Following Antigenic Restimulation In Vitro Capability Improved Survival and Expansion Maintaining CD28 Expression Have Tumor-Infiltrating Lymphocytes Specific Melanoma - MART-1

We determined how CD8 + melanoma tumor–infiltrating lymphocytes (TILs) isolated from two distinct phases of expansion in preparationforadoptiveTcelltherapyrespondtomelanomaAgrestimulation.WefoundthatTILsisolatedaftertherapidexpansion protocol (REP) phase, used to generate the final patient TIL infusion product, were hyporesponsive to restimulation with MART-1 peptide-pulsed dendritic cells, with many CD8 + T cells undergoing apoptosis. Telomere length was shorter post-REP, but of sufficient length to support further cell division. Phenotypic analysis revealed that cell-surface CD28 expression was significantly reduced in post-REP TILs,whereasCD27 levelsremainedunchanged.Trackingpost-REPTILproliferationby CFSE dilution,aswell assorting for CD8 + CD28 + and CD8 + CD28 2 post-REP subsets, revealed that the few CD28 + TILs remaining post-REP had superior survival capacity and proliferated after restimulation with MART-1 peptide. An analysis of different supportive cytokine mixtures during the REP found that a combination of IL-15 and IL-21 facilitated comparable expansion of CD8 + TILs as IL-2, but prevented the loss of CD28 expression with improved responsiveness to antigenic restimulation post-REP. These results suggest that current expansion protocolsusingIL-2formelanomaadoptiveT celltherapy yieldslargelyCD8 + T cellsunable topersistanddivideinvivofollowing Ag contact. The few CD8 + CD28 + T cells that remain may be the only CD8 + TILs that ultimately survive to repopulate the host and mediate long-term tumor control. A REP protocol using IL-15 and IL-21 may as data from supervised group analysis. Microarray data sets were explored further using a heatmap and Onto-Tool (12; Real-time PCR was conducted to validate key genes of interest.

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