STAT3-mediated up-regulation of BLIMP1 Is coordinated with BCL6 down-regulation to control human plasma cell differentiation

Abstract

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.

FIGURE 1

Sustained STAT3 phosphorylation after IL-21 activation in primary human B cells. A, Peripheral blood (PB) CD19⁺ B cells were freshly isolated and rested at 37°C for 2 h. Cells were then stimulated for 15 min with either medium alone (−, thin histogram), or rmIL-21 (200 ng/ml, shaded histogram). Levels of phospho-STAT3 (Y705), phospho-STAT1 (Y701), or phospho-STAT5 (Y694) were determined by FACS. Isotype control staining is depicted by a dashed line. B, CD19⁺ B cells were activated for 36 h on CD40L-transfected mouse L cell fibroblasts (CD40L-L cells). Cells were then rested followed by stimulation with medium alone (thin line) or IL-21 (shaded) for 15 or 60 min and levels of phospho-STAT3, STAT1, and STAT5 were determined by flow cytometry. C, CD19⁺ B cells were cultured with CD40L stimulation with medium alone (thin line) or with IL-21 (shaded) and STAT activation was measured at the indicated times.

FIGURE 2

STAT3 activation leads to acquisition of plasma cell phenotype in vitro. A, Immunoblot analysis of 293T cells transfected with LZRS-IRES-STAT3ER. probed with anti-ERα or anti-STAT3 Abs. B, STAT3ER-transduced CD19⁺ B cells were cultured overnight with or without 1 μM 4-HT and then analyzed with anti-ERα Ab and 4′,6′-diamidino-2-phenylindole to show nuclear localization by confocal microscopy. C, Total PB CD19⁺ B cells were transduced with LZRS-control-IRES-GFP (Control GFP, ◇), LZRS-STAT3ER-IRES-GFP (STAT3ER, ○), or LZRS-STAT5b-ER-IRES-GFP (STAT5b-ER, □). Transduced cells were cultured on CD40L-L cells in the presence of IL-2 (40U/ml) and IL-4 (10 ng/ml) and in the presence (closed symbols) or absence (open symbols) of 1 μM 4-HT. Upper panel, Percentages of transduced cells were determined over time and the relative increase in GFP from the start of the culture is expressed. Lower panel, Absolute numbers of GFP⁺ cells were determined and the cumulative expansion is shown. D, At day 17 GFP⁺ cells were gated and analyzed for CD38, CD20, CD19, HLA-DR, and CD138 expression. Data are representative of three independent experiments using different donors. E, Tonsil CD19⁺ B cells were transduced with LZRS-control-IRES-GFP or LZRS-STAT3ER-IRES-GFP and cultured on CD40L-L cells with IL-2 plus IL-4 in the presence of 4-HT (1 μM). The percentage of GFP⁺ cells was determined continuously throughout the culture period. Data are means ± SD of two individual donors. F, At day 17 after transduction tonsil STAT3ER-B cells exhibited increased CD38 and decreased CD19 expression.

FIGURE 3

STAT3 activation leads to BLIMP1 up-regulation and Ig secretion. A, PB CD19⁺ B cells were transduced with either LZRS-control-IRES-GFP (Control, □) or LZRS-STAT3ER-IRES-GFP (STAT3ER, ■). GFP⁺ cells were sorted and cultured on CD40L-L cells in the presence of IL-2 (40U/ml) and in the presence (+) or absence (−) of 1 μM 4-HT for 5 days. ivPC made from total PB CD19⁺ B cells (see Results) and nontransduced CD19⁺ cells (None, ) cultured in the presence of IL-2 or rmIL-21 (50 ng/ml) for 5 days were included for comparison. mRNA expression of BLIMP1, IRF4, XBP1, BCL6, and PAX5 were determined by quantitative (q)RT-PCR. Results represent means ± SD. *, p < 0.05 vs STAT3ER −4-HT or vs control +4-HT by Student's t test (n = 4). n.s. denotes not significant. B, GFP⁺ sorted control– or STAT3ER-transduced CD19⁺ B cells were cultured on CD40L and with IL-2 or IL-21, or with IL-2 in the presence (+) or absence (−) of 4-HT for 5 days. Whole cell extracts were analyzed for BLIMP1, IRF4, BCL6, PAX5, and tubulin expression by immunoblotting. C, Nonswitched CD19⁺IgM⁺CD27– and (D) switched CD19⁺IgM–CD27⁺ B cells were isolated by FACS from PB and transduced with LZRS-control-IRES-GFP (□) or LZRS-STAT3ER-IRES-GFP (■) virus. Sorted GFP⁺ cells were cultured with CD40L stimulation in the presence of IL-2 and with (+) or without (−) of 1 μM 4-HT for 5 days. Nontransduced cells of the corresponding phenotype () were cultured in the presence of IL-2 or IL-21. IgM (left panel) and IgG (right panel) were then measured in culture supernatants by ELISA. *, p < 0.05 vs STAT3ER −4-HT or control +4-HT. Results represent means ± SD. *, p < 0.05 vs STAT3ER −4-HT or vs control + 4-HT by Student's t test (n = 4). E, Absolute cell numbers of cells from B and C. F, Nonswitched PB CD19⁺IgM⁺CD27– and (G) switched CD19⁺IgM–CD27⁺ were transduced with control- or STAT3ER virus, GFP-sorted, and cultured with IL-2, IL-21, or IL-2 in the presence or absence of 4HT. After 7 days, equal numbers of cells were plated onto membranes in serial dilutions and in triplicate and IgM (F) and IgG (G) secretion were determined by ELISPOT. Representative individual wells are shown.

