Journal: Nature Communications
Article Title: Developmental self-reactivity determines pathogenic Tc17 differentiation potential of naive CD8 + T cells in murine models of inflammation
doi: 10.1038/s41467-024-47144-4
Figure Lengend Snippet: a Histograms (left) and MFI levels (right) for IRF4 expression among CD8 + T N subsets stimulated with either anti-CD3 in the presence or absence of TGF-β (left) or with various concentrations of anti-CD3 (right). b B6 CD8 + T N subsets were activated under Tc17-polarizing conditions for 24 h and subjected to ChIP using IRF4 antibody. Eluted DNA was analyzed by qPCR. c Endogenous levels of SMAD3 and p-SMAD3 in ex vivo B6 CD8 + T N subsets shown in histogram (left) and MFI (right) ( n = 5 mice/group). d B6 CD8 + T N subsets were activated under Tc17-polarizing conditions for 24 h and subjected to ChIP using SMAD3 antibody. Eluted DNA was analyzed by qPCR. e Representative FACS plots for IL-17A/IFN-γ production of Tc17-polarized CD8 + T N subsets transduced with MigR-1 vector encoding SMAD3 (top) and the percentage of IL-17A + (bottom left) and IFN-γ + cells (bottom right) in untranduced GFP − and tranduced GFP + cells. f Representative FACS plots for IL-17A/IFN-γ production of Tc17-polarized CD8 + T N subsets transduced with LMP vector containing SMAD3 shRNA (top) and the percentage of IL-17A + (bottom left) and IFN-γ + cells (bottom right) in untranduced GFP − and tranduced GFP + cells. g Experimental scheme for h − j . h Expression levels of SMAD3 in donor cells (from LI) transduced with either MigR-1 vector encoding SMAD3 (left) or LMP vector containing SMAD3 shRNA (right). i , j Percentage of IL-17A + cells ( i ) and IL-17A − IFN-γ + cells ( j ) in transduced GFP + donor cells from LI. Data are representative of three independent experiments ( a , b , d − f , n = 3; c , n = 5 per experiment ) or pooled from two to independent experiments ( h−j , n = 8) and presented as the mean ± SD ( a − f ), and ± SEM ( h−j ). Statistical significance by two-way ANOVA Multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data and exact P value are provided as a Source Data file.
Article Snippet: For over-expression, full-length SMAD3 cDNA from mouse lymphocyte was cloned into the EcoRI and XhoI sites of the MigR1 plasmid (Addgene; #27490). shRNA targeting SMAD3 was cloned in to retroviral shRNA LMP plasmid (Addgene; #36955).
Techniques: Expressing, Ex Vivo, Transduction, Plasmid Preparation, shRNA
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