Soluble immunosuppressive checkpointome of mesenchymal stromal cells: Immunoregulatory mechanisms and a conceptual framework for potency assessment

Table 5 Research agenda for qualifying the mesenchymal stromal cell soluble checkpointome as a potency and pharmacodynamic marker

Priority	Open question	Proposed approach	Criterion for success
Analyte definition	What fraction of measured soluble checkpoint is free protein rather than vesicle-associated?	Size-exclusion chromatography or ultracentrifugation of licensed conditioned medium before multiplex immunoassay	Reproducible partition ratio across donors and licensing conditions
Candidate membership	Do MSCs release soluble CD25, HVEM, BTLA, CD200 or CD276 in functionally relevant amounts?	Targeted proteomic interrogation of licensed and unlicensed secretome with orthogonal immunoassay confirmation and functional blockade	Detection above a pre-specified limit of quantification, with a demonstrable functional effect
Donor-level correlation	Does a composite checkpoint score correlate with independent measures of suppressive capacity?	Paired measurement of the panel and a qualified immunopotency assay across a donor panel of adequate size	Correlation demonstrated in a pre-registered analysis with correction for multiple comparisons
Pre-analytical harmonisation	How much of the observed inter-study variance is attributable to licensing and handling?	Multi-laboratory ring study using a shared protocol and a common reference batch	Inter-laboratory coefficient of variation within a pre-defined acceptance limit
Clinical qualification	Does the score predict clinical response, and does it add information beyond existing assays?	Prospective correlative study nested within an ongoing MSC trial, with pharmacodynamic sampling after infusion	Incremental predictive value over the existing release assay, assessed prospectively

MSC: Mesenchymal stromal cell; HVEM: Herpesvirus entry mediator; BTLA: B- and T-lymphocyte attenuator.