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MSC‐EVs suppress the migration of activated T cells. (a) RT‐PCR assays for chemokine mRNA levels with the left eyes of mice on day 14 after CFA only or IRBP immunization. (b), (c) The top wells were loaded with IRBP reactive T cells (2.5 × 10 5 ) from EAU mice with or without EVs (3 × 10 9 particles/mL) and the lower chambers contained recombinant mouse CXCL9 (50–200 ng/mL) or IRBP peptides (250–1000 ng/mL). After 3‐h incubation at 37°C, the cells that migrated to the lower chamber were counted (b). After 6 h, the cells that migrated to the lower chamber were counted (c). * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; by one‐way ANOVA with Tukey's multiple comparison tests. (d), (f) The top wells were loaded with anti‐CD3/CD28 microbead‐stimulated T cells (2.5 × 10 5 ) from naïve C57BL/6 mice and the lower chambers contained recombinant mouse CCL2 or CCL19 (20 ng/mL). After 3‐ or 6‐h incubation at 37°C, the cells that migrated to the lower chamber were counted. *** p < 0.005; **** p < 0.001; by two‐way ANOVA. The top wells were loaded with IRBP‐specific T cells isolated from C57BL/6 mice with EAU (f) or B10 RIII mice with EAU (g) with or without MSC‐EVs (1.5–3 × 10 9 particles/mL) and the cells that migrated to the lower chamber were counted after 3 h. *** p < 0.005; by one‐way ANOVA with Dunnett's multiple comparison tests.

Journal: Journal of Extracellular Vesicles

Article Title: Biopotency and surrogate assays to validate the immunomodulatory potency of extracellular vesicles derived from mesenchymal stem/stromal cells for the treatment of experimental autoimmune uveitis

doi: 10.1002/jev2.12497

Figure Lengend Snippet: MSC‐EVs suppress the migration of activated T cells. (a) RT‐PCR assays for chemokine mRNA levels with the left eyes of mice on day 14 after CFA only or IRBP immunization. (b), (c) The top wells were loaded with IRBP reactive T cells (2.5 × 10 5 ) from EAU mice with or without EVs (3 × 10 9 particles/mL) and the lower chambers contained recombinant mouse CXCL9 (50–200 ng/mL) or IRBP peptides (250–1000 ng/mL). After 3‐h incubation at 37°C, the cells that migrated to the lower chamber were counted (b). After 6 h, the cells that migrated to the lower chamber were counted (c). * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; by one‐way ANOVA with Tukey's multiple comparison tests. (d), (f) The top wells were loaded with anti‐CD3/CD28 microbead‐stimulated T cells (2.5 × 10 5 ) from naïve C57BL/6 mice and the lower chambers contained recombinant mouse CCL2 or CCL19 (20 ng/mL). After 3‐ or 6‐h incubation at 37°C, the cells that migrated to the lower chamber were counted. *** p < 0.005; **** p < 0.001; by two‐way ANOVA. The top wells were loaded with IRBP‐specific T cells isolated from C57BL/6 mice with EAU (f) or B10 RIII mice with EAU (g) with or without MSC‐EVs (1.5–3 × 10 9 particles/mL) and the cells that migrated to the lower chamber were counted after 3 h. *** p < 0.005; by one‐way ANOVA with Dunnett's multiple comparison tests.

Article Snippet: The top wells were loaded with stimulated T cells (2.5 × 10 5 ) from naïve C57BL/6 mice or EAU mice and the lower chambers contained recombinant mouse CCL2 (20 ng/mL, R&D systems), CXCL9 (50–200 ng/mL, R&D systems), IRBP (250–1000 ng/mL) or CCL19 (20 ng/mL, R&D systems).

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Recombinant, Incubation, Comparison, Isolation