Evidence of systemic Th2-driven chronic inflammation in patients with metastatic melanoma

Abstract

Purpose: Immunotherapeutic modalities are commonly used for treatment of patients with melanoma. The therapeutic success in preclinical models has not yielded the expected clinical results. To understand this discrepancy, we attempted to define immune homeostasis of 209 patients with melanoma across stages of disease relative to normal controls.

Experimental design: Peripheral blood mononuclear cells (PBMC) and plasma were collected from patients and healthy donors. PBMC were analyzed for frequencies of natural killer, dendritic, and T cells and their functional status. Matched plasma samples were analyzed for the concentrations of 27 cytokines, chemokines, and growth factors. RNA was isolated from 24 metastatic melanoma tumor biopsies and profiled by microarray analysis.

Results: The frequency of natural killer, T, and dendritic cells in patients does not significantly change across stages of melanoma. However, plasma concentrations of Th2 cytokines [interleukin (IL)-4, IL-5, IL-10, and IL-13] in tumor-bearing patients were significantly higher than those with resected melanoma. Expression array analysis of metastatic melanoma revealed that the malignant melanocytes were not the source of the Th2 cytokines but did highly up-regulate vascular endothelial growth factor (VEGF) transcripts, consistent with plasma VEGF concentrations. In vitro VEGF exposure of normal PBMC lead to repolarization from Th1 to Th2 emulating the state of metastatic melanoma.

Conclusions: Patients with metastatic melanoma exist in a state of Th2-mediated "chronic inflammation" as a result of at least VEGF overproduction by malignant tumors. These data support prior observations regarding the effect of VEGF on immune cell function and suggests consideration of VEGF inhibitors in future cancer immunotherapy clinical studies in metastatic melanoma.

Figure 1

Principal component analysis of cytokine levels across all stages of disease. Plasma collected from patients diagnosed with atypical nevi (red), benign nevi (blue), melanoma in situ (green), stage I melanoma (purple), stage II melanoma (orange), stage III melanoma (yellow) and stage IV melanoma (brown) was tested in the 27-plex cytokine array to determine plasma levels of 27 cytokines. Using principal component analysis no difference in cytokine levels was noted among the early stages of disease (non-tumor bearing) patients, however cytokine levels were shown to be different in stage IV, tumor bearing patients, which is represented as separation of the brown spheres from all the others.

Figure 2

Assessment of T-cell phenotype and function in healthy donors and stage IV melanoma patients. The number of T-cells exhibiting the FoxP3 (Treg) or PD-1 phenotype in peripheral blood was determined in healthy donors and stage IV melanoma patients (A). The frequency of FoxP3 positive cells were measured by 3-color flow cytometry, CD4-PC5, CD25-PE and FoxP3-Alexa flour 488. The mean percent (+/− SD) of FoxP3 positive were determined from the CD4 and CD25 double positive population. The mean frequency (+/− SD) of PD-1+ cells was measured from the CD8+ population. The frequency (+/− SD) of tetramer positive (CMV or MART-1) CD8+ T cells was compared among normal volunteers and patients with stage IV melanoma (B).

Figure 3

VEGF levels in patients with metastatic melanoma. (A) RNA expression of cytokines in human metastatic melanoma tissue. Twenty-four frozen biopsies of metastatic melanoma tumor tissues was used to extract RNA for expression array analysis. Illustrated are the RNA expression intensity profiles of 45 probes for 24 cytokines. (B) Comparison of expression intensities between genes coding for Th1 (IFN-γ and IL-2), Th2 (IL-4, IL-5, IL-10, and IL-13) cytokines and VEGF. There were no statistically significant differences when comparing Th1 vs Th2 cytokine expression levels (p=0.04); there was a statistically significant difference when comparing VEGF expression with Th1 or Th2 cytokines (p<0.001). Levels of significance were determined using the Wilcoxin signed-rank test. (C ) ELISA (mean concentration +/−SD) for VEGF-A was performed on plasma samples from healthy donors (n=30) and stage IV melanoma patients (n=40).

Figure 4

Co-culture with recombinant human VEGF shifts T-helper polarity from Th1 (IFN-γ) to Th2 (IL-4) predominance. PBMC (A) isolated from healthy donors were stimulated with PMA and ionomycin in the presence of brefeldin-A, permeabolized and intracellularly stained for human IFN-γ (FITC) and human IL-4 (PE). PBMC were exposed to increasing concentrations of VEGF (0–16pg/mL) without/with IL-12. All cells were immunostained with PC5 anti-human CD4. Purified CD4+ T-cells (B) were negatively isolated using Miltenyi beads, cultured and stained in the same fashion as PBMC (A). Similar results were observed in 5 different experiments.