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Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy

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Science Translational Medicine  17 Aug 2016:
Vol. 8, Issue 352, pp. 352ra110
DOI: 10.1126/scitranslmed.aaf6843
  • Fig. 1.

    Anti-CD25-F(ab′)2 lacks Treg depletion effect, but NIR-PIT with anti-CD25-F(ab′)2–IR700 kills target cells. (A) Intravenously injected anti-CD25-IgG (100 μg) systemically depleted CD4+CD25+Foxp3+ Tregs, but anti-CD25-F(ab′)2 (100 μg) did not significantly deplete these cells within the CD4 T cell population 1 day after injection (n = 3) [*P < 0.0001, one-way analysis of variance (ANOVA) with Dunnett’s test; **P = not significant (ns)]. (B) HT-2-A5E cells (mouse T lymphocytes) incubated with anti-CD25-F(ab′)2–IR700 for 6 hours were examined under a microscope before and 0.5 hour after NIR light irradiation (4 J/cm2). NIR light irradiation induced cellular swelling, bleb formation, and necrosis of the cells, as indicated with propidium iodide (PI) staining (scale bar, 10 μm; right magnified view, ×4). DIC, differential interference contrast. (C) Cell necrosis induced by NIR-PIT increased in a NIR light dose–dependent manner, as determined by flow cytometry analysis with PI staining (left graph, n = 3; *P < 0.0001 versus 0 J/cm2, unpaired t test). No significant cell killing was detected when a control-F(ab′)2–IR700 was used (right graph, n = 3; **P = ns versus 0 J/cm2, unpaired t test).

  • Fig. 2.

    In vivo local CD25-targeted NIR-PIT induces regression of treated LL/2-luc tumors. (A) An increased CD4+CD25+Foxp3+ Treg population among CD4 T cells was observed in tumors (LL/2-luc or MC38-luc) compared to that in the spleen (n = 3) (*P < 0.001, **P < 0.001 versus control, one-way ANOVA with Dunnett’s test). (B) Regimen used for local NIR-PIT. (C) In vivo BLI is shown for tumor-bearing mice that were untreated, received control-F(ab′)2–IR700 followed by NIR-PIT, received CD25-F(ab′)2–IR700 alone, or treated with local CD25-targeted NIR-PIT. Before NIR-PIT, tumors sizes were equivalent, exhibiting similar bioluminescence, but only the CD25-targeted NIR-PIT group showed a decrease in BLI. iv, intravenously. (D) Quantitative relative light units (RLU) (before PIT is set to 100) showed a significant RLU decrease in the experimental tumors [n = 6 mice in each group; *P = 0.0217 (day 1) and 0.0243 (day 2), PIT versus control, Tukey’s test with ANOVA]. (E) Local CD25-targeted NIR-PIT reduced tumor volume (n = 8 mice in each group; *P < 0.0001, PIT versus others, Tukey’s test with ANOVA). Treatment schedule indicated below the graph corresponds to that in (B). (F) Local CD25-targeted NIR-PIT prolonged the survival of the mice (n = 8 mice in each group; *P < 0.0001, PIT versus control, log-rank test and Wilcoxon test). (G) The body weight of mice not receiving CD25-targeted NIR-PIT gradually increased because of tumor growth, in contrast to the PIT group showing negligible body weight increase (n = 8 mice in each group) (*P = 0.0128, PIT versus control at day 14). (H) Local CD25-targeted NIR-PIT resulted in a depletion of CD4+CD25+Foxp3+ Tregs in tumors, but not in the spleen (n = 5) (*P < 0.008, Mann-Whitney test; P = ns, Mann-Whitney test). (I) Local CD25-targeted NIR-PIT did not significantly affect the number of CD8 T cells (CD3+CD8+) or NK cells (CD3NK1.1+) (n = 5; P = ns, Mann-Whitney test).

  • Fig. 3.

