1044. Identification and Characterization of an Unconventional NK Subset in COVID-19
Authors: Platt Andrew , Lach Izabella , Strich Jeffrey R , Wigerblad Gustaf , Curto Ryan , Singireddy Shreya , Wu Jocelyn , Raja Katherine , Raja Katherine , Saharia Kapil K , Kaplan Mariana J , Chertow Daniel S 
Published: December 2022

Abstract

Background
Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16- NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset.

Methods
Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n=15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n=7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis.

Results
The CD56bright CD16- NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. Figure 1.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset in patients with severe COVID-19.
a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n=8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p&lt; 0.05, ns: not significant Figure 2.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro
a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. **: p&lt; 0.01, ***: p&lt; 0.001, ns: not significant.

Conclusion
We demonstrate and characterize a nonclassical population of CD56dim CD16- NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19.

Disclosures
All Authors: No reported disclosures.