Journal: Frontiers in Cell and Developmental Biology

Article Title: Prolonged TNF-α stimulation induces a PD-1–associated exhaustion-like phenotype in mesenchymal stromal cells

doi: 10.3389/fcell.2026.1680076

Figure Lengend Snippet: Immunomodulatory exhaustion-like phenotype of MSCs under prolonged TNF-α stimulation. (A) Schematic representation of indirect co-culture system. MSCs were repeatedly exposed to inflammatory stimulation by transferring them to freshly polarized M1 macrophages every 12 h using Transwell inserts. (B) Time-course analysis of immunoregulatory gene expression ( Tgf-β , Il-10 , and Fasl ) in MSCs during co-culture. Tgf-β expression peaked at 12 h, while Il-10 and Fasl were transiently upregulated and then declined. (C) Progressive increase in the expression of immune checkpoint genes ( Pd-1 and Ctla-4 ) over time. (D) Representative immunofluorescence images showing co-expression of PDGFRα and PD-1 in MSCs after 48 h of co-culture with M1 macrophages (Scale bar = 20 µm). Images are representative of independent experiments and are shown to illustrate the spatial distribution and co-expression of PDGFRα and PD-1, rather than serving as quantitative evidence. (E) Increased Tnf-α and iNos expression in the bone marrow following intraperitoneal injection of LPS-PG (5 mg/kg) for 7 days. (F) Immunohistochemistry of bone marrow showing decreased PDGFRα + TGF-β + cells and increased PDGFRα + PD-1 + cells in LPS-treated mice compared to untreated controls (Scale bar = 100 µm). Quantitative analysis was performed at this magnification, which allowed for the reliable identification of PD-1–positive MSC-like cells. Data represent mean ± SD of independent biological replicates derived from separately prepared MSC cultures (n = 4–5). One-way ANOVA with Tukey’s post-hoc test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To assess the role of TNF-α secreted by M1 macrophages, a TNF-α neutralizing antibody (Cell Signaling Technology, MA, United States) was added to the MSC culture medium at a final concentration of 1 μg/mL during co-culture experiment.

Techniques: Co-Culture Assay, Transferring, Gene Expression, Expressing, Immunofluorescence, Injection, Immunohistochemistry, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Prolonged TNF-α stimulation induces a PD-1–associated exhaustion-like phenotype in mesenchymal stromal cells

doi: 10.3389/fcell.2026.1680076

Figure Lengend Snippet: MSC dysfunction persists after withdrawal of TNF-α stimulation. (A) Schematic of the withdrawal experiment: MSCs were stimulated with TNF-α for 36 h, followed by culture in TNF-α–free medium. (B) Expression of Tgf-β, Il-10, and Fasl remained suppressed after TNF-α withdrawal. (C) Pd-1 and Ctla-4 expression declined slightly but did not return to baseline. (D) Expression of TNF-α signaling genes (Tradd, Ikkα, and Nf-κb) decreased gradually but remained elevated. (E) Apoptosis-related genes ( Casp3 and Bax ) remained upregulated, indicating sustained cellular stress associated with persistent MSC dysfunction. Data represent mean ± SD of independent biological replicates derived from separately prepared MSC cultures (n = 4−5). One-way ANOVA with Tukey’s post-hoc test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To assess the role of TNF-α secreted by M1 macrophages, a TNF-α neutralizing antibody (Cell Signaling Technology, MA, United States) was added to the MSC culture medium at a final concentration of 1 μg/mL during co-culture experiment.

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Prolonged TNF-α stimulation induces a PD-1–associated exhaustion-like phenotype in mesenchymal stromal cells

doi: 10.3389/fcell.2026.1680076

Figure Lengend Snippet: TNF-α neutralization partially restores immunoregulatory gene expression in MSCs. (A) Expression of Tgf-β , Il-10 , and Fasl in MSCs co-cultured with M1 macrophages for 12 and 48 h, with or without TNF-α neutralizing antibody. Neutralization inhibited early upregulation (12 h) and prevented the subsequent downregulation observed at 48 h. (B) TNF-α neutralization suppressed the late-phase induction of Pd-1 and Ctla-4 . (C) Expression of TNF-α signaling-related genes ( Tradd , Ikkα , and Nf-κb ) was also reduced after antibody treatment. Data represent mean ± SD of independent biological replicates derived from separately prepared MSC cultures (n = 4–5). One-way ANOVA with Tukey’s post-hoc test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To assess the role of TNF-α secreted by M1 macrophages, a TNF-α neutralizing antibody (Cell Signaling Technology, MA, United States) was added to the MSC culture medium at a final concentration of 1 μg/mL during co-culture experiment.

Techniques: Neutralization, Gene Expression, Expressing, Cell Culture, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Prolonged TNF-α stimulation induces a PD-1–associated exhaustion-like phenotype in mesenchymal stromal cells

doi: 10.3389/fcell.2026.1680076

Figure Lengend Snippet: Prolonged TNF-α stimulation induces an exhaustion-like phenotype in MSCs. (A) Time-course expression of Tgf-β , Il-10 , and Fasl in MSCs stimulated with TNF-α (10 ng/mL) for up to 48 h. An initial upregulation at the indicated time points was observed, followed by significant downregulation. (B) Immune checkpoint genes ( Pd-1 and Ctla-4 ) were progressively upregulated over time. (C) Immunofluorescence revealed decreased numbers of TGF-β + and IL-10 + MSCs and increased PD-1 + MSCs with prolonged TNF-α stimulation (Scale bar = 20 µm). (D) Expression of Tradd , Ikkα , and Nf-κb remained elevated throughout stimulation. (E) Western blot analysis showed sustained phosphorylation of p65 in MSCs treated with TNF-α. Quantification was performed by densitometric analysis of independent biological replicates (n = 3), each derived from separately prepared MSC cultures. (F) Prolonged TNF-α stimulation led to an increased number of ROS-positive MSCs, indicating elevated intracellular oxidative stress (Scale bar = 100 µm). ROS positivity was defined using a fixed fluorescence intensity threshold, and all samples were processed in parallel, under identical acquisition settings. ROS data are presented as supportive evidence rather than definitive mechanistic proof. Data represent mean ± SD of independent biological replicates derived from separately prepared MSC cultures (n = 4–5). One-way ANOVA with Tukey’s post-hoc test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To assess the role of TNF-α secreted by M1 macrophages, a TNF-α neutralizing antibody (Cell Signaling Technology, MA, United States) was added to the MSC culture medium at a final concentration of 1 μg/mL during co-culture experiment.

Techniques: Expressing, Immunofluorescence, Western Blot, Phospho-proteomics, Derivative Assay, Fluorescence