IL-15 Upregulates Telomerase Expression and Potently Increases Proliferative Capacity of NK, NKT-Like, and CD8 T Cells

Abstract

Interleukin-15 (IL-15) is a cytokine that has been shown to expand CD8 T cell and natural killer (NK) cell populations, and therefore has potential for potentiating adoptive immune cell therapy for cancer. Previously, IL-15 has been shown to induce proliferation of CD8 memory T cells through activation of telomerase. Here, we investigated whether telomerase is also activated during the IL-15 mediated proliferation of NK and NKT-like (CD56+CD3+) cells. We also examined the extent that each of the three signaling pathways known to be stimulated by IL-2/IL-15 (JAK-STAT, PI3K-AKT Ras-RAF/MAPK) were activated and involved in the telomerase expression in the three cell types NK, NKT, or CD8 T cells. To assess cell proliferation and doubling, peripheral blood mononuclear cells (PBMCs) or isolated NK, NKT-like or CD8 T cells were incubated with varying concentrations of IL-15 or IL-2 for 7 days. CD8 T, NK, and NKT cell expansion was determined by fluorophore-conjugated antibody staining and flow cytometry. Cell doubling was investigated using carboxyfluorescein-succinimidyl-ester (CFSE). Telomerase expression was investigated by staining cells with anti-telomerase reverse transcriptase (anti-TERT). Telomerase activity in CD56+ and CD8 T cells was also measured via Telomerase Repeat Amplification Protocol (TRAP). Analysis of cellular expansion, proliferation and TERT expression concluded that IL-15 increased cellular growth of NK, NKT, and CD8 T cells more effectively than IL-2 using low or high doses. IL-15, increased TERT expression in NK and NKT cells by up to 2.5 fold, the same increase seen in CD8 T cells. IL-2 had effects on TERT expression only at high doses (100-1000 ng/ml). Proteome profiling identified that IL-15 activated selected signaling proteins in the three pathways (JAK-STAT, PI3K-AKT, Ras-MAPK) known to mediate IL-2/IL-15 signaling, more strongly than IL-2. Evaluation by signaling pathway inhibitors revealed that JAK/STAT and PI3K/AKT pathways are important in IL-15's ability to upregulate TERT expression in NK and NKT cells, whereas all three pathways were involved in CD8 T cell TERT expression. In conclusion, this study shows that IL-15 potently stimulates TERT upregulation in NK and NKT cells in addition to CD8 T cells and is therefore a valuable tool for adoptive cell therapies.

Keywords: adoptive cell therapy; cell-signaling; interleukin-15; interleukin-2; telomerase.

Figure 1

Expansion of immune effector cell populations treated for 7 days with IL-15 at concentrations from 0.01 ng/ml to 100 ng/ml or IL-2 from 0.1 to 1000 ng/ml with PBS as a control. (A) Gating strategy for NK cells, NKT-like cells and CD8 T cells in the PBMC population showing the significant increase in CD56+CD3- cells (NK cells), CD56+CD3+ (NKT cells) and (CD3+CD8+) (CD8 T) cells in non-adherent PBMCs treated with IL-15. (B) displays quantitated expression of NK, NKT, CD8 T cells and CD4 T cells in lymphocytes treated with different doses of IL-15 or IL-2. (C) shows increased total percentage of purified NK, NKT, and CD8 T cells upon incubation with IL-2 or IL-15. (D) shows the expansion of the three cell types over a time period with time points at days 2, 7, and 14 using IL-15 at 2.5 ng/ml for NK cells and 25 ng/ml with a more pronounced expansion with IL-15 compared to IL-2 in NK and NKT-like cells. Results are expressed as % of the expansion of the untreated lymphocytes and represent means ± SEM from six separate experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA and Newman Keuls post-hoc analysis).

Figure 2

Effects of IL-15 and IL-2 on cell doubling capacity. Non-adherent PBMCs premixed with CFSE were treated for 7 days with concentrations of IL-15 from 0.1–100 ng/ml and IL-2 from 0.1 to 1000 ng/ml with PBS as a control. (A) displays the dot plots and a representative histogram showing the representative CFSE staining of a population of CD56+CD3- NK cells in the PBMCs. CFSE staining is measured as the geometric mean of the gated NK cell population and a decrease in the geometric mean signifies an increase in cell division. (B) shows the effects of IL-15 and IL-2 on NK, NKT-like and CD8 T cell proliferation as expressed by the normalized geometric mean fluorescence of CFSE after 7 days. (C) shows the number of cell divisions observed above that of the control cell population (with no cytokines). The numbers of cell divisions were calculated from the geometric mean fluorescence intensity of the cells by using 1 cell division as equivalent to a 50% decrease in the geometric mean of CFSE. Results represent means ± SEM from six separate experiments (* p < 0.05 by one-way ANOVA and Newman Keuls post-hoc analysis).