    In vivo local CD25-targeted NIR-PIT induces rapid activation and cytotoxicity of intratumoral CD8 T and NK cells. (A) Cytotoxic action of CD8 T and NK cells infiltrating LL/2-luc or MC38-luc tumors was examined by flow cytometry with or without local CD25-targeted NIR-PIT. CD8 T and NK cells collected 1.5 hours after PIT were producing IFN-γ and IL-2 and had CD107a exposed on the cell surface, whereas the cells from nontreated tumors did not (n = 5; *P < 0.008, Mann-Whitney test). (B) One day after local CD25-targeted NIR-PIT, up-regulation of activation markers, CD69 and CD25, and continued production of IL-2 in both CD8 T and NK cells were observed (n = 5; *P < 0.008, Mann-Whitney test).

  • Fig. 4.

    The therapeutic effects of local CD25-targeted NIR-PIT extend to distant nonirradiated tumors. (A) Regimen of NIR-PIT. (B) Mice with bilateral flank tumors were either not injected (control) or injected with control-F(ab′)2–IR700 or anti-CD25-F(ab′)2–IR700, followed by NIR light irradiation of only the right tumor. (C) In vivo BLI showed changes in bioluminescence signals in the tumors in response to local CD25-targeted NIR-PIT only. Before NIR-PIT, tumors were about the same size and exhibited similar bioluminescence. (D) Quantitative RLU showed a significant decrease in signal in NIR-PIT–treated right-side tumors and even in nonirradiated left-side tumors [n = 6 mice in each group; *P < 0.001, **P < 0.01, ***P = 0.0197 (PIT:R) and 0.0142 (PIT:L), PIT versus cont-F(ab′)2–IR700 iv:R, Tukey’s test with ANOVA]. (E) Local CD25-targeted NIR-PIT reduced the size of NIR-PIT–treated right tumors and nonirradiated left tumors (n = 8 mice in each group; *P < 0.0001, **P < 0.0005, PIT versus others, Tukey’s test with ANOVA). Time of treatments is indicated below the graph. (F) Local CD25-targeted NIR-PIT prolonged the survival of the mice (n = 8 mice in each group; *P < 0.0001, PIT versus control, log-rank test and Wilcoxon test). (G) Body weight changes of tumor-bearing mice were followed. After NIR-PIT, both right and left dorsa became edematous and mice gained weight (n = 8 mice in each group; *P < 0.001, PIT versus others, Tukey’s test with ANOVA), which started to disappear by day 10. (H) Local CD25-targeted NIR-PIT of the right dorsal tumor caused edema bilaterally (arrow). (I) NIR-PIT depleted CD4+CD25+Foxp3+ Tregs within the irradiated tumor on the right dorsum, but not Tregs in the left nonirradiated tumors. Mice not injected (control) or injected with control-F(ab′)2–IR700 showed no significant difference in Treg populations between the right and left tumors (n = 5 in each group; *P < 0.0001, Tukey’s test with ANOVA). (J) Local CD25-targeted NIR-PIT of the right dorsal LL/2-luc tumor caused regression of multiple additional LL/2-luc tumors by 1 day after the treatment. (K) Local CD25-targeted NIR-PIT of the right dorsal LL/2-luc tumor had negligible antitumor effects on the left dorsal MC38-luc tumor 1 day after PIT.

  • Fig. 5.

    Activated CD8 T and NK cells are present in the contralateral tumor after ipsilateral CD25-targeted NIR-PIT. (A to D) CD8 T and NK cells collected from nonirradiated left dorsal tumors in mice receiving local CD25-targeted NIR-PIT of the right dorsal tumors were analyzed for their expression of activation markers 1 day after the treatment. CD8 T and NK cells producing IFN-γ (A) and IL-2 (B) and with up-regulated CD25 (C) and CD69 (D) expression were present in the nonirradiated left tumor after CD25-targeted NIR-PIT of the right-side tumor [n = 5; *P = 0.0021 (A: CD8 T cells), P = 0.0023 (B: CD8 T cells), P = 0.0012 (D: CD8 T cells), P < 0.008 (others), Mann-Whitney test].

