Project description:Cell-based immunotherapy can control bulky tumors, but effector cell persistence, tumor resistance, inconsistencies of the manufactured product, and treatment cost continue to pose challenges. To address these limitations, we developed a triple gene-edited induced pluripotent stem cell (iPSC) platform for broad patient-based adoptive cell therapy. iPSCs were engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a to augment antibody-mediated activity and a membrane-bound IL-15/IL-15R fusion (IL-15RF) protein to promote survival and maturation. The third edit was knockout of the ecto-enzyme CD38 that hydrolyzes NAD+ and is the target of the therapeutic antibody daratumumab. Natural killer (NK) cells derived from these iPSCs displayed metabolic features and gene expression profiles similar to those of adaptive NK cells that arise in response to cytomegalovirus (CMV) infection. These engineered iPSC-derived NK cells, termed iADAPT, persisted in vivo in the absence of exogenous cytokine and could be combined with daratumumab for efficient killing of multiple myeloma and acute myeloid leukemia cells both in vitro and in vivo. This strategy has broad off-the-shelf potential for the treatment of patients with advanced cancer.
Project description:Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.
Project description:Natural killer (NK) cells are a type of innate lymphocytes that play key roles in immune surveillance against tumors and viral infection. NK cells distinguish abnormal cells from healthy cells by cell-cell interaction with cell surface proteins and then attack target cells via multiple mechanisms involving TRAIL, Fas Ligand, cytokine secretion, perforin, and granzymes. In addition, extracellular vesicles (EVs), including exosomes derived from NK cells (NK-EVs), possess cytotoxic capacity against tumor cells, but their characteristics and regulation by cytokines remain unknown. Here, we report that EVs derived from human NK-92 cells stimulated with IL-15 + IL-21 show enhanced cytotoxic capacity against tumor cells in a granzyme B independent manner. In addition, small RNA-seq and mass spectrometry analyses indicate that miRNA and protein profiles in EVs are altered by cytokine stimulation. We also show NK-EVs are taken up by target cells via macropinocytosis. Collectively, our findings reveal novel characteristics of NK-EVs and the mechanism of their incorporation into target cells.
Project description:Natural killer (NK) cells are circulating lymphocytes that possess both innate and adaptive features, the latter including antigen-specific clonal expansion and long-lived memory responses. Unlike other adaptive lymphocytes like T and B cells, NK cells are not thought to require priming in lymphoid organs during activation. However, although NK cells respond in multiple tissue sites during cytomegalovirus (CMV) infection, here we observed that early activation and virus-specific expansion occurs predominantly in the spleen. These splenic NK cells exhibited heightened TNF-a signaling, which we identify as a novel and critical regulator of both innate and adaptive responses through engagement of distinct NF-kB signaling arms downstream of TNFR2. These findings highlight the central role of the spleen as a lymphoid organ in facilitating the innate-to-adaptive transition NK cells undergo during viral infection, and provide insight into how we can better generate innate and adaptive NK cell immunity across diverse settings. Bulk RNA-Seq data of cDC1, pDC, red pulp macrophages from spleen at different time points post MCMV infection
Project description:The mechanisms underlying human NK cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins following cytomegalovirus (CMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hypermethylation. Intriguingly, geneome-wide analyses revealed patterns of DNA methylation that were strikingly similar between CMV-associated adaptive NK cells and cytotoxic effector CD8+ T cells, but differed from those of canonical NK cells. A total of 23 samples were analyzed (4 sorted NK cell subsets and 2 sorted T cell subsets each from 4 individual donors). In one donor only 5 subsets were analyzed. Bisulfite-converted genomic DNA was hybridized to the Illumina Human Methylation450 BeadChip
Project description:Natural killer (NK) cells are circulating lymphocytes that possess both innate and adaptive features, the latter including antigen-specific clonal expansion and long-lived memory responses. Unlike other adaptive lymphocytes like T and B cells, NK cells are not thought to require priming in lymphoid organs during activation. However, although NK cells respond in multiple tissue sites during cytomegalovirus (CMV) infection, here we observed that early activation and virus-specific expansion occurs predominantly in the spleen. These splenic NK cells exhibited heightened TNF-a signaling, which we identify as a novel and critical regulator of both innate and adaptive responses through engagement of distinct NF-kB signaling arms downstream of TNFR2. These findings highlight the central role of the spleen as a lymphoid organ in facilitating the innate-to-adaptive transition NK cells undergo during viral infection, and provide insight into how we can better generate innate and adaptive NK cell immunity across diverse settings. Bulk RNA-Seq data of WT or TNFR2-/- Ly49H+ NK from spleen at different time points post MCMV infection.
