A Human Melanoma Cell Line Transduced with an Interleukin-4 Gene by a Retroviral Vector Releases Biologically Active IL-4 and Maintains the Original Tumor Antigenic Phenotype

Abstract

Experimental models of vaccination with tumor cells engineered to produce interleukin-4 (IL-4) have shown that the local release of this cytokine is associated with the development of antitumor immunity that may induce regression of established cancer. The aim of this study was to transduce a human melanoma cell line with the gene coding for human IL-4, and to analyze cytokine production, phenotypic characteristics, and antigen expression after transduction. A retroviral vector, constructed by inserting IL-4 cDNA into the LXSN vector, was used to infect the human melanoma cell line Me14932, known to express the MHC class I HLA-A2 and the melanoma-associated antigen Melan-A/MART-1, recognized by HLA-A2-restricted T-cells. The confluence of all G418-resistant cells (Me14932/IL-4) was then analyzed for proviral integration and IL-4 mRNA expression. Substantially stable IL-4 release was detected by ELISA in the supernatant of transduced cells, ranging from 1.6 to 4.6 ng/ml per 10⁵ cells per 24 hr; such a cytokine displayed a specific biologic activity, as revealed by the stimulation of blast cell proliferation and the inhibition of lymphokine activated killer cell (LAK) induction by IL-2. After 200 Gy irradiation, IL-4 release remained detectable for 5 weeks, whereas cell proliferation ceased within 7 days. Morphology and immunophenotypic characteristics of the parental cell line (expression of MHC classes I and II, ICAM-1, LFA 3, melanoma-associated antigens, etc.) were retained by the IL-4 gene-transduced melanoma as assayed by microscopy and immunofluorescence; likewise, susceptibility to lysis by LAK cells as well as a T-cell clone recognizing the Melan-A/MART-1 antigen did not change. These results, together with the lack of replication-competent retrovirus, suggest that the Me14932/IL-4 cell line displays suitable characteristics for its use in the treatment of HLA-matched melanoma patients.

Overview summary

Transduction of the interleukin-4 (IL-4) gene into a human melanoma cell line could provide tumor cells with a new immunostimulatory function, as shown by murine experimental models of vaccination with tumor cells engineered to release IL-4. To this aim, a retroviral vector carrying the human IL-4 gene has been used to infect a human melanoma cell line known to express a T cell-recognized antigen. A stable and prolonged secretion of biologically active IL-4 was obtained from the infected melanoma cell line, which retained the phenotypic characteristics of the parental cells, including the expression of surface molecules (HLA, ICAM-1, LFA 3, etc.) necessary for melanoma antigen(s) presentation, and remained susceptible to lysis by an HLA-A2-restricted cytolytic T cell clone and by LAK cells. These results suggest the potential use of this IL-4-transduced melanoma cell line as vaccine for active immunization of HLA-matched melanoma patients.