Fig. 5: Normal NF-κB activation but defective regulation of extrinsic cell death pathways in Tnfaip3 deficient T cells.

A Lymph nodes and spleen were recovered from Tnfaip3ΔT^CD4 (n = 6) and Cre –ve litter mates (n = 15) aged between 8 and 12 weeks, and T cell phenotype and number determined by flow cytometry. Density plots are of CD4 vs CD8 expression by total live lymph node cells, CD25 vs CD44 by the indicated subset. Bar charts summarise total numbers of TCR^hiCD4⁺ memory phenotype (CD4⁺CD44^hiFoxp3^–), T_reg (CD25⁺Foxp3⁺ Treg), CD4⁺TCR^hiCD44^lo naive T cells (CD4 naive) and CD8⁺TCR^hiCD44^lo naive T cells (CD8 naive) recovered from total lymph nodes and spleen combined. Total numbers of CD25⁺Foxp3⁺ thymic Treg are from thymus. Bar chart of IL-7R expression by naive CD4 and naive CD8 T cells is expressed as fraction of MFI normalised to MFI of corresponding subset from Cre –ve controls. B Thymocytes from Tnfaip3ΔT^CD4 and Cre –ve litter mates were stimulated with FLAG-TNF or hTNF for different times, lysed and immunoprecipitated with either anti-FLAG-sepharose or HALO-NEMO beads and analysed by immunoblotting for RIPK1, M1 ubiquitin and IKK1. Uncropped immunoblots are shown in Supplementary Fig. 1. C Total thymocytes from Tnfaip3ΔT^CD4 and Cre –ve litter mates were stimulated with TNF for upto 16 hr, nuclear extracts prepared and RELA levels determined by ELISA. D–F Lymph node cells from Tnfaip3ΔT^CD4 and Cre –ve litter mates were cultured overnight with titrations of TNF, IKK2 inhibitor (IKK2i), in the presence or absence of fixed concentrations of TNF (20 ng/ml), Nec1 (10 µM), pan IKK inhibitor (IKK16, 0.25 µM). Line graphs show fraction of dead cells amongst TCR^hi naive CD4⁺CD44^lo and TCR^hi naive CD8⁺CD44^lo T cells from Tnfaip3ΔT^CD4 and Cre –ve litter mates from cultures titrating TNF (D), in cultures titrating IKK2i in the presence or absence of Nec1 and/or TNF (E) and in cultures titrating TNF with addition of Nec1 and or IKK16 (F). Data are representative (A–C) or show average of three or more independent experiments. Error bars show SEM. Uncropped gels from (B) are in Supplementary Fig. 1.