Biomimetic vascularized adipose-derived mesenchymal stem cells bone-periosteum graft enhances angiogenesis and osteogenesis in a male rabbit spine fusion model.

Aims: Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration. Methods: A total of 24 male New Zealand white rabbits were divided into four groups according to the experimental materials. Allogenic adipose-derived mesenchymal stem cells (AMSCs) were cultured and seeded evenly in the collagen/chitosan sheet to form cell sheet as periosteum. Simultaneously, allogenic AMSCs were seeded onto alginate beads and were cultured to differentiate to endothelial-like cells to form vascularized bone construct (VBC). The cell sheet was wrapped onto VBC to create a vascularized bone-periosteum construct (VBPC). Four different experimental materials - acellular construct, VBC, non-vascularized bone-periosteum construct, and VBPC - were then implanted in bilateral L4-L5 intertransverse space. At 12 weeks post-surgery, the bone-forming capacities were determined by CT, biomechanical testing, histology, and immunohistochemistry staining analyses. Results: At 12 weeks, the VBPC group significantly increased new bone formation volume compared with the other groups. Biomechanical testing demonstrated higher torque strength in the VBPC group. Notably, the haematoxylin and eosin, Masson's trichrome, and immunohistochemistry-stained histological results revealed that VBPC promoted neovascularization and new bone formation in the spine fusion areas. Conclusion: The tissue-engineered VBPC showed great capability in promoting angiogenesis and osteogenesis in vivo. It may provide a novel approach to create a superior blood supply and nutritional environment to overcome the deficits of current artificial bone graft substitutes.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

Total intravenous anesthesia for geriatric hip fracture with severe systemic disease.

PURPOSE: Our study aimed to determine the impact of a novel technique of anesthesia administration on the clinical outcomes and complications in geriatric patients with severe systemic disease undergoing hip surgery. METHODS: We retrospectively identified patients aged > 65 years with severe systemic disease that was a constant of life [American Society of Anesthesiologists (ASA) IV] who underwent surgery for hip fracture between January 2018 and January 2020. The patients were divided into two groups: Group I [fascia iliaca compartment block plus propofol-based total intravenous anesthesia (FICB + TIVA)] and Group II [general anesthesia (GA)]. The primary outcomes were 30-day and 1-year mortality. The secondary outcomes included length of hospital stay, length of intensive care unit (ICU) stay, postoperative morbidity, Visual Analog Scale score, and consumption of analgesics. RESULTS: There was no significant difference in the 30-day mortality (5 vs. 3.8%, p = 0.85) and 1-year mortality (15 vs. 12%, p = 0.73) between the groups. Group I had significantly lower ICU requirements (p = 0.01) and shorter lengths of ICU stay (p < 0.001) and hospital stay (p < 0.001). Moreover, a smaller proportion of patients in Group I required postoperative morphine or oral opiates. CONCLUSION: Geriatric patients who underwent hip surgery under FICB + TIVA required fewer ICU admissions, shorter lengths of ICU and hospital stay, and had lesser postoperative opioid consumption than those who were under GA. Hence, we recommend the novel FICB + TIVA technique for hip fracture surgery in geriatric patients with poor general health status and high surgical risks (ASA IV).

The Use of Ultrasonic Bone Scalpel (UBS) in Unilateral Biportal Endoscopic Spine Surgery (UBESS): Technical Notes and Outcomes.

STUDY DESIGN: Case Series and Technical Note, Objective: UBS has been extensively used in open surgery. However, the use of UBS during UBESS has not been reported in the literature. The aim of this study was to describe a new spinal surgical technique using an ultrasonic bone scalpel (UBS) during unilateral biportal endoscopic spine surgery (UBESS) and to report the preliminary results of this technique. METHODS: We enrolled patients diagnosed with lumbar spinal stenosis who underwent single-level UBESS. All patients were followed up for more than 12 months. A unilateral laminotomy was performed after bilateral decompression under endoscopy. We used the UBS system after direct visualization of the target for a bone cut. We evaluated the demographic characteristics, diagnosis, operative time, and estimated blood loss of the patients. Clinical outcomes included the visual analog scale (VAS), the Oswestry Disability Index (ODI), the modified MacNab criteria, and postoperative complications. RESULTS: A total of twenty patients (five males and fifteen females) were enrolled in this study. The mean follow-up period was 13.2 months (range 12-17 months). The VAS score, ODI, and modified MacNab criteria classification improved after the surgery. A minimal mean blood loss of 22.1 mL was noted during the operation. Only one patient experienced neuropraxia, which resolved within 2 weeks. There was no durotomy, iatrogenic pars fracture, or infection. CONCLUSIONS: In conclusion, our study represents the first report of the use of UBS during UBESS. Our findings demonstrate that this technique is safe and efficient, with improved clinical outcomes and minimal complications. These preliminary results warrant further investigation through larger clinical studies with longer follow-up periods to confirm the effectiveness of this technique in the treatment of lumbar spinal stenosis.

