Melanoma induces immunosuppression by up-regulating FOXP3(+) regulatory T cells

Abstract

Background: The immune response to melanoma is rarely curative, suggesting the emergence of immunosuppression. FOXP3-expressing regulatory T cells (T(reg) cells) function to suppress immune responses. The objective of this study was to determine if melanoma evades immune surveillance, in part, by inducing T(reg) cells.

Material and methods: Peripheral blood mononuclear cells (PBMCs) were isolated and exposed to melanoma-conditioned media (MCM) or control media for 1 week. The induction of T(reg) cells in these PBMCs was determined by measuring the proportion of CD25(+)FOXP3(+) T cells in all CD4(+) T cells by flow cytometry. FOXP3 expression was determined by mean fluorescence intensity (MFI) and Western blot. Supernatant cytokines were determined by ELISA.

Results: Normal PBMCs exposed to MCM revealed higher proportions of T(reg) cells than those exposed to control media after 6 days (3.4% versus 1.3%, respectively, P < 0.02). The expression of FOXP3 in T(reg) cells from PBMCs exposed to MCM increased over time by MFI and Western blot but was not significantly different than those exposed to control media. The level of IL-10 and TGF-beta in supernatants after 6 days growth was higher in MCM than control media, but this did not reach statistical significance.

Conclusion: Exposure of PBMCs to melanoma results in induction of FOXP3(+) T(reg) cells.

FIG. 1

Gating strategy used for CD4⁺CD25⁺FOXP3⁺ T_reg cells. Representative examples of flow cytometry plots and gating strategies of T_reg cells from PBMCs grown in control media and MCM. T_reg cells were those in the CD25+/FOXP3+ double-positive quadrant after gating for live lymphocytes and CD3+/CD4+ T cells, respectively. The quadrant labels showed the proportion of single or double-positive cells.

FIG. 2

Proportion of T_reg cells from normal PBMCs in MCM versus control media (n = 5). (A) Representative graph of the percentage of CD25+/FOXP3+ T_reg cells in control media versus MCM over time. (B) There was a significantly higher percentage of T_reg cells in MCM versus control after six days (3.4% v. 1.3%, respectively, *P < 0.02). Graph represents mean values ± SEM of the percentage of CD25⁺FOXP3⁺ T_reg cells in CD3⁺CD4⁺ T cells from PBMCs grown in control media versus MCM after six days.

FIG. 2

Proportion of T_reg cells from normal PBMCs in MCM versus control media (n = 5). (A) Representative graph of the percentage of CD25+/FOXP3+ T_reg cells in control media versus MCM over time. (B) There was a significantly higher percentage of T_reg cells in MCM versus control after six days (3.4% v. 1.3%, respectively, *P < 0.02). Graph represents mean values ± SEM of the percentage of CD25⁺FOXP3⁺ T_reg cells in CD3⁺CD4⁺ T cells from PBMCs grown in control media versus MCM after six days.

FIG. 3

MCM increases expression of FOXP3 with time. Typical Western blot analysis showed increasing amounts of FOXP3 expression in PBMCs over time cultured in MCM. Lanes were loaded with equal volume of cell lysate and probed for GAPDH as loading controls.

FIG. 4

Cytokine levels in control media versus MCM after six days growth (n = 6). Graphs represent mean values ± SEM of levels of TGF-β and IL-10 as determined by ELISA. (A) There was an increased level of TGF-β in supernatants from PBMCs grown in MCM versus control media after six days (3410 pg/ml and 2862 pg/ml, respectively, P = 0.70), but this did not reach statistical significance. (B) Concurrently, there was no significant difference in the level of IL-10 in MCM v. control media after six days growth (114 pg/ml and 49 pg/ml, respectively, P = 0.37).

FIG. 4

Cytokine levels in control media versus MCM after six days growth (n = 6). Graphs represent mean values ± SEM of levels of TGF-β and IL-10 as determined by ELISA. (A) There was an increased level of TGF-β in supernatants from PBMCs grown in MCM versus control media after six days (3410 pg/ml and 2862 pg/ml, respectively, P = 0.70), but this did not reach statistical significance. (B) Concurrently, there was no significant difference in the level of IL-10 in MCM v. control media after six days growth (114 pg/ml and 49 pg/ml, respectively, P = 0.37).