Development of Limiting Dilution Analysis as a Method for Analyzing Tumor Infiltrating Lymphocyte Trafficking and Survival in Vivo and in Vitro
Retroviral mediated transduction and expression of a bacterial gene for Neomycin resistance (Neo R) labels T cells with a unique gene sequence and allows them to grow in the Neomycin analog (G418. Recently, this approach has been utilized to identify cell survival and trafficking in both animal and human systems. In this study we examined whether the selectable growth advantage of T cells transduced with the Neo R gene could be exploited in Limiting Dilution Analysis (LDA) to quantitatively assess the frequency of labeled cells present in a population. A dose of G418 (400 µg/ml active concentration) was established which prevented growth of nontransduced cells. Both normal spleen cells and cultured T cells were found to be adequate feeder cells and the addition of mitogens to the LDA did not increase the cloning frequency of gene modified cells directly out of culture. The observed frequency of clones growing in LDA was a good approximation to the number of transduced cells plated. Twenty million Neo R T cells were adoptively transferred i.v. into mice, and the mice received 30,000 U IL-2 i.p. After 48 hours, the mice were sacrificed and their organs were processed. Limiting dilution analysis of the organ preparations has demonstrated recovery of the adoptively transferred TIL (as these cells grew in G418). Recovery of these cells was documented at frequencies ranging from 1/200 to >1/5000. Cells from control animals or recipients of nontransduced T cells fail to grow in LDA with G418. Therefore, the combination of LDA and Neo R T cells may provide an additional approach to quantitate trafficking of gene modified T cells and might be utilized to analyze qualitative differences of the isolated cells.