IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells - PubMed (original) (raw)
IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells
Sean A Diehl et al. Immunol Cell Biol. 2012 Sep.
Abstract
Interleukin (IL)-21-producing CD4(+)T cells are central to humoral immunity. Deciphering the signals that induce IL-21 production in CD4(+) T cells and those triggered by IL-21 in B cells are, therefore, of importance for understanding the generation of antibody (Ab) responses. Here, we show that IL-6 increased IL-21 production by human CD4(+) T cells, particularly in those that express the transcriptional regulator B cell lymphoma (BCL)6, which is required in mice for the development of C-X-C chemokine receptor type 5 (CXCR5(+)) IL-21-producing T follicular helper (T(FH)) cells. However, retroviral overexpression of BCL6 in total human CD4(+) T cells only transiently increased CXCR5, the canonical T(FH)-defining surface marker. We show here that IL-21 was required for the induction of Ab production by IL-6. In IL-21-treated B cells, signal transducer and activator of transcription (STAT)3 was required for optimal immunoglobulin production and upregulation of PR domain containing 1 (PRDM1(+)), the master plasma cell factor. These results, therefore, demonstrate the critical importance of STAT3 activation in B cells during IL-21-driven humoral immunity and suggest that BCL6 expression, although not sufficient, may serve as a platform for the acquisition of a T(FH)-like phenotype by human CD4(+) T cells.
Figures
Figure 1. Interleukin-6 induces Ig secretion in a multicellular context
PBMCs (5 × 105/ml) were stimulated with (PHA (2 μg/ml) and the indicated cytokines: IL-2 (40 U/ml), IL-4 (10 ng/ml), IL-6 (100 ng/ml, or IL-21 (50 ng/ml) for 7 days. Cells were counted and IgG production relative to cell number was determined by ELISA. Results are means ± SD of two independent experiments using three different donors. Statistical significance (*, P < 0.05; ***, P < 0.001) was determined using one-way ANOVA with Dunnett’s multiple comparison test.
Figure 2. IL-21, but not IL-6 directly initiates plasma cell differentiation
Total CD19+ B cells were stimulated for 5 days with CD40L-L cells in the presence of the indicated cytokines (concentrations as in Fig. 1). (A) Staining for CD38, CD20, CD19, and CD138 in activated B cells. (B) Total cell numbers were determined. (C) IgG and IgM were measured by ELISA in supernatants from B and values are shown as production per 104 cells. (D) CD19+ B cells were stimulated with CD40L-L cells for 5 days in the presence of cytokines and PRDM1 mRNA levels were determined by qRT-PCR. ACTB levels were used for normalization. Results are means ± SD of three independent experiments using different donors. Statistical significance (*, P < 0.05) was determined by one-way ANOVA with Dunnett’s multiple comparison test.
Figure 3. IL-6 induces IL-21 and BCL6 in human CD4+ T cells
(A) CD4+ T cells were activated for 2 days with PHA (2 μg/ml) in the presence of IL-6 (100 ng/ml) or IL-21 (50 ng/ml) and RT-PCR for IL21 was performed using HPRT as a control. (B) Human CD4+ T cells from donors were activated with PHA in the presence or absence of human IL-6 (100 ng/ml) for 2 days. IL-21 production was determined by ELISA. (C) CD4+ T cells were activated for 2 days with PHA in the presence of IL-6 (100 ng/ml) or IL-21 (50 ng/ml) and qRT-PCR for BCL6 and ACTB was performed. Values were normalized to ACTB and expressed relative to control. Results are means ± SD of duplicates of two independent donors. Statistical significance (*, P < 0.05) was determined using unpaired Student’s t-tests.
