LYMPHOKENE GENE EXPRESSION IN ANTI-CD3 STIMULATED HUMAN T-LYMPHOCYTES

Abstract

In an attempt to evaluate cytokine gene expression in humans with cancer, PBLs were obtained from patients participating in an immunotherapy Phase I trial using anti-CD3 mAb (in vitro) in conjunction with IL-2 (in vivo). The peripheral blood lymphocytes containing the anti-CD3 activated T-lymphocytes were obtained from 4 patients at 7 different timepoints in the 2 cycles of treatment. A system was developed in which purified RNA, from both fresh and cultured PBLs, as well as CD4+ and CD8+ enriched subsets, was reverse transcribed and PCR amplified with specific cytokine primers. General trends of mRNA expression were observed amongst the four cytokines that were studied; TGF-β1, TNF-α, IFN-γ, and IL-1β. There was an induction of mRNA expression of TNF-α, IFN-γ, and IL-1β after the in vitro anti-CD3 activation on day 1 of their treatment. This coincides with a slight reduction of TGF-β1 mRNA expression on day 1. The cytokine mRNA expression at the peak of T lymphocyte proliferation in vivo illustrated potential synergistic mechanisms. The decreasing trend of both TNF-α and IL-1β may indicate that the expression of these cytokines was necessary in the early stages of the anti-CD3 T-cell activation, to upregulate the IL-2 receptor for further activation and proliferation. TGF-β1 and IFN-γ, on the other hand, demonstrated increasing trends of mRNA expression coinciding with the proliferation peak. It is possible that the TGF-β1 protein is downregulating the IL-2 receptor expression, while IFN-γ may be downregulating the proliferation. These synergistic effects would help control the proliferation of the T lymphocytes in vivo. It was also observed that there was a difference in IL-1β mRNA expression between PBLs maintained in culture when compared to fresh PBLs. While IL-1β was not detected in the cultured samples after day 4, there was still some expression in the fresh cells. This may be due to a potential depletion of IL-1β producing macrophages from the culture. On the other hand, IFN-γ did not show significant differences when comparing the in vitro and in vivo samples where the levels remained fairly stable throughout the patients' treatments. This may be explained by the fact that both the fresh and cultured samples received IL-2 for a total of 28 days in the treatment. RNA from CD4+ and CD8+ enriched populations was also analyzed, and indicated that both subsets were found to express IFN-γ and TGF-βl mRNA. On the other hand, mainly the CD8+ cells expressed TNF-α (in vitro), and IL-1β (in vivo). These are only initial experiments as it is not known whether cytokine protein secretion coincided with cytokine gene expression. The results, however, do indicate that there are general trends in cytokine mRNA expression which are more distinguishable in the first cycle of their treatment.