Article Figures & Data
Figures
- Figure 1.
Stereotactic radiotherapy increases Tregs in tumors. A, Experimental design. 5 × 105 B16/F10, 1.5 × 106 MC38, or 3 × 105 RENCA cells were implanted subcutaneously (s.c.) to the flank of wild-type C57BL/6J mice (in B16/F10 or MC38 experiments) or BALB/c mice (in RENCA experiment), respectively on day 0. Mice received 10 Gy of stereotactic radiotherapy (RT) via SARRP on day 7 (B16/F10), day 16 (RENCA), day 10 (MC38), respectively. Tumors, draining lymph nodes (DLN), and spleens were harvested 7 days after the radiation. B, Tumor growth curves of B16/F10-bearing mice as in A. Nonirradiated tumors (control) in black line and irradiated tumors (RT) in red line, respectively. C, The absolute number of TIL-Tregs per gram tumor weight in the B16/F10 model. D–F, Representative flow plots (D) and quantitative bar graph of % Foxp3+ cells (E) and MFI of Foxp3 (F) in CD4+ cells from TILs. N = 7–15 per group, repeated at least 4×. G, Immunohistochemistry of Foxp3 in tumors (B16/F10, RENCA, MC38), 20× and 40× magnification as indicated. Foxp3+ cells are stained red in B16/F10 (due to the brown pigment in melanoma), and brown in RENCA and MC38 tumors. Error bars, SEM; ***, P < 0.001; **, P < 0.01; *, P < 0.05, determined by two-way ANOVA (B) or unpaired Student t test (C, E and F).
- Figure 2.
Stereotactic radiotherapy increases the activation/suppression markers of TIL-Tregs. A and B, Representative flow plots (A) and quantitative scatterplot (B) depicting of the percentages of CCR4+, CTLA4+, 4-1BB+ or Helios+ TIL-Treg, from day 14 B16/F10 tumor–bearing mice. N = 8 per group, repeated 3×. Error bars, SEM; ***, P < 0.001; **, P < 0.01; *, P < 0.05, determined by an unpaired Student t test (B).
- Figure 3.
Radiated tumor-infiltrating Tregs (TIL-Treg) are functionally suppressive. A, Representative figure of the in vitro suppression assay. Responder T cells were gated based on the CD45.1 congenically marked and their proliferation was analyzed based on the dilution of CTV dye. Solid lines show the conditions with spleen Tregs (black line), nonirradiated control TIL-Tregs (blue line), and RT TIL-Tregs (red line); filled green histograms shows the T responder–only condition. B, Quantitative plots represent percent suppression at the indicated Treg:T responder ratio. A representative experiment of a total of three independent replicates is shown (n = 2–3 per group, repeated 3×).
- Figure 4.
The effect of TGFβ on the post-RT increase of Tregs in tumors. A, Experimental design. C57BL/6 mice were injected s.c. with 5 × 105 B16/F10 cells on day 0. Mice received 10 Gy of RT on day 7. Mice received galunisertib (300 mg/kg/day, LY2157299) or vehicle via oral gavage every 12 hours, starting a day before RT. Tumors, DLNs and spleens were harvested on day 14. B, Tumor growth curves of vehicle versus galunisertib treated mice (dashed lines vs. real line, respectively), with or without radiation (black line or red line, respectively, n = 5–6 per group, repeated ×2). C, Representative flow plot of TIL-Tregs. D and E, Quantitative scatter plots of (D) the percent of tumor-infiltrating CD4+ cells that were Foxp3+ and (E) the absolute number of TIL-Tregs per gram tumor weight, in galunisertib-treated versus vehicle-treated groups (pooled data from two experiments; Error bars, SEM). ***, P < 0.001; **, P < 0.01; *, P < 0.05, determined by two-way ANOVA (B) and an unpaired Student t test (D, E).
- Figure 5.
