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

Table 2 Routes of generation of soluble immune checkpoint proteins and their analytical implications

Route	Molecular mechanism	Effect on surface pool	Representative evidence	Implication for assay design
Ectodomain shedding	Cleavage of the juxtamembrane stalk by ADAM10, ADAM17 or matrix metalloproteinases	Reduced	ADAM10 and ADAM17 cleave PD-L1 from tumour cells and vesicles, yielding a fragment that induces CD8+ T-cell apoptosis[19] (note: This evidence derives from tumour-cell and non-MSC systems and is therefore indirect for MSC biology)	Surface staining alone underestimates total checkpoint output; sheddase activity should be reported alongside the analyte
Alternative splicing	Transcript lacking the transmembrane exon, secreted constitutively	Unchanged	HLA-G5 secretion by MSCs is required for T-cell and NK-cell suppression and for regulatory T-cell expansion[12]	Secretion may proceed independently of protease inhibitors, so pharmacological blockade of sheddases is not a valid specificity control
Vesicle association	Checkpoint retained in the vesicle membrane	Redistributed	Wharton jelly MSC vesicles are enriched in PD-L1; blockade or gene editing abolishes their suppressive effect[20]	Requires ultracentrifugation or size-exclusion fractionation before immunoassay if free and vesicle-bound pools are to be distinguished

ADAM: A disintegrin and metalloproteinase; PD-L1: Programmed death ligand-1; MSC: Mesenchymal stromal cell; HLA: Human leukocyte antigen; NK: Natural killer.