Project description:Natural killer (NK) cells are circulating lymphocytes that possess both innate and adaptive features, the latter including antigen-specific clonal expansion and long-lived memory responses. Unlike other adaptive lymphocytes like T and B cells, NK cells are not thought to require priming in lymphoid organs during activation. However, although NK cells respond in multiple tissue sites during cytomegalovirus (CMV) infection, here we observed that early activation and virus-specific expansion occurs predominantly in the spleen. These splenic NK cells exhibited heightened TNF-a signaling, which we identify as a novel and critical regulator of both innate and adaptive responses through engagement of distinct NF-kB signaling arms downstream of TNFR2. These findings highlight the central role of the spleen as a lymphoid organ in facilitating the innate-to-adaptive transition NK cells undergo during viral infection, and provide insight into how we can better generate innate and adaptive NK cell immunity across diverse settings. Bulk RNA-Seq data of WT Ly49H+ NK from spleen or liver on day 0 and day 1 post MCMV infection.
Project description:The mechanisms underlying human NK cell phenotypic and functional heterogeneity are unknown. Here, we describe the emergence of diverse subsets of human NK cells selectively lacking expression of signaling proteins following cytomegalovirus (CMV) infection. The absence of B and myeloid cell-related signaling protein expression in these NK cell subsets correlated with promoter DNA hypermethylation. Intriguingly, geneome-wide analyses revealed patterns of DNA methylation that were strikingly similar between CMV-associated adaptive NK cells and cytotoxic effector CD8+ T cells, but differed from those of canonical NK cells.
Project description:Adaptive human natural killer (NK) cells display significantly enhanced responsiveness to a broad-range of antibody-bound targets through the engagement of CD16 compared to conventional NK cells, yet direct reactivity against tumor targets is generally reduced. Adaptive NK cells also display a distinct phenotype and differential expression of numerous genes, including reduced expression of signaling adapter FcRγ and transcription factor PLZF. However, it is unclear whether differential expression of specific genes is responsible for the characteristics of adaptive NK cells. Using CRISPR-Cas9, we show deletion of FcRγ in conventional NK cells led to enhanced CD16 responsiveness, abolished cell surface expression of natural cytotoxicity receptors, NKp46 and NKp30, and dramatically reduced responsiveness to K562 and Raji tumor cells. However, deletion of PLZF had no notable effects. These results suggest multiple roles for FcRγ and identify its deficiency as an important factor responsible for the functional and phenotypic characteristics exhibited by adaptive NK cells.
Project description:In recent years, tumor immunotherapy has produced remarkable results in tumor treatment. Nevertheless, its effects are severely limited in patients with low or absent pre-existing T cell immunity. Accordingly, metastasis remains the major cause of tumor-associated death. On the other hand, natural killer (NK) cells have the unique ability to recognize and rapidly act against tumor cells and surveil tumor cell dissemination. The role of NK cells in metastasis prevention is undisputable as an increase in the number of these cells mostly leads to a favorable prognosis. Hence, it is reasonable to consider that successful metastasis involves evasion of NK-cell-mediated immunosurveillance. Therefore, harnessing NK cells to control metastasis is promising. Circulating tumor cells (CTCs) are the seeds for distant metastasis, and the number of CTCs detected in the blood of patients with tumor is associated with a worse prognosis, whereas NK cells can eliminate highly motile CTCs especially in the blood. Here, we review the role of NK cells during metastasis, particularly the specific interactions of NK cells with CTCs, which may provide essential clues on how to harness the power of NK cells against tumor metastasis. As a result, a new way to prevent or treat metastatic tumor may be developed.