Figure 1.
Expression of SOX5 transcript variants in human B cells.
(A) Schematic representation of human SOX5 transcript variants. Non-coding exons are depicted as open rectangles, partial coding exons - as half open rectangles and coding exons - as filled rectangles. Primer regions are indicated with appropriate arrows. Exons and coding exons are numbered according to their location along the genomic sequence, which are drawn as black lines. (B) RT-PCR analysis for the expression of β-actin, CD19 genes and SOX5 transcript variants. HD PBMCs were separated into: A – PBMCs; B – B cells and C – non-B lymphocytes. Except for SOX5 transcript variant 3 (SOX5-var 3) in which human testis RNA sample served as a control, human costal cartilage cells used as a positive control in all RT-PCR reactions. In agarose gel pictures DNA markers were cut out, since they were loaded between the tested samples and the control sample. (C) RT-PCR assay performed to discriminate between SOX5 transcript variant 2 and variant 4 in samples of peripheral blood lymphocytes: A – PBMCs; B – B cells and C – non-B lymphocytes.
Figure 2.
Human B cells express at least three different transcript variants of SOX5.
(A) Schematic representation of sequence verified human SOX5 transcript variants in B lymphocytes. Non-coding exons are depicted as open rectangles, partial coding exons - as half open rectangles and coding exons - as filled rectangles. Cloning primer locations are indicated with appropriate arrows. Exons and coding exons are numbered according to their location along the genomic sequence, which are drawn as black lines. (B) PCR analysis of SOX5 transcript expression in B cells and in single clones picked for sequence analysis. B lymphocytes express at least three different SOX5 transcript splice variants as evidenced by representative single TOPO clones 1, 2 and 3.
Figure 3.
Expression of SOX5 in human B cell subpopulations.
(A) Relative quantification of SOX5 by RT-qPCR in peripheral blood naive, MZ-like, switched memory (sw mem), non-classical memory (nc mem) and CD21low B cells. (B) Relative quantification RT-qPCR assay for SOX5 expression in follicular naive, germinal center B cells (GC), memory B cells and plasma cells (PC) from tonsils. T-test p-values indicate the significance of differences between the samples. Relative expression levels of SOX5 are shown as mean ± SD. RPLP0 gene was used as an internal control in the samples. (C) Immunofluorescence staining for the expression of SOX5 protein in tonsillar tissues. IgD staining was used to stain mantle zones, Ki67 staining for proliferating centroblasts within germinal centers and CD138 as a marker for extrafollicular plasma cells.
Figure 4.
Induction of SOX5 during in vitro B cell differentiation.
(A) Isolated peripheral blood B cells were either left for 3 days without any stimulus or stimulated for 9 days in vitro either with a single stimulus (IL4, IL21 and CD40L) or the combination of these (CD40L+IL4+/− IL21). The cells were analyzed by FACS for the plasma cell markers CD138 and CD38 at days 3, 6 and 9. Gates indicate the frequency of CD138+CD38hi plasmablasts in each plot. CFSE at day 9 was measured as an indicator of proliferation in the cells. Representative FACS plots of five independent experiments are shown. (B) RT-qPCR analysis of SOX5 expression in ex vivo B cells and samples either unstimulated (unstim.) for 3 days or stimulated with a single stimulus (IL4, IL21 and CD40L) or the combination of these (CD40L+IL4+/− IL21). Significant differences are depicted and appropriate t-test p-values (n = 5; p<0.05) are indicated. Relative expression levels of SOX5 are shown as mean ± SD. RPLP0 gene served as an internal control in the samples.
Figure 5.
Expression of “SOX-trio” and SOX13 genes in ex vivo B cells and during in vitro differentiation of human primary B cells.
(A) PCR analysis for the expression of RPLP0, SOX6, SOX9 and SOX13 genes in human peripheral blood lymphocyte populations: A – PBMCs; B – B cells and C – non-B cell fractions. (B) RPLP0, SOX6, SOX9 and SOX13 expression in isolated peripheral blood B cells stimulated with IL4+CD40L+IL21 for 9 days in vitro. The analysis was performed at days 3, 6 and 9. In agarose gel pictures DNA markers were cut out, since they were loaded between the tested samples and the control sample. Human costal cartilage cells served as a control for SOX6 and SOX9 expression, whereas BEAS-2B cells were used for SOX13 as a control.
Figure 6.
SOX5 modulates in vitro terminal B cell differentiation.
(A) Proliferation measured as the ratio of absolute numbers of GFP+ cells in the samples. Absolute cell counts of GFP+ cells on day 0 are taken as 1.0 and the data are expressed as mean ± SD. Summary of three independent experiments are depicted. (B) Frequencies of GFP+ DAPI+ cells within GFP (control) and SOX5-GFP-transduced peripheral blood B cells cultivated in vitro. (C) Plasma cell differentiation analyzed by FACS in peripheral blood B cells stably transduced either with GFP (control) or SOX5-GFP fusion construct upon in vitro stimulation with IL4+CD40L+IL21. DAPI-negative GFP+ gated cells were analyzed by FACS for the plasma cell markers CD138 and CD38 at days 3, 6 and 9. Gates indicate the frequencies of CD138+CD38hi plasmablasts in each FACS plot. Representative FACS plots of three independent experiments are shown. (D) Numbers of CD138+CD38hi plasmablasts in GFP (control) and SOX5-GFP-transduced B cells at days 3, 6 and 9 referring to CD38hi CD138+ cells per 1000 GFP+ cells at day 0. Cell numbers are depicted as mean ± SD and t-test p-values indicate the significant differences. Summary of three independent experiments are shown.