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:Natural killer (NK) cells are innate lymphocytes that play a critical role in host defense against viral infection. In addition to rapid effector cytokine production and direct cytotoxicity, NK cells exhibit features of adaptive immunity, including the capacity to undergo robust antigen-specific clonal proliferation and to generate immunological memory. However, the transcriptional programs and regulators governing dynamic NK cell responses to viral infection have not been fully uncovered. In this study, we identified Transcription factor 19 (TCF19) as a key driver of NK cell proliferation and calcium signaling in the context of mouse cytomegalovirus infection. Ablation of TCF19 was detrimental to NK cell clonal expansion and host protection against viral infection. Tcf19-/- NK cells were also unable to properly mobilize calcium downstream of antigen signaling to mediate cytotoxicity. Altogether, we find that TCF19 drives a transcriptional program that coordinates the innate and adaptive NK cell responses against viral infection.
Project description:Natural killer (NK) cells are innate lymphocytes that play a critical role in host defense against viral infection. In addition to rapid effector cytokine production and direct cytotoxicity, NK cells exhibit features of adaptive immunity, including the capacity to undergo robust antigen-specific clonal proliferation and to generate immunological memory. However, the transcriptional programs and regulators governing dynamic NK cell responses to viral infection have not been fully uncovered. In this study, we identified Transcription factor 19 (TCF19) as a key driver of NK cell proliferation and calcium signaling in the context of mouse cytomegalovirus infection. Ablation of TCF19 was detrimental to NK cell clonal expansion and host protection against viral infection. Tcf19-/- NK cells were also unable to properly mobilize calcium downstream of antigen signaling to mediate cytotoxicity. Altogether, we find that TCF19 drives a transcriptional program that coordinates the innate and adaptive NK cell responses against viral infection.