Journal: Nature immunology
Article Title: Apoptosis in response to microbial infection induces autoreactive TH17 cells
Figure Lengend Snippet: Detection of self-reactive CD4 + T cells without an effector phenotype in DTg mice ( a ) Frequency ( left ; numbers adjacent to blue outlined areas), and absolute number (middle) and frequency ( right ) of Vα2 hi Vβ5 + CD4 + T cells in the thymus of OT-II and DTg mice. ( b ) CD5 expression on I-A b –OVA(328–337) tetramer–positive CD4 + T cells from the MLNs and LI LP of OT-II and DTg mice. Isotype, isotope-matched control antibody. ( c ) Flow cytometry ( left ) of CD45 + CD8 − Vα2 + Vβ5 + CD4 + T cells from the thymus of OT-II and DTg mice. Numbers adjacent to outlined areas indicate percent Foxp3 + CD4 + T cells. Right, frequency of Foxp3 + Vα2 + Vβ5 + CD4 + T cells in the thymus of OT-II or DTg mice. ( d ) Frequency of Foxp3 + Vα2 + Vβ5 + CD4 + T cells in the LI LP of OT-II and DTg mice ( left ), and flow cytometry of Vα2 − Vβ5 − or Vα2 hi Vβ5 + CD4 + T cells ( right margin) from the LI LP of the DTg mice at left ( right ). Numbers above bracketed lines ( right ) indicate percent Foxp3 + cells. ( e ) Expression of RORγt, T-bet and GATA-3 in Vα2 hi Vβ5 + LI LP CD4 + T cells from uninfected DTg mice, assessed by flow cytometry. ( f ) Proliferation (CFSE dilution) of splenic CD25 − CD4 + T cells from OT-II or DTg mice (key) after 5 d of culture with BMDCs pulsed with non-cognate peptide or OVA(329–337) (each at 10 μg/ml) or with BMDCs that had phagocytosed LPS-stimulated (LPS-blasts) apoptotic B cells isolated from Act-mOVA BALB/c mice. ( g ) Secretion of IL-17 by splenic T cells as in f, as well as T cells stimulated with BMDCs that had phagocytosed unstimulated apoptotic B cells from Act-mOVA BALB/c mice (B cells), assessed after 48 h of secondary re-stimulation with OVA(329–337). Data are representative of two independent experiments ( a–c, d, right, e–g ; mean + s.d. in a,c,d,g ) with n = 5 ( a–c and d, right ) or n = 4 ( e ) mice, or three independent experiments with n = 9 mice ( d, left ; mean + s.d.).
Article Snippet: Antibodies, tetramers, 5-Bromo-2’-deoxyuridine (BrdU) and CFSE labeling For flow cytometry, the following antibodies were purchased from eBioscience and were all used at a 1:100 dilution: anti-mouse CD45.1 (clone A20), CD4 (clone RM4-5), CD44 (clone IM7), Vα2 (clone B20.1), CD5 (clone 53-7.3), CD25 (clone PC61.5), CD8β (clone eBioH35-17.2), CD16/32 (clone 93), Thy 1.1 (clone HIS51), TCRβ (clone H57-597), IL17A (clone eBio17B7), IFN-γ (clone XMG1.2), Foxp3 (clone FJK-16s), RORγt (clone B2D), and T-bet (clone 4B10).
Techniques: Mouse Assay, Expressing, Flow Cytometry, Cytometry, Isolation, Activated Clotting Time Assay