  • Fig. 6.

    Antitumor effect of local CD25-targeted NIR-PIT partially depends on each of NK cells, CD8 T cells, and IFN-γ production. (A) Regimen of NIR-PIT against LL/2-luc tumors subjected to depletion of NK or CD8 T cells or neutralization of IFN-γ (Depletion Abs i.p. at 4 and 7 days after tumor inoculation). (B and C) In vivo BLI (B) and quantitative RLU (C) showed a significant decrease of bioluminescence signals in NIR-PIT–treated tumors; however, depletion of NK (anti-NK1.1) or CD8 T (anti-CD8) cells or neutralization of IFN-γ (anti–IFN-γ) reduced the effect (n = 5 mice in each group; *P = 0.0158, PIT versus anti-NK1.1 + PIT; *P < 0.0001, PIT versus control, Tukey’s test with ANOVA). (D and E) Similarly, the effect of local CD25-targeted NIR-PIT in suppressing tumor growth was inhibited by adding the depletion or neutralization antibodies (n = 7 mice in each group; *P < 0.0001, PIT versus others, Tukey’s test with ANOVA; treatments are indicated below the graph) (D), resulting in shorter survival of these groups of mice compared to the PIT group (n = 7 mice in each group; *P < 0.0001 versus control, log-rank test and Wilcoxon test) (E). (F) Body weights showed no significant difference among groups (n = 7 mice in each group; Tukey’s test with ANOVA).

  • Fig. 7.

    Scheme indicates the proposed mechanism of local CD25-targeted NIR-PIT–induced immunotherapy. Tregs suppress CD8 T cell and NK cell activation, providing a permissive environment for tumor growth (upper panel). After Tregs are selectively depleted by NIR-PIT, CD8 T and NK cells are activated against the tumor (middle panel). Activated CD8 T and NK cells can also leave the treated tumor to attack distant tumors, accompanied by released cytokines and chemokines (lower panel).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/352/352ra110/DC1

    Fig. S1. Anti-CD25-F(ab′)2 and control-F(ab′)2 are generated, characterized, and conjugated with IR700 dye.

    Fig. S2. NIR-PIT with anti-CD25-F(ab′)2–IR700 induces necrosis of CD25-expressing cells in a NIR light dose–dependent manner.

    Fig. S3. Tumor-infiltrating CD8 T and NK cells do not express CD25.

    Fig. S4. Anti-CD25-F(ab′)2–IR700 does not bind to the tumor cells.

    Fig. S5. Depletion of tumor-infiltrating CD4+CD25+Foxp3+ Tregs with local CD25-targeted NIR-PIT is NIR light dose–dependent.

    Fig. S6. Tumor-infiltrating CD4+Foxp3+ cells are depleted by local CD25-targeted NIR-PIT.

    Fig. S7. Local CD25-targeted NIR-PIT of an MC38-luc tumor induces regression of the tumor.

    Fig. S8. Local CD25-targeted NIR-PIT induces regression of TRAMP-C2-luc flank tumors.

    Fig. S9. Repeated local CD25-targeted NIR-PIT enables prolonged suppression of tumor growth.

    Fig. S10. Repeated local CD25-targeted NIR-PIT induces up-regulation of CD25 on CD8 T and NK cells at each treatment.

    Fig. S11. Local CD25-targeted NIR-PIT induces activation of tumor-infiltrating dendritic cells and other APCs.

    Fig. S12. Local CD25-targeted NIR-PIT increases the number of granulocytes in treated tumors.

    Fig. S13. NIR light irradiation of tumors induces negligible production of cytokines and chemokines.

    Fig. S14. Local CD25-targeted NIR-PIT induces systemic and intratumoral cytokine storm.

