Chronic graft-versus-host disease suppressing CD56^brightPerforin^neg regulatory-like NK cells inhibit CD4⁺ T cells via PD-1, LAG-3, and TRAIL.

Abstract

Chronic graft-versus-host disease (cGvHD) is a significant cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Previously, in large human cohorts of HSCT patients we identified increased numbers of CD56^brightPerforin^- NK cells ([regulatory-like NK cells] [NK_reg-like]) to associate with cGvHD suppression. Our further studies demonstrated that NK_reg-like cells can be characterized according to a unique phenotype and transcriptome, distinct from classic cytolytic NK cells, and can induce immune tolerance by selectively suppressing CD4⁺ T cells through a non-cytolytic, contact-dependent mechanism. Further, we demonstrated that the NK_reg-like cells can be expanded up to 300-fold while maintaining their regulatory phenotype, function, and transcriptomic characteristics. We aimed to further elucidate the mechanism of human NK_reg-like cells towards inflammatory immune cells to better understand their immunoregulatory function while also determining the frequency of such cells in human blood. CD56^brightCD16^- NK_reg-like cells were sterile sorted from healthy human donors, and co-cultured with cell proliferation dye stained and activated CD4⁺ T cells, T_reg cells ± dendritic cells, B cells, or NK cells for 96 hours to measure responder cell proliferation in the presence of NK_reg-like cells (N = 3-5). For proteomics analysis, supernatant from 24 hr or 20-day cultured/expanded NK_reg-like cells and classic cytolytic NK cells (CD56^dimCD16⁺ NK cells) were collected and sent to Olink Proteomics for analysis (N=6). For extracellular vesicle analysis, supernatant from NK_reg-like cells expanded for 20 days underwent differential ultracentrifugation, small particle flow cytometry analysis, and nanoLC-MS/MS proteomics analysis. Lastly, NK_reg-like cell frequency was measured via FACS cell sorting analysis according to sex and age demographics (N=20). Statistical analyses were performed using Microsoft Excel version 2110 and a 2-tailed T test - 2-sample assuming unequal variance, or the Welch 2 Sample t-test. NK_reg-like cells were found to secrete several regulatory proteins, including IL-10, TGF-β, adenosine, and TRAIL, and extracellular vesicles enriched in EOMES. Further, NK_reg-like cells were found to selectively suppress CD4⁺ T cells with no impact on B cells, NK cells, or T_reg cells, mediated by the PD-1, LAG-3, and TRAIL pathways. Finally, we determined no significant difference in NK_reg-like cell frequency according to age or sex demographics. The results of these studies contribute to our understanding of how NK_reg-like cells induce a selective suppressive function to promote tolerance. Further, this data provides applications for enhancing NK_reg-like cell function, such as through increasing PD-1, LAG-3, TRAILR ligand, and/or EOMES expression/secretion, and the ability to obtain consistent cell numbers from donors, regardless of age and sex.