Figure 4. IL-6 increases expression of IL-21 and BCL6, but not surface TFH markers
Total CD4+ T cells were stimulated with CD3/CD28 beads for 5 days in the presence of no cytokine (–), IL-6, IL-21, or IL-21. Immediately after culture, CD4+ T cells were analyzed for surface CXCR5, ICOS, and PD1 expression by flow cytometry (see Fig _S_1 for representative FACS plots). Frequencies were determined from isotype control stainings using a sample pooled from the shown experiments). Quantitation of CXCR5+ cells within the CD3+CD4+–gated cells (A) and ICOS/PD1 (B) expression on CXCR5+CD4+–gated T cells is shown. (C) Intracellular BCL6 expression in CD3/CD28-activated CXCR5+CD4+–gated cells. Immediately after culture, cells were surface stained, fixed, permeablized and stained for intracellular BCL6. Number on graph indicates percent BCL6–positive cells. Dashed histogram is isotype control. (D) For intracellular IL-21 detection, CD3/CD28-stimulated cells were washed and restimulated with PMA and ionomycin in the presence of monensin for 6 hrs, surface stained, fixed, permeabilized, and stained for intracellular BCL6 and IL-21. Isotype control staining was used to determine frequencies. (E) Frequency of IL-21+BCL6+ cells (gated on CXCR5+CD4+ cells) and (F) and the fold difference in IL-21+BCL6+ cells as compared to no cytokine control. Graphical results show mean values and are derived from 7 donors in two separate experiments and FACS plots are representative. Statistical significance across donors (*, P < 0.05; **, P < 0.01; #, P = 0.06 vs no cytokine control) was determined by pairwise Mann-Whitney tests.
Figure 5. Enforced BCL6 expression enhances lymphoid/follicular markers, including CXCR5 in primary human CD4+ T cells
Total CD4+ T cells from peripheral blood were stimulated with CD3/CD28 beads for 4 days and transduced (A) with retroviral vectors encoding either LZRS-IRES-ΔNGFR (Control) or LZRS-BCL6-IRES-ΔNGFR (BCL6). (B) CXCR5 surface expression on resting, activated (4 days), or Control and BCL6-transduced CD4+ T cells at the indicated timepoints after transduction. Transduced cells were cultured on irradiated allogeneic PBMC feeders with PHA (2 μg/ml) and IL-2 (20 U/ml) (methods). (C) Percent CXCR5+ and (D) CXCR5 mean fluorescence intensity (MFI) of CD4+NGFR+ cells over time. (E) CCR7/CXCR4 expression in CD4+NGFR+ cells at Days 14 and 21 after transduction. (F) CD80 expression on CD4+NGFR+ cells 21 days after transduction. For panels E and F, Gates were set using isotype control stainings and total tonsil cells to define positive populations as described for CXCR4 and CD80. CCR7 gates in panel E were defined as described. Graphical results are means ± SD of two independent experiments each containing two donors and FACS plots are representative. Two-way ANOVA was used to calculate statistical significance (_P_-values shown on graphs) in panels C and D. Quantitated data for CXCR4/CCR7 and CD80 are shown in Fig _S_2.
Figure 6. IL-21 is required for IL-6-induced Ig production during T-B cell collaboration
(A) Mitomycin-c–treated CD4+ T cells (1 × 105) were cultured with 1 × 105 CD19+ B cells in 0.2 ml in the presence of PHA with medium, IL-6, or IL-21 and Ig production was determined after five days by ELISA. (B) IL-6–stimulated co-cultures of T and B cells were treated for five days with either control Ig (10 μg/ml) or IL-21R:Fc fusion protein (10 μg/ml). Results are means ± SD of three independent donors. Statistical significance (***, P < 0.001) was determined using unpaired Student’s t-tests.
Figure 7. STAT3 is required for IL-21-induced plasma cell differentiation
(A) Raji B cells were transduced with retrovirus encoding control shRNA targeting firefly Renilla luciferase (shRen) or with STAT3i (shSTAT3). Whole cell extracts from GFP-sorted cells were analyzed for STAT3 protein expression by immunoblot. Non-transduced (none) cells are shown as control. (B) Total CD19+ B cells were activated with CD40L, transduced with shRen or shSTAT3, and after 2 days GFP sorted, cultured with CD40L-cells and IL-2 or IL-21 (or: for 3 days). After three additional days, total RNA was isolated and PRDM1 and ACTB mRNA expression were analyzed by quantitative RT-PCR. PRDM1 expression was normalized to ACTB expression within each sample and the means +/− SD of triplicate measurements in two different donors is shown. (C to F) Naïve IgM+CD27− B cells (C,D) or total CD19 B cells (E,F) from peripheral blood were transduced with the indicated retrovirus, GFP-sorted and cultured on CD40L-L cells in the presence of IL-2 or IL-21 for three days. IgM (C,E) and IgG (D,F) were measured in the supernatant by ELISA. Results are means ± SD of two independent experiments each containing two donors. Statistical significance (*, P < 0.05; **, P < 0.01, ***, P < 0.001) was determined by one-way ANOVA with Tukey’s post-test for differences.
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