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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25844_sm_SuppFig1.tif1100KSupporting Information Figure 1: Uptake of circulating CEA and OVA in antigen-presenting cells in the spleen. Alexa647-labelled CEA (A) or OVA (B) protein was injected intravenously into C57BL/6 and MR-/- mice. 5 minutes after injection splenocytes were isolated stained for CD11b or CD11c and analyzed by flow cytometry. Mean fluorescence intensity of splenic CD11c+ or CD11b+ from C57BL/6 or MR-/- mice previously injected with CEAAlexa647 (A) or OVAAlexa647 (B). Shown are cumulative results from 3 independent experiments.
HEP_25844_sm_SuppFig2.tif1128KSupporting Information Figure 2: CEA does not affect antigen presenting cells. (A) LSEC from C57BL/6 or TLR4-/- mice were stimulated with LPS (0.1μg/ml) or with indicated amount of CEA or OVA in vitro. IL-6 secretion was measured by ELISA. Shown are cumulative results from 3 independent experiments.
HEP_25844_sm_SuppFig3.tif1287KSupporting Information Figure 3: Growth of MC38 tumors not expressing CEA is not affected by CEA-specific T cells. C57BL/6 (A), B7H1-deficient (B) or MR-deficient (C) mice received 2 mg of CEA protein i.v. every second day, were immunized with the pGT64 CEA plasmid or were left untreated. After 2 weeks splenic CD8 T cells from these C57BL/6 (A) B7H1-/- (B) or MR-/- mice (C) were obtained and 5x106 total CD8 T cells were adoptively transferred into Rag2-deficient mice. Three days later, 5x106 MC-38 cells were injected into the left flank. Graphs show tumor growth of MC38 in Rag2-/- mice that received CD8 T cells from C57BL/6 (A), B7H1-/- (B) or MR-/-mice (C).
HEP_25844_sm_SuppFig4.tif769KSupporting Information Figure 4: Phenotypical tolerant CD8 T cells do not proliferate or degranulate upon stimulation. PBMC from healthy individuals were isolated from blood and sorted for CD8, CD45RO, CD62L and CD25 using FACS-Sort. Isolated T cells labeled with CFSE (A, B) or not (C) and were stimulated with anti-CD3ε/CD28 beads(A-C). Proliferation was measured by CFSE dilution using flow cytometry. A: % of proliferated CD8 T cells. B: CFSE dilution of sorted CFSE-labeled CD8 T cells. C: Surface CD107a expression on sorted CD8 T cells 1h after stimulation. Shown are results from at least 3 independent experiments.
HEP_25844_sm_SuppInfo.doc45KSupporting Information
HEP_25844_sm_SuppTable1.doc30KSupporting Information Table 1.

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