Molecular states during acute COVID-19 reveal distinct etiologies of long-term sequelae.

Post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are debilitating, clinically heterogeneous and of unknown molecular etiology. A transcriptome-wide investigation was performed in 165 acutely infected hospitalized individuals who were followed clinically into the post-acute period. Distinct gene expression signatures of post-acute sequelae were already present in whole blood during acute infection, with innate and adaptive immune cells implicated in different symptoms. Two clusters of sequelae exhibited divergent plasma-cell-associated gene expression patterns. In one cluster, sequelae associated with higher expression of immunoglobulin-related genes in an anti-spike antibody titer-dependent manner. In the other, sequelae associated independently of these titers with lower expression of immunoglobulin-related genes, indicating lower non-specific antibody production in individuals with these sequelae. This relationship between lower total immunoglobulins and sequelae was validated in an external cohort. Altogether, multiple etiologies of post-acute sequelae were already detectable during SARS-CoV-2 infection, directly linking these sequelae with the acute host response to the virus and providing early insights into their development.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.

Does Direct Surgical Repair Benefit Pars Interarticularis Fracture? A Systematic Review and Meta-analysis.

BACKGROUND: Promising results have been shown in previous studies from direct pars interarticularis repair. These include Scott wiring, Buck repair, pedicle screw repair, and Morscher techniques. In addition, several minimally invasive techniques have been reported to show high union rates, low rates of implant failure and wound complications, shorter length of stay, a lower postoperative pain score with faster recovery, and minimal blood loss. OBJECTIVES: To compare the evidence on techniques for direct pars interarticularis repair. STUDY DESIGN: Systematic review and meta-analysis. SETTING: Review article. METHODS: We conducted a comprehensive search of databases to identify studies assessing outcomes of direct pars interarticularis defect repair. Two authors independently screened electronic search results, performed study selection, and extracted data for meta-analysis. Sensitivity and subgroup analyses were performed to assess risk of bias. RESULTS: Forty studies were included in the final analysis. Union rate was higher in the pedicle screw repair group (effect size [ES] 95%; 95% CI, 86% to 100%), followed by the Buck repair group (ES 93%; 95% CI, 86% to 98%), Scott wiring (ES 85%; 95% CI, 63% to 99%), and Morscher method group (ES 63%; 95% CI, 2% to 100%). Positive functional outcome was higher for the Morscher method (ES 91%; 95% CI, 86% to 96%), followed by the Buck repair group (ES 85%; 95% CI, 68% to 97%), pedicle screw repair (ES 84%; 95% CI, 59% to 99%) and Scott repair group (ES 80%; 95% CI, 60% to 95%). Complication rates were highest among the Scott repair group (ES 12%; 95% CI, 4% to 22%) and Morscher method group (ES 12%; 95% CI, 0% to 34%). LIMITATIONS: Heterogeneity of the included studies were noted. However, we performed sensitivity analyses from the available data to address this issue. CONCLUSION: Our results indicate that pedicle screw repair and Buck repair may be associated with a higher union rate and lower complication rates compared to the Scott repair and Morscher method. Ultimately, the choice of technique should be based on the surgeon's preference and experience.

Minimally Invasive Percutaneous Vertebroplasty for Thoracolumbar Instrumented Vertebral Fracture in Patients With Posterior Instrumentation.

BACKGROUND: The traditional treatment for an instrumented vertebral fracture involves removing the loosened pedicle screws and extending the posterior instrumentation cephaladly or caudally. There has been a recent trend of performing minimally invasive fluoroscopy-guided percutaneous vertebroplasty as a salvage procedure. OBJECTIVE: The aim of this study was to compare the outcomes of surgical interventions for instrumented vertebral fracture. STUDY DESIGN: Retrospective assessment. SETTING: All data came from Chang Gung Memorial Hospital, Taiwan. METHODS: We retrospectively reviewed 35 patients with an instrumented vertebral fracture who underwent fluoroscopy-guided percutaneous vertebroplasty (Group I, n = 16) or extension of the posterior instrumentation (Group II, n = 19). Demographic data were recorded. The operating time, amount of intraoperative blood loss, time to postoperative ambulation, and duration of hospital stay were also evaluated. The visual analog scale (VAS) score, kyphotic angle on radiological images, Kirkaldy-Willis functional score, complications, and revision surgery were evaluated at one week and one, 3, 6, and 12 months postoperatively. RESULTS: Group I had a shorter operating time (P < 0.001), less intraoperative blood loss (P < 0.001), earlier postoperative ambulation (P < 0.001), and a shorter hospital stay (P < 0.001). The mean VAS score improved significantly after surgery in both groups (P = 0.001). The postoperative kyphotic angle was better in Group II (P < 0.05). There was no significant between-group difference in the Kirkaldy-Willis functional score at the last follow-up (P = 0.91). There was no significant between-group difference in the need for revision surgery (Group I, n = 4; Group II, n = 5; P = 0.93). LIMITATION: This study is a retrospective cohort. CONCLUSIONS: Minimally invasive fluoroscopy-guided percutaneous vertebroplasty can be used as an alternative to extension of posterior instrumentation for instrumented vertebral fracture. It has several advantages, including a shorter operating time, earlier postoperative ambulation, less blood loss, and a shorter hospital stay. The clinical outcomes of these 2 treatment approaches were similar.

