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Blood, Vol. 113, Issue 11, 2426-2433, March 12, 2009
Interleukin-6/STAT3 signaling regulates the ability of naive T cells to acquire B-cell help capacities
Blood Eddahri et al.
113: 2426
Supplemental materials for: Eddahri et al
Files in this Data Supplement:
- Figure S1. Th-cell deliver help to B cells through cognate and non cognate T–B-cell interactions (JPG, 70 KB)
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(A) Delivery of T-cell help to B cells requires T–B-cell contacts. Naïve CD62L+ CD4+ T cells were stimulated for 48h with plastic-coated anti-CD3 and anti-CD28 mAbs and rested 24h in fresh medium. Serial dilutions of irradiated Th cells were then incubated in Transwell plates (lower chamber) with purified B cells (5 × 105 cells/well) and plastic-coated anti-CD3 mAbs (5 µg/ml). B cells were either placed in the lower or upper chamber, as indicated. Culture supernatants were tested on day 7 for IgG1 and IgG2a contents. Results are expressed as mean +∕− SD of triplicates (SD < 5% did not appear in the figure) and indicate that in vitro antibody production was only observed when T and B cells were cultured in the same compartment. This experiment also clearly demonstrates that antibody secretion is not due to the activation of B cells contaminating the T-cell preparation. (B) Delivery of T-cell help to B cells requires CD40L-CD40 interaction. CD4+ T cells were cultured with WT or CD40-deficient CD19+ B cells in the presence or absence of anti-CD3 mAbs. Culture supernatants were tested on day 7 for IgG1 and IgG2a contents. Results are expressed as mean +∕− SD of triplicates. (C) Delivery of T-cell help to B cells through cognate T–B-cell interaction. CD62L+ CD4+ DO11.10 T cells were stimulated in the presence of CD19+ B cells purified from TNP-KLH–immunized mice and OVA-TNP. Culture supernatants were tested for TNP-specific IgG1 and IgG2a antibody contents on day 7.
- Figure S2. Th cells promote naïve B-cell proliferation and IgG secretion (JPG, 96.6 KB)
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(A) Th cells promote contact-dependent, B-cell proliferation. CFSE-labelled CD45.1 and CD45.2 B cells were cultured with CD4+ T cells and plastic-coated anti-CD3 mAbs in Transwell plates. CD45.2+ B cells and Th cells are plated in the lower chamber while CD45.1+ B cells are plated either in the lower or upper chamber, as indicated. Cells are gated on CD45R (B220+) B cells and data show CFSE dilution in CD45.2+ and CD45.1+ B cells. The experiment demonstrates that proliferation of CD45.1+ B cells is only observed when these cells are cultured in the lower, T cell containing chamber. (B) FACS analysis of the purified CD19+ and CD43− B-cell populations. Splenic B cells were purified according to CD19 or CD43 expression and analysed for IgM/IgD, CD43/CD45R and CD5/IgM expression. Numbers indicated the percentage of cells. Note that the number of proliferating cells in T/B-cell co-cultures outnumbered the number of potentially contaminating memory cells, clearly indicating that naïve B cells respond to T-cell help in this setting (see also below). (C) Activated Th cells deliver B helper signals to naïve CD43− B cells. Irradiated CD4+ T cells were cultured with B cells purified according to CD19 or CD43 expression, in the presence of anti-CD3 mAbs. Culture supernatants were tested on day 7 for IgG1 and IgG2a contents. Results are expressed as mean +∕− SD of triplicates. Despite a reduced number of “memory-like” B cells in the CD43− population, both B cells preparations produced antibodies in vitro in response to T-cell help, indicating that the responses observed in vitro cannot be ascribed to a small population of contaminating memory B cells.
- Figure S3. Induction of T-helper differentiation in vitro (JPG, 88.6 KB)
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Following culture in control or polarizing conditions as indicated (see methods section), cells were restimulated with plastic coated anti-CD3. (A) Cells were fixed, stained for intracellular cytokine determination and analyzed by flow cytometry. (B) Culture supernatants were tested for cytokines contents by ELISA.
- Figure S4. IL-6 promotes B-cell helper function, while a combination of IL-6 and TGF-β is required for Th17 differentiation (JPG, 55 KB)
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(A) CD4+ CD62L+ T cells were stimulated in the presence of medium, IL-6, or IL-6 + TGFβ. Recovered cells were rested 1 day in fresh medium and tested for B-cell help capacity. (B) Flow cytometric analysis of IL-17 production for each population obtained in A.
- Figure S5. Effects of IL-6 on IL-4 and IFN-γ expression (JPG, 78.5 KB)
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CD4+ CD62L+ T cells were stimulated in the presence of medium or IL-6 as indicated, and analyzed by flow cytometry as described in the methods section.
- Figure S6. B-helper function of IFN-γ–deficient Th cells (JPG, 35.1 KB)
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Naïve T cells from wild type or IFN-γ−∕− mice were stimulated for 48 h with plastic-coated anti-CD3 and anti-CD28 mAbs in the presence of IL-6, rested 24h in fresh media and tested for B-cell help activity in the presence of B cells (5 × 105 cells/well) and anti-CD3 mAbs (500 ng/ml). Culture supernatants were tested on day 7 for IgG1 and IgG2a contents. Results are expressed as mean +∕− SD of triplicate cultures.
- Figure S7. IL-21 secretion by Th0, Th1, Th2, or Th17 cells (JPG, 41.8 KB)
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