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. 2010 Jun;59(6):921-31.
doi: 10.1007/s00262-010-0818-0. Epub 2010 Jan 26.

Combination of IL-21 and IL-15 enhances tumour-specific cytotoxicity and cytokine production of TCR-transduced primary T cells (V体育官网入口)

Nadine Pouw  1 , Elike Treffers-Westerlaken, Jaco Kraan, Floyd Wittink, Timo ten Hagen, Jaap Verweij, Reno Debets
Affiliations

Combination of IL-21 and IL-15 enhances tumour-specific cytotoxicity and cytokine production of TCR-transduced primary T cells

Nadine Pouw et al. Cancer Immunol Immunother. 2010 Jun.

Abstract

IL-21, and to a lesser extent IL-15, inhibits differentiation of antigen-primed CD8 T cells and promotes their homeostasis and anti-tumour activity. Here, we investigated molecular mechanisms behind tumour-specific responses of primary murine T lymphocytes engineered to express a TCR directed against human gp100/HLA-A2 following short-term exposure to IL-15 and/or IL-21. We demonstrated that IL-15 + IL-21, and to a lesser extent IL-21, enhanced antigen-specific T-cell cytotoxicity, which was related to enhanced expression of granzymes A and B, and perforin 1. Furthermore, IL-15 + IL-21 synergistically enhanced release levels and kinetics of T-cell IFNgamma and IL-2, but not IL-10. Enhanced secretion of IFNgamma was accompanied by increased gene expression and cytosolic protein content, and was restricted to effector memory T cells. To summarize, we show that IL-15 + IL-21 improves antigen-specific responses of TCR-transduced effector T cells at multiple levels, which provides a rationale to treat T cells with a combination of these cytokines prior to their use in adoptive TCR gene therapy VSports手机版. .

