Increased expression of anti-apoptotic molecules and improved survival in post-REP TIL that received 4-1BB co-stimulation during the REP.

<p>RNA from post-REP TIL that were rapidly expanded with or without anti-4-1BB were subjected to real-time PCR (qRT-PCR) analysis for bcl-2, bcl-xL, bim, and survivin (<b>A</b>). The results of two representative patient TIL lines are shown (#2392 and #2396). Each PCR reaction was ran in triplicate with a CV of <5% for all assays. The results with the IL-2+4-1BB REP were normalized against the levels of gene expression in the control (IL-2) REP, as indicated with the 1-fold expression for the control REP for each gene. The increase in bcl-2 expression in the IL-2+4-1BB group was confirmed in three different TIL samples by intracellular staining for bcl-2 using flow cytometry (<b>B</b>). The post-REP TIL were also analyzed for their ability to survive and further expand when re-cultured with or without IL-2 (100 U/ml) for 5 days, as described in the Materials and Methods (<b>C</b> and <b>D</b>). Viable cell counts together with flow cytometry analysis for CD8 expression was performed to calculate the fold change in CD8⁺ T cells after the re-culturing period (<b>C</b>). TIL from 4-1BB REP (IL-2+4-1BB) or control REP (IL-2) cultures from 4 different patients were also subjected to staining with Annexin V and 7-AAD after re-plating without or with 200 IU/ml IL-2 for 5 days and the frequency of Annexin V⁺7-AAD⁻ (apoptotic cells) were determined (<b>D</b>).