Long-lived “memory-like” NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have ...shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.
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•NK cells with multiple protein deficiencies are present in HCMV-infected individuals•SYK deficiency is associated with hyper-methylation of the gene promoter•Memory-like NK cells have protein deficiencies in combination with FcRγ deficiency•FcRγ-deficient NK cells expand preferentially in an antibody-dependent manner
Long-lived “memory-like” NK cells have been identified in HCMV-infected individuals at variable frequencies, but little is known about how this NK cell pool is formed. Kim and colleagues show data that support epigenetic modifications and antibody-dependent expansion as mechanisms underlying the formation of this memory-like NK cell pool.
Because NK cells lack gene-recombination machinery and are thought to be relatively short-lived, it is unclear whether NK cells can mount long-term effective recall responses to reinfections by ...diverse pathogens. In this article, we report that FcRγ-deficient NK cells, which we recently identified and termed g(-)NK cells, possess distinct memory features directed by FcR-mediated Ab-dependent target recognition. The presence of g(-)NK cells was associated with prior human CMV (HMCV) infection, yet g(-)NK cell responses were not restricted to HCMV-infected target cells. In the presence of virus-specific Abs, g(-)NK cells had greatly enhanced functional capabilities, superior to conventional NK cells, and were highly responsive to cells infected with either HCMV or HSV-1. Remarkably, the g(-)NK cell subset persisted long-term at nearly constant levels in healthy individuals. Therefore, FcRγ deficiency distinguishes an Ab-dependent memory-like NK cell subset with enhanced potential for broad antiviral responses.
During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms ...leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.
NK cells respond to tumor and virus-infected cells directly through several activation receptors, including natural cytotoxicity receptors, or indirectly through the activating Fc receptor CD16 for ...antibody-coated cells. Triggering of NK-cell effector functions through these receptors depends on physically associated transmembrane signaling adaptors, such as FcRγ (also known as FcεRIγ) and CD3ζ, both of which have been traditionally believed to be expressed by all mature NK cells. However, we have identified a distinct subset of human NK cells that are deficient for FcRγ expression but express normal levels of CD3ζ. FcRγ-deficient NK cells were readily detectable in about one-third of the healthy individuals examined. The deficiency was confined to the CD56(dim) population and was due to low FcRγ mRNA. FcRγ-deficient NK cells displayed dramatically reduced expression of the natural cytotoxicity receptors NKp46 and NKp30 but still expressed substantial levels of CD16. Compared to FcRγ-expressing NK cells, FcRγ-deficient NK cells showed poor direct reactivity toward tumor targets as measured by cytokine production and degranulation. Unexpectedly, however, FcRγ-deficient NK cells exhibited significantly more robust responsiveness upon stimulation through CD16, particularly for cytokine production, compared to FcRγ-expressing NK cells. Thus, our study reveals FcRγ-deficient NK cells as a novel subset of human NK cells that have remarkably potent responses toward antibody-coated targets. These findings also illustrate a differential contribution of FcRγ and CD3ζ for the expression and functional activity of their associated receptors.
Background and Aims
Upper GI bleeding (UGIB) is a common indication for inpatient esophagogastroduodenoscopy (EGD). Guideline adherence improves post-EGD care, including appropriate medication ...dosing/duration and follow-up procedures that reduce UGIB-related morbidity. We aimed to optimize and standardize post-EGD documentation to improve process and clinical outcomes in UGIB-related care.
Methods
We performed a prospective quality improvement study of inpatient UGIB endoscopies at an academic tertiary referral center during 6/2019–7/2021. Guidelines were used to develop etiology/severity-specific electronic health record note templates. Participants (39 faculty/15 trainees) completed 10-min training in template content/use. We collected pre/post-intervention process data on “Minimal Standard Report” (MSR) documentation including patient disposition, diet, and medications. We also recorded documentation of re-bleed precautions and follow-up procedures. Study outcomes included guideline-based medication prescriptions, ordering of follow-up EGD, and post-discharge re-bleeding. Pre/post-intervention analysis was performed using chi-square tests.
Results
From a pre-intervention baseline of 199 patients to 459 patients post-intervention, compliance improved with inpatient PPI (53.4–77.9%,
p
< 0.001) and discharge PPI (31.3–61.0%,
p
< 0.001) prescriptions. There was improvement in MSR completion (28.6–42.5%,
p
< 0.001). Compliance improved with octreotide prescriptions (75.0–93.6%,
p
= 0.002) and follow-up EGD order (61.3–87.1%,
p
< 0.001). There was no change in post-discharge re-bleeding. 82.6% of cases used templates.
Conclusions
Our project leveraged endoscopy software to standardize documentation, resulting in improved clinical care behavior and efficiency. Our intervention required low burden of maintenance, and sustainability with high utilization over 9 months. Similar endoscopy templates can be applied to other health systems and procedures to improve care.
"Adaptive" NK cells, characterized by FcRγ deficiency and enhanced responsiveness to Ab-bound, virus-infected cells, have been found in certain hCMV-seropositive individuals. Because humans are ...exposed to numerous microbes and environmental agents, specific relationships between hCMV and FcRγ-deficient NK cells (also known as g-NK cells) have been challenging to define. Here, we show that a subgroup of rhesus CMV (RhCMV)-seropositive macaques possesses FcRγ-deficient NK cells that stably persist and display a phenotype resembling human FcRγ-deficient NK cells. Moreover, these macaque NK cells resembled human FcRγ-deficient NK cells with respect to functional characteristics, including enhanced responsiveness to RhCMV-infected target in an Ab-dependent manner and hyporesponsiveness to tumor and cytokine stimulation. These cells were not detected in specific pathogen-free (SPF) macaques free of RhCMV and six other viruses; however, experimental infection of SPF animals with RhCMV strain UCD59, but not RhCMV strain 68-1 or SIV, led to induction of FcRγ-deficient NK cells. In non-SPF macaques, coinfection by RhCMV with other common viruses was associated with higher frequencies of FcRγ-deficient NK cells. These results support a causal role for specific CMV strain(s) in the induction of FcRγ-deficient NK cells and suggest that coinfection by other viruses further expands this memory-like NK cell pool.
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.
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•FcRγ deletion leads to increased cytokine production in response to CD16 stimulation•FcRγ deletion abolishes cell surface expression of NKp46 and NKp30•FcRγ deletion results in reduced responsiveness to K562 and Raji cells•PLZF deletion does not change responsiveness to CD16 and cytokine stimulation
Molecular Biology; Immunology; Cancer
Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide ...binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the
S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only ∼30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.
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