CD56 regulates human NK cell cytotoxicity through Pyk2

Human natural killer (NK) cells are defined as CD5

CD56 regulates human NK cell cytotoxicity through Pyk2

Human natural killer (NK) cells are defined as CD56+CD3. Despite its ubiquitous expression on human NK cells the role of CD56 (NCAM) in human NK cell cytotoxic function has not been defined. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis through interactions with focal adhesion kinase (FAK). Here we demonstrate that deletion of CD56 on the NK92 cell line leads to impaired cytotoxic function. CD56-knockout (KO) cells fail to polarize during immunological synapse (IS) formation and have severely impaired exocytosis of lytic granules. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 is decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 are rescued by the reintroduction of CD56. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells

Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells

From in situ to in vitro: measuring contact-dependent determinants of human natural killer cell development

Human natural killer (NK) cells are found in virtually all tissues where they act as a first line defense against malignant and virally infected cells. The development of NK cells from CD34+ hematopoietic progenitors is a complex process that involves navigating through different microenvironments and requires contact-dependent interactions with stromal cells. The molecular mediators of NK cell developmental subset trafficking, cell-cell interactions, and maturation have not been fully characterized. This thesis presents 3 studies that aim to uncover contact-dependent interactions that drive human NK cell development. Chapter 2 focuses on defining the adhesome profile of human NK cells from in vitro derived populations, tonsil, and peripheral blood. This study reveals that the tissue origin and developmental stage of NK cells influence the expression of adhesome-associated genes and proteins, as well as the content of cortical actin, which suggests a link between adhesome expression and actin regulation in NK cells. Chapter 3 presents the first comprehensive study on human NK cell development in pediatric tonsil using cyclic immunofluorescence microscopy and imaging mass cytometry. We reveal that NK cell subset localization and interactions are dependent on NK cell developmental stage and tissue residency. Chapter 4 demonstrates that neural cell adhesion molecule (NCAM) on stromal cells promotes maintenance of a mesenchymal-like state and subsequently the survival and proliferation of human NK cell precursors. Overall, this thesis provides new insights on previously unknown mediators of NK cell contact-dependent interactions and unveils the first road map of in situ NK cell development

Viral-host interactome evolution compensates for an array of host gene deletions

In order to carry out successful infections, bacteriophage lambda depends on many proteins in its host, the bacterium Escherichia coli. lambda proteins and lambda DNA interact with host molecules to facilitate infection and replication. These interactions form a molecular network known as the interactome. Viral evolution is therefore largely governed by changes that optimize the productivity of the interactome. Here, I present studies where we perturbed lambda’s interactome and allowed it to regain functionality through adaptive evolution. These studies provide insight into how viruses adapt, and more generally, how gene networks evolve. Previous studies have identified host genes used by lambda during infection. We used this knowledge in order to perturb the interactome by culturing lambda on a variety of host strains with one of these genes deleted. We proceeded to study lambda evolution to the 16 KOs and a wild type control to distinguish between specialized adaptations to specific KOs, or general adaptations to suboptimal laboratory conditions. Overall, most perturbations to the interactome could be solved with generic mutations that improve lambda growth, although, there are a few host gene deletions that require specialized mutations. Our study shows that viruses can easily adapt to perturbations in their interactomes through different adaptive solutions. While mutations with general benefits aid the correction of node deletions by providing disproportionate benefits for especially destructive deletions, some host gene deletions require specialized mutations

Viral-host interactome evolution compensates for an array of host gene deletions

In order to carry out successful infections, bacteriophage lambda depends on many proteins in its host, the bacterium Escherichia coli. lambda proteins and lambda DNA interact with host molecules to facilitate infection and replication. These interactions form a molecular network known as the interactome. Viral evolution is therefore largely governed by changes that optimize the productivity of the interactome. Here, I present studies where we perturbed lambda’s interactome and allowed it to regain functionality through adaptive evolution. These studies provide insight into how viruses adapt, and more generally, how gene networks evolve. Previous studies have identified host genes used by lambda during infection. We used this knowledge in order to perturb the interactome by culturing lambda on a variety of host strains with one of these genes deleted. We proceeded to study lambda evolution to the 16 KOs and a wild type control to distinguish between specialized adaptations to specific KOs, or general adaptations to suboptimal laboratory conditions. Overall, most perturbations to the interactome could be solved with generic mutations that improve lambda growth, although, there are a few host gene deletions that require specialized mutations. Our study shows that viruses can easily adapt to perturbations in their interactomes through different adaptive solutions. While mutations with general benefits aid the correction of node deletions by providing disproportionate benefits for especially destructive deletions, some host gene deletions require specialized mutations

Table_3_Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.xlsx

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.</p

Table_2_Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.xlsx

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.</p

Table_1_Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.xlsx

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.</p

DataSheet_1_Comparative transcriptomics of porcine liver-resident CD8αdim, liver CD8αhigh and circulating blood CD8αhigh NK cells reveals an intermediate phenotype of liver CD8αhigh NK cells.docx

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.</p