    Fig. S15. IFN-γ production by CD8 T and NK cells in the lungs is not detected 1 day after CD25-targeted NIR-PIT.

    Fig. S16. CD25-targeted NIR-PIT reduces IR700 fluorescence of the treated tumor, but not that of the contralateral nonirradiated tumor.

    Fig. S17. Local CD25-targeted NIR-PIT of the right dorsal tumor induces reduction of multiple tumors at distant sites.

    Fig. S18. Local CD25-targeted NIR-PIT inhibits the growth of tumor challenged on the contralateral side.

    Fig. S19. The concentrations of cytokines and chemokines in the contralateral nonirradiated tumor are increased after CD25-targeted NIR-PIT.

    Fig. S20. Antitumor effect of local CD25-targeted NIR-PIT is at least partly IFN-γ–dependent.

  • Supplementary Material for:

    Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy

    Kazuhide Sato, Noriko Sato, Biying Xu, Yuko Nakamura, Tadanobu Nagaya, Peter L. Choyke, Yoshinori Hasegawa, Hisataka Kobayashi*

    *Corresponding author. Email: kobayash{at}mail.nih.gov

    Published 17 August 2016, Sci. Transl. Med. 8, 352ra110 (2016)
    DOI: 10.1126/scitranslmed.aaf6843

    This PDF file includes:

    • Fig. S1. Anti-CD25-F(ab′)2 and control-F(ab′)2 are generated, characterized, and conjugated with IR700 dye.
    • Fig. S2. NIR-PIT with anti-CD25-F(ab′)2–IR700 induces necrosis of CD25-expressing cells in a NIR light dose–dependent manner.
    • Fig. S3. Tumor-infiltrating CD8 T and NK cells do not express CD25.
    • Fig. S4. Anti-CD25-F(ab′)2–IR700 does not bind to the tumor cells.
    • Fig. S5. Depletion of tumor-infiltrating CD4+CD25+Foxp3+ Tregs with local CD25-targeted NIR-PIT is NIR light dose–dependent.
    • Fig. S6. Tumor-infiltrating CD4+Foxp3+ cells are depleted by local CD25-targeted NIR-PIT.
    • Fig. S7. Local CD25-targeted NIR-PIT of an MC38-luc tumor induces regression of the tumor.
    • Fig. S8. Local CD25-targeted NIR-PIT induces regression of TRAMP-C2-luc flank tumors.
    • Fig. S9. Repeated local CD25-targeted NIR-PIT enables prolonged suppression of tumor growth.
    • Fig. S10. Repeated local CD25-targeted NIR-PIT induces up-regulation of CD25 on CD8 T and NK cells at each treatment.
    • Fig. S11. Local CD25-targeted NIR-PIT induces activation of tumor-infiltrating dendritic cells and other APCs.
    • Fig. S12. Local CD25-targeted NIR-PIT increases the number of granulocytes in treated tumors.
    • Fig. S13. NIR light irradiation of tumors induces negligible production of cytokines and chemokines.
    • Fig. S14. Local CD25-targeted NIR-PIT induces systemic and intratumoral cytokine storm.
    • Fig. S15. IFN-γ production by CD8 T and NK cells in the lungs is not detected 1 day after CD25-targeted NIR-PIT.
    • Fig. S16. CD25-targeted NIR-PIT reduces IR700 fluorescence of the treated tumor, but not that of the contralateral nonirradiated tumor.
    • Fig. S17. Local CD25-targeted NIR-PIT of the right dorsal tumor induces reduction of multiple tumors at distant sites.
    • Fig. S18. Local CD25-targeted NIR-PIT inhibits the growth of tumor challenged on the contralateral side.
    • Fig. S19. The concentrations of cytokines and chemokines in the contralateral nonirradiated tumor are increased after CD25-targeted NIR-PIT.
    • Fig. S20. Antitumor effect of local CD25-targeted NIR-PIT is at least partly IFN-γ–dependent.

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