Journal: Molecular Therapy. Nucleic Acids

Article Title: IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism

doi: 10.1016/j.omtn.2023.02.016

Figure Lengend Snippet: IL-10 modRNA transcript produces functional protein in vitro (A–D) IL-10 modRNA produced IL-10 protein in mouse embryonic fibroblasts (MEFs), C2C12 mouse muscle myoblasts, human embryonic kidney 293 (HEK293) cells, and Jurkat cells (human T cells) at different time points by IL-10 ELISA. MEF (2 × 10 4 ), C2C12 (1 × 10 5 ), and 293 cells (1 × 10 5 ) were transfected with 100 ng modRNA. Jurkat cells (5 × 10 5 ) were transfected with 200 ng modRNA. All transfection experiments were performed in 96-well flat plate using Lipofectamine 3000 reagent. (E) Jurkat cells expressed IL-10 protein when transfected with IL-10 modRNA. Jurkat cells (12 × 10 6 ) were transfected without or with 5,000 ng IL-10 modRNA in 6-well plate. Cells were harvested one day after transfection, and 20 μg protein extract was analyzed using western blot assay. β-Actin was used as loading control. (F and G) Jurkat cells treated with IL-10 modRNA secreted functional IL-10 protein, which inhibited IL-2 and IFN-γ production in vitro . Jurkat cells (3 × 10 5 cells per well) transfected with 200 ng IL-10 modRNA were seeded in the top chambers of Transwell 96-well plates. Splenocytes (SPL; 3 × 10 5 cells per well) were seeded in bottom reservoir and stimulated with coated anti-CD3 (2 μg/mL) and soluble anti-CD28 (2 μg/mL). Supernatants in bottom reservoir were collected at different time points and analyzed using IL-2 and IFN-γ ELISA. Statistical data are represented as mean ± SD. The IL-10 modRNA group was compared with the buffer group at different time points (∗∗p < 0.005 and ∗∗∗p < 0.0005, Student’s t test).

Article Snippet: Primary antibodies were used against CD3 (SP7 clone; dilution 1:100; GeneTex) or FoxP3 (FJK-16 s clone; dilution 1:200; Thermo Fisher Scientific for 60 min.

Techniques: Functional Assay, In Vitro, Produced, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: IL-10 modified mRNA monotherapy prolongs survival after composite facial allografting through the induction of mixed chimerism

doi: 10.1016/j.omtn.2023.02.016

Figure Lengend Snippet: Degree of lymphocytic infiltration into facial allograft (A) Macroscopic and histological changes (indicated by hematoxylin and eosin staining) in facial OMC allografts of IL-10 modRNA and buffer groups on PODs 10–14. Facial OMC allografts from allotransplanted mice receiving single 10 μg dose of IL-10 modRNA or saline buffer on POD 3 were analyzed. Infiltrating lymphocytes into skin and muscle layers of facial OMC allografts are shown by dense violet staining. Histological Banff classification of facial OMC allografts between buffer and IL-10 modRNA groups is as shown in the right of (A). Each group contains four mice. Scale bars, 50 μm. (B) Immunohistochemistry in facial OMC allografts of IL-10 modRNA and buffer groups on PODs 10–14. Infiltrating CD3 + T cells or FoxP3 + cells into skin and muscle layers of facial OMC allografts are shown by red staining. Because CD3 is expressed in the cell surface of T cells, the staining displays a circle red. FoxP3 is a critical transcription factor and marker for mouse CD4 + CD25 + natural Tregs, and the staining displays a dense red. Scale bars, 50 μm.

Article Snippet: Primary antibodies were used against CD3 (SP7 clone; dilution 1:100; GeneTex) or FoxP3 (FJK-16 s clone; dilution 1:200; Thermo Fisher Scientific for 60 min.

Techniques: Staining, Saline, Immunohistochemistry, Marker