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Blood, Vol. 111, Issue 8, 4209-4219, April 15, 2008
Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients
Blood Cassani et al.
111: 4209
Supplemental material for: Cassani et al
Files in this Data Supplement:
- Document 1. Supplementary material and methods (PDF, 22.6 KB)
- Figure S1. Restored ADA and SAHH activity in T cells from ADA-SCID patients after gene therapy (PDF, 22.6 KB) -
(A) ADA protein expression detected by intracellular staining in bulk T-cell lines established from ADA-SCID patients, before (ADA-/-, black line) and after HSC gene therapy (GT, grey line), and from healthy controls (ND). The analysis was performed in T cells cultured in FBS-free medium, to avoid the potential interference of bovine ADA in the determination. M.F.I. values are indicated in the histogram plots. (B) ADA and (C) SAHH activity were measured in T cells from five patients and ND (n≥5). The square symbol indicates Pt5.
- Figure S2. Reduced IkBα phosphorylation in ADA-deficient T cells (PDF, 53 KB) -
Anti-phospho-IkB immunoblots of CD4+ T cells stimulated with cross-linked (1 µg/ml) anti-CD3 plus (10 µg/ml) anti-CD28 mAbs for the indicated time points. Filters were stripped and re-probed with anti-G3PDH. Upper panel: two patients (ADA-/- and GT) and two ND. Lower panel: cumulative data of densitometric and statistical analysis (* p<0.05) from four patients (ADA-/- and GT) and four ND are presented. Values corresponding to unstimulated cells were subtracted.
- Figure S3. Analysis of TCR-elicited Ca2+ flux in CD4+ T-cell lines (PDF, 23.7 KB) -
Ca2+ flux in CD4+ T-cell lines from five ADA-SCID patients and five healthy controls measured by Fluo3-AM dye. FACS analysis of Ca2+ flux was performed after stimulation with cross-linked anti-CD3 mAb. Levels of fluorescence were recorder for 400 ms ca., then ionomycin was added. Representative dot plots from Pt4 (ADA-/- and GT) and one healthy control are shown. Analysis of the MFI calculated at the peak and after 400 ms of stimulation is shown for CD4+ T cells of five patients and five healthy controls in one out two independent determinations.
- Figure S4. Expression of A2A adenosine receptor in T-cell lines (PDF, 53.7 KB) -
(A) Expression of A2A adenosine receptor in gated CD4+ T cells. Intracytoplasmic staining was performed in resting cells and M.F.I. values were indicated in the histogram plots. (B) Levels of A2A R mRNA in CD4+ T cells from patients and healthy controls. Total RNA was isolated from either resting or stimulated (1µg/ml antiCD3 plus 10 µg/ml antiCD28) cells and adenosine receptor gene expression was measured by quantitative RT-PCR using the standard curve of a reference sample. Values were measured in duplicates, and normalized by the expression of the housekeeping gene HPRT.
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