Chronic graft-versus-host disease suppressing CD56brightPerforinneg regulatory-like NK cells inhibit CD4+ T cells via PD-1, LAG-3, and TRAIL

Background
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 CD56brightPerforin− NK cells ((regulatory-like NK cells) (NKreg-like)) to associate with cGvHD suppression. Our further studies demonstrated that NKreg-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 NKreg-like cells can be expanded up to 300-fold while maintaining their regulatory phenotype, function, and transcriptomic characteristics.
Objective(s)
We aimed to further elucidate the mechanism of human NKreg-like cells towards inflammatory immune cells to better understand their immunoregulatory function while also determining the frequency of such cells in human blood.
Study Design
CD56brightCD16− NKreg-like cells were sterile sorted from healthy human donors, and co-cultured with cell proliferation dye stained and activated CD4+ T cells, Treg cells ± dendritic cells, B cells, or NK cells for 96 hrs to measure responder cell proliferation in the presence of NKreg-like cells (N=3-5). For proteomics analysis, supernatant from 24 hr or 20-day cultured/expanded NKreg-like cells and classic cytolytic NK cells (CD56dimCD16+ NK cells) were collected and sent to Olink Proteomics for analysis (N=6). For extracellular vesicle analysis, supernatant from NKreg-like cells expanded for 20 days underwent differential ultracentrifugation, small particle flow cytometry analysis, and nanoLC-MS/MS proteomics analysis. Lastly, NKreg-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 two-tailed T test – two-sample assuming unequal variance, or the Welch Two Sample t-test.
Results
NKreg-like cells were found to secrete several regulatory proteins, including IL-10, TGF-β, adenosine, and TRAIL, and extracellular vesicles enriched in EOMES. Further, NKreg-like cells were found to selectively suppress CD4+ T cells with no impact on B cells, NK cells, or Treg cells, mediated by the PD-1, LAG-3, and TRAIL pathways. Finally, we determined no significant difference in NKreg-like cell frequency according to age or sex demographics.
Conclusion(s)
The results of these studies contribute to our understanding of how NKreg-like cells induce a selective suppressive function to promote tolerance. Further, this data provides applications for enhancing NKreg-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.