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.
Highlights
Advances in the understanding of anti-cancer immunity has resulted in the successful use of immunotherapies against a number of cancers with checkpoint inhibitor antibodies such as the anti-CTLA-4 antibody Ipilumimab,(YervoyTM) and anti-PD1 antibodies Pembrolizumab, (KetrudaTM) and Nivolumab, (OpdivoTM) becoming FDA approved for a number of cancers [1]
IL-2 incubation resulted in a more modest increase in expansion with a 5-fold increase occurring from 100–1000 ng/ ml in NK and NKT-like cells whereas CD8 T cells were expanded by 1.3 fold at 1000 ng/ml
IL-2 and IL-15 have been identified as agents that can expand these cells in protocols where they are either injected systemically or used to treat effector cells that are adoptively transferred
Summary
Advances in the understanding of anti-cancer immunity has resulted in the successful use of immunotherapies against a number of cancers with checkpoint inhibitor antibodies such as the anti-CTLA-4 antibody Ipilumimab,(YervoyTM) and anti-PD1 antibodies Pembrolizumab, (KetrudaTM) and Nivolumab, (OpdivoTM) becoming FDA approved for a number of cancers [1]. IL-2 and IL-15 are cytokines that have been used to expand these immune cells before adoptive transfer, or for use as adjuvants to these cells once administered. Both cytokines can stimulate the proliferation of NK cells. IL-2 has been FDA approved for single agent administration to patients with renal cell carcinoma and melanoma [4], while, IL-15 has been administered in a number of first in man studies to patients with metastatic cancer [5, 6]. IL-15 was recently selected from a large panel of cytokines as the only agent that can significantly expand NK and CD8 T effector populations in the presence of prostate tumor cells [16]
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