Regulatory T cell frequency in patients with melanoma with different disease stage and course, and modulating effects of high-dose interferon-alpha 2b treatment

Abstract

Background: High-dose interferon-alpha 2b (IFN-alpha 2b) is the only approved systemic therapy in the United States for the adjuvant treatment of melanoma. The study objective was to explore the immunomodulatory mechanism of action for IFN-alpha 2b by measuring serum regulatory T cell (Treg), serum transforming growth factor-beta (TGF-beta), interleukin (IL)-10, and autoantibody levels in patients with melanoma treated with the induction phase of the high-dose IFN-alpha 2b regimen.

Methods: Patients with melanoma received IFN-alpha 2b administered intravenously (20 MU/m2 each day from day 1 to day 5 for 4 consecutive weeks). Serum Treg levels were measured as whole lymphocytes in CD4+ cells using flow cytometry while TGF-beta, IL-10, and autoantibody levels were measured using enzyme-linked immunosorbent assays.

Results: Twenty-two patients with melanoma received IFN-alpha 2b treatment and were evaluated for Treg levels. Before treatment, Treg levels were significantly higher in patients with melanoma when compared with data from 20 healthy subjects (P = 0.001; Mann-Whitney test). Although a trend for reduction of Treg levels following IFN-α 2b treatment was observed (average decrease 0.29% per week), statistical significance was not achieved. Subgroup analyses indicated higher baseline Treg levels for stage III versus IV disease (P = 0.082), early recurrence versus no recurrence (P = 0.017), deceased versus surviving patients (P = 0.021), and preoperative neoadjuvant versus postoperative adjuvant treatment groups (not significant). No significant effects were observed on the levels of TGF-beta, IL-10, and autoantibodies in patients with melanoma treated with IFN-alpha 2b.

Conclusions: Patients with melanoma in this study showed increased basal levels of Treg that may be relevant to their disease and its progression. Treg levels shifted in patients with melanoma treated with IFN-alpha 2b, although no firm conclusions regarding the role of Tregs as a marker of treatment response or outcome can be made at present.

Figure 1

Flow cytometry: percentage of CD4⁺CD25^+HFFoxp3⁺ cells in interferon-α 2b-treated patients with melanoma. Flow cytometric gating strategy to identify Treg cells. (A) Dot plot of forward scatter (FSC) versus side scatter (SSC) for all events: all peripheral blood cell populations are shown in red; the population of lymphocytes is shown in green. (B) Lymphocytes (green) were analyzed on the basis of surface markers CD4 and CD25; the P3 gate identifies the percentage of CD4⁺CD25^+HF cells. (C) The P4 gate identifies the percentage of Foxp3⁺CD4⁺CD25^+HF cells; this represents the region used to calculate the final percentage of Treg cells in CD4⁺ lymphocytes.

Figure 2

Comparison of regulatory T cell levels at baseline in patients with melanoma and healthy subjects. (A) Box plot showing baseline regulatory T cell (Treg) levels in 22 patients with melanoma prior to interferon-α 2b (IFN-α 2b) treatment compared with 20 healthy subjects (HS) (P = 0.001). (B) Box plot showing Treg levels by disease stage in 20 healthy subjects and 44 patients with melanoma (P <0.01 for increased Tregs in all melanoma patients vs. healthy subjects); P = not significant for Treg increase by disease stage. Horizontal lines inside the boxes = median values; upper and lower boundaries of the boxes = first and third quartiles of the distribution; whiskers = mild outliers; open dots = outliers; and asterisks = extreme outliers.

Figure 3

Subgroup comparisons: regulatory T cell levels at baseline in patients with melanoma before treatment with interferon-α 2b. Box plot subgroup comparisons of regulatory T cell (Treg) levels (%) at baseline in 22 patients with melanoma before treatment with interferon-α 2b (IFN-α 2b). (A) Adjuvant (ADJ) versus neoadjuvant (NEO) IFN-α 2b (P = not significant); (B) stage III versus stage IV (P = 0.082); (C) early recurrence (ER) versus no recurrence (NR) (P = 0.017); (D) surviving (ALV) versus deceased (DCD) (P = 0.021). Horizontal lines inside the boxes = median values; upper and lower boundaries of the boxes = first and third quartiles of the distribution; whiskers = mild outliers; open dots = outliers; and asterisks = extreme outliers.

Figure 4

Regulatory T cell levels by week in interferon-α 2b-treated patients with melanoma. (A) Individual regulatory T cell (Treg) levels by week in the 22 patients with melanoma treated with interferon-α 2b. (B)Box plot showing circulating regulatory T cell (Treg) levels by week in the 22 patients with melanoma treated with interferon-α 2b (P = not significant). Horizontal lines inside the boxes = median values; upper and lower boundaries of the boxes = first and third quartiles of the distribution; whiskers = mild outliers; and open dots = outliers.

Figure 5

Determination of transforming growth factor-β, interleukin-10 and autoantibody in interferon-α 2b-treated patients with melanoma. (A) Transforming growth factor-β (TGF-β), (B) interleukin (IL)-10, and serum autoantibodies: (C) antinuclear antibody (ANA), (D) anti-cardiolipin (ACA), (E) anti-double stranded DNA (anti-dsDNA), and (F) anti-thyroglobulin (anti-HTG) levels by week in 14/22 patients with melanoma treated with interferon-α 2b. Horizontal lines inside the boxes = median values; upper and lower boundaries of the boxes = first and third quartiles of the distribution; whiskers = mild outliers; open dots = outliers; and asterisks = extreme outliers.

Figure 6

Kaplan-Meier relapse-free survival curves. Kaplein-Meier relapse-free survival curves for (A) all 22 interferon-α 2b-treated patients with melanoma and (B) patients receiving neoadjuvant versus adjuvant therapy.