Acute COVID-19 gene-expression profiles show multiple etiologies of long-term sequelae.

Two years into the SARS-CoV-2 pandemic, the post-acute sequelae of infection are compounding the global health crisis. Often debilitating, these sequelae are clinically heterogeneous and of unknown molecular etiology. Here, a transcriptome-wide investigation of this new condition was performed in a large cohort of acutely infected patients followed clinically into the post-acute period. Gene expression signatures of post-acute sequelae were already present in whole blood during the acute phase of infection, with both innate and adaptive immune cells involved. Plasma cells stood out as driving at least two distinct clusters of sequelae, one largely dependent on circulating antibodies against the SARS-CoV-2 spike protein and the other antibody-independent. Altogether, multiple etiologies of post-acute sequelae were found concomitant with SARS-CoV-2 infection, directly linking the emergence of these sequelae with the host response to the virus.

Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and pathology of multiple organs in individuals under 21 years of age in the weeks following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although an autoimmune pathogenesis has been proposed, the genes, pathways and cell types causal to this new disease remain unknown. Here we perform RNA sequencing of blood from patients with MIS-C and controls to find disease-associated genes clustered in a co-expression module annotated to CD56dimCD57+ natural killer (NK) cells and exhausted CD8+ T cells. A similar transcriptome signature is replicated in an independent cohort of Kawasaki disease (KD), the related condition after which MIS-C was initially named. Probing a probabilistic causal network previously constructed from over 1,000 blood transcriptomes both validates the structure of this module and reveals nine key regulators, including TBX21, a central coordinator of exhausted CD8+ T cell differentiation. Together, this unbiased, transcriptome-wide survey implicates downregulation of NK cells and cytotoxic T cell exhaustion in the pathogenesis of MIS-C.

Molecular evidence of SARS-CoV-2 in New York before the first pandemic wave.

Numerous reports document the spread of SARS-CoV-2, but there is limited information on its introduction before the identification of a local case. This may lead to incorrect assumptions when modeling viral origins and transmission. Here, we utilize a sample pooling strategy to screen for previously undetected SARS-CoV-2 in de-identified, respiratory pathogen-negative nasopharyngeal specimens from 3,040 patients across the Mount Sinai Health System in New York. The patients had been previously evaluated for respiratory symptoms or influenza-like illness during the first 10 weeks of 2020. We identify SARS-CoV-2 RNA from specimens collected as early as 25 January 2020, and complete SARS-CoV-2 genome sequences from multiple pools of samples collected between late February and early March, documenting an increase prior to the later surge. Our results provide evidence of sporadic SARS-CoV-2 infections a full month before both the first officially documented case and emergence of New York as a COVID-19 epicenter in March 2020.

Negative feedback control of neuronal activity by microglia.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

Cytotoxic lymphocytes are dysregulated in multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) presents with fever, inflammation and multiple organ involvement in individuals under 21 years following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To identify genes, pathways and cell types driving MIS-C, we sequenced the blood transcriptomes of MIS-C cases, pediatric cases of coronavirus disease 2019, and healthy controls. We define a MIS-C transcriptional signature partially shared with the transcriptional response to SARS-CoV-2 infection and with the signature of Kawasaki disease, a clinically similar condition. By projecting the MIS-C signature onto a co-expression network, we identified disease gene modules and found genes downregulated in MIS-C clustered in a module enriched for the transcriptional signatures of exhausted CD8 + T-cells and CD56 dim CD57 + NK cells. Bayesian network analyses revealed nine key regulators of this module, including TBX21 , a central coordinator of exhausted CD8 + T-cell differentiation. Together, these findings suggest dysregulated cytotoxic lymphocyte response to SARS-Cov-2 infection in MIS-C.

Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia.

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

Multiscale causal networks identify VGF as a key regulator of Alzheimer's disease.

