Journal: Science Advances

Article Title: Skin and heart allograft rejection solely by long-lived alloreactive T RM cells in skin of severe combined immunodeficient mice

doi: 10.1126/sciadv.abk0270

Figure Lengend Snippet: ( A ) Flow cytometric analysis of cytokines IL-17a and IL-17f in CD4 + T RM cells in skin grafts in SCID mice that received a piece of MHC-matched, immunized BALB/c skin tissue during the secondary allograft rejection. The percentages of IL-17a + or IL-17f + in CD4 + CD69 + T RM cells and the cell number of these cells in skin are shown. ( B ) mRNA expressions of IL-17A, IL-17F, and IL-22 in skin grafts in the corn oil control group and RORγt inhibitor group. RORγt inhibitor (10 mg/kg) was given once every 2 days via oral gavage. ( C ) Photos of C57BL/6 skin grafts in the control group and RORγt inhibitor group. ( D ) C57BL/6 skin graft survival in control and RORγt inhibitor–treated SCID mice ( n = 5 per group). ( E ) Pathological examination of the skin grafts in the control group and RORγt inhibitor group on days 8 and 13. ( F ) Quantification of H&E staining corresponding to the control group and RORγt inhibitor group on days 8 and 13. Data are representative of three independent experiments ( n ≥ 3). Data are means ± SD. ** P < 0.01 and *** P < 0.001.

Article Snippet: RORγt inhibitor (catalog no. S6767) and corn oil (catalog no. S6701) were purchased from Selleckchem (Houston, TX, USA).

Techniques: Control, Staining

Journal: Cell Death & Disease

Article Title: Th17-driven CD8 + T cells in hUC-MSC and CAR T-cell dual immunotherapy for superior anti-tumor efficacy

doi: 10.1038/s41419-026-08656-7

Figure Lengend Snippet: CD19 CAR T-cells were co-cultured with Raji cells at a E: T ratio of 1: 5, in the presence or not of hUC-MSCs. CAR T-cells were collected for assessment by flow cytometry at 48 h; A Flow cytometry analysis of CD4 + IL17A + CCR6 + cells, demonstrating a significant increase in the Th17 cell subset in the mCAR group, with subsequent downregulation after RORγt inhibition with GSK805. Data are representative of at least three independent experiments ( n = 3); B Increase of CD19 CAR T-cell cytolysis by MSCs was partially reversed by GSK805. ( n = 3); C MSC-upregulated CD94 and NKG2A expression in CD8 + CAR T-cells is reversed by the Th17 differentiation inhibitor GSK805. Bar graph shows the proportional distribution of CD8⁺CD94⁺NKG2A⁺, CD8⁺CD94⁺NKG2A⁻, and CD8⁺CD94⁻ subpopulations within CD8⁺ CAR T-cells across experimental groups. Scatter plots represent schematic distributions of individual data points. D CD19 CAR T-cells were co-cultured with Raji cells at a E: T ratio of 1: 5 in 6-well plates for 48 h. Live cells (GFP⁻FVD⁻) were sorted into three distinct populations based on CD8, CD94, and NKG2A expression: CD8⁺CD94⁺NKG2A⁺, CD8⁺CD94⁺NKG2A⁻, and CD8⁺CD94⁻. RNA was extracted from each population, reverse transcribed into cDNA, and subjected to quantitative PCR (qPCR) analysis to determine the relative mRNA expression levels of TNF, IFN-γ, GZMB, PRF1, NKG7, and GNLY. E Experimental design of the in vivo experiment on the antitumor effect of MSC on CD19 CAR T-cells, which were purified by sorting after lentiviral transfection and expansion in vitro for 7 days before being i.v. injection into mice. The Th17 differentiation inhibitor GSK805 was given by gavage at 10 mg/kg/day ( n = 5); F Tumor growth monitored by BLI at the indicated time points ( n = 4 or 5); G Flow cytometric analysis of human CD8⁺ T cell subsets in BM at day 7 post CAR-T infusion. MSC treatment increased CD8⁺CD94⁺ CAR T-cell frequencies in BM, consistent with in vitro findings. Data represent mean ± SD ( n = 4). One-way ANOVA was used to assess significance in ( A–D , G ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Where indicated, the RORγt inhibitor GSK805 (10 μM, MCE, NJ, USA), purified anti-human IL-1β (1 μg/mL, BioLegend), purified anti-human IL-6 (1 μg/mL, BioLegend), recombinant human IL-1β (1 ng/mL, Pepro Tech) and recombinant human IL-6 (10 ng/mL, Pepro Tech) were added to the co-culture system [ ].

Techniques: Cell Culture, Flow Cytometry, Inhibition, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, In Vivo, Purification, Transfection, In Vitro, Injection