Fig. 4

Production of TNF-α in mBSA-specific CD4 T cells regulated by deficiency or ectopic expression of SIGIRR. A, B Intracellular production of TNF-α and IL-17 among memory CD4 T cells (CD4⁺ CD44^hi) sorted from immunized Rag1^−/− mice transferred with SIGIRR (WT to Rag1 ^−/−) or SIGIRR KO (KO to Rag1 ^−/−) CD4 T cells and then cultured by stimulation with mBSA in the presence of mitomycin C-treated syngeneic splenocytes for 3 days. A Numbers in quadrants indicate percent TNF-α⁺ IL-17A⁻ cells (top left) or TNF-α⁻ IL-17A⁺ cells (bottom right) or TNF-α⁺ IL-17A⁺ (top right) gated in CD4⁺ CD44^hi cells. B Each symbol represents an individual mouse; small horizontal lines indicate the mean ± SD. **P < 0.01, *P < 0.05 (n = 5, unpaired t test); Data are from one experiment representative of two independent experiments with similar results. C Intracellular TNF-α production in sorted memory CD4 T cells isolated from wild-type C57BL/6 mice and transduced with retroviral vectors encoding IRES-GFP (MIG) and SIGIRR-IRES-GFP (SIGIRR). Cells were cultured with antigen mBSA for 3 days and analyzed with flow cytometry following intracellular staining. D Relative expression levels of TNF-α mRNAs in retrovirally transduced memory CD4 T cells cultured in the presence of mBSA. Expression levels were monitored by quantitative real-time RT–PCR. The relative fold changes were calculated using the ΔΔCt method; data from all the samples were normalized to the SIGIRR group, and ACTB served as an endogenous control. E Knee joint swelling of Sigirr ^+/+ (WT) and SIGIRR KO mice (KO) that received 500 μg of anti-TNF-α monoclonal antibody (clone XT3.11, Bio X Cell) or rat IgG1 κ isotype control antibody (Bio X Cell) intraperitoneally at days 0 and 3 during AIA. AIA was induced as described in the Materials and Fig. 3. Statistical significance was determined by multiple t tests using the Holm‒Sidak method, with alpha = 0.05 (* indicates significance at *P < 0.05 and n = 4)