Increased frequency of CD4+ICOShi and CD8+ICOShi T cells in tumor tissues after treatment with 10 mg/kg/dose of anti–CTLA-4. A, representative patient samples showing that CD4+ICOShi (top) and CD8+ICOShi (bottom) T cells were increased in frequency in tumor tissues after treatment with 10 mg/kg/dose of anti–CTLA-4 as compared with untreated tumor tissues. B, compilation of data showing statistically significant increased frequencies of CD4+ICOShi and CD8+ICOShi T cells in tumor tissues from anti–CTLA-4 treated patients (n = 5) as compared with tumor tissues obtained from untreated patients (n = 10).
Perivascular infiltration of cells into tumor tissues after treatment with 10 mg/kg/dose of anti–CTLA-4. Representative pictures showing an absence of perivascular infiltration of cells in untreated tumor tissues (0 of 11) and tumor tissues obtained from patients treated with 3 mg/kg/dose of anti–CTLA-4 (0 of 6) as compared with the presence of perivascular infiltration noted in tumor tissues obtained from patients treated with anti–CTLA-4 at 10 mg/kg/dose (2 of 5; top). Immunohistochemistry revealed that the infiltrating cells were positive for CD3, CD8, CD4, and granzyme, but were predominantly negative for CD20 and CD56 (bottom).
Increased frequency of CD4+ICOShi and CD8+ICOShi T cells in the peripheral blood after treatment with 10 mg/kg/dose of anti–CTLA-4. A, representative peripheral blood samples taken after the first dose of anti–CTLA-4 at week 3 and after the second dose of anti–CTLA-4 at week 7 showed an increased frequency of CD4+ICOShi (top) and CD8+ICOShi T cells (bottom) as compared with baseline (pretherapy) samples. Compilation of data showing pretherapy values for frequency of CD4+ICOShi T cells (B) and CD8+ICOShi T cells (C) in bladder cancer patients (n = 6) and average frequency in healthy donors (HD; n = 10) with statistically significant increases in frequencies of ICOShi T cells at weeks 3 and 7 after treatment with anti–CTLA-4.
Increased frequency of CD4+ICOShi T cells is detected after patients with metastatic melanoma are treated with anti–CTLA-4. A, representative dot plot of ICOS staining in a melanoma patient at baseline and after anti–CTLA-4 therapy, as well as the mouse IgG1 isotype control. B, all samples analyzed for % CD4+ICOShi expression in the different donor and patient groups at different time points. HD, healthy donor; Mel-Pt, control melanoma patients; Pre-tx, pretreatment; SAV, streptavidin.
Correlation of clinical outcome with frequency of CD4+ICOShi T cells after patients were treated with anti–CTLA-4. A, multiparameter flow cytometric analyses were done on fresh patient PBMCs at baseline, and on weeks 7 and 12. Seven of seven patients with clinical benefit at week 24 had persistent elevation in % of CD4+ICOShi cells, defined as a ≥2-fold increase in % CD4+ICOShi expression at week 7 or 12 over baseline that was sustained at week 12. In comparison, one of seven patients without clinical benefit, i.e., with progressive disease or death at week 24, had persistent elevation in % of CD4+ICOShi cells (*). B, Kaplan-Meier curve showing the difference in overall survival for patients with and without persistent % CD4+ICOShi increase is statistically significant (median overall survival not reached versus 27 weeks, P = 0.03).