using a digital caliper, and tumor sizes are presented as mean ± SEM. Statistical differences in tumor sizes between cIg-, anti-PD1-, anti-PDL1-, and anti-PD1 + anti-PDL1–treated mice were determined by an unpaired t test (day 16 MC38: cIg vs. anti-PD1 P = 0.0035; cIg vs. anti-PDL1 P = 0.0010; cIg vs. anti-PD1 + PDL1, P = 0.0006; day 28 AT3: cIg vs. anti-PDL1, P = 0.0008; cIg vs. anti-PD1 + anti-PDL1, P = 0.0007). Data are representative of two independent experiments. Tumor growth data for cIg- and anti-PD1–treated mice shown in D are the same dataset as shown in Supplementary Fig. S5D.
- Figure 6.
PD1 MFI on CD8+ T cells predicts tumor aggressiveness and response to anti-PD1 mAb. ANCOVA was applied to test the association between the PDL1 or PD1 MFI on various TIL subsets (CD4+ T, CD8+ T, CD11b+/Gr1+, CD45.2−) and the tumor weight and the interaction between the two slopes. Plots of PD1 MFI (A–D) and PDL1 MFI (E–H) on CD4+ (left) and CD8+ T cells (right) against the weight of tumors (monitored 2 to 3 days after the first injection of mAb) from mice inoculated with MC38 (A and B; E and F) and AT3 (C and D; G and H) tumor cells and treated with cIg (solid circles) or anti-PD1 mAb (open circles). Solid (cIg) and dashed (anti-PD1 mAb) lines correspond to the slopes and associated SE as estimated by the ANCOVA model for each treatment group. All experiments described in this manuscript comprising n = 34 to 36 (AT3) and 79 to 86 (MC38) mice/group were gathered for this analysis. The analyses pertaining to the CD45.2− and myeloid cell fractions were not significant in this context and therefore not included. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
- Figure 7.
Schematic diagram of the PD1/PDL1 axis in anti-PD1–sensitive and –resistant tumors. Left, in a tumor microenvironment with PD1lo T cells, an increased level of PDL1 expression is present that increases the probability of PD1 ligation on T cells. Right, in contrast, in a tumor microenvironment with PD1hi T cells, a low level of PDL1 expression is sufficient to ensure the ligation of PD1 on T cells.
Additional Files
Supplementary Data
- Supplementary Figure Legend - Supplementary Figure Legend. Legends for Supplementary Figures S1-S8.
- Supplementary Figures S1-S8 - Supplementary Figures S1-S8. Anti-PD-1 reduces tumor weight and modulates PD-1 expressing intratumor T cells in anti-PD-1-sensitive tumor (S1); Anti-PD-1 therapy mAb does not compete for the binding of PD-1 with the anti-PD-1 flow cytometry staining mAb (S2); Anti-PD-1 mAb reduces PD-1 expression on PD-1-expressing T cells (S3); Frequency of PD-1+ CD8+ T cells maintained in anti-PD1-resistant Renca tumors (S4); Anti-Tim3 and anti-PD1 sensitivity correlates with reduced frequency of PD1 expressing CD8+ T cells (S5); Anti-PD-1/PD-L1 anti-tumor effect is FcR-independent (S6); PD-1-expressing T cells are predominantly found in the tumor microenvironment (S7); PD1 MFI on CD8+ T cells predicts tumor aggressiveness and response to anti-PD1 mAb in MC38 tumor (S8).
Supplementary Data
- Supplementary Figure Legend - Supplementary Figure Legend. Legends for Supplementary Figures S1-S8.
- Supplementary Figures S1-S8 - Supplementary Figures S1-S8. Anti-PD-1 reduces tumor weight and modulates PD-1 expressing intratumor T cells in anti-PD-1-sensitive tumor (S1); Anti-PD-1 therapy mAb does not compete for the binding of PD-1 with the anti-PD-1 flow cytometry staining mAb (S2); Anti-PD-1 mAb reduces PD-1 expression on PD-1-expressing T cells (S3); Frequency of PD-1+ CD8+ T cells maintained in anti-PD1-resistant Renca tumors (S4); Anti-Tim3 and anti-PD1 sensitivity correlates with reduced frequency of PD1 expressing CD8+ T cells (S5); Anti-PD-1/PD-L1 anti-tumor effect is FcR-independent (S6); PD-1-expressing T cells are predominantly found in the tumor microenvironment (S7); PD1 MFI on CD8+ T cells predicts tumor aggressiveness and response to anti-PD1 mAb in MC38 tumor (S8).
Volume 75, Issue 18
