Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Unregulated antigen-presenting cell activation by T cells breaks self tolerance

doi: 10.1073/pnas.1818624116

Figure Lengend Snippet: MHCII ligands plus raised B7 levels induce proliferation of naive CD4 T cells in auto-MLR. Shown are auto-MLR following coculture of syngeneic purified CD11c+ MHCII+ PLN DCs with CTV-labeled CD44lo CD62Lhi B6 naive CD4 T cells from Foxp3-GFP mice in vitro. (A) Comparison of coculturing 1 × 105 B6 (H2b) naive CD4 T cells with 5 × 103 BALB/C (H2d) or B6 (H2b) PLN DCs. Shown are representative FACS plots of proliferation of T cells in 3-d culture (Left), and percentage of divided cells at various time points (Right). (B) Effect of MHCII or B7 blockade on auto-MLR. A total of 5 × 103 PLN B6 DCs was cocultured with 1 × 105 B6 naive CD4 T cells in the presence of PBS (n = 10), MHCII blocking mAb (10 μg/mL Y3P; n = 5), or B7 (CD80 + CD86) blocking mAbs (5 ug/mL 1G10 and GL-1; n = 10) for 7 d. Shown is percentage of divided cells for each group. (C) Auto-MLR for 7 d following coculture of 1 × 104 GF unseparated Rag1−/− PLN cells (B6 APCs) or 1 × 104 H2m−/− Rag1−/− PLN cells (H2M APCs) with 1 × 105 CTV-labeled naive CD4 T cells from B6 Foxp3-GFP mice or from H2m−/− Foxp3-GFP mice (n = 8∼10). Shown are representative CTV histogram of cocultured T cells (Left) and percentages of divided cells among total cocultured T cells (Right). (D) Auto-MLR for 7 d with coculture of 1 × 104 B6 APCs with 1 × 106 or 1 × 105 CTV-labeled CD5hi or CD5lo B6 naive CD4 T cells from Foxp3-GFP mice (100/1, or 10/1 T:APC ratio; n = 10). For purification, upper 25% or lower 25% of naive CD4 T cells were purified for CD5 expression. Shown are representative histogram for CTV-dilution profile of total cocultured T cells (Left) and percentages of CTV− cells of total cocultured T cells (Right). (E) Suppressive effect of nTregs on auto-MLR. Total of 1 × 106, 1 × 105, or 1 × 104 CD45.1 B6 naive CD4 T cells were cocultured with 1 × 104 B6 APCs for 8 d in the absence (n = 5) or presence (n = 3) of CD90.1-marked nTregs. The ratio of naive T cells (CD45.1):nTregs (CD90.1) was 2:1 in each situation. Shown are representative histogram of CTV dilution on cocultured CD45.1 T cells (Left) and percentages of CTV− cells (Right). (F) Shown are mean fluorescence intensities (MFIs) of CD80 and CD86 on cultured DCs when purified PLN DCs from B6 mice were incubated alone for 24 h. CD80 and CD86 levels of PLN DCs from fresh Rag1−/− or B6 mice are shown as controls. (G) Comparison of pTreg induction when 1 × 105 B6 naive CD4 T cells were cocultured with 5 × 103 BALB/c (H2d) or B6 (H2b) PLN DCs for 5 d (see above) (H2d DCs, n = 5; H2b DCs, n = 10). P values were determined by Student’s t test or one-way ANOVA with Newman–Keuls multiple-comparison test. Error bars show mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For MHCII blockade, clone Y3P mAb specific for I-A b (Bio X Cell) was used at 10 μg/mL.

Techniques: Purification, Labeling, In Vitro, Comparison, Blocking Assay, Expressing, Fluorescence, Cell Culture, Incubation