Project description:NK cells from healthy donors were isolated from peripheral blood and stimulated overnight with IL-12/15/18 to induce a memory-like phenotype. After 7 days of culture, the phenotype and degranulation potential of CIML NK cells was characterized in two distinct NK cell subsets: CD16+/CD56+ and CD16−/CD56. Finally, NK cell subsets were purified using fluorescence-activated cell sorting (FACS) and subjected to high-throughput multiplexed quantitative proteomics.
Project description:NK cells are innate lymphoid cells that protect the host against malignant and infected cells. Activation with the cytokines IL-12, IL-15, and IL-18 induces NK cells to differentiate into memory-like NK cells that have enhanced function compared to conventional NK (cNK) cells. However, mechanisms governing their biology and whether all cNK cells become memory-like are unclear. We identified that IL-12/15/18 activation results in two main fates: reprogramming into enriched memory-like (eML) or priming into effector (eff)cNK cells. eML NK cells have distinct epigenetics, phenotype, and enhanced function (IFNγ, cytotoxicity) compared to cNK and effcNK cells. In contrast, effcNK cells transcriptionally and epigenetically resemble cNK cells. Furthermore, we identify that within cNK cells CD56bright and CD56dim NK cells are the origin of distinct subsets of eML NK cells. Moreover, these two subsets of eML NK cells persist within patients receiving ML NK cell therapy for several months. Thus, IL-12/15/18 activation of NK cells results in multiple cell fates, with epigenetic and transcriptional mechanisms orchestrating eML NK cell differentiation and function. These mechanistic insights provide new strategies to enhance NK cellular therapy.
Project description:NK cells are innate lymphoid cells that protect the host against malignant and infected cells. Activation with the cytokines IL-12, IL-15, and IL-18 induces NK cells to differentiate into memory-like NK cells that have enhanced function compared to conventional NK (cNK) cells. However, mechanisms governing their biology and whether all cNK cells become memory-like are unclear. We identified that IL-12/15/18 activation results in two main fates: reprogramming into enriched memory-like (eML) or priming into effector (eff)cNK cells. eML NK cells have distinct epigenetics, phenotype, and enhanced function (IFNγ, cytotoxicity) compared to cNK and effcNK cells. In contrast, effcNK cells transcriptionally and epigenetically resemble cNK cells. Furthermore, we identify that within cNK cells CD56bright and CD56dim NK cells are the origin of distinct subsets of eML NK cells. Moreover, these two subsets of eML NK cells persist within patients receiving ML NK cell therapy for several months. Thus, IL-12/15/18 activation of NK cells results in multiple cell fates, with epigenetic and transcriptional mechanisms orchestrating eML NK cell differentiation and function. These mechanistic insights provide new strategies to enhance NK cellular therapy.
Project description:NK cells are innate lymphoid cells that protect the host against malignant and infected cells. Activation with the cytokines IL-12, IL-15, and IL-18 induces NK cells to differentiate into memory-like NK cells that have enhanced function compared to conventional NK (cNK) cells. However, mechanisms governing their biology and whether all cNK cells become memory-like are unclear. We identified that IL-12/15/18 activation results in two main fates: reprogramming into enriched memory-like (eML) or priming into effector (eff)cNK cells. eML NK cells have distinct epigenetics, phenotype, and enhanced function (IFNγ, cytotoxicity) compared to cNK and effcNK cells. In contrast, effcNK cells transcriptionally and epigenetically resemble cNK cells. Furthermore, we identify that within cNK cells CD56bright and CD56dim NK cells are the origin of distinct subsets of eML NK cells. Moreover, these two subsets of eML NK cells persist within patients receiving ML NK cell therapy for several months. Thus, IL-12/15/18 activation of NK cells results in multiple cell fates, with epigenetic and transcriptional mechanisms orchestrating eML NK cell differentiation and function. These mechanistic insights provide new strategies to enhance NK cellular therapy.
Project description:It is known that NK cells are a heterogeneous population of functionally distinct NK cell subsets. Here we report on different genomic, phenotypic and functional properties of four murine NK cell subsets distinguished by CD117 (c-kit), CD27 and CD11b expression. Gene expression was measured in NK cell subsets freshly sorted from murine C57Bl/6 splenocytes. Two to three different batches were analysed.
Project description:Cytomegalovirus (CMV) infection is ubiquitous; though often unnoticed in healthy adults it is a leading cause of neurodisability in neonates/ infants. NK and CD8 T cell cytolysis control CMV; yet their coordinate response to CMV remains uncharacterized, particularly in infants. We interrogated how NK and CD8 T cell responses differed between adults and infants in a murine CMV (MCMV) infection model. NK and CD8 T cells both limited infant MCMV infection morbidity. Notably, adult and infant NK cells effectuate discrete responses to MCMV infection with the latter preferentially forming memory associated with transcriptional distinctions prior to infection. Adoptive transfer studies further revealed that infant T cells are effector-biased but acquire the capacity to form memory in an age-dependent manner. Infant T cells' restricted ability to form memory was associated with a unique transcriptional state before and after infection. Further, acquisition of T cell memory capacity coincided with an age-dependent waning in the induction of adaptive NK cells. Our data suggests non-redundant roles for NK and CD8 T cells in MCMV infection during infancy, as NK cells compensate for the limited establishment of memory T cell subsets.
Project description:In this study we have compared the proteomic profile of subsets of extracellular vesicles (EVs) prepared from the human NK cell line NK-92 cultured for 48hrs in serum-free conditions supplemented with 10 ng/ml human recombinant IL-15. The aim was to isolate and define an EV subset with cytolytic activity against tumor cells. Fort his purpose, bulk EVs were separated according to size (via size exclusion chromatography; SEC) or density (density gradient ultracentrifugation; DG-UC).
Project description:In this study we have compared the proteomic profile of subsets of extracellular vesicles (EVs) prepared from primary human NK cells cultured for 48hrs in serum-free conditions supplemented with 10 ng/ml human recombinant IL-12, IL-15, and IL-18. The aim was to isolate and define an EV subset with cytolytic activity against tumor cells. For this purpose, bulk EVs were separated according to density (density gradient ultracentrifugation; DG-UC) to yield 3 distinct subsets.
Project description:The mechanisms underlying human natural killer (NK) cell phenotypic and functional heterogeneity are unknown. Here, we have described 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. Genome-wide DNA methylation patterns were strikingly similar between CMV-associated adaptive NK cells and cytotoxic effector T cells, but differed from those of canonical NK cells. Functional interrogation demonstrated altered cytokine responsiveness in adaptive NK cells that was linked to reduced expression of the transcription factor PLZF. Furthermore, subsets of adaptive NK cells demonstrated significantly reduced functional responses to activated autologous T cells. The present results uncover a spectrum of epigenetically unique adaptive NK cell subsets that diversify in response to viral infection and have distinct functional capabilities compared to canonical NK cell subsets.