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Blood, Vol. 112, Issue 2, 362-373, July 15, 2008
Tumor-specific Th17-polarized cells eradicate large established melanoma
Blood Muranski et al.
112: 362
Supplemental materials for: Muranski et al
Files in this Data Supplement:
- Figure S1. Minimal epitope recognized by the TRP-1 T cell receptor (JPG, 95.9 KB)
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TRP-1–specific CD4+ hybridoma clone 7A6 generated from Bw mouse after multiple rounds of vaccination against TRP-1 recognizes the minimal epitope corresponding to amino acids 113-127 of TRP-1 protein as tested by IL-2 release in ELISA using differently truncated TRP-1 peptides.
- Figure S2. TRP-1 CD4+ T cells are negatively selected in tyrp1 antigen positive RAG1−/− transgenic hosts and can be detected only in RAG1−/−Bw TRP-1 transgenic mice (JPG, 70.3 KB)
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Lymph nodes from RAG1−/− TRP-1 TCR transgenic wt (black, tyrp1+/+,) and Bw (cappuccino, tyrp1−/−,) mice were analyzed for presence of CD4+ and CD8+ cells (upper row) and CD4+ and Vb14+ cells (lower row). C57/BL6 wild type mice is shown as a positive control for antibody staining. Only in the absence of the antigen TRP-1 CD4+ T cells are selected and detectable in secondary lymphoid organs.
- Figure S3. Intracellular staining for cytokines by polarized TRP-1 cells (JPG, 92.8 KB)
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TRP-1 CD4+ T cells were cultured in vitro under neutral (Th0) or polarizing (Th1, Treg and Th17) conditions for 1 week. They were re-stimulated with PMI/ionomycin in the presence of brefeldin A and analyzed by flow cytometry for intracellular IFN- and IL-17A. Non-stimulated cells are shown as negative control. The results are representative of multiple experiments.
- Figure S4. Expression of Foxp3 in Th0, Th1 and Th17-skewed populations (JPG, 33.5 KB)
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TRP-1 CD4+ T cells were cultured under different polarizing conditions. Intracellular staining for Foxp3 expression was performed on day 8 of culture. Percentage of positive cells was calculated based on the comparison with an isotype control antibody.
- Figure S5. Th17 TRP-1 cells demonstrate persistence advantage after transfer into tumor bearing non-irradiated RAG1−/− mice (JPG, 75.7 KB)
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Mice were injected with 106 Th0, Th1 or Th17 cells or left untreated as a control (NT). Spleens, lymph nodes and tumors were harvested 6 days after transfer analyzed for presence of V 14+ CD4+ cells. The frequency of V14+ CD4+ double positive cells was higher in Th17 treated animals. The results are representative of two independent experiments.
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