FIGURE 4

IL-21 drives partial plasma cell differentiation in BCL6-transduced B cells. A, PB CD19⁺ B cells expressing BCL6-IRES-YFP were cultured in the presence of IL-2 and IL-4 or with rmIL-21 alone. At the indicated time points expression of BLIMP1 and ACTB were measured by qRT-PCR. BLIMP1 expression was normalized to ACTB expression within each sample and the means ± SD of triplicate measurements in two different donors is plotted. B, BCL6-YFP expressing B cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21 and BLIMP1, BCL6, and tubulin protein expression were determined by immunoblotting 3 or 7 days after IL-21 treatment. C, CD19⁺ B cells were transduced with LZRS-BCL6-IRES-ΔNGFR and cultured for 10 days on CD40L in the presence of IL-2 and IL-4 or with IL-21 alone. Cells were stained with anti-CD19, anti-NGFR, anti-CD38, and anti-CD20 and analyzed by flow cytometry. The rightmost four plots show CD38/CD20 expression on CD19⁺BCL6–ΔNGFR– and CD19⁺BCL6–ΔNGFR⁺ cells. D, CD38/CD20 expression on BCL6-IRES-YFP-transduced B cells after 21 days of culture with IL-2 and IL-4 or with IL-21 alone. E, Surface Igλ and Igκ BCR expression on cells cultured as in C. F, Expression of CD138, HLA-DR, CD86, CD27, CD25, and CD3 on BCL6⁺ B cells cultured as in C. Dotted histogram is iso-type control. G, CD19⁺ BCL6-overexpressing B cells maintained on CD40L with either IL-2 and IL-4 (○)or with IL-21 (•). Cumulative expansion was calculated based on absolute cell numbers over a 17-day period. Data shown are means ± SD of three independent experiments involving different donors and constructs (BCL6-IRES-ΔNGFR or BCL6-IRES-GFP). H, BCL6-IRES-ΔNGFR⁺ cells were cultured for 6 days on CD40L-L cells in the presence of IL-2 and IL-4 or with IL-21. Cell numbers were counted and IgM and IgG in culture supernatants were measured by ELISA to obtain per cell values of IgM and IgG. Data are representative of three independent experiments. I, Total PB CD19⁺ BCL6-GFP⁺ cells were cultured with CD40L in the presence of IL-2 and IL-4 or with IL-21. After 7 days, equal numbers of cells were plated in serial dilutions and in triplicate and IgG and IgM production were determined by ELISPOT.

FIGURE 5

Specific activation of STAT3 in BCL6-transduced B cells. 100% pure PB CD19⁺ BCL6-IRES-YFP⁺ B cells were transduced with LZRS-STAT3ER-IRES-GFP and expanded on CD40L-L cells with IL-2 and IL-4. Double positive GFP⁺YFP⁺ cells were sorted by FACS. A, Cumulative expansion of BCL6/ STAT3ER cells cultured with medium only, with IL-2 and IL-4, or with only 1 μM 4-HT for 22 days. B, IgG and IgA production in BCL6-YFP⁺/STAT3ER-GFP⁺ cells treated for 4 days in the presence or absence of 4-HT. IgM was not detected in these cultures. C, Semi-quantitative RT-PCR for BLIMP1 and HPRT1 mRNA expression in BCL6-YFP⁺/STAT3ER-GFP⁺ cells cultured for 4 days in the presence or absence of 4-HT. Triangles represent 5-fold dilutions of cDNA in the PCR reaction. Data are representative of two independent experiments. D, PB CD19⁺ B cells coexpressing BCL6 and STAT3ER were cultured in the presence or absence of 4-HT and at different time points after 4-HT addition, BLIMP1, BCL6, and tubulin protein levels were determined by immunoblot analysis. E and F, Raji cells expressing control-IRES-GFP or STAT3ER-IRES-GFP were cultured for 4 days in the presence of 1 μM 4-HT. E, BLIMP1 and BCL6 (F) mRNA levels were determined by qRT-PCR and normalized to ACTB expression.