Human Regulatory-like Natural Killer Cells Associated with Chronic Graft-Versus-Host Disease Suppression Produce Regulatory Extracellular Vesicles.

Introduction Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity after Hematopoietic Stem Cell Transplantation (HCT). Previously, in large human cohorts of HCT patients we identified CD56 bright CD16 - Perforin - NK cells (NK reg -like) to be associated with the suppression of cGvHD. Functional analysis has suggested that the NK reg -like population selectively suppresses CD4 + T cells through a non-cytolytic, contact-dependent mechanism, partially reliant on PD-1, LAG-3, and TRAIL inhibitory pathways. As other groups have demonstrated NK reg -like suppressive function through various mechanisms including direct contact and soluble factors, we hypothesized that NK reg -like cells may also contribute to suppression via extracellular vesicle (EV) production. Objective We aimed to determine whether healthy donor NK reg -like cells produce EVs and identify the genes and proteins of interest for future functional analysis. Methods The supernatant of healthy donor (N=3) NK reg -like cells expanded for 20 days via complete vesicle-free Immunocult NK Cell Expansion medium supplemented with IL-2, TGF-β1 (StemCell Technologies, Vancouver, BC, Canada), and a pulse of rapamycin (Sigma Aldrich, Oakville, CA, USA) were collected, and compared to vesicle-free control medium (N=3). Large and small EVs were isolated by differential ultracentrifugation at 50,000xg for large EVs and 200,000xg for small EVs. The supernatant was sent for small particle flow cytometry (sp-FACs) analysis (Boilard lab, Quebec City). Aco490 dye was used for membrane and intracellular EV labeling. The resulting pellets after ultracentrifugation were sent for nanoLC-MS/MS proteomics analysis (IRIC, University of Montreal). Results sp-FACs analysis demonstrated that NK reg -like cells produce Aco490 + vesicles with lipid membranes sensitive to Triton and that can be pooled down by ultracentrifugation (Figure 1). Proteomics analysis identified the NK reg -like cells to produce EVs, including both large and small EVs. Proteomics analysis suggested that NK reg -like cells significantly produce the small EVs EOMES, IGKV2-30, GLUD1, IGKV2D-30, GLUD2, and the large EVs H3F3A, ACTA2, ACTA1, HIST2H3A, ACTB, ATRN (Log 2 EF > 9, p < 0.05) (Figure 2). Conclusion There is increasing evidence that EVs are involved in the function of cytolytic NK cells , and studies have demonstrated that multiple mechanisms may be responsible for NK reg -like cell function. Our studies are the first to show that the NK reg -like population associated with cGvHD suppression are substantial producers of EVs, including EOMES, which has been demonstrated to associate with GvHD inhibition by Tr1 cells. Future studies will focus on further investigating whether blockade or promotion of such EVs may significantly affect NK reg -like cell function, with the goal of harnessing these cells and/or molecules as a therapeutic target for cGvHD.

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