Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma

Abstract

Background: Dysfunction of the immune system has been documented in many types of cancers. The precise nature and molecular basis of immune dysfunction in the cancer state are not well defined.

Methods and findings: To gain insights into the molecular mechanisms of immune dysfunction in cancer, gene expression profiles of pure sorted peripheral blood lymphocytes from 12 patients with melanoma were compared to 12 healthy controls. Of 25 significantly altered genes in T cells and B cells from melanoma patients, 17 are interferon (IFN)-stimulated genes. These microarray findings were further confirmed by quantitative PCR and functional responses to IFNs. The median percentage of lymphocytes that phosphorylate STAT1 in response to interferon-alpha was significantly reduced (Delta = 16.8%; 95% confidence interval, 0.98% to 33.35%) in melanoma patients (n = 9) compared to healthy controls (n = 9) in Phosflow analysis. The Phosflow results also identified two subgroups of patients with melanoma: IFN-responsive (33%) and low-IFN-response (66%). The defect in IFN signaling in the melanoma patient group as a whole was partially overcome at the level of expression of IFN-stimulated genes by prolonged stimulation with the high concentration of IFN-alpha that is achievable only in IFN therapy used in melanoma. The lowest responders to IFN-alpha in the Phosflow assay also showed the lowest gene expression in response to IFN-alpha. Finally, T cells from low-IFN-response patients exhibited functional abnormalities, including decreased expression of activation markers CD69, CD25, and CD71; TH1 cytokines interleukin-2, IFN-gamma, and tumor necrosis factor alpha, and reduced survival following stimulation with anti-CD3/CD28 antibodies compared to controls.

Conclusions: Defects in interferon signaling represent novel, dominant mechanisms of immune dysfunction in cancer. These findings may be used to design therapies to counteract immune dysfunction in melanoma and to improve cancer immunotherapy.

Figure 1. Hierarchical Clustering of the Microarray Data Using ISGs

Hierarchical clustering of the microarray data was performed using the ten ISGs with lowest adjusted p-values in combined data from B cells, CD8 T cells, and CD4 T cells (A) and CD56dim NK cells (B) from patients with melanoma versus healthy controls. White indicates highest expression, red indicates lowest, and yellow/orange indicates intermediary expression in melanoma versus healthy control.

Figure 2. Percentage of STAT1-pY701-Positive Cells in Lymphocytes from Patients with Melanoma and Healthy Controls in Response to IFN Stimulation

PBMCs from patients with melanoma and healthy controls were stimulated with 1,000 IU/ml IFN-α, IFN-β, or IFN-γ. The percentage of STAT1-pY701-positive cells was measured by Phosflow. p-Values were calculated using the Wilcoxon rank sum test (one-sided). Medians are indicated by the bar in each scatter column. H, healthy; M, melanoma. (A–D) IFN-α-stimulated PBMCs. Lymphocytes (A); CD8 T cells (B); CD4 T cells (C); B cells (D). (E–H) IFN-β-stimulated PBMCs. Lymphocytes (E); CD8 T cells (F); (G) CD4 T cells (G); B cells (H). (I–L) IFN-γ-stimulated PBMCs. Lymphocytes (I); CD8 T cells (J); CD4 T cells (K); B cells (L).

Figure 3. Fold Change in Mean Fluorescence Intensity of STAT1-pY701-Alexa Fluor 647 Staining in Lymphocytes from Patients with Melanoma and Healthy Controls in Response to IFN Stimulation

The mean fluorescence intensity (MFI) of STAT1-pY701-Alexa Fluor 647 staining was measured by Phosflow in IFN-stimulated (1,000 IU/ml) and unstimulated cells. Total fold change = MFI stimulated cells/MFI-unstimulated cells; positive cell fold change = MFI STAT1-pY701-positive cells/MFI-unstimulated cells. p-Values were calculated using the Wilcoxon rank sum test (one-sided). Medians are indicated by the bar in each scatter column. Healthy (□; n = 9); melanoma (▪; n = 9). (A) IFN-α-stimulated cells. (B) IFN-β-stimulated cells. (C) IFN-γ-stimulated cells. (D) Positive cell fold change in lymphocytes.

Figure 4. Expression of ISGs in Lymphocytes from Patients with Melanoma and Healthy Donors Stimulated with IFN-α

Lymphocytes from patients with melanoma and healthy controls were stimulated with 1,000 IU/ml for 14 h. The expression of STAT1, IFIT1, IFI44, and MX2 were measured by qPCR. Data are shown on a log₂ scale. Medians are indicated by the bar in each scatter column. p-Values are from one-sided Wilcoxon rank sum test. H, healthy; M, melanoma. (A) Gene expression in IFN-α-stimulated cells, sample sizes were n = 13 (H) and n = 12 (M) for each gene. (B) Fold change in gene expression in IFN-α–stimulated cells versus unstimulated control cells. STAT1: n = 13 (H), n = 11 (M); IFIT1: n = 12 (H), n = 12 (M); IFI44: n = 13 (H), n = 11 (M); MX2: n = 12 (H), n = 12 (M).

Figure 5. Activation and Survival of CD8 and CD4 T Cells in Response to Activating Stimulus

Lymphocytes from patients with IFN-nonresponsive melanoma and healthy donors were stimulated with beads coated with anti-CD3 and anti-CD28 antibodies. (A) Percentage of CD8 and CD4 T cells expressing CD69 at 6 h following stimulation (mean and standard error). (B) Percentage of CD8 and CD4 T cells expressing CD69, CD25, and CD71 (data combined) at 24 h following stimulation (mean and standard error). (C) Percentage of CD8 and CD4 T cells expressing IL-2, TNF-α, and IFN-γ (data combined) at 24 h following stimulation. (D) Percentage of surviving (Annexin V-negative 7-AAD-negative) cells at 4 d following stimulation. Means are indicated by the bar in scatter columns.