CTLA-4 blockade increases IFNgamma-producing CD4+ICOShi cells to shift the ratio of effector to regulatory T cells in cancer patients

Abstract

Significant anti-tumor responses have been reported in a small subset of cancer patients treated with the immunotherapeutic agent anti-CTLA-4 antibody. All clinical trials to date, comprising over 3,000 patients, have been conducted in the metastatic disease setting, which allows for correlation of drug administration with clinical outcome but has limited analyses of intermediate biomarkers to indicate whether the drug has impacted human immune responses within the tumor microenvironment. We conducted a pre-surgical clinical trial in six patients with localized bladder cancer, which allowed for correlation of drug administration with biomarkers in both blood and tumor tissues but did not permit correlation with clinical outcome. We found that CD4 T cells from peripheral blood and tumor tissues of all treated patients had markedly increased expression of inducible costimulator (ICOS). These CD4(+)ICOS(hi) T cells produced IFN-gamma (IFNgamma) and could recognize the tumor antigen NY-ESO-1. Increase in CD4(+)ICOS(hi) cells led to an increase in the ratio of effector to regulatory T cells. To our knowledge, these are the first immunologic changes reported in both tumor tissues and peripheral blood as a result of treatment with anti-CTLA-4 antibody, and they may be used to guide dosing and scheduling of this agent to improve clinical responses.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

ICOS expression by CD4 T cells in the blood of bladder cancer patients was increased after treatment with anti-CTLA-4. (A) ICOS expression on peripheral blood CD4 T cells was increased after anti-CTLA-4 treatment (patient 1). (B) Summary of the effect of anti-CTLA-4 therapy on CD4+ICOShi T cells in the blood of all six treated patients (with the exception of the week 7 sample for patient 2, who did not have blood drawn at week 7) as compared with the average frequency of CD4+ICOShi T cells in 10 healthy donors (HD).

Fig. 2.

Coexpression of ICOS and FOXP3 by CD4 T cells from peripheral blood. (A) The populations of CD4+ICOShi and CD4+ICOSlow T cells had subsets of both FOXP3+ and FOXP3− cells in healthy donors and untreated bladder cancer patients. Data shown are from one representative sample; numbers shown are mean percentages ± standard deviations calculated from samples from 10 healthy donors and 10 patients with untreated bladder cancer. (B) CD4+ICOShi and CD4+ICOSlow cells in pre- and post-therapy (weeks 3 and 7) samples from one patient (patient 1) treated with anti-CTLA-4 antibody also had subsets of both FOXP3+ and FOXP3− cells. (C) FOXP3 expression in total measured CD4 T cells (CD4+FOXP3+ cells) in pre- and post-therapy samples from one patient (patient 1) treated with anti-CTLA-4 therapy. (D) Variable effects of anti-CTLA-4 therapy on CD4+FOXP3+ T cells in the blood of all six treated patients (patient 2 did not have blood drawn at week 7).

Fig. 3.

Increased production of IFNγ by CD4 and CD4+ICOShi T cells and recognition of tumor antigen NY-ESO-1 by CD4+ICOShi T cells. (A) Increased IFNγ production by CD4 T cells from the peripheral blood of patients treated with anti-CTLA-4 therapy. (B) Fold induction of IFNγ, IL-10, IL-4, IL-2, and FOXP3 mRNA levels relative to CD3-ε mRNA expression in CD4 T cells from peripheral blood. (C) Increased IFNγ production by CD4+ICOShi T cells from the peripheral blood of anti-CTLA-4-treated patients. (D) Tumor antigen NY-ESO-1 mRNA detected by RT-PCR and gel electrophoresis. Lane 1 shows the NY-ESO-1 mRNA detected in a positive control cell line, SK-Mel 37, which has high expression of NY-ESO-1; lane 2 shows the absence of NY-ESO-1 mRNA in the negative control cell line SK-Mel 23; lanes 3, 5, and 7 show the absence of NY-ESO-1 expression in tumor tissues from patients 1, 3, and 5, respectively; and lanes 4, 6, and 8 show the NY-ESO-1 expression in tumor tissues from patients 2, 4, and 6, respectively. B-actin is shown to be present in all samples. (E) CD4+ICOShi T cells from the pre-therapy blood sample of patient 6 and the post-therapy blood samples of patients 2, 4, and 6 produced IFNγ on recognition of APCs pulsed with overlapping NY-ESO-1 tumor antigen peptides encompassing the entire protein (all peptides) but did not respond to APCs without peptide (no peptide).

Fig. 4.

Effects of anti-CTLA-4 therapy on tumor tissues. (A) ICOS expression on tumor-infiltrating CD4 T cells was increased after anti-CTLA-4 treatment. Data shown are from representative samples. (Left) Nonmalignant urothelial tissues from an untreated bladder cancer patient. (Center) Urothelial carcinoma tissues from an untreated bladder cancer patient. (Right) Urothelial carcinoma tissues from a bladder cancer patient treated with anti-CTLA-4 antibody. (B) Plot of percentage of CD4+ICOShi T cells from individual tissue samples including nonmalignant tissue samples (n = 4), untreated tumor tissues (n = 10), and anti-CTLA-4-treated tumor tissues (n = 6). (C) Plot of percentage of CD4+FOXP3+ T cells from individual tissue samples including nonmalignant tissue samples (n = 4), untreated tumor tissues (n = 10), and anti-CTLA-4-treated tumor tissues (n = 6). (D) Fold induction of CD3-ε, IFNγ, IL-10, IL-4, IL-2, T-bet, GATA-3, and FOXP3 mRNA levels relative to GAPDH mRNA expression in surgical samples of nonmalignant urothelial tissues and bladder cancer tissues from untreated and anti-CTLA-4-treated patients.