Though discovered over 100 years ago, the molecular foundation of sporadic Alzheimer's disease (AD) remains elusive. To better characterize the complex nature of AD, we constructed multiscale causal networks on a large human AD multi-omics dataset, integrating clinical features of AD, DNA variation, and gene- and protein-expression. These probabilistic causal models enabled detection, prioritization and replication of high-confidence master regulators of AD-associated networks, including the top predicted regulator, VGF. Overexpression of neuropeptide precursor VGF in 5xFAD mice partially rescued beta-amyloid-mediated memory impairment and neuropathology. Molecular validation of network predictions downstream of VGF was also achieved in this AD model, with significant enrichment for homologous genes identified as differentially expressed in 5xFAD brains overexpressing VGF. Our findings support a causal role for VGF in protecting against AD pathogenesis and progression.

Prognostic factors and outcomes of secondary surgery after plate fixation for midshaft clavicle fracture: Comparison of traditional DCP and pre-contoured locking plate.

OBJECTIVES: This study aimed to evaluate the prognostic factors of reoperation after plate fixation for midshaft clavicle fracture and compare outcomes of dynamic compression plates (DCP) and pre-contoured locking plates. DESIGN: Retrospective comparative study. SETTING: Level I trauma center. PATIENTS/PARTICIPANTS: We recruited 274 consecutive patients who underwent plate fixation for midshaft clavicle fracture from 2007 to 2017 and completed at least 1 year of follow-up. INTERVENTION: 235 patients underwent surgery with DCP, while 39 patients were treated with locking plate. MAIN OUTCOME MEASUREMENTS: We reviewed the possible variables of secondary surgeries, and documented complications from the medical records. Patients with a DCP or locking plate were further divided for outcome comparisons. RESULTS: 150 (54.7%) patients underwent reoperation after the initial surgery, and plate removal represented the major etiology. On multivariate analysis, superficial wound infection and prominent implant were identified as significant risk factors for reoperation, while aging, locking plate usage, and higher body mass index (BMI) were protective factors (all P < 0.05). The locking plate featured a significantly decreased implant removal rate (7.7% vs. 60.9%, P < 0.05). CONCLUSIONS: Patients with risk factors for superficial wound infection or a prominent implant tended to require a secondary operation after surgery for midshaft clavicle fracture, while aging, pre-contoured locking plate usage, and higher BMI decreased the risk. Compared with DCP, patients with locking plates tended to have better prognosis and significantly lower implant removal rates. LEVEL OF EVIDENCE: Prognostic III.

Cement Augmentation for Single-Level Osteoporotic Vertebral Compression Fracture: Comparison of Vertebroplasty With High-Viscosity Cement and Kyphoplasty.

BACKGROUND: Although the majority of available evidence suggests that vertebroplasty and kyphoplasty (KP) can relieve pain associated with vertebral compression fractures (VCFs) and improve function, evidence of clinical and radiographic outcome in highly viscous cement vertebroplasty (HVC) or KP for the treatment of VCFs is limited. The purpose of this study was to compare the clinical effects between HVC and KP in the treatment of single-level osteoporotic VCFs including radiographic and clinical outcomes. METHODS: From January 2017 to October 2018, 96 patients with single-level osteoporotic vertebral compression fracture who had undergone either KP or HVC surgery at our hospital were reviewed retrospectively, with at least 1 year follow-up. All patients were divided into the HVC group (n = 50) or the KP group (n = 46). Clinical data including clinical and radiologic evaluation results were performed pre- and postoperatively. RESULTS: The operation time of the HVC group (32.24 ± 10.08 minutes) was less than that of the KP group (40.76 ± 9.49 minutes), with significant differences. Compared with preoperative data, the visual analog scale scores, Oswestry disability index scores, vertebral body height, and local kyphotic angle were improved after surgery. There were no significant differences between the 2 groups in local kyphotic angle, vertebral body height, leakage rate of bone cement, and incidence of adjacent-level vertebra fracture. CONCLUSIONS: Restoring the vertebral height and local kyphotic angle corrections of HVC are similar with those of KP. Additionally, KP is not superior in the leakage rate of bone cement and incidence of adjacent-level vertebra fracture compared to HVC.

Introductions and early spread of SARS-CoV-2 in the New York City area.

New York City (NYC) has emerged as one of the epicenters of the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To identify the early transmission events underlying the rapid spread of the virus in the NYC metropolitan area, we sequenced the virus that causes coronavirus disease 2019 (COVID-19) in patients seeking care at the Mount Sinai Health System. Phylogenetic analysis of 84 distinct SARS-CoV-2 genomes indicates multiple, independent, but isolated introductions mainly from Europe and other parts of the United States. Moreover, we found evidence for community transmission of SARS-CoV-2 as suggested by clusters of related viruses found in patients living in different neighborhoods of the city.

Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing.

Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.