CD4+CD25+ suppressor lymphocytes in the circulation of patients immunized against melanoma antigens

Abstract

Murine studies have suggested that a population of CD4+ T cells expressing the alpha chain of the interleukin (IL)-2 receptor (CD25+) are phenotypically anergic in response to T cell receptor stimulation and can suppress the function of CD4+ and CD8+ T cells. Recent studies of peripheral lymphocytes from healthy human volunteers have identified a similar population, although little is known about the presence and activity of these cells in patients with cancer and their possible impact on anticancer immunization strategies. Thus, the authors have undertaken these studies in patients with metastatic melanoma undergoing immunizations with known melanoma antigens. CD4+ CD25+, CD4+ CD25-, and a 1:1 ratio of these isolated T cells were stimulated with soluble anti-CD3 antibody in the presence of irradiated T cell-depleted PBMCs, and proliferation was assessed by measuring [3H]thymidine incorporation. In 13 patients, isolated CD4+CD25+ T cells proliferated 68% (+/- 5.8%) less than separately cultured CD4+ CD25- T cells. Moreover, CD4+ CD25+ T cells suppressed the proliferation of an equal number of cocultured CD4+ CD25+ T cells in 11 of 13 patients by an average of 60% (+/- 4.9%). Suppression was not seen at day three of culture and became apparent at days five through nine. The degree of suppression was proportional to the numbers of CD4+ CD25+ T cells. Addition of high-dose IL-2 reversed the hypoproliferative phenotype of the CD4+ CD25+ T cells and abrogated their suppressive function. These studies demonstrate that anergic and functionally suppressive CD4+ CD25+ T cells exist in patients with melanoma undergoing tumor antigen immunization and thus may play a role in modifying the magnitude of the T cell response to immunization.

FIG. 1

CD4+CD25+ T cells are present in patients with melanoma. Cells were labeled with PE-conjugated CD25 antibody and FITC-conjugated CD4 antibody. Proper isotype controls were also done, but are not presented here. (A) CD4+CD25+ T cells represent a significant portion of total PBMC in patients with melanoma. CD4+CD25+ T cells were 16.6% (± 5.4, n = 20) of total PBMC and 34.2% (± 8.0, n = 20) of CD4+ T cells. (B) Moreover, using the MACS system by Miltenyi Biotec, CD4+CD25+ T cells were purified with 74% purity (SDEV = ± 16.8, n = 16) and CD4+CD25⁻ T cells were purified with 74% purity (SDEV = ± 14.3, n = 16).

FIG. 2

Kinetics of suppression. (A) CD4+CD25+ (5 × 10³), CD4+CD25⁻ (5 × 10³), and a 1:1 ratio of CD4+CD25+:CD4+CD25⁻ (5 × 10³:5 × 10³) T cells were isolated from three different patients with melanoma. 1.5 μg/mL anti-CD3 and 1 × 10⁴ autologous, irradiated T cell-depleted accessory cells were used to stimulate the cells. A control for overcrowding was included whereby 1 × 10⁴ CD4+CD25⁻ T cells were incubated under identical conditions. [³H]thymidine was added to cultures for the final 18 hours of culture. Cultures in sextuplicate were harvested at days three, five, seven, and nine. CD4+CD25+ T cells proliferated far less than CD4+CD25⁻ T cells over the entire length of the time course. The 1:1 ratio of CD4+CD25+:CD4+CD25⁻ T cells proliferated at a low level over the time course in patients 1 and 3, whereas it increased up to day five and then declined in patient 2. (B) Percent suppression on each day of harvest is shown. Negative % suppression values were denoted as 0% suppression. Suppression was not seen at day three in any patient. Peak suppression was seen at day five to seven. Error bars represent SEM.

FIG. 3

Effect of adding different numbers of CD4+CD25+ T cells upon degree of suppression. Various numbers of CD4+CD25+ T cells were added to a constant number of CD4+CD25⁻ T cells (5 × 10³). Cells were stimulated with 1.5 μg/mL anti-CD3 and 1 × 10⁴ T cell-depleted accessory cells for 7 days. [³H]thymidine was added for the final 18 hours of culture. The degree of suppression increased as the number of CD4+CD25+ T cells increased. All points were done in sextuplicate, and error bars represent SEM.

FIG. 4

Effects of IL-2 on CD4+CD25+ T cell-mediated suppression. (A) CD4+CD25+, CD4+CD25⁻, or a 1:1 ratio of CD4+CD25+:CD4+CD25⁻T cells were stimulated with 5 μg/mL anti-CD3 and 1 × 10⁴ T cell-depleted accessory cells for 7 days. (B) Identically treated cells were also incubated with 300 IU/mL of human recombinant IL-2. [³H]thymidine was added to culture for the final 18 hours of culture. IL-2 reversed the hyporesponsive state of CD4+CD25+ T cells and largely abrogated suppression mediated by these cells. Conditions were done in sextuplicates, and error bars represent SEM.