CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion

Abstract

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4(+) T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4(+) T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4(+) T cells, but also in the number of CD4(+) FoxP3(+) Tregs. Although the effects were dose-dependent, CD4(+) FoxP3(+) regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4(+) FoxP3(+) regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4(+) FoxP3(+) Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.

Figures

Figure 1

Administration of anti-CTLA4 antibody and GM-CSF. (A) The dose of anti-CTLA4 antibody (mg/kg) and number of patients (n) on each dose level are presented. (B) The counts of CD4+ T cells per volume of blood were calculated by multiplying the percentage of CD4+ T cells by the absolute lymphocyte counts measured simultaneously. Each row represents the corresponding dose level. Each line represents the counts for each evaluable subject within the specified dose level.

Figure 2

Activation of CD4+ T cells with escalating doses of anti-CTLA4 antibody. (A) PBMCs at baseline, week 4, and week 16 of treatment from study subjects in each dose level were stained with antibodies to CD4, CD25, and CD69. Stained cells were then assessed by flow cytometry and gated on CD4+ T cells. Each row represents a different dose level. Numbers on plots represent the percentage of cells for each quadrant. Individual study subjects are presented from each dose level. Gating for CD25 and CD69 expression was set with results from staining with isotype-matched control IgG. (B) The counts of CD25+CD69+ CD4+ T cells per volume of blood was calculated by multiplying the percentage of CD25+CD69+CD4+ T cells by the absolute lymphocyte counts measured simultaneously. Each row represents the corresponding dose level. Each line represents the counts for each evaluable subject within the specified dose level.

Figure 3

Expansion of FoxP3+ CD4+ T cells after treatment. (A) PBMCs from baseline and at week 4 from a subject in dose level 5 were stained for CD4, CD25, and CD69 with fluorescence-labeled antibodies. Anti-FoxP3 antibody staining was performed after cell permeabilization. Cells were then analyzed by flow cytometry and gated on CD4+ T cells. CD4+ T cells were gated (top panels) and analyzed for CD25, CD69, and FoxP3 expression. The numbers on plots represent the percentage of cells in each of the quadrants. Data are derived from 1 subject treated in dose level 5 and are representative of 6 subjects in this cohort. Gating for CD4+ FoxP3+ staining was set with results from isotype-matched control IgG staining. (B) PBMCs from baseline and at week 4 from a subject who received anti-CTLA4 antibody at 3 mg/kg alone in a separate clinical trial for metastatic hormone-refractory prostate cancer were also stained for CD4 and intracellular FoxP3. Results are representative of 3 patients assessed. (C) PBMCs from baseline and at week 4 from a subject in dose level 5 were stained for CD4 and FoxP3 as described. Phycoerythrin-labeled anti-CTLA4 antibody, which is not blocked by the study drug, was also added either before (surface staining, top panels) or after (intracellular staining, bottom panels) cell permeabilization. FoxP3+ CD4+ T cells (solid line) or FoxP3− CD4+ effector T cells (dashed line) were gated and assessed for CTLA4 expression. Staining with an isotype-matched control antibody is also shown (shaded histogram). (D) Dynamics of the intracellular pool of CTLA4 were assessed by surface capture staining. PBMCs from a representative subject in cohort 5 at week 4 of treatment were cultured for 6 hours and stained with antibody for surface CTLA4 or intracellular CTLA4 (after permeabilization) at the conclusion of culture, or the cells were cultured in a cocktail containing monensin, brefeldin A, and an anti-CTLA4 antibody to capture surface CTLA4 that translocates to the cell membrane over this time. PBMCs were also stained for CD4 and FoxP3 with fluorescently labeled antibodies, assessed by flow cytometry, and gated on CD4+ T cells. Results are representative of PBMCs from 3 different patients in cohort 5 at week 4 or at baseline.

Figure 4

Expansion of FoxP3+ CD4+ T cells across dose levels of anti-CTLA4 antibody. (A) PBMCs from baseline, week 4, and week 16 were stained for CD4 and intracellular FoxP3. Stained cells were then assessed by flow cytometry and gated on CD4+ T cells. Each row represents results from individual patients corresponding to subjects presented in Figure 2A at different dose levels assessed longitudinally at each of the time points. Numbers on plots represent the percentage of cells for each quadrant. (B) The counts of FoxP3+ CD4+ T cells per volume of blood was calculated by multiplying the percentage of FoxP3+ CD4+ T cells by the absolute lymphocyte counts measured simultaneously in a complete blood count. Each row represents the corresponding dose level. Each line represents the counts for each evaluable subject within the specified dose level. (C) The change in the number of FoxP3+ CD4+ T cells per volume of blood is compared by plotting the baseline count (x-axis) versus the week-16 measurement (y-axis). The solid line indicates no change in cell count between the 2 time points. There was a significant increase in cell count among patients treated on dose levels 2 to 5, with different symbols indicating the different dose cohorts (Wilcoxon matched pairs test: P = .001). All data points residing above the line indicate an increase in the number of cells.

Figure 5

Function of expanded Tregs. (A) CD4+ T cells were assessed for cytokine production before and after treatment by intracellular staining with fluorescence-labeled antibodies. PBMCs from before and after treatment were cultured with or without anti-CD3 and anti-CD28 antibodies for 8 hours. The cells were stained with anti-CD4 followed by intracellular staining for FoxP3, IFN-γ, and IL-2. For analysis, the PBMCs were gated on CD4+ T cells. Numbers on plots represent the percentages for each quadrant. Data shown are derived from 1 subject and are representative experiments from 3 different subjects. (B) PBMCs from baseline and at week 8 were stained for CD4, CD25, and CD127 followed by intracellular staining for FoxP3. Gating for FoxP3+ CD4+ cells was set with results from isotype-matched control IgG staining. Tregs were sorted from PBMCs derived from a study subject (C) in dose level 5 at week 8 and from a healthy control individual (D) based on the expression of CD4, CD127, and CD25. Thirty thousand autologous CD4+CD127+CD25− T cells (responders) were cocultured with 100 000 irradiated allogeneic PBMCs and anti-CD3 antibody. Where indicated, CD4+CD127−CD25+ Tregs were added at the indicated ratios of responders/Tregs. 3H-thymidine incorporation was assessed after 5 days of culture. Results are representative experiments from 3 different subjects. Error bars represent SD.

Figure 6

Proliferation of FoxP3+ CD4+ T cells with treatment. (A) PBMCs from a representative study subject in dose level 2 obtained before and after treatment were stained with CD4 and intracellular FoxP3 as well as for (B) CD71 and intracellular Ki67 expression with fluorescently labeled antibodies. Stained cells were assessed by flow cytometry and gated on CD4+ T cells for FoxP3, Ki67, and CD71 expression. Numbers on plots represent the percentages for each quadrant. The percentage of CD4 T cells that are (C) FoxP3+Ki67+ or (D) FoxP3+ CD71+ are shown at baseline and week 4 for the 9 patients assessed in dose levels 2 (▵), 3 (□), 4 (◇), and 5 (•). (E) PBMCs from before (top panels) and after (bottom panels) treatment were also stained with CD4 as well as stained intracellularly for FoxP3, Bcl-2, and Bcl-xl expression with fluorescently labeled antibodies. Stained cells were again assessed by flow cytometry and gated on FoxP3+ CD4+ T cells. Staining with anti-Bcl2 and anti–Bcl-xl antibodies (open histograms) and an isotype-matched control IgG (filled histogram) is shown. Data are derived from individual subjects and representative of subjects assessed and were consistent across dose levels 2 to 5.