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IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells.

ABSTRACT: Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.

SUBMITTER: Mao Y

PROVIDER: S-EPMC5025899 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells.

Mao Yumeng Y van Hoef Vincent V Zhang Xiaonan X Wennerberg Erik E Lorent Julie J Witt Kristina K Masvidal Laia L Liang Shuo S Murray Shannon S Larsson Ola O Kiessling Rolf R Lundqvist Andreas A

Blood 20160727 11

Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenge ...[more]

PMID: 27465917

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Project description:Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor post-infusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK cell functions following cytokine withdrawal to model post-infusion performance. In contrasts to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided functional advantages. Genome-wide analysis of cytosolic and polysome-associated mRNA revealed cytokine dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mTOR signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced functional advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15 stimulated NK cells was also observed using a clinically applicable protocol for NK cell expansion. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK cell cancer therapy. Freshly isolated NK cells from 6 donors were activated with IL-2 or IL-15 for 48 hours, followed by cytokine withdrawal for 24 hours, resulting in four RNA samples per donor. From each sample, both the cytosolic as well as the polysomal fraction were collected. Donor 3 contains activation and post withdrawal data from two different donors due to poor RNA-quality obtained for some samples which did not allow for processing of the complete set of 6 donors (resulting in a total of 40 samples).

Project description: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.

Project description:NK cells have potent antitumor capacity. They are enriched in the human liver, with a large subset specialized for tissue-residence. The potential for liver-resident versus liver-infiltrating NK cells to populate, and exert antitumor functions in, human liver tumors has not been studied. We examined liver-resident and liver-infiltrating NK cells directly ex vivo from human hepatocellular carcinomas (HCCs) and liver colorectal (CRC) metastases, compared with matched uninvolved liver tissue. We found that NK cells were highly prevalent in both HCC and liver CRC metastases, although at lower frequencies than unaffected liver. Up to 79% of intratumoral NK cells had the CXCR6+CD69+ liver-resident phenotype. Direct ex vivo staining showed that liver-resident NK cells had increased NKG2D expression compared to their non-resident counterparts, but both subsets had NKG2D downregulation within liver tumors compared to uninvolved liver. Proliferation of intratumoral NK cells (identified by Ki67) was selectively impaired in those with the most marked NKG2D downregulation. Human liver tumor NK cells were functionally impaired, with reduced capacity for cytotoxicity and production of cytokines, even when compared to the hypo-functional tissue-resident NK cells in unaffected liver. Coculture of human liver NK cells with the human hepatoma cell line PLC/PRF/5, or with autologous HCC, recapitulated the defects observed in NK cells extracted from tumors, with downmodulation of NKG2D, cytokine production, and target cell cytotoxicity. Transwells and conditioned media confirmed a requirement for cell contact with PLC/PRF/5 to impose NK cell inhibition. IL-15 was able to recover antitumor functionality in NK cells inhibited by in vitro exposure to HCC cell lines or extracted directly from HCC. In summary, our data suggest that the impaired antitumor function of local NK cells reflects a combination of the tolerogenic features inherent to liver-resident NK cells together with additional contact-dependent inhibition imposed by HCC itself. The demonstration that IL-15 can recover hepatic NK cell function following tumor exposure supports its inclusion in immunotherapy strategies.

Dataset Information

IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells.

Publications

IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells.

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