Effects of conventional therapeutic interventions on the number and function of regulatory T cells

Abstract

Several lines of investigation have revealed the apparent interplay between the immune system of the host and many conventional, "standard-of-care" anticancer therapies, including chemotherapy and small molecule targeted therapeutics. In particular, preclinical and clinical studies have demonstrated the important role of regulatory T cells (Tregs) in inhibiting immune responses elicited by immunotherapeutic regimens such as those based on anticancer vaccines or checkpoint inhibitors. However, how the number and immunosuppressive function of Tregs change in cancer patients undergoing treatment with non-immune anticancer therapies remains to be precisely elucidated. To determine whether immunostimulatory therapies can be employed successfully in combination with conventional anticancer regimens, we have investigated both the number and function of Tregs obtained from the peripheral blood of carcinoma patients before the initiation and during the course of chemotherapeutic and targeted agent regimens. Our studies show that the treatment of breast cancer patients with tamoxifen plus leuprolide, a gonadotropin releasing hormone agonist, has minimal effects on Tregs, while sunitinib appears to exert differential effects on Tregs among patients with metastatic renal carcinoma. However, the administration of docetaxel to patients with metastatic prostate or breast cancer, as well as that of cisplatin plus vinorelbine to non-small cell lung cancer patients, appears to significantly increase the ratio between effector T cells and Tregs and to reduce the immunosuppressive activity of the latter in the majority of patients. These studies provide the rationale for the selective use of active immunotherapy regimens in combination with specific standard-of-care therapies to achieve the most beneficial clinical outcome among carcinoma patients.

Keywords: T lymphocytes; Tregs; carcinoma; chemotherapy; drug therapy.

Figure 1. Circulating immune cell subsets in patients with prostate cancer or non-small cell lung cancer before and during the course of chemotherapy. (A–C) Patients (n = 15) with hormone refractory metastatic prostate cancer were treated with docetaxel and evaluated before and during therapy for the number of peripheral blood mononuclear cells (PBMCs) (A), the frequency of CD4⁺ T cells among PBMCs (B), and the frequency of regulatory T cells (Tregs) among CD4⁺ T cells (C). Peripheral blood samples were collected at the indicated time points. Statistical analyses were performed using Friedman and Dunn’s multiple comparison tests and revealed a significant decrease in the frequency of Tregs at post-cycle I (**P < 0.01) and pre-cycle II (***P < 0.001). (D–F) Patients (n = 14) with stage IB, II, and IIIA non-small cell lung cancer (NSCLC) were evaluated before and during therapy with cisplatin plus vinorelbine for the number of PBMCs (D), the frequency of CD4⁺ T cells among PBMCs (E), and the frequency of Tregs among CD4⁺ T cells (F). Peripheral blood samples were collected at the indicated time points. Statistical analyses were performed using Friedman and Dunn’s multiple comparison tests; * P < 0.05, *** P < 0.001. In all dot plots, the median and interquartile range are shown.

Figure 2. Docetaxel-induced changes in the ratio of effector to regulatory T cells and in the immunosuppressive activity of the latter in hormone-refractory prostate cancer patients. (A) Waterfall plot of the change in the ratio between effector T cells and regulatory T cells (Teff:Treg ratio) in the course of docetaxel therapy in patients with hormone-refractory prostate cancer (n = 15). Patients were treated with docetaxel weekly for 3 weeks followed by 1 week of rest; this comprised a 4-week cycle of therapy. Peripheral blood samples were collected prior to therapy (baseline, day 0) and at the end of the week of rest before starting cycle II. (B) Waterfall plot of the change in suppressive activity of Tregs in the course of docetaxel therapy in patients with hormone-refractory prostate cancer (n = 11).

