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Blood, Vol. 111, Issue 3, 1464-1471, February 1, 2008
CD28 provides T-cell costimulation and enhances PI3K activity at the immune synapse independently of its capacity to interact with the p85/p110 heterodimer
Blood Garçon et al.
111: 1464
Supplemental materials for: Garçon et al
Files in this Data Supplement:
- Figure S1 (JPG, 51.4 KB)
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(A) Purified WT or AktPH-GFP+ CD4+ T cells were stimulated with anti-CD3 and anti-CD28 mAb for 5 min, and phosphorylation of Akt was assessed by Western blotting of whole cell lysates with antibody to phosphorylated Akt (Ser473) (top panel). Blots were also probed with antibody to total Akt (bottom panel). (B) Purified C57BL/6 or AktPH-GFP+CD4+ T cells were stimulated with B cells loaded with the indicated concentration of OVA323-339 peptide. Proliferation was measured by 3HThymidine incorporation during the last 16 h of a 66 h culture. (C–D) Antibody-stimulated phosphorylation of Akt in CD4+ T cells. Purified CD4+ T cells were incubated with anti-CD3 and anti-CD28 on ice, followed by cross-linking with anti-hamster Ig for the indicated times at 37°C. Lysates were separated by SDS-PAGE and transferred to membranes. (C) T cells were stimulated with 10 µg/ml anti-CD3 and 10 µg/ml anti-CD28 for 1-30 min. (D) T cells were stimulated with 0, 0.1 or 10 µg anti-CD3 +/− 10 µg/ml anti-CD28 for 5 min.
- Figure S2. Surface phenotype of B cells used as APCs for conjugates experiments (JPG, 24.9 KB)
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Purified splenic B cells were analyzed for surface expression of CD80 and CD86. Solid color, isotype control; solid line, APC-conjugated antibody.
- Figure S3. Quantification of AktPH-GFP redistribution at the membrane in individuals WT, p110δD910A/D910A, p110γ−/−, CD28−/− and CD28Y170F CD4+ T cells used to calculate average values in Fig 1C (JPG, 70.7 KB)
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The outliers and the average values are highlighted.
Supplementary Videos
AktPH-GFP relocalization during immunological synapse formation. Representative video versions as presented in Fig 1A. Time-lapse series of GFP-fluorescent (left) and fluorescent-DIC overlay (right) images of CD4+ T cells interacting with APCs loaded with 10 µM of OVA323-339 peptide.
- Video 1. WT CD4+ T cells (AVI, 5.15 MB)
- Video 2. WT CD4+ T cells pre-treated with the PI3K inhibitor LY294002 (AVI, 8.28 MB)
- Video 3. p110δD910A/D910A CD4+ T cells (AVI, 8.08 MB)
- Video 4. p110γ−/− CD4+ T cells (AVI, 7.60 MB)
- Video 5. CD28−/− CD4+ T cells (AVI, 2.61 MB)
- Video 6. CD28Y170F CD4+ T cells (AVI, 4.70 MB)
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In videos 7–9, AktPH-GFP relocalization in response to stimulation with the chemokine CXCL-12 (shown in Fig 2A).
- Video 7. WT CD4+ T cells stimulated with CXCL12 (AVI, 335 KB)
- Video 8. p110δD910A T cells (AVI, 411 KB)
- Video 9. p110γ−/− T cells (AVI, 565 KB)
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