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Blood, Vol. 112, Issue 5, 1759-1766, September 1, 2008
Locally produced C5a binds to T cell–expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis
Blood Lalli et al.
112: 1759
Supplemental materials for: Lalli et al
Files in this Data Supplement:
- Figure S1. Serum C3 in bone marrow chimeric animals (JPG, 30.7 KB)
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Serum from each BM chimeric mouse in Fig 1 was obtained and C3 was detected by zymosan uptake assay/flow cytometry. Representative flow cytometry plots are shown.
- Figure S2. DAF and complement influence T-cell apoptosis (JPG, 43 KB)
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Flow cytometry plots showing TUNEL staining of Mar T cells on d 4 after in vitro stimulation with WT, Daf1−/−, C3−/− or Daf1/C3−/− macrophages plus HYDby peptide. Representative of 3 individual experiments. TUNEL assay methods: Terminal dUTP nick-end labeling (TUNEL) was used to detect apoptosis using the APO-DIRECT apoptosis detection kit from BD Biosciences (San Jose, CA) and performed according to the manufacturer’s specifications.
- Figure S3. Local complement activation influences Fas expression on polyclonal alloreactive T cells but does not influence FasL expression (JPG, 55.6 KB)
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(A) Relative Fas expression (MFI, Isotype control---, anti-Fas—) on polyclonal CD4+ H-2b T cells stimulated in vitro with allogeneic H-2d WT, Daf1−/− or C3−/− macrophages HYDby peptide. Results are mean plus s.d. of triplicate wells. (B) Representative flow plots of FasL on the surface of Mar T cells stimulated with WT, Daf1−/− or C3−/− macrophages (d 3). No significant differences among groups. The experiment was repeated 3 times with identical results.
- Figure S4. Complement regulated apoptosis involves PI3-Kγ (JPG, 43.9 KB)
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Mar T cells were stimulated with HYDby + WT macrophages +/− C5a +/−0.1µM PI3-Kγ inhibitor AS252424 as noted and Annexin V staining (left), Fas expression (middle), and total Mar cells per well (right) were determined on day 3. 300,000 Mar T cells were added to each culture at time 0. * p< 0.05 vs. AS252424.
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