Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease
Bibliographic record
Abstract
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 bright Perforin − 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".