Retroviral Replicating Vector Transduced Alloresponsive T Lymphocytes Retain Mobility And Cytotoxic Functionalities

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The radial monolayer cell migration assay was originally developed to measure the infiltrative properties of adherent tumor cells on individual extracellular matrix (ECM) proteins, or on ECM extracted from tumor cell monolayers. For this assay a single cell suspension of tumor cells is seeded using a cell sedimentation manifold (CSM), precisely into the center of Teflon-masked, ECM-coated slide wells. The adherent tumor cells establish a focal, circular monolayer from which horizontal motility can be microscopically-viewed and photographed over time. We adapted use of the CSM to evaluate the in vitro mobility of non-adherent effector T cells on confluent tumor cell monolayers or on ECM extracts from them. Cytotoxic T lymphocytes (CTL) sensitized to the human leukocyte antigens (HLA) on 13-06-MG glioma cells or on brain-tropic breast MDA-MB-231 carcinoma cells were generated by one-way mixed lymphocyte tumor cell reactions. These alloresponsive CTL were then transduced with retroviral replicating vectors (RRV) coding for green fluorescent protein (GFP). Using the CSM, the RRV-GFP transduced CTL were seeded into the center of wells with a confluent monolayer of glioma cells that were partially HLA matched to 13-06-MG. By light and fluorescence microscopy, we visualized not only the a) mobility of individual GFP+ T cells, but b) the cytotoxicity engendered to the underlying adherent glioma cell monolayer by the overlaid nonadherent CTL, in addition to c) transfer of RRV from the CTL to tumor cells. The in vitro cytotoxic functionality of the RRV-transduced CTL was confirmed by real-time xCELLigence impedance cytotoxicity assays. In other experiments, the RRV-GFP transduced CTL were also stained with a vital fluorescent red dye, CMPTX, to distinguish transduced from nontransduced CTL. The CTL were placed into the center of wells coated with ECM harvested from MDA-MB-231BR cells and radial movement of the transduced and nontransduced CTL was also observed over 72 hr time. We conclude that effector T cells transduced with RRV maintain mobility and cytotoxic functionality, and further, are capable of transmitting RRV to tumor cells. Thus, transduced alloresponsive CTL can facilitate immunogene therapy by their capability of gene delivery to infiltrating tumor cells in the brain. Citation Format: Kate L. Erickson, Colin C. Malone, Michelle J. Hickey, Geoffrey C. Owens, Yuki Kato, Robert M. Prins, Noriyuki Kasahara, Carol A. Kruse. Retroviral replicating vector transduced alloresponsive T lymphocytes retain mobility and cytotoxic functionalities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1660. doi:10.1158/1538-7445.AM2014-1660