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. 2005 Apr 18;201(8):1243–1255. doi: 10.1084/jem.20042323

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Copyright © 2005, The Rockefeller University Press

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Figure 6. — Enhanced in vivo immunogenicity of ESO 9V vaccines. (A) A2K^b transgenic mice were immunized with plasmid DNA followed by recombinant vaccinia virus encoding either ESO 9C or ESO 9V peptides. Splenocytes from immunized mice were stained ex vivo 1 wk after vaccinia injection with A2K^b tetramers loaded with ESO 9C peptide (57). Results from four separate experiments were combined, each experiment includes five to eight mice (P = 0.0001). (B and C) Splenocytes from ESO 9V– immunized mice are capable of recognizing ESO 9C peptide. B shows that PBL from mice immunized with ESO 9V vaccines were ex vivo stained with A2K^b tetramers containing either ESO 9C (top row) or ESO 9V (bottom row) peptides. Percentage of tetramer⁺ of CD8⁺ cells is shown in each panel. C shows lysis of target cells (Jurkat cells transfected with A2K^b cDNA) pulsed with either ESO 9V or ESO 9C peptide. Target cells pulsed with the irrelevant peptide flu matrix58–66 (Flu) were used as negative controls. To ensure optimal presentation of the ESO 9C peptide, lysis of target cells pulsed with ESO 9C peptide was compared in the presence or absence of the reducing agent TCEP. In agreement with our previous results (19), ESO 9C pulsed target cells were killed more efficiently in the presence of the reducing agent TCEP (ESO 9C+TCEP) than in the absence of TCEP (ESO 9C). (D) Enhanced IFN-γ response by T cells from ESO 9V–primed mice. Splenocytes from either ESO 9V– or ESO 9C–immunized mice were tested for their ability to recognize target cells pulsed with ESO 9C peptide. Duplicate samples were used. Peptide concentrations and numbers of tetramer positive T cells are shown.