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

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.