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Blood, Vol. 112, Issue 13, 5254-5258, December 15, 2008
STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease
Blood Lu et al.
112: 5254
Supplemental materials for: Lu et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 3.35 MB)
- Figure S1. Validation and characterization of antibody reagents used for the flow cytometric analysis of intracellular phosphorylation via Western Blot assays, hydrogen peroxide treatment, and T-cell stimulation (JPG, 99.1 KB)
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(A) A431, HEL, PC12, U937, Jurkat, HepG2, and NIH3T3 cells were stimulated with a variety of cytokines and growth factors for the indicated durations. Cells were then lysed, and antibodies used in flow cytometry applications in this manuscript were used to detect protein phosphorylation via western blot. This revealed specific staining corresponding to the phosphorylation of the molecules ERK 1/2, STAT-1, STAT-3, and STAT-5A, which corresponded to independent analysis of these cells by flow cytometry. (B) Normal B6 splenocytes were incubated either with PBS or with hydrogen peroxide at 0.5 or 5 mM concentration for 5 minutes, and analyzed by flow cytometry for intracellular phosphorylation in CD4 and CD8 T cells. Signals were compared with the PBS control. This demonstrated the wide dynamic range of antibodies in increases of phosphorylation of up to 100-fold over PBS control. (C) Splenocytes from normal B6 mice were stimulated with anti-CD3 antibody, PMA, or PBS control for 5 minutes; subsequently, levels of intracellular phosphorylated ERK 1/2 in CD8 T cells were measured by flow cytometry, demonstrating an increase in ERK1/2 phosphorylation above basal level in response to challenge with anti-CD3 or PMA. One of two independent experiments.
- Figure S2. Splenic T cells from non-transplanted mice demonstrate a near-global increase in signaling with the naïve → effector → memory transition (JPG, 47.2 KB)
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Splenic T cells from non-transplanted B6 mice (n=5) were analyzed by flow cytometry for basal levels of phosphorylated signaling proteins and total levels of each signaling protein (i.e. regardless of phosphorylation state). One of three independent experiments.
- Figure S3. In vitro interrogation of murine splenocytes with cytokines reveals the specific phosphorylation of STAT molecules in T-cell subsets (JPG, 78.9 KB)
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(A) Purified splenic T cells from normal B6 mice were stimulated for one hour with IL-2, IL-7, IL-12, or IL-15. CD4 and CD8 CD44lo naïve and CD44hi effector/memory splenic T cells demonsrated a differential response to cytokines by phosphorylating the appropriate STAT molecules (n=5 pooled). (B) Analyses of bivariate plots from (a) revealed a bimodal distribution for the response of naïve CD4 T cells to IL-2, but a unimodal response to IL-7. (C) Normal splenocytes were treated with IFNγ for the durations indicated. IFNγ caused a highly-specific phosphorylation of STAT-1 in CD44lo naïve CD4 and CD8 T cells (n=24, pooled n=4).
- Figure S4. The signaling profile of expanding donor T cells after adoptive transfer into lethally irradiated allogeneic and syngeneic recipients reveals that STAT-3 is prominently phosphorylated in allogeneic but not syngeneic recipients (JPG, 62.1 KB)
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Lethally irradiated BALB/c or B6 CD45.1 recipients were infused with CFSE-labeled donor T cells and splenocytes analyzed on day 3 (allogeneic, BALB/c) or day 7 (syngeneic, B6 CD45.1) for the phosphorylation patterns of donor T cells. Numbers at the top 0,1,2 etc. represent individual peaks of cell proliferation as measured by CFSE dilution. A comparison reveals that STAT-3 phosphorylation at both Tyr705 and Ser727 is increased in allogeneic but not syngeneic recipients.
- Figure S5. Transient exposure to cucurbitacin I can durably inhibit STAT-3 phosphorylation and T-cell activation for more than 24 hours post-exposure (JPG, 61.6 KB)
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(A) B6 splenocytes were stimulated with plate-bound anti-CD3+CD28 (both coated at 5 µg/mL) or PBS, in the presence or absence of cucurbitacin I in culture media. At 24 hours of stimulation, cells were analyzed for levels of phosphorylated STAT-3 (pS727) by flow cytometry. Fold changes relative to isotype control were determined (N=5 per condition). (B) B6 splenocytes were incubated with cucurbitacin I at 37°C for one hour, washed clean of drug, rested for two hours, and then stimulated and analyzed as above (N=5 per condition). (C) B6 splenocytes were stimulated as above with plate-bound anti-CD3+CD28 with cucurbitacin I constantly in the culture media, or incubated at 37°C for one hour, washed clean of drug and rested for 24 hours before stimulation. Proliferation and thymidine incorporation was measured from day 1 to day 2 of the assay beginning at 24 hours after stimulation (N=24 per condition).
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