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. 2016 Jul 19;14(1):214.
doi: 10.1186/s12967-016-0973-y.

A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

C Alvarez-Fernández  1   2   3 , L Escribà-Garcia  1   2   3 , S Vidal  2   4 , J Sierra  1   2 , J Briones  5   6
Affiliations

A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

C Alvarez-Fernández et al. J Transl Med. .

Abstract

Background: Immunotherapy based on the adoptive transfer of gene modified T cells is an emerging approach for the induction of tumor-specific immune responses. Memory stem T cells, due to their enhanced antitumor and self-renewal capacity, have become potential candidate for adoptive T cell therapy of cancer. Methods to generate memory stem T cells ex vivo 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 memory stem T cells to effector memory T cells. Here we show that manipulation of the length of the costimulation and addition of IL-21 enhance the ex vivo expansion of memory stem T 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 (25 ng/ml). T cells phenotype from the different memory and effector subpopulations were analyzed by multiparametric flow cytometry.

Results: A short anti-CD3/CD28 costimulation of naïve T cells, combined with IL-7 and IL-15 significantly increased the frequencies of CD4(+) and CD8(+) memory stem T cells ex vivo, compared to a prolonged costimulation (34.6 ± 4.4 % vs 15.6 ± 4.24 % in CD4(+); p = 0.008, and 20.5 ± 4.00 % vs 7.7 ± 2.53 % in CD8(+); p = 0.02). Moreover, the addition of IL-21 to this condition further enhanced the enrichment and expansion of CD4(+) and CD8(+) memory stem T cells with an increase in the absolute numbers (0.7 × 10(6) ± 0.1 vs 0.26 × 10(6) ± 0.1 cells for CD4(+); p = 0.002 and 1.1 × 10(6) ± 0.1 vs 0.27 × 10(6) ± 0.1 cells for CD8(+); p = 0.0002; short + IL-21 vs long).

Conclusions: These new in vitro conditions increase the frequencies and expansion of memory stem T cells and may have relevant clinical implications for the generation of this memory T cell subset for adoptive cell therapy of patients with cancer.

Keywords: IL-21; Immunotherapy; Memory stem T cell.

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Figures

Fig. 1
Fig. 1
Gating strategy for the identification of human TSCM. T cells were identified by gating on lymphocytes (SSC versus FSC), singlets (FSC-H versus FSC-A) and live T cells (SSC versus LIVE/DEAD). CD4+ and CD8+ T cells were simultaneously identified with anti-CD4 and anti-CD8 antibodies. a Gating strategy of fresh blood cells. After gating on CD4+ and CD8+ cells, TCM and TEM subpopulations were identified based on CCR7 and CD45RO expression. In the gated CCR7+CD45RO− population, cells expressing CD45RA and CD27 were further analyzed. In this later population (CCR7+CD45RO−CD45RA+CD27+), TN and TSCM were identified based on the CD95 expression. TN is defined as CCR7+CD45RO−CD45RA+CD27+CD95− whereas TSCM subpopulation is defined as CCR7+CD45RO−CD45RA+CD27+CD95+. Red arrows indicate the sequential gating strategy. b Gating strategy of 10 days culture cells. After gating on CD4+ and CD8+ cells, TCM and TEM subpopulations were identified based on CCR7 and CD45RO expression. In the gated CCR7+CD45RO− population, cells expressing CD45RA and CD27 were further analyzed. In this later population (CCR7+CD45RO−CD45RA+CD27+), TSCM were identified based on the CD95 expression. TSCM subpopulation is defined as CCR7+CD45RO−CD45RA+CD27+CD95+. Similarly, in the gated CCR7+CD45RO+ population, cells expressing CD45RA, CD27 and CD95+ identify a TSCM-like subpopulation, which is defined as CCR7+CD45RO+CD45RA+CD27+CD95+. Red arrows indicate the sequential gating strategy
Fig. 2
Fig. 2
Short CD3/CD28 costimulation increases CD4+ and CD8+ TSCM frequencies compared with long costimulation. Naïve T cells from healthy donors (n = 6) were cultured for 10 days with short (48 h) (solid black line) or long (solid grey line) costimulation. a, b Frequency of CD4+ (a) and CD8+ (b) TSCM cell subset (mean ± SEM). c, d Frequencies of total TSCM (TSCM + TSCM-like) CD4+ (c) and CD8+ (d) (mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 3
Fig. 3
IL-21 enhances CD4+ and CD8+ TSCM frequencies. Naïve T cells from healthy donors (n = 6) were cultured for 10 days with short (solid black line) or long (solid grey line) costimulation and in the presence (black dashed line) or absence (grey dashed line) of IL-21. a, b Frequencies of CD4+ and CD8+ TSCM (mean ± SEM) are shown in (a) and (b), respectively. c, d Frequencies of CD4+ and CD8+ Total TSCM (TSCM + TSCM-like) (mean ± SEM) are shown in (c) and (d), respectively. *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 4
Fig. 4
Analysis of CD4+ and CD8+ T cell subsets composition. a, b Column graphs showing the relative frequencies (mean ± SEM) of CD4+ (a) and CD8+ (b) T cell subsets respectively, for each condition tested (long costimulation, short costimulation, long + IL-21, and short + IL-21). TN (T Naïve), TCM (T central memory), TEM (T effector memory), TEMRA (T effector memory-RA+ cells)
Fig. 5
Fig. 5
IL-21 enhances CD4+ and CD8+ TSCM expansion. Naïve T cells from healthy donors (n = 3) were cultured for 10 days and fold expansion were analyzed in short costimulation (solid black line), long costimulation (solid grey line) short + IL-21 (black dashed line) and long + IL-21 (grey dashed line) conditions. a, b Absolute fold expansion of CD4+ (a) and CD8+ (b) (mean ± SEM), expressed in increment of cells from the starting culture time point is shown. c, d Absolute fold expansion of CD4+ (c) and CD8+ (d) total TSCM (TSCM + TSCM-like) (mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001
Fig. 6
Fig. 6
Analysis of transduction efficiency with a GFP-expressing lentivirus. Lentivirus transduction efficiency measured in CD4+ and CD8+ TSCM and TCM subsets by flow cytometry (% of cells expressing GFP; mean ± SEM)
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