CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients

Abstract

Regulatory T cells (T(regs)) are key mediators of immune tolerance and feature prominently in cancer. Depletion of CD25(+) FoxP3(+) T(regs) in vivo may promote T cell cancer immunosurveillance, but no strategy to do so in humans while preserving immunity and preventing autoimmunity has been validated. We evaluated the Food and Drug Administration-approved CD25-blocking monoclonal antibody daclizumab with regard to human T(reg) survival and function. In vitro, daclizumab did not mediate antibody-dependent or complement-mediated cytotoxicity but rather resulted in the down-regulation of FoxP3 selectively among CD25(high) CD45RA(neg) T(regs). Moreover, daclizumab-treated CD45RA(neg) T(regs) lost suppressive function and regained the ability to produce interferon-γ, consistent with reprogramming. To understand the impact of daclizumab on T(regs) in vivo, we performed a clinical trial of daclizumab in combination with an experimental cancer vaccine in patients with metastatic breast cancer. Daclizumab administration led to a marked and prolonged decrease in T(regs) in patients. Robust CD8 and CD4 T cell priming and boosting to all vaccine antigens were observed in the absence of autoimmunity. We conclude that CD25 blockade depletes and selectively reprograms T(regs) in concert with active immune therapy in cancer patients. These results suggest a mechanism to target cancer-associated T(regs) while avoiding autoimmunity.

Fig. 1

CD25 blockade in vitro mediates change in expression of FoxP3 and CD25 by human CD45RA^neg T_regs. Flow cytometric cell sorting was used to purify four CD4⁺ T cell populations from normal human peripheral blood: naïve (CD25^neg CD45RA⁺), memory (CD25^neg CD45RA^neg), activated T_regs (CD25^high CD45RA^neg), and resting T_regs (CD25^high CD45RA⁺). Equal numbers of each cell population were incubated in vitro for 4 days with IL-2 (20 U/ml) and either human IgG1 (10 μg/ml) or daclizumab (Dac, 10 μg/ml). (A) Cell viability at day 4. (B and C) FoxP3 and CD25 expression for each cell population at (B) baseline and (C) at day 4 for each experimental condition, shown for a representative experiment. The percentage of cells in each quadrant is shown. (D) Quantification of mean fluorescence index (MFI) and percent positivity for FoxP3 and CD25 expression on T_regs at day 4, comparing IgG1 to daclizumab conditions. Data are from four or five independent experiments and shown as means ± SD. *P < 0.05; ***P < 0.001.

Fig. 2

CD25 blockade in vitro mediates functional reprogramming of FoxP3 and CD25 human CD45RA^neg T_regs. Equal numbers of each cell population were incubated in vitro with IgG1 or daclizumab (Dac), as in Fig. 1, and then tested for function. (A to D) Suppressive capability (A and B) and IFN-γ expression (C and D) upon phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation for each T cell population. Data shown in (A) and (C) are representative of three independent experiments. In (A), numbers shown on the left of each plot indicate percent suppression. In (C), the percentage of cells in each quadrant is shown. Summary data are shown in (B) and (D) as means ± SD. *P < 0.05; ***P < 0.001.

Fig. 3

CD25 blockade in vivo depletes systemic T_regs in cancer patients. Peripheral blood samples obtained from patients before and at various times after a single infusion of CD25 mAb daclizumab were analyzed by flow cytometry. (A) Representative data from one patient comparing baseline (before) to 5 weeks after daclizumab. (B and C) Relative changes in the fraction (B) or absolute counts (C) of various T cell subsets over time, shown as the means for all patients normalized to individual baseline values. Daclizumab was given on week 0. Red diamonds, total CD4⁺ T cells; blue squares, FoxP3⁺ CD4 T cells; purple triangles, CD25⁺ FoxP3⁺ CD4 T cells as identified by the nonblocked monitoring CD25 mAb 4E3; green circles, CD25⁺ FoxP3⁺ CD4 T cells as identified by the monitoring CD25 mAb 2A3, which is blocked by daclizumab. For total FoxP3⁺ CD4 T cells and CD25⁺ (4E3) FoxP3⁺ CD4 T cells, P < 0.001 at weeks 1, 2, and 5; for CD25⁺ (2A3) FoxP3⁺ CD4 T cells, P < 0.001 at weeks 1, 2, 5, and 7 (statistical details are provided in table S3). (D) Peptide vaccination of breast cancer patients without daclizumab administration does not alter T_regs. Peripheral blood T cell populations were analyzed as fractions before and after vaccination by flow cytometry in a cohort of patients (n = 7) with metastatic breast cancer on a previously described clinical trial (29). Nonblocked mAb 4E3 was used to evaluate CD25. Data are shown as the means of all patients normalized to individual baseline values with error bars representing SD. P > 0.05, comparing T_reg fractions at the time of the second or third vaccine to baseline. Similar data were obtained if T cell populations were analyzed as absolute counts.

Fig. 4

Impact of CD25 blockade on vaccine immune responses in patients in vivo. (A to G) Peripheral blood T cells obtained from patients at baseline and at various times after treatment with daclizumab plus an experimental vaccine were analyzed for (A and B) peptide/MHC tetramer reactivity, (C) peptide-specific CD8 T cell responses as measured by CD107a mobilization and IFN-γ expression, (D) CMV tetramer reactivity, (E) CMV-specific CD8 T cell responses as measured by CD107a mobilization and IFN-γ expression, and (F and G) CRM197-specific responses for CD8 and CD4 T cells as measured by antigen-induced CFSE dilution. Representative data for each assay are shown in (A), (C), (E), and (F). Summary data for all patients are shown as heat maps of the best responses in (B), (D), and (G). V3 corresponds to the time of the third vaccine. In (C) and (E), an HLA-A2–binding peptide from HTLV-1 tax (Tax) was used as a negative control, and in (F) and (G), Staphylococcus enterotoxin B (SEB) was used as a positive control. In (D), patient CMV status was determined by seropositivity at baseline; all CMV-seronegative patients remained seronegative at the end of the study.