IL-21 augments rapamycin in expansion of alpha fetoprotein antigen specific stem-cell-like memory T cells in vitro

Abstract

IntroductionAlloreactive 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.MethodsWe 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.ResultsOur 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.ConclusionIL-21 and Rapamycin can be used concurrently to raise and maintain antigen specific Tscm cells in vitro for purposes of augmenting immunotherapy strategies against cancers.

Memory T cell, exhaustion, and tumor immunity

“…Several in vitro methods for T SCM generation have been reported, but most of them generate T SCM cells from naïve T cells [47,50,55,[63][64][65][66]. We recently reported that co-culture with NOTCH ligand-expressing feeder cells converts fully activated or more differentiated memory cells into induced T SCM -like (iT SCM ) cells [67,68] (Figure 5).…”

“…Less-differentiated CAR-T induction was obtained by inhibiting the Akt-mTOR pathway, enhancing FOXO1 activity, and potentiating mitochondrial function [68,[71][72][73][74][75]. Additionally, IL-21 in combination with rapamycin has been reported to enhance generation of tumor antigen-specific T SCM -like cells [64], and IL-21 enhances T CM cell formation in a FAO-dependent manner [76]. We found that FOXM1 plays an important role of the induction of the FAO-dependent OXPHOS-related genes in NOTCH-induced iT SCM cells [69].…”

Generation and application of human induced‐stem cell memory T cells for adoptive immunotherapy

“…These methods could generate T SCM cells in vitro, but these methods still generate T SCM cells from na€ ıve T cells ( Table 1). [16][17][18][19][20] We have established a novel two-step culture system for T SCM induction, which is constituted by a "prime" step and an "induction" step, and have named the induced-T SCM "induced-T SCM (iT SCM )" cells. 12 In this paper, we describe a detailed methodology of iT SCM generation and report optimal conditions of priming and cytokine treatment.…”

“…The strength of TCR stimulation, cytokine effects, and signaling inhibitors were associated with T SCM generation from na€ ıve T cells and are summarized in Table 1. 11,[16][17][18][19][20][21][22][23][24][25] In our two-step culture system, T cells are first primed and expanded by strong TCR stimulation, and are then cocultured with OP9-hDLL1 cells to convert into iT SCM cells (Figure 1A). To acquire a large number of antigen-specific CD8a + T cells, we first isolated peripheral human CD8 + T cells, labeled with cell trace dye and co-cultured them with autologous Epstein-Barr virus (EBV)transformed lymphoblastoid cell lines (LCLs) for 7 days in the "prime"…”

“…Alternative methods have been proposed by optimizing TCR strength, cytokine supplement (including interleukin [IL]‐7, IL‐15, and IL‐21), and drug treatment. These methods could generate T SCM cells in vitro, but these methods still generate T SCM cells from naïve T cells (Table ) . We have established a novel two‐step culture system for T SCM induction, which is constituted by a “prime” step and an “induction” step, and have named the induced‐T SCM “induced‐T SCM (iT SCM )” cells .…”