The effect of IL33 on the post-RT increase of Tregs in tumors. A, The time course of the IL33 level in tumor lysates from different time points (1, 6, 24, 48, and 72 hours and 7 days post RT) measured by ELISA (n = 5 per group). B, Experimental design. C57BL/6 mice were injected subcutaneously with 5 × 105 B16/F10 cells on day 0. Mice received 10 Gy of RT on day 7. Mice received a mAb to ST2 (200 μg/mouse, Anti-ST2 Ab) or vehicle intraperitoneally (i.p.) every 3 days, starting 1 day before RT. Tumors, DLNs, and spleens were harvested on day 14. C, Tumor growth curves of vehicle control- (dashed line) versus ST2 Ab-treated mice (solid line), with or without radiation (black or red, respectively). D, Representative flow plot of TIL-Tregs. E and F, Quantitative scatter plots of % Foxp3+ cells of tumor-infiltrating CD4+ cells (E), and the absolute number of TIL-Tregs per gram tumor weight (F) in ST2 Ab-treated versus vehicle-treated group (n = 5 per group, repeated ×2). Error bars, SEM; ***, P < 0.001; **, P < 0.01; *, P < 0.05, determined by two-way ANOVA (A, C), and an unpaired Student t test (E, F).
- Figure 6.
Stereotactic radiation enhances preferential Treg proliferation in the tumor microenvironment (TME). A, Experimental design. C57BL/6 mice were injected subcutaneously with 5 × 105 B16/F10 cells on day 0. Mice received 10 Gy of RT on day 7. Mice received Fingolimod (25 μg, FTY720) or PBS containing DMSO as control via oral gavage on days 6, 9, and 12. Peripheral blood was collected for monitoring lymphocyte counts. Tumors, DLNs and spleens were harvested on day 14. B, Tumor growth curves of vehicle versus FTY720-treated mice (dashed line vs. solid line, respectively), with or without radiation (black or red line respectively). C, Representative flow plot of TIL-Tregs (CD4+ Foxp3+). D, Absolute number Tregs per gram tumor weight in FTY720-treated versus vehicle-treated group. E and F, Representative flow plot (E) and quantitative scatter plot (F) of tumor-infiltrating Ki-67+ Tregs (CD4+ Foxp3+ cells). G, Quantitative scatter plot of Ki-67 expression in tumor-infiltrating CD3+, CD4+, CD8+ T cells, and Tregs (n = 9–10 per group, repeated ×2). D, Pooled data from two experiments. Error bars, SEM; ***, P < 0.001; **, P < 0.01; *, P < 0.05, determined by two-way ANOVA (B), an unpaired Student t test (D, F, and G).
Additional Files
Supplementary Data
- Supplementary Figures and Legends - Supplementary Figure 1: Radiotherapy suppresses tumor growth. Supplementary Figure 2: Characterization of the TME of B16/F10 tumors post-RT. Supplementary Figure 3: Stereotactic radiation does not change Treg in draining lymph nodes nor in spleens of treated mice. Supplementary Figure 4: Persistently increased Treg Post-RT. Supplementary Figure 5: Stereotactic radiation increases the suppressive markers of Treg in the RENCA tumor model. Supplementary Figure 6: Stereotactic radiation increases the suppressive markers of Treg in the MC38 tumor model. Supplementary Figure 7: Stereotactic radiation does not change the expression of the suppressive markers of Treg in DLNs. Supplementary Figure 8: Stereotactic radiation does not change the expression of the suppressive markers of Treg in spleens. Supplementary Figure 9: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the B16/F10 model. Supplementary Figure 10: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the RENCA model. Supplementary Figure 11: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the MC38 model. Supplementary Figure 12: Expression of 4-1BB on TIL-CD8+ cells. Supplementary Figure 13: The effect of TGF-beta blockade on different T cell subsets. Supplementary Figure 14: TGF-beta expression in the tumor microenvironment. Supplementary Figure 15: The effect of Fingolimod (FTY720) on peripheral blood lymphocytes counts. Supplementary Figure 16: Chemokine/cytokine expression of a selected panel in the tumor microenvironment post-radiation.
Volume 5, Issue 11
