A Short CD3/CD28 Costimulation Combined with IL-21 Enhance the Generation of Human Memory Stem T Cells for Adoptive Immunotherapy
Authors: Alvarez-Fernandez Carmen , Escribà-Garcia Laura , Sierra Jorge , Briones Javier 
Published: October 2019

Abstract
Introduction:
Adoptive transfer of gene modified T cells (ACT) with chimeric antigen receptors (CARs) is becoming a clinically relevant immunotherapy approach for cancer treatment. One important question involves the selection of the T cell subpopulation for gene-modified ACT. Recent preclinical studies have shown that memory stem T cells (TSCM) (with a phenotype CD45RO-, CD45RA+, CCR7+, CD62L+, CD95+) have higher in vivo persistence, self-renewal, proliferative and antitumor capacities compared to other memory and effector T cell subpopulations. For these reasons, TSCM may represent an ideal candidate for ACT with CARs.
Ex vivo generation of TSCM rely on CD3/CD28 costimulation and the use of cytokines such as IL-7 and IL-15 during the entire culture period. However, a strong costimulation may induce differentiation of TSCM to effector memory T cells. In this study, we have analyzed the impact of the costimulation length and the addition of the IL-21 on the ex vivo generation of TSCM cells.
Methods:
Purified naïve T cells from healthy donors were cultured in the presence of anti-CD3/CD28 coated beads, IL-7, IL-15 and/or IL-21 (25ng/ml). T cells phenotype from the different memory (TCM, TSCM and TEM) and effector subpopulations were analyzed by multiparametric flow cytometry. Particularly, TSCM were identified by a specific plotting strategy. Briefly, CD4+ and CD8+ cells were gated and a CCR7 against CD45RO plot were used to select CCR7+ CD45RO- cells in each subpopulation. Within these cells, a CCR7+ CD45RO- CD45RA+ cell population was identified by plotting CCR7 against CD45RA. Then, CD27 was used to detect CCR7+ CD45RO- CD45RA+ CD27+ cells. Finally, a plot of CD95 against CCR7 was used to identify the CCR7+ CD45RO- CD45RA+ CD27+ CD95+ cell population. Cellular proliferation was analyzed by flow cytometry using CFSE.
Results:
A short (48 hours) anti-CD3/CD28 costimulation of naïve T cells combined with IL-7 and IL-15 significantly increased the frequencies of CD4+ and CD8+ TSCMex vivo, compared to a long (10 days) costimulation (34.6%±4.4% vs 15.6%±4.24% for CD4+; p=0.008, and 20.5%±4.00% vs 7.7%±2.53% for CD8+; p=0.02). Moreover, the addition of IL-21 to this condition further enhanced the enrichment (47.9%±4.1% vs 34.6%±4.4% for CD4+; p=0.006, and 34.1%±3.5% vs 20.5%±4% for CD8+; p<0.0005) and expansion of CD4+ and CD8+ TSCM, with an increase in absolute numbers (311.3±39 vs 192.8±58.6 fold expansion in CD4+; p=0.04, and 728.1±53 vs 442.7±122 fold expansion in CD8+; p=0.04). In addition, IL-21 did not preferentially enriched for the CD8+ population since the CD4+ TSCM were maintained over the cell culture period in all the conditions tested (CD4+/CD8+ TSCM ratio:1.03±0.28 vs 1.34±0.19 for long costimulation versus long + IL-21, and 0.68±0.14 vs 0.72±0.002 for short vs short + IL-21; p=0.23).
Conclusion:
We show that these refined in vitro conditions significantly increase the frequencies and expansion of TSCM. Since the reagents used are available in a clinical grade setting, these data may have relevant clinical implications for the generation of this memory T cell subset for adoptive cell therapy with CAR-T cells of patients with cancer.

Disclosures
No relevant conflicts of interest to declare.