Analysis of the cellular mechanism of antitumor responses and autoimmunity in patients treated with CTLA-4 blockade

Abstract

We have demonstrated previously that the administration of CTLA-4 blockade has mediated objective cancer regression and autoimmunity in patients with metastatic melanoma. To explore the mechanism of these in vivo effects, we have studied the changes in lymphocyte phenotype and function in patients receiving anti-CTLA-4 Ab (MDX-010). Patients with stage IV melanoma or renal cell cancer were treated every 3 wk with an anti-CTLA-4 Ab with or without peptide immunization. Pheresis samples were analyzed using flow cytometry to determine lymphocyte cell surface markers. Gene expression analyses and proliferation assays were conducted on purified T cell subsets. Anti-CTLA-4 Ab did not inhibit the suppressive activity of CD4+CD25+ cells in vitro or in vivo. In addition, there was no decrease in the expression of CD4+CD25+ cells in whole PBMC, nor a decrease in Foxp3 gene expression in the CD4+ or CD4+CD25+ purified cell populations posttreatment. The percentage of CD4+, CD8+, CD4+CD25+, and CD4+CD25- T cells in PBMC expressing the activation marker HLA-DR increased following anti-CTLA-4 Ab administration. Therefore, our results suggest that the antitumor effects of CTLA-4 blockade are due to increased T cell activation rather than inhibition or depletion of T regulatory cells.

FIGURE 1

The effect of in vitro CTLA-4 blockade on the suppressive ability of CD4⁺CD25⁺ cells. Fresh lymphocytes from five patients were separated into CD4⁺CD25⁺ and CD4⁺CD25⁻ cell populations and treated in vitro with 0, 10, or 100 μg/ml anti-CTLA-4 Ab in a coculture suppression assay. The percent suppression of CD25⁻ proliferation by CD25⁺ cells was determined in the presence and in the absence of anti-CTLA-4 Ab. PBMC were obtained from patients A, B, and C after treatment in vivo with two or more doses of anti-CTLA-4 Ab and from patients F and G who were not treated with Ab in vivo. *, Anti-CTLA-4 Ab was added on days 0, 2, and 4 of culture. For all other cultures Ab was added on day 0. **, Cells were pulsed with [³H]thymidine on day 4 of culture and harvested on day 5. All other cultures were pulsed on day 5 and harvested on day 6.

FIGURE 2

Expression of CD4⁺ CD25⁺ cells in PBMC of patients treated in vivo with anti-CTLA-4 Ab. Patients were treated in vivo with escalating doses of anti-CTLA-4 Ab every 3 wk. PBMC were evaluated pretreatment and after two doses of Ab at each dose level of 3, 5, and 9 mg/kg. The percentage of CD25⁺ cells was determined by gating on the isotype control (lower panel), or based on the upper 10% of CD4⁺CD25⁺-expressing cells on the pretreatment sample (upper panel). Examples of pretreatment sample FACS gating of these two populations from patient 4 are shown in the corresponding panels to the right of the histogram. There were no consistent changes in the percentage of CD25⁺ cells in the CD4⁺ cell population resulting from the administration of anti-CTLA-4 Ab.

FIGURE 3

Relative Foxp3 levels in CD4⁺ cells from patients treated with CTLA-4 blockade. CD4⁺ lymphocytes were separated from PBMC obtained from patients pre- and posttreatment with anti-CTLA-4 Ab at 3, 5, and 9 mg/kg. Foxp3 gene expression relative to β-actin did not change consistently across doses, although there was a trend toward higher relative levels at some Ab doses compared with pretreatment levels (relative Foxp3 levels at 3, 5, and 9 mg/kg: +2.96, p = 0.24; +3.26, p = 0.18; +4.61, p = 0.05; paired t tests, not corrected for multiple analyses).

FIGURE 4

Relative Foxp3 levels in CD4⁺CD25⁺ and CD4⁺CD25⁻ cells from patients treated with CTLA-4 blockade. Lymphocytes from patients treated with at least one dose of anti-CTLA-4 Ab at 3 mg/kg were highly purified by flow cytometry into CD4⁺CD25⁺ high and CD4⁺CD25⁻ low populations. Cell acquisition was accomplished by flow sorting the upper 10% of CD4⁺CD25⁺ cells and the lower 10% of CD4⁺CD25⁻ cells (right panel). Foxp3 gene expression relative to β-actin was higher in CD25⁺ compared with CD25⁻ cells and significantly increased in CD4⁺CD25⁺ cell subpopulations posttreatment with anti-CTLA-4 Ab compared with pretreatment (p = 0.05, paired t test).

FIGURE 5

Suppression of CD25⁻ cell proliferation by CD25⁺ cells in patients after in vivo CTLA-4 blockade. Fresh pheresis samples from five patients were bead purified into CD4⁺CD25⁺ and CD4⁺CD25⁻ subpopulations for use in suppression assays. Samples were taken from patients after receiving at least four doses of anti-CTLA-4 Ab. CD4⁺CD25⁺ cells displayed a persistent ability to suppress proliferation of CD4⁺CD25⁻ cells after in vivo treatment with CTLA-4 blockade. *, Cultures were pulsed with [³H]thymidine on day 5 and harvested on day 6. All other cultures were pulsed on day 4 and harvested on day 5. †, A total of 5000 cells/population was cultured. All other cultures were plated with 10,000 cells/population.

FIGURE 6

HLA-DR expression on CD4⁺ lymphocytes in a patient treated with escalating doses of anti-CTLA-4 Ab. Whole PBMC from patient 4 were analyzed by FACS for expression of the lymphocyte activation marker HLA-DR on CD4⁺ cells. This patient was treated with escalating doses of anti-CTLA-4 Ab (3, 5, and 9 mg/kg) and tested before and after each dose level as indicated. Expression of HLA-DR on CD4⁺ cells increased over pretreatment with administration of Ab at all doses.

FIGURE 7

HLA-DR expression on CD4⁺ and CD8⁺ lymphocytes in patients treated with escalating doses of anti-CTLA-4 Ab. PBMC from patients treated with escalating doses of anti-CTLA-4 Ab (3, 5, and 9 mg/kg) were analyzed by FACS for expression of the activation marker HLA-DR on CD4⁺ and CD8⁺ cells before and after treatment at each Ab dose level. HLA-DR expression on both CD4⁺ and CD8⁺ lymphocytes increased with escalating Ab doses. The change was statistically significant in the CD8⁺ population after receiving treatment at both 5 and 9 mg/kg anti-CTLA-4 Ab (p = 0.02 and p = 0.03, paired t test; not corrected for multiple analyses).

FIGURE 8

HLA-DR expression on CD4⁺CD25⁺ and CD4⁺CD25⁻ lymphocytes in patients treated with escalating doses of anti-CTLA-4 Ab. PBMC from patients treated with escalating doses of anti-CTLA-4 Ab (3, 5, and 9 mg/kg) were analyzed by FACS for expression of the activation marker HLA-DR on CD4⁺CD25⁺ and CD4⁺CD25⁻ cells before treatment and after administration at each dose level. There was a trend toward increased HLA-DR expression on both subpopulations after treatment, with a significant increase observed after Ab administration at 5 mg/kg vs pretreatment (p = 0.05; not corrected for multiple analyses).