IL-21 Augments Rapamycin in Expansion of Alpha Fetoprotein Antigen Specific Stem-Cell-like Memory T Cells in vitro

Alloreactive tumor specific T cells are important arsenals of the adaptive immune system in the fight against tumors. However, stem cell-like memory T cells (Tscm) provide the key to effective elimination of tumor cells. Methods for generating these T cell subsets already exist. However, they could be made more efficient. Further, they are expensive and unattainable to the resource poor laboratories. In this regard, we are hereby describing a novel in vitro allogeneic co-culture method for raising allo-restricted tumor specific Tscm cells that we developed. We started by obtaining PBLs that screened negative for HLA-A2 molecules from healthy donors followed by co-culture with T2/AFP cells to generate AFP peptide specific tumor-reactive T cells. Controls, IL-21 and/or rapamycin were applied to samples in 24 well plates. Samples were harvested and stained with anti-human CD3, CD8, CD44, CD62L, and HLA-A2/AFP dimer followed by flow cytometry analysis. Cell viability was measured by Trypan blue exclusion assay. One Way ANOVA and independent t test were used to compare the mean differences among and between groups where P values less than 0.05 were considered significant. Our results show that rapamycin arrests the differentiation of, and expands AFP specific Tscm cells. Further, the expansion of Tscm cells is augmented in the presence of IL-21. IL-21 and Rapamycin can be used concurrently to raise and maintain antigen specific Tscm cells for purposes of augmenting immunotherapy strategies against cancers.