PMC

A. Ly7 cells were subjected to culture conditions as described for . After 46 h, whole cell lysates were prepared and analyzed by Western Blot for the expression of indicated protein markers. B. Forty-eight hours after being transfected with either control (ctrl) or STAT3-specific siRNA oligos, Ly7 cells were stimulated as indicated for another 44 h. Whole cell lysates were prepared and subject to Western Blot analysis for the indicated protein markers. The RNAi-mediated knock-down effect of total STAT3 protein was shown in the bottom panel. n.s., non-specific band.

Ly7 cells were cultured for 16 h on either the control 3T3 (3T3-ctrl) or 3T3-CD40L feeder cells prior to IL-21 stimulation. A. Whole cell lysates were prepared at the indicated time points during IL-21 treatment and analyzed by Western Blot for the indicated protein markers. B. Normalized intensity of PY-STAT3 bands were plotted against IL-21 treatment time. The graph shows the mean +/− S.D. of two independent experiments. C–D. Aliquots of cells that were either untreated or exposed to varying concentrations of IL-21 for 1 h were analyzed for surface IL-21R expression. FACS profiles for untreated cells and those treated with 100 ng/ml IL-21 are shown in C. Mean fluorescence intensity (MFI) values for all samples are listed in D.

Ly7 cells were cultured for 14 days in the presence or absence of IL-21 on either control 3T3 fibroblasts or 3T3-CD40L stable transfectants. A. On day 7 and 14, aliquots of cells were stained and phenotypic changes were examined based on CD20 and CD38 expression. B. Culture supernatants taken on day 0, 4, 7 and 14 were analyzed by ELISA for production of total IgM and IgG antibodies. Note the different y-axis scale for IgM and IgG. Student t-tests were performed to compare the Ig titer from a given treatment to that of the control culture (3T3) on the same day. *, P < 0.05; **, P < 0.01. Results are representative of 4 independent experiments.

Purified CD20^hiCD38^hi centroblasts (CB) were differentiated in vitro in the presence of IL-2, IL-4, and CD40L-CD8 fusion on HK cells for 12 days. In parallel cultures, AG490 (5 uM) or IL-21 (50 ng/ml) was included throughout the assay to either inhibit or further activate Jak/STAT signaling, respectively. A. On indicated days, aliquots of cells from the control (ctrl) and AG490 cultures were stained and B cell subsets identified based on surface CD20 and CD38 expression. B. Changes in the expression of indicated transcription factors were analyzed by Western Blot. n.s., a non-specific band recognized by the polyclonal Blimp-1 Ab. The Mum1 Ab recognizes a conformational epitope on the IRF4 protein enriched in the nucleus (). GAPDH is used as a loading control. C. Culture supernatants were harvested on the days indicated from control, AG490, and IL-21 cultures, and analyzed by ELISA for total IgG and IgM Ab production. Plotted in the graph are mean ± s.d. of two duplicate cultures in the same experiment. Note the different y-axis scale for IgM and IgG. D. Expression of Jak3 and IRF4 mRNA was measured by qRT-PCR on RNA samples prepared from the control and AG490 cultures on the days indicated. Data are representative of 3 or more independent experiments.

A. qRT-PCR analysis of Blimp-1 mRNA in Ly7 cells treated as indicated during a period of 1–4 days. All values were normalized to the pre-treatment level defined as 1.0. B. Schematic representation of the 5′ regulatory region of the human PRDM1 gene and its third intron showing a shared BCL6/STAT3 site. Transcriptional start site +1 is defined as in GenBank accession number AY198414 (http://www.ncbi.nlm.nih.gov/genbank/). Graph is not drawn to scale. C. Ly7 cells pretreated with either control or 3T3-CD40L feeder overnight were left either untreated (ctrl and CD40L) or treated with 100 ng/ml IL-21 for 1 h (IL-21 and IL-21+CD40L). Chromatin was cross-linked and purified from cells subjected to different treatment conditions. Binding of endogenous STAT3 and BCL6 to the indicated regions of the PRDM1 gene were analyzed by qChIP. Signals enrichment by normal rabbit IgG was also measured and used as background control (set as 1.0 in the graphs). D. IL-21/CD40L stimulated transcriptional response from the 3 Blimp-1 Luciferase reporters illustrated at the top. Ly7 cells transiently transfected with the reporters were either left untreated or subject to IL-21/CD40L costimulation as in . Cells were harvested for reporter assays 44 h later. Plotted in the graph are the means ± SD of duplicate tests with the mean from the untreated promoter only construct set as 100. E. A working model depicting regulatory relationships between the signaling molecules and transcriptional factors that we have analyzed in this study. Plain lines with arrow heads represent sequential events or positive regulation; blunt bars represent negative regulation. The dashed arrow with a circled plus sign depicts a potentiation effect of activated CD40 on IL-21R/Jak/STAT3 signaling. Roles of the molecules in grey, CD40 and NF-κB, are based on published literature. For simplicity, the direct influence of IRF4 on Blimp-1 as well as inhibitory effects of BCL6 on IRF4 and STAT3 are not shown. PC diff., PC differentiation.