Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease

Background: Chronic graft-versus-host disease (cGvHD) is a major cause of morbidity and mortality after Hematopoietic Stem Cell Transplantation (HSCT). Previously, in large patient cohorts, we identified increased numbers of CD56^brightPerforin⁻ regulatory-like NK cells (NK_reg-like) associated with cGvHD suppression. Thus, we hypothesized that NK_reg-like cells may be a potential candidate for cGvHD cell therapy. Aim: To expand NK_reg-like cells while maintaining regulatory phenotype and function. Methods: Total NK cells were first expanded with IL-2, which was then combined with rapamycin, Transforming Growth Factor Beta 1 (TGF-β1), NECA (Adenosine A2A receptor (A2AR) agonist), metformin, or dexamethasone, to prevent change in cell phenotype/function. The functional characteristics were evaluated via T cell suppression assays and the phenotype was measured using flow cytometry. The optimal expansion protocol was compared in terms of function and metabolism for three NK expansion media, and cells from cord vs. peripheral blood. Further, expanded NK_reg-like cell gene expression was characterized using bulk RNA sequencing. Finally, NK_reg-like cells were differentiated from CD34⁺ hematopoietic stem and progenitor cells (HSPCs) and compared in terms of proliferation and function. Results: The expansion of total NK cells found the addition of TGF-β1 and/or NECA with the pulsing of rapamycin in IL-2 containing media to prevent NK_reg-like differentiation (up to 200-fold expansion). Expanded NK_reg-like cells maintained a phenotype, transcriptome, and T cell suppression similar to freshly isolated NK_reg-like cells. NK_reg-like expansion was greatest in the Immunocult media (up to 300-fold), and NK_reg-like cells from peripheral blood demonstrated significantly greater proliferation than cells isolated from cord blood (65-fold). The metabolic profile of NK_reg-like and cytolytic NK cells appeared similar at baseline, though rapamycin induced a shift to oxidative over glycolytic metabolism. Further, we demonstrated that suppressive NK_reg-like cells may alternatively be expanded from CD34⁺ cells isolated from cord blood, reaching an average 340-fold expansion. Conclusions: In conclusion, our studies have optimized two alternative expansion approaches for deriving functional NK_reg-like cells. Additionally, evaluating the transcriptomic and metabolic characteristics provides useful information regarding NK_reg-like cell function and differentiation. With further optimization and in vivo validation, we may work towards preparing these cells as a therapy for cGvHD.