Figure 3. Docetaxel treatment of metastatic breast cancer patients increased the ratio of CD4⁺ or CD8⁺ T lymphocytes vs. regulatory T cells. (A–F) Cytofluorometric analysis of peripheral blood mononuclear cells (PBMCs) from patients (n = 8) with metastatic breast cancer treated with docetaxel. PBMCs were analyzed before treatment (baseline) and at the time of first re-staging near day 91 (at the second week of cycle III). (A) Percentage of CD4⁺ T lymphocytes among PBMCs. (B) Percentage of CD8⁺ T lymphocytes among PBMCs. (C) Percentage of CD4⁺CD25^highCD127^–FoxP3⁺ regulatory T cells (Tregs) among PBMCs. (D) Ratio of CD4⁺ effector T cells to Tregs (CD4⁺ Teff:Treg ratio). (E) Ratio of CD8⁺ effector T cells to Tregs (CD8⁺ Teff:Treg ratio). (F) Percentage of CD3^–CD56⁺ natural killer (NK) cells among PBMCs. Medians at baseline and cycle III are shown. Statistical analyses were performed by Wilcoxon matched-pairs signed rank test and P < 0.05 was considered statistically significant.

Figure 4. Cisplatin plus vinorelbine-induced changes in the ratio of effector T cells to regulatory T cells and in the immunosuppressive activity of the latter in non-small cell lung cancer patients. (A) Waterfall plot of the change in the ratio between CD4⁺ effector T cells and regulatory T cells (Teff:Treg ratio) in non-small cell lung cancer (NSCLC) patients before and during therapy with cisplatin plus vinorelbine (n = 14). Patients with NSCLC (stages IB, II, and IIIA) were treated in the adjuvant setting, post-surgery, with cisplatin plus vinorelbine at day 1 and vinorelbine at day 8. This was followed by 14 days of rest, which comprised the 3-week cycle of therapy. PBMC were collected from peripheral blood at baseline and post-cycle III. (B) Waterfall plot of the change in immunosuppressive activity of Tregs from NSCLC patients before and during therapy with cisplatin plus vinorelbine (n = 10). The Treg suppressive activity was evaluated baseline and post-cycle III.

Figure 5. Circulating immune cell subsets in patients with breast cancer and metastatic renal cell carcinoma before and during therapy. (A–C) Premenopausal (< 50 y) breast cancer patients (n = 8) were treated upon surgery with tamoxifen plus a gonadotropin-releasing hormone (GnRH) agonist, and evaluated before and during therapy for the number of circulating peripheral blood mononuclear cells (PBMCs; A), the frequency of CD4⁺ T cells among PBMCs (B), and the frequency of regulatory T cells (Tregs) among CD4⁺ T cells (C). Peripheral blood samples were collected at the indicated time points. (D–F) Metastatic renal cell carcinoma patients on sunitinib monotherapy (n = 10) were evaluated before and during treatment for the frequency of CD4⁺ T cells among PBMCs (D), the frequency of Tregs among CD4⁺ T cells (E), and the absolute number of Tregs (F). Peripheral blood was collected at the indicated time points. Statistical analyses were performed using Friedman and Dunn’s multiple comparison test; * P < 0.05, ** P < 0.01, *** P < 0.001. In all dot plots, the median and interquartile ranges are shown.

Figure 6. Effects of targeted anticancer therapeutics on the ratio between effector and regulatory T cells and on the immunosuppressive activity of the latter in patients with breast cancer or metastatic renal cell carcinoma. (A–B) Waterfall plots of the change in the ratio between CD4⁺ effector T cells and regulatory T cells (Teff:Treg ratio) (A), and in the immunosuppressive activity of Tregs (B), in breast cancer patients receiving tamoxifen plus a gonadotropin-releasing hormone (GnRH) agonist upon surgery (n = 8). Patients of premenopausal status were treated in the adjuvant setting post-surgery with tamoxifen daily and GnRH agonist every 3 months. Peripheral blood samples were collected prior to therapy (baseline, day 0) and at post 3 months. (C–D) Waterfall plots of the change in CD4⁺ Teff:Treg ratio (C), and Treg-mediated immunosuppression (D), in metastatic renal cell carcinoma patients receiving sunitinib monotherapy (n = 10). Patients with metastatic renal cell carcinoma were treated with sunitinib monotherapy daily for 4 weeks followed by 2 weeks of rest; this comprised a 6-week cycle of therapy. Peripheral blood was collected prior to therapy, and at the end of the second round of chemotherapy in cycle II.