T Cell Activation Modulates Retrovirus-Mediated Gene Expression

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Abstract
Important considerations for T lymphocyte-based gene therapy include efficient gene delivery and expression in primary, human T cells. In this study, retrovirus-mediated gene transfer and the fate of proviral gene expression were evaluated in human T cells activated using (1) immobilized anti-CD3 monoclonal antibody (MAb) plus interleukin 2, or (2) cis costimulation using beads carrying coimmobilized anti-CD3 and anti-CD28 MAbs. By cross-linking the CD3 and CD28 receptors, these MAbs mimic in vivo signaling events, leading to cytokine production and proliferation. A modified human interleukin 1β (IL-1β) cDNA inserted into the MFG retroviral vector served as an indicator gene. Retroviral transduction frequencies were similar for T lymphocytes activated by the respective methods. However, early after MAb stimulation and virus exposure, proviral gene expression was greater at the RNA and protein levels in optimized anti-CD3/anti-CD28 bead-activated T cells, corresponding with augmented endogenous cytokine responses and mitogenesis. Proviral gene expression was not regulated by extrinsic cell factors present in activated T cell supernatants. Regardless of the MAb stimulation method, proviral IL-1β expression declined in later T cell cultures concomitant with a decrease in cellular cytokines. Restimulation by either method reinduced both T cell activity and vector expression. Our finding that proviral gene regulation is downmodulated in the absence of T cell signaling events has implications for clinical strategies using retrovirus-modified T cells. Gene-modified T lymphocytes hold promise for treatments of cancer, AIDS, and T cell disorders. With the advent of retrovirus-mediated gene transfer, emphasis has been placed on improving retroviral vector expression and enhancing retrovirus gene transfer into human T cells. A clinically relevant consideration is the potential effect of biological T cell activity on the expression status of the introduced proviral gene. T cells remain quiescent in vivo until activated in response to antigen displayed on the surface of an antigen-presenting cell (APC). Using monoclonal antibodies that signal T cell receptors and mimic varying degrees of T cell activation, we demonstrate that proviral gene expression (1) corresponds with the magnitude of T cell activation, (2) downregulates in the absence of T cell signaling, and (3) is reinduced on secondary activation. Because the amount of provirus-encoded product can determine therapeutic efficacy, our findings have important implications for clinical applications of retrovirus gene-modified T cells.