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

Table 1 The classical mediator set and the soluble checkpointome compared

Property	Classical mediators (IDO, PGE2, nitric oxide)	Soluble checkpointome
Molecular class	Enzymes, lipid and gaseous messengers[5-7]	Immunoglobulin superfamily ligands, lectins and MHC class Ib isoforms[9-12]
Receptor engaged	No dedicated inhibitory receptor; act on metabolism or signaling tone	Defined inhibitory receptors: PD-1, TIM-3, ILT2 and ILT4[9,11,12]
Inducibility	Licensed by IFN-γ and TNF-α[5,8]	Mixed: PD-L1 and galectin-9 induced, galectin-1 constitutive[9-11]
Measured in the product	Indirectly, through kynurenine or nitrite	Directly, as protein in cell-free conditioned medium[9,11]
Precedent as a potency marker	IDO proposed and used[39]	Galectin-9 predicts preparation-level potency[16]; PD-L1 proposed alongside IDO1[39]
Suitability for a release assay	Enzymatic readouts are stable but reflect a single pathway	Multi-analyte panel, matches the assay matrix logic already advocated[14,15]

IDO: Indoleamine 2,3-dioxygenase; PGE2: Prostaglandin E2; MHC: Major histocompatibility complex; PD-1: Programmed death 1; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; ILT: Immunoglobulin-like transcript; IFN-γ: Interferon gamma; TNF-α: Tumor necrosis factor alpha; PD-L1: Programmed death ligand-1.

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.

Table 3 Membership of the Mesenchymal stromal cell soluble checkpointome and the strength of supporting evidence

Molecule	Principal receptor	Regulation in MSCs	Documented functional consequence	Evidence status
Soluble PD-L1 and PD-L2	PD-1	Induced by IFN-γ and TNF-α synergistically; further enhanced by interleukin-1 beta[9,21,22]	Neutralisation restores T-cell interleukin-2 production and PD-1 surface expression; PD-1-dependent regulatory T-cell induction[9,22]	Established; demonstrated in transwell and in vivo models. Evidence level: Human and murine in vivo plus in vitro; applies to secreted PD-L1 and PD-L2 (free forms) as distinct from vesicle-associated PD-L1 (separate row)
Galectin-9	TIM-3	Absent at rest; strongly induced by IFN-γ, TNF-α and Toll-like receptor 2, 3 and 4 ligands[11]	Knockdown removes a significant fraction of antiproliferative activity; suppresses T and B cells; mediates benefit in experimental endotoxaemia[11,16,23,24]	Established; already shown to predict preparation-level potency[16]. Evidence level: In vitro (human MSC); in vivo support in murine endotoxaemia
Galectin-1	CD45 and CD43 glycan ligands	Constitutive; released into supernatant[10]	Knockdown partially restores CD4+ and CD8+ proliferation and alters cytokine release; no effect on non-alloreactive NK cells[10]	Established; effect is population-selective. Evidence level: In vitro (human MSC)
HLA-G5	ILT2 and ILT4	Secreted isoform; full secretion requires contact with allostimulated T cells[12]	Necessary for suppression of alloproliferation and NK cytolysis and for regulatory T-cell expansion[12]	Established; antibody neutralisation abrogates suppression. Evidence level: In vitro (human MSC)
Vesicle-associated PD-L1	PD-1	Enriched on small extracellular vesicles from clinical-grade Wharton jelly MSCs[20]	Blockade or gene editing abolishes suppression of T-cell activation; rises in patient circulation after infusion[20]	Established as a vesicular rather than a freely soluble species. Evidence level: In vitro (human MSC) plus correlational human data after infusion; vesicle-associated, not freely soluble
Soluble CD25	Interleukin-2 (decoy capture)	Not characterised in MSCs	Sequesters interleukin-2 and exacerbates autoimmunity when supplied exogenously[25,26]	Candidate; MSC release unproven. Evidence level: No MSC data; in vivo evidence is from non-MSC systems
Soluble HVEM and BTLA	BTLA, CD160 and HVEM	Not characterised in MSCs	Not determined for an MSC-derived protein	Candidate; MSC release unproven. Evidence level: No MSC data
Soluble CD200 and CD276	CD200R and cognate receptors	Membrane expression reported; shedding not established	Not determined for an MSC-derived protein	Candidate; MSC release unproven. Evidence level: MSC membrane expression only; no release data
CD80/CD86	CTLA-4; CD28	CD80/CD86 expression is inconsistent among MSCs preparations, and there is no direct evidence for soluble CD80/CD86	Potentially context-dependent: CTLA-4 engagement is inhibitory, whereas CD28 engagement is costimulatory	Candidate; soluble MSC-derived forms require direct validation. Evidence level: MSC membrane expression and receptor-context data only; no MSC-derived soluble form demonstrated
CD155 (PVR)	TIGIT; CD96; CD226	Relevant to MSC immune co-signaling, but direct evidence defining an MSC-derived soluble CD155 pool and its contribution to MSC immunosuppression remains insufficient	Receptor-dependent; TIGIT-associated signaling is inhibitory, whereas CD226 can support immune activation	Candidate; MSC release and soluble-form function require validation. Evidence level: MSC membrane expression and receptor-context data only; no MSC-derived soluble form demonstrated

MSC: Mesenchymal stromal cell; PD-L1: Programmed death ligand-1; PD-1: Programmed death 1; IFN-γ: Interferon gamma; TNF-α: Tumor necrosis factor alpha; TIM-3: T cell immunoglobulin and mucin domain-containing protein 3; MSC: Mesenchymal stromal cell; NK: Natural killer; HLA: Human leukocyte antigen; ILT: Immunoglobulin-like transcript; HVEM: Herpesvirus entry mediator; BTLA: B- and T-lymphocyte attenuator; CTLA-4: Cytotoxic T-lymphocyte antigen-4; PVR: Poliovirus receptor; TIGIT: T cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domain.

Table 4 Determinants shaping the composition of the mesenchymal stromal cell soluble checkpointome and the corresponding standardisation requirement

Determinant	Reported influence	Standardisation requirement
Tissue source	Bone marrow, adipose and birth-associated MSCs differ in immunomodulatory behaviour; direct comparative data remain limited[32]	Report source explicitly; do not pool analyte reference ranges across sources
Licensing regimen	IFN-γ is the canonical licensing signal[8]; IFN-γ and TNF-α act synergistically on PD-L1[9,21]; interleukin-1 beta further enhances ligand expression and in vivo benefit[22]; licensed cells acquire a distinct potency marker profile[36]	Fix cytokine identity, concentration and exposure duration; treat licensing as part of the assay, not as pre-treatment
Culture and manufacturing	Medium composition, supplement choice and passage number alter MSC function; standardisation is a recognised prerequisite for immunotherapeutic use[37]	Define passage window and medium; qualify reagents, since biological variability of assay reagents degrades reproducibility[38]
Cryopreservation and cell state	Freezing alters functional properties; apoptotic MSCs nevertheless retain efficacy[29,30]	Report the interval between thaw and sampling; viability alone is an inadequate surrogate for potency
MSC subpopulation heterogeneity	Distinct MSC subpopulations may differ in transcriptional state, secretory activity and immunomodulatory function	Characterize cellular composition using subpopulation-resolved/single-cell approaches and determine its relationship with checkpoint output

MSC: Mesenchymal stromal cell; IFN-γ: Interferon gamma; TNF-α: Tumor necrosis factor alpha; PD-L1: Programmed death ligand-1.

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.