Journal: Nature immunology
Article Title: Increased Cathepsin S in Prdm1−/− dendritic cells alters TFH repertoire and contributes to lupus
Figure Lengend Snippet: Preferential differentiation of IL-21 producing T cells by co-culture with Blimp-1 deficient DCs in vitro DCs (spleens of female wild-type (open symbol) or CKO (closed symbol) mice) and OT-II CD4 + T cells were co-cultured for 3 days at various ratios of DCs to T cells with OVA protein (10 µg/ml). (a) To assess proliferation, OT-II T cells were labeled with CFSE (10 µM) before co-culture with DCs. Proliferation was measured by calculation of division index (total number of divisions/number of starting cells). A representative flow image is presented on the left (red line represents wild-type DCs and filled blue represents Blimp-1 deficient DCs). The box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25 th to 75 th percentile, and whiskers indicate 10 th and 90 th percentile (n=4). (b) To assess cytokine expression, T cells were harvested after co-culture with DCs (either unfixed DCs or fixed DCs as designated in figure) and total RNA was purified. Cytokine mRNA was measured by qPCR and the relative expression was calculated by normalization to Polr2a . In the box-and-whisker plots, horizontal bars indicate the median, boxes indicate 25 th to 75 th percentile, and whiskers indicate 10 th and 90 th percentile (unfixed DCs (n=12) and fixed DCs (n=10)). (c) Protein level of cytokines was measured by intracellular staining of T cells. After co-culture, T cells were stimulated with PMA (100 ng/ml)/ionomycin (1 µg/ml) for 6 hours. BFA (20 µg/ml) was added during the last 4 hours of stimulation. Cytokine-positive cells were identified by flow cytometry and their frequency was calculated. In the box-and-whisker plot, horizontal bars indicate the median, boxes indicate 25 th to 75 th percentile, and whiskers indicate 10 th and 90 th percentile (n=8). For both (b) and (c), 1 nM CTSS inhibitor was added during the OVA protein incubation with DCs or cells were cultured in transwell plates. Flow images are from one representative set from 4 individual experiments. (d) DQ-OVA protein was incubated with DCs from wild-type (open circle) or CKO (closed circle) mice overnight. After incubation, DCs were extensively washed and OVA uptake was measured by flow cytometry (upper panel, a representative image from 3 individual experiments). The Geo MFI is presented as a dot plot graph (bottom graph). Each do represents an individual experiment and the bar represents the mean ± SEM (n=3).
Article Snippet: Antibodies and reagents Taqman primers were purchased from Applied Biosystems: Mm00515580_m1 (Ctsc ), Mm00515586_m1 (Ctsd ), Mm00456010_m1 (Ctse ), Mm00456011_m1 (Ctsg ), Mm00484039_m1 (Ctsk ), Mm00617413_m1 (Ctso ), Mm00515599_m1 (Ctsw ), Mm01255859_m1 (CtsS ), Mm00515597_m1 (Ctsl ), Mm00613524_m1 (March1 ), Mm00439211_m1 (H2-Aa ), Mm00482914_m1 (Ciita ), Mm00783707_s1 (H2-dmb2 ), Mm00511327_m1 (Zbtb46 ), Mm00658576_m1 (CD74 ), Mm00468476_m1 (H2-oa ), Mm01325350_m1 (Lgmn ), Mm00434455_m1 (Itgam ), Mm00438347_m1 (Cst3 ), Mm00499585_m1 (CD209b ), Mm00516431_m1 (Irf4 ), Mm00477633_m1 (Bcl6 ), Mm00581355_s1 (Maf ), Mm00479410_m1 (Batf ), Mm00839502_m1 (Polr2a ), and Mm03024075_m1 (Hprt ).
Techniques: Co-Culture Assay, In Vitro, Mouse Assay, Cell Culture, Labeling, Flow Cytometry, Whisker Assay, Expressing, Purification, Real-time Polymerase Chain Reaction, Staining, Cytometry, Incubation