Baseline clinical, serological, and immunophenotypic characterization dataset of healthy donors

Description

Abstract

Relapse and opportunistic viral infections remain major causes of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this context, adoptive transfer of natural killer (NK) cells is a promising strategy to enhance post-transplant immune reconstitution. Adaptive NKG2C+ NK cells exhibit enhanced cytotoxicity and persistence,
but the influence of baseline donor immunophenotype on GMP manufacturing has not been systematically investigated. We evaluated whether donor NKG2A/NKG2C immunophenotypes
are associated with successful manufacture of adaptive NK-cell products. To address this, circulating NK-cell subsets from eighty-three healthy donors from the ReDoCel registry were
characterized by multiparametric flow cytometry. Donors were stratified by unsupervised hierarchical clustering according to NKG2A/NKG2C expression.
Representative donors from NKG2C- and NKG2A-dominant clusters underwent feeder-free GMP-compliant expansion using the automated CliniMACS Prodigy® platform
(Miltenyi Biotec, Bergisch Gladbach, Germany). Expanded products were evaluated for manufacturing efficiency, immunophenotype, cytotoxic function, and post-thaw stability.
Baseline NKG2C frequencies showed marked inter-donor variability, allowing identification of four immunophenotypic clusters. Only the NKG2C-dominant donor
achieved successful GMP manufacturing, exceeding the predefined expansion threshold while maintaining high viability and purity. Both NKG2A-dominant donors showed
limited proliferative capacity under identical manufacturing conditions. In addition, expanded NK cells acquired an activated phenotype characterized by increased expression of DNAM-1,
NKG2D, NKp30, NKp46, and TIM-3 while preserving mature differentiation and KIR expression. Functional analyses demonstrated potent degranulation against leukemia
targets with minimal autoreactivity, resulting in a predominantly cytotoxic effector profile. The successfully expanded product maintained viability, phenotype, and function after
long-term cryopreservation. Overall, this proof-of-concept study suggests that baseline donor NKG2A/NKG2C immunophenotype may influence GMP manufacturing of
adaptive NK-cell products. These findings support prospective evaluation of donor immunophenotyping as a biomarker for donor qualification and manufacturing optimization
to facilitate standardized off-the-shelf adaptive NK-cell therapies after allo-HSCT.