Figure 3

Effects of IL-15 and IL-2 on the expression of TERT in NK, NKT, and CD8 T cells in non-adherent PBMCs after 7 days culture. (A) shows dot plots and histograms displaying TERT as detected by flow cytometric analysis using Alexa-Fluor647 conjugated anti-TERT antibodies. Cells were also stained with fluorophore conjugated anti-CD8, anti-CD56, and anti-CD3 to distinguish the NK, NKT-like, and CD8 T cell populations. In (B), results are expressed as the TERT geometric mean values from each experiment normalized to the PBS control. IL-15 induces significant increases in TERT in NK, NKT, and CD8 T cells starting at concentrations of 2.5 ng/ml. IL-2 also significantly increases TERT expression at concentrations from 100 ng/ml to 1000 ng/ml. Results represent means ± SEM from six separate experiments (six donors)(* p < 0.05 by one-way ANOVA and Newman Keuls post-hoc analysis). (C) shows telomerase activity in CD56⁺ and CD8+ T cells. measured via a TRAP assay after 7 days incubation with IL-15. Four concentrations of IL-15 were used: 0, 2.5, 25 and 100 ng/ml, with six repeats carried out. Results showed that as the concentration of IL-15 increased there was a rise in absorbance, indicating telomerase activity in the CD56+ and CD8 T cells.

Figure 4

The effects of IL-15 and IL-2 on the expression of phosphorylation of proteins from the three signaling pathways JAK/STAT, P13/AKT, and RAS/MAPK. (A) shows membrane dot images developed from probing of the three cell types (NK, NKT, and CD8 T cells) treated with 1ng/ml IL-2 with the two membranes from a representative donor. The intensity of the dots relates to the detection of phosphorylated protein intensity within a certain sample. (B) displays a heatmap of the protein array collated from repeated experiments (n=4), with the proteins arranged by their signaling pathways and with colors indicating the degree of upregulation of phosphorylated proteins as calculated by densitometry carried out on the developed membranes. Yellow represents no change in expression compared to the low dose IL-2 control. Light green = protein expression increased by 1.1 fold -1.5 fold. Dark green = expression increased by more than 1.5 fold. Cream indicates no protein expression. (C) displays western blots of proteins selected from the array that showed increased phosphorylation (or upregulation of protein in the case of Hsp60).

Figure 5

Effects of inhibitors of JAK1, JAK3, MEK/ERK, and PI3/AKT on the upregulation of expression of TERT (A), cell expansion (B), and cell divisions from CFSE data (C) in NK, NKT-like and CD8 T cells. Non-adherent PBMCs were aliquoted at 8 x 10⁵ cells/ml in 24-well plates together with the optimal concentrations of cytokines found to increase TERT expression for each cell type (2.5 ng/ml IL-15 and 1000 ng/ml IL-2 for NK cells, 25 ng/ml IL-15 and 1000 ng/ml IL-2 for NKT-like cells and 25 ng/ml IL-15 and 1000 ng/ml IL-2 for CD8 T cells), or controls (PBS)]. Cells were incubated for 7 days either with cytokines/control only or in the presence of inhibitors of each of the three signaling pathways known to be impacted by IL-2/IL-15 [CP690550 for JAK1 (J1) and JAK3 (J3) inhibition dependent on concentration, LY-294002 hydrochloride (P) for PI3K/AKT inhibition and U0126 (M) for inhibition of MEK/ERK]. Results are expressed as the normalized geometric mean expressions of TERT, normalized cell expansion, and normalized numbers of cell divisions and are the means +/- SEM from six separate experiments. The comparisons are between the each of the no inhibitor conditions (PBS, low IL-2, high IL-2 and IL-15) and the corresponding condition with each of the inhibitors. (*p < 0.05, **p < 0.01 by one-way ANOVA and Newman Keuls post-hoc analysis).