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"V体育2025版" Figures

Fig. 1
Fig. 1
IL-15 decreases the number of TCR transgene-positive T cells, whereas IL-21 increases the expression levels of TCR transgenes per cell. Primary murine T cells were retrovirally transduced with TCRαβ directed against human gp100280–288/HLA-A2 (gp100/A2) and subsequently treated either with IL-2 (100 U/ml), IL-15 (50 ng/ml), IL-21 (50 ng/ml) or IL-15 + IL-21 (50 ng/ml each). T cells were analysed by flow cytometry for expression of TCR-Vβ14 within the FSC/SSC lymphocyte gate. Mock-transduced T cells were used as negative controls and showed <5% TCR-Vβ14 staining (data not shown). Transduction efficiencies at day 5 after start of culture (i.e., 3 days after addition of various cytokines) are indicated as percentages (a) or mean fluorescence intensities (MFI) (b) of T cells expressing TCR-Vβ14 (mean ± SEM, n = 18, *p < 0.01 compared to IL-2)
Fig. 2
Fig. 2
T cells treated with a combination of IL-15 and IL-21 show enhanced antigen-specific cytotoxicity. Murine splenocytes were gp100/A2 TCR-transduced and cultured with IL-2, IL-15, IL-21 or IL-15 + IL-21 as described in legend to Fig. 1. Cytokine-stimulated T cells were co-cultured for 4 h with 51Cr-labelled target cells. Target cells included B16A2 cells loaded with human gp100 peptide (a), B16gp100/A2 cells (b) or B16A2 cells (c). Effector to target ratios and percentages of specific 51Cr-release are indicated in the figures. Mock-transduced T cells served as negative controls. Only in case of mock-transduced T cells cultured with IL-15 + IL-21 there was some background activity towards B16gp100/A2 (not towards B16A2 with or without gp100 peptide) up to 20% of 51Cr-release at E:T ratio’s of >40:1. At the moment of cytotoxicity assay, 2 weeks after start of culture, TCR-Vβ14 surface expression was around 40% (data not shown). Results are from a representative experiment out of 6 experiments with similar results
Fig. 3
Fig. 3
A combination of IL-15 and IL-21 enhances antigen-specific Gzma, Gzmb and Prf1 gene expression in T cells. Murine splenocytes were gp100/A2 TCR-transduced and cultured with IL-2, IL-15, IL-21 or IL-15 + IL-21 as described in legend to Fig. 1. Five days after start of culture, T cells were stimulated with B16gp100/A2 or B16 cells for 20 h. Stimulated T cells were analysed by micro-array for gene expression of Granzyme A (Gzma, a), Granzyme B (Gzmb, b) or Perforin 1 (Prf1, c). See  “Materials and methods” for details about micro-array procedures. Y-axes show relative gene expression for cytokine cultures compared to a common reference. Two independent experiments (starting from T-cell cultures) gave similar results (mean ± SD, n = 3, *p < 0.05 compared to IL-2)
Fig. 4
Fig. 4
Antigen-specific secretion of IFNγ is enhanced upon combined treatment with IL-15 and IL-21. Murine splenocytes were gp100/A2 TCR-transduced, cultured with cytokines and stimulated with B16 target cells as described in legend to Fig. 3. Stimulated T cells were analysed by commercial ELISA for the secretion of IFNγ (a), IL-2 (b) and IL-10 (c). During the stimulation assay, no exogenous cytokines were added to the T-cell:target cell co-cultures. T cells cultured with medium or Con A served as negative and positive controls, respectively. Mock-transduced T cells showed no cytokine secretion in any condition tested (data not shown). The different T-cell cultures and the levels of cytokines that were present in supernatants are indicated on the X- and Y-axis, respectively (mean ± SEM, n = 4 (IL-2 and IL-10) or n = 10 (IFNγ), *p < 0.05 and **p < 0.005 compared to IL-2)
Fig. 5
Fig. 5
Antigen-specific secretion of IFNγ and IL-2, but not IL-10, shows accelerated kinetics upon combined treatment with IL-15 and IL-21. Murine splenocytes were gp100/A2 TCR-transduced and cultured with cytokines as described in legend to Fig. 1. Cytokine cultured T cells were stimulated with B16gp100/A2 and B16 cells for 1, 4 or 20 h, and analysed by commercial ELISA for the secretion of IFNγ (a), IL-2 (b) or IL-10 (c). Mock-transduced T cells showed no cytokine secretion at any time-point tested (data not shown). Time points following target cell stimulations are indicated at the X-axes. Absolute levels of cytokines present in supernatants are indicated at the Y-axes (mean ± SEM, n = 3, *,# p < 0.05 compared to IL-2 for B16gp100/A2 and B16, respectively)
Fig. 6
Fig. 6
The combination of IL-15 and IL-21 enhances antigen-specific Ifng gene expression in T cells. Murine splenocytes were gp100/A2 TCR-transduced, cultured with cytokines as described in legend to Fig. 1. Five days after start of culture, T cells were stimulated with B16gp100/A2 or B16 cells for 20 h. Stimulated T cells were analysed by micro-array for gene expression of IFNγ (a), IL-2 (b) or IL-10 (c). See  “Materials and methods” for details about micro-array procedures. Y-axes show relative gene expression for cytokine cultures compared to a common reference. Two independent experiments (starting from T-cell cultures) gave similar results (mean ± SD, *p < 0.05 compared to IL-2)
Fig. 7
Fig. 7
IL-15 + IL-21-induced IFNγ production is restricted to CD62L+/CD44− effector memory T cells. Murine splenocytes were gp100/A2 TCR-transduced and cultured with cytokines as described in legend to Fig. 1. Five days after start of culture, T cells were stimulated with medium, Con A (10 ng/ml) or B16gp100/A2 cells, and analysed by flow cytometry for the expression of IFNγ (a). In (b) IFNγ staining was combined with the T-cell differentiation markers CD62L and CD44. In the latter case, T cells were first stained with anti-CD62L mAbFITC and anti-CD44 mAbAPC, followed by intracellular staining with anti-IFNγ mAbPE. Cells were FSC/SSC gated on viable lymphocytes and percentages of IFNγ+ T cells (in red) were determined. The results for IL-2 and IL-15 + IL-21 cultures are shown. Results are from a representative experiment out of 3 independent experiments with similar results
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