Gene expression profiling reveals similarities between the in vitro and in vivo responses of immune effector cells to IFN-alpha

Abstract

Purpose: The precise molecular targets of IFN-alpha therapy in the context of malignant melanoma are unknown but seem to involve signal transducers and activators of transcription 1 signal transduction within host immune effector cells. We hypothesized that the in vitro transcriptional response of patient peripheral blood mononuclear cells (PBMC) to IFN-alpha would be similar to the in vivo response to treatment with high-dose IFN-alpha.

Experimental design: The gene expression profiles of PBMCs and immune cell subsets treated in vitro with IFN-alpha were evaluated, as were PBMCs obtained from melanoma patients receiving adjuvant IFN-alpha.

Results: Twenty-seven genes were up-regulated in PBMCs from normal donors after treatment with IFN-alpha in vitro for 18 hours (>2-fold, P < 0.001). A subset of these genes (in addition to others) was significantly expressed in IFN-alpha-treated T cells, natural killer cells, and monocytes. Analysis of gene expression within PBMCs from melanoma patients (n = 13) receiving high-dose IFN-alpha-2b (20 MU/m(2) i.v.) revealed significant up-regulation (>2-fold) of 21 genes (P < 0.001). Also, the gene expression profile of in vitro IFN-alpha-stimulated patient PBMCs was similar to that of PBMCs obtained from the same patient after IFN-alpha therapy.

Conclusions: This report is the first to describe the transcriptional response of T cells, natural killer cells, and monocytes to IFN-alpha and characterize the transcriptional profiles of PBMCs from melanoma patients undergoing IFN-alpha immunotherapy. In addition, it was determined that microarray analysis of patient PBMCs after in vitro stimulation with IFN-alpha may be a useful predictor of the in vivo response of immune cells to IFN-alpha immunotherapy.

Figures

Figure 1. Real Time PCR analysis of select genes identified by microarray analysis of normal PBMCs following 1 hour in vitro IFN-α stimulation

Real Time PCR was used to validate the expression of genes in PBMCs (CXCL10, CCL8, IFIT1). Data were expressed as the mean fold increase relative to baseline levels (PBS treatment). All real time PCR data were normalized to the level of β-actin mRNA. Error bars denote the standard deviations of triplicate experiments.

Figure 2. Real Time PCR analysis of select genes identified by microarray analysis of PBMCs from melanoma patients receiving IFN-α

Real Time PCR was used to validate the expression of representative genes (IRF7, OASL, TAP1). Data were expressed as the mean fold increase relative to baseline levels (pre-treatment). All real time PCR data were normalized to the level of β-actin mRNA. Patient gene expression estimates were pooled and error bars denote the standard deviations of 7 melanoma patients.