Single-cell profiling identifies a CD8bright CD244bright Natural Killer cell subset that reflects disease activity in HLA-A29-positive birdshot chorioretinopathy

Birdshot chorioretinopathy uveitis (BCR-UV) is strongly associated with HLA-A29 which implicates MHC-I pathway mediated perturbation of natural killer (NK) cells as a potential disease mechanism. We profiled blood NK cells at single-cell resolution in a cohort of patients and healthy controls and investigated the links between NK cell subpopulations and disease activity. Flow cytometry analysis of major immune cell lineages revealed substantial expansion of the CD56dim CD16+ NK cells in BCR-UV compared to healthy controls and to other types of non-infectious uveitis. Ex vivo restimulation showed that NK cells from BCR-UV patients exhibit increased secretion of TNF-alpha, a cytokine considered central to the pathogenesis of BCR-UV. Unbiased transcriptomic characterization at single-cell resolution established that the expanded CD16+ (i.e., FCGR3A+ ) NK cells also co-express high levels of CD8A and CD244 , indicating expansion of a subset of CD56dim CD16+ CD8+ NK cells in patients. Confirmation of these results by high-dimensional flow cytometry further showed that the BCR-UV-associated CD8bright CD244bright NK cells displayed activation receptors including CD314 (NKG2D), and cytotoxicity receptor CD337 (NKp30). Finally, longitudinal monitoring of patients showed that clinical remission after systemic immunomodulatory treatment correlated with a significant decrease in CD8bright CD244bright NK cells. In conclusion, there is an expansion of CD8bright CD244bright NK cells during active disease in BCR-UV patients which decrease upon successful systemic immunomodulatory treatment, suggesting that CD8bright/CD244bright NK cells may be a pro-inflammatory NK subset involved in the underlying disease mechanism. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT02656381 ### Funding Statement This study was funded by the NEI intramural research program (project number 1ZIAEY000556-04) and made possible by the Lasker Clinical Research Grant to HNS, and the Vision Grant from Prevention of Blindness Society of Metropolitan Washington to PRN. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was conducted in compliance with the Declaration of Helsinki and ethical principles regarding human experimentation. All samples were obtained under a National Institutes of Health (NIH) Institutional Review Board (IRB) approved protocol (Uveitis/Intraocular Inflammatory Disease Biobank (iBank); [NCT02656381][1]). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. * BCR-UV : birdshot chorioretinopathy uveitis; NIU : non-infectious uveitis; KIR : killer cell immunoglobulin-like receptors; scRNAseq : single-cell RNA sequencing; MFI : mean fluorescent intensity; SUN : standardization of uveitis nomenclature. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02656381&atom=%2Fmedrxiv%2Fearly%2F2022%2F09%2F15%2F2022.09.11.22279821.atom