Fig. 2. In vivo local CD25-targeted NIR-PIT induces regression of treated LL/2-luc tumors.

(A) An increased CD4⁺CD25⁺Foxp3⁺ T_reg 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⁺ T_regs 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 (CD3⁻NK1.1⁺) (n = 5; P = ns, Mann-Whitney test).