RESUMO
Manipulation of human natural killer (NK) cell repertoires promises more effective strategies for NK cell-based cancer immunotherapy. A subset of highly differentiated NK cells, termed adaptive NK cells, expands naturally in vivo in response to human cytomegalovirus (HCMV) infection, carries unique repertoires of inhibitory killer cell immunoglobulin-like receptors (KIR), and displays strong cytotoxicity against tumor cells. Here, we established a robust and scalable protocol for ex vivo generation and expansion of adaptive NK cells for cell therapy against pediatric acute lymphoblastic leukemia (ALL). Culture of polyclonal NK cells together with feeder cells expressing HLA-E, the ligand for the activating NKG2C receptor, led to selective expansion of adaptive NK cells with enhanced alloreactivity against HLA-mismatched targets. The ex vivo expanded adaptive NK cells gradually obtained a more differentiated phenotype and were specific and highly efficient killers of allogeneic pediatric T- and precursor B-cell acute lymphoblastic leukemia (ALL) blasts, previously shown to be refractory to killing by autologous NK cells and the NK-cell line NK92 currently in clinical testing. Selective expansion of NK cells that express one single inhibitory KIR for self-HLA class I would allow exploitation of the full potential of NK-cell alloreactivity in cancer immunotherapy. In summary, our data suggest that adaptive NK cells may hold utility for therapy of refractory ALL, either as a bridge to transplant or for patients that lack stem cell donors. Cancer Immunol Res; 5(8); 654-65. ©2017 AACR.
Assuntos
Imunoterapia , Células Matadoras Naturais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores KIR/imunologia , Imunidade Adaptativa , Linhagem Celular Tumoral , Terapia Baseada em Transplante de Células e Tecidos , Criança , Citomegalovirus/imunologia , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/uso terapêutico , Humanos , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores KIR/uso terapêutico , Antígenos HLA-ERESUMO
Infection by human cytomegalovirus (HCMV) leads to NKG2C-driven expansion of adaptive natural killer (NK) cells, contributing to host defense. However, approximately 4% of all humans carry a homozygous deletion of the gene that encodes NKG2C (NKG2C(-/-)). Assessment of NK cell repertoires in 60 NKG2C(-/-) donors revealed a broad range of NK cell populations displaying characteristic footprints of adaptive NK cells, including a terminally differentiated phenotype, functional reprogramming, and epigenetic remodeling of the interferon (IFN)-γ promoter. We found that both NKG2C(-) and NKG2C(+) adaptive NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Notably, CD2 co-stimulation was critical for the ability of adaptive NK cells to respond to antibody-coated target cells. These results reveal an unexpected redundancy in the human NK cell response to HCMV and suggest that CD2 provides "signal 2" in antibody-driven adaptive NK cell responses.
Assuntos
Imunidade Adaptativa/imunologia , Antígenos CD2/imunologia , Citomegalovirus/imunologia , Células Matadoras Naturais/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Interferon gama/genética , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/deficiência , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteína S6 Ribossômica/metabolismoRESUMO
OBJECTIVE: Interleukin (IL)-15 is a promising novel cytokine for natural killer (NK) cell activation and survival. We studied the effects of IL-15 compared to IL-2 on NK cells in long-term cultures for clinical translation. MATERIALS AND METHODS: CD56(+)CD3(-) NK cells were expanded with IL-2 or IL-15 for 2 to 4 weeks within lymphokine-activated killer (LAK) cell cultures (LAK-NK) in serum-enriched AIM V or CellGro Stem Cell Growth Medium (SCGM). Cell growth, viability, and NK cell content were monitored and cytotoxicity assessed in a flow cytometric cytotoxicity assay. RESULTS: IL-15 (100-1000 U/mL) could replace IL-2 (1000 U/mL) in AIM V cultures to achieve efficient LAK cell expansion. However, IL-15-stimulated LAK cells exceeded cytotoxicity of IL-2-stimulated LAK cells against K562, notably at later culture points. In the powerful CellGro SCGM, LAK cells expanded over 28 days an average of 905-fold ± 320-fold standard error of the mean (SEM) for IL-2 (500 U/mL) and 484-fold ± 98-fold SEM for IL-15 (500 U/mL), and NK cells within such LAK cultures expanded an average of 2320-fold ± 975-fold SEM for IL-2 and 1084-fold ± 309-fold SEM for IL-15. Importantly, such IL-15-activated LAK-NK cells retained enhanced cytotoxicity at later culture points against K562 as well. IL-15-stimulated effectors were also highly cytotoxic against hematological targets MOLT-4 and KU812 and nontoxic against autologous nonmalignant cells. Interestingly, IL-15-LAK-NK cells showed overall significant upregulation of the main activating and inhibitory NK cell receptors after long-term cytokine stimulation. CONCLUSIONS: Our results demonstrate the potential for IL-15 to support large-scale expansion of clinical-grade LAK-NK effectors, which could retain enhanced longer-term potency and preserve activation receptors in therapy of hematological malignancies. Protocols are readily clinically translatable.