Natural Killer Cell Dysfunction in Severe Pediatric RSV Infection

The purpose of this study was to identify natural killer (NK) cell immune signatures in patients with severe pediatric respiratory syncytial virus (RSV) by comparing NK cell characteristics from airways samples and peripheral blood and assess their association with clinical severity.The study enrolled 47 RSV-positive children admitted to the pediatric intensive care unit (PICU) at Boston Children’s Hospital between 2015 and 2022. Patients ranged in age from infancy to 2 years, with a few older children. The cohort included 15 patients who required advanced respiratory support, 17 who required noninvasive ventilation with positive pressure, and 15 who required high-flow nasal cannula. Age-matched healthy uninfected controls were enrolled during minor surgical procedures (n = 18), and uninfected ventilated controls, intubated for noninfectious reasons (n = 4), were included for comparison. Patients with immunodeficiency, malignancy, and/or chronic lung disease and/or on immunosuppressants were excluded.Peripheral blood from 37 RSV-positive patients and endotracheal aspirate samples from 10 intubated patients (moderate n = 5; severe n = 5) were collected within 24 hours of PICU admission. Flow cytometry was used to evaluate NK cell subsets, specific NK cell markers (CD69, NKG2D, NKp46), and cytotoxic function in peripheral blood samples. Functional assays included NK cell coculture with K562 target cells and confocal microscopy to evaluate NK cell–oriented cytokines and cytotoxic mediator levels. Statistical analyses included use of appropriate parametric or nonparametric tests based on normality; assessment of correlations with Pearson correlation coefficient; comparison of categorical variables using χ2 or Fisher exact tests; and evaluation of associations between clinical variables and NK cell parameters using principle component analysis and multivariable linear regression.Severe RSV infection (defined by vasopressor use, duration of mechanical ventilation duration, duration of ICU stay, and pSOFA score [assesses organ failure]) was associated with increased NK cell frequency in the airway. Circulating NK cells were decreased in RSV-positive patients, with a larger proportion presenting with increased CD69 and decreased NKG2D expression, particularly in ventilated patients. A distinct CD69+NKG2D− subpopulation was enriched in severe cases. RSV-positive patients’ NK cells exhibited impaired cytotoxicity despite increased perforin release. Disrupted immunologic synapse formation was also noted, including F-actin accumulation decrease microtubule organizing center polarization impairment. RSV-positive patients had decreased counts of circulating lymphocytes compared with healthy controls. In a comparison between steroid-treated and untreated RSV-positive patients, no significant differences were found in NK cell counts, distribution, or phenotypes.Severe RSV infection in children is associated with aberrations of NK cell regulation, characterized by an increase in airway NK cell accumulation, peripheral depletion, altered receptor expression, and impairment in cytotoxicity. These immune alterations were associated with clinical indicators of critical illness and may contribute to RSV pathogenesis. Although there have been recent advances to RSV prevention and treatment including Food and Drug Administration–approved maternal vaccines and long-acting monoclonal antibodies, challenges remain due to uncertainties in access as well as ongoing RSV surges. Continued evaluation of immune mechanisms associated with severe pediatric RSV infection, such as NK cell dysfunction, may highlight screening for risk stratification and opportunities for targeted therapies.This study highlights NK cells as potential contributors in the immunopathology of severe pediatric RSV infection. Strengths of the study include the analysis of NK cells in both airway and blood samples, prospective study design, inclusion of multiple control groups to ensure broad comparisons, and sample collections within 24 hours to best capture early, timely immune responses. Limitations of the study include limited cohort size, small functional assay sample sizes, absence of serial sampling, and inability to obtain airway samples from patients that were not intubated. Overall, this study provides insight into the possible role that alterations in NK cell expression and function may play in the severity of RSV infections in pediatric patients.