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Blood, Vol. 107, Issue 12, 4597-4605, June 15, 2006
Chemokine receptor targeting efficiently directs antigens to MHC class I pathways and elicits antigen-specific CD8+ T-cell responses
Blood Schiavo et al.
107: 4597
Supplemental materials for: Schiavo et al
Files in this Data Supplement:
- Figure S1A. Chemokine-Ag is internalized into lysosomes (PDF, 49.5 KB) -
B6/129 Macrophages were treated with MIP3 -gp100 for 30 min at 37°C or 4°C and processed for immunofluorescence as described in the Methods section. Cells were stained with rabbit anti-LAMP-1(red) to indicate lysosomes and mouse anti-myc mAb (green) in order to detect fusion proteins. Merge, overlay of both channels showing co-localization in yellow. No significant binding was detected when cells were incubated with mutant chemokine fusion (MC148-D-gp100, Data not shown). The experiment was repeated three times with similar results and a representative experiment is shown.
- Figure S1B (PDF, 20 KB) -
Naïve C57BL/6 splenocytes were incubated overnight with 1μg/ml MIP3-gp100 or the gp100 protein alone. The cells were subsequently washed, irradiated and then co-cultured with immune effector splenocytes from C57BL/6 mice (immunized with hgp10025-33/IFA). IFN- release was measured after overnight incubation. Effector cell specificity was validated using splenocytes pulsed with 1g/ml of MHC class I human or murine gp100 peptide (hgp10025-33 and mgp10025-33, respectively), or an irrelevant A20 peptide; or incubating with B16 melanoma or control A20 lymphoma cells.
- Figure S1C (PDF, 21.1 KB) -
Naïve mice immunized with DNA encoding MIP3-gp100 or mDF2  -gp100 (mDF2 is an antimicrobial peptide that also binds to CCR6), but not with pMIP3-D-gp100, a fusion with mutant MIP3, or PBS, induce specific T cell in vivo. Splenocytes from immunized mice (effector cells) produced significant amount of IFN- when stimulated with APC pulsed with hgp10025-33 peptide. The specificity of the effector cells were tested using as target cells splenocytes pulsed with irrelevant MHC peptide (A20 peptide). IFN- release was measured in supernatants of cells cultured for 24 hours by ELISA. A representative experiment is shown from two independent experiments, all yielding similar results.
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