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Blood, Vol. 113, Issue 13, 3008-3016, March 26, 2009
Heat shock protein 70 (HSP70) induces cytotoxicity of T-helper cells
Blood Figueiredo et al.
113: 3008
Supplemental materials for: Figueiredo et al
Files in this Data Supplement:
- Figure S1. Granzyme B expression is affected by sHSP70 but no by the stimulation with sSema7A and sCMVpp65 (JPG, 66.1 KB)
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Secretion of granzyme B by CD3+ (A) and CD4+ (B) T cells obtained from CMV-seronegative donors in the absence of target cells was assessed by ELISA (pg/ml) after 7 days of stimulation with the different proteins alone or in combination with IL-2. (C) mRNA expression of granzyme B by CD4+ T cells was assessed by real-time PCR after 7 days of stimulation with the different stimulation combinations and in the absence of target cells. Results of five independent experiments are expressed as mean ± SD. For statistical analyses (two-tailed t-tests) the values obtained for the recombinant control proteins sCMVpp65 and sSema 7A were compared to the values obtained for sHP70. Statistically significant differences are indicated with asterisks (* p<0.05 or ** p<0.01).
- Figure S2. Secretion of IFN-γ by T cells is target-independent and could be induced by recombinant HSP70 (JPG, 46 KB)
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The capacity of T-cell subpopulations (A: CD3+ T cells, B: CD4+ T cells) from five healthy donors to secrete IFN-γ in the presence of the control proteins (sCMVpp65, sSema 7A) and sHSP70 alone and in combination with 100 U/ml IL-2 was assessed by ELISA (pg/ml). Stimulation with sHSP70 resulted in a significant increase of IFN-γ secretion compared to the secretion levels achieved in the presence of the control proteins sCMVpp65 and sSema7A. Statistically significant differences between the stimulation with sHSP70, sCMVpp65 and sSema7A are indicated with asterisks (* p<0.05 or ** p<0.01).
- Figure S3. sHSP70 in combination with IL-2 induces T-cell proliferation and expression of CD25 (JPG, 78.2 KB)
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CD3+ T cells were labeled with CSFE and stimulated under different conditions during one week. Cell proliferation within the CD4+ T-cell populations was evaluated by CFSE dilution. Cells were stained with monoclonal anti-CD25 APC antibody. One representative experiment of CD4+ T-cell proliferation under different stimulation conditions and using the control proteins is shown. The percentage (%) of CD25+ cells is given.
- Figure S4. Analysis of sHSP70 uptake by in vitro polarized Th1 and Th2 cells using fluorescent microscopy (JPG, 89.4 KB)
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Representative results of fluorescent microscopic images of in in vitro polarized Th1 (cultured in the presence of IL-12, IFN-γ and anti-IL-4) versus Th2 (cultured in the presence of IL-4 and anti IL-12) CD4+ T-cell populations incubated with sHSP70-FITC. CD3-PE-stained polarized Th1 cells (A), polarized Th2 cells (D) and after sHSP70-FITC internalization (Th1: B, Th2: E) are depicted separately, as well as with the merged images (Th1: C, Th2: F).
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