Pulsing the recipient-derived DCs with tumor lysates before priming affects the response to stimulation and the phenotype of the ETCs. Donor-derived ETCs primed with R-DCspulsed (solid) or R-DCsnonpulsed (hollow) were characterized in vitro. (A) The ETCs were stained to study expression of CXCR3 and α4β7. Mean fluorescent intensity of the expression on CD8+ live cells is shown. Values are shown plus or minus SE. (B) ETCs either primed on R-DCspulsed (◆) or R-DCsnonpulsed (◇) were tested for their alloreactivity either in a MLR (left panel) at decreasing stimulator/responder ratios or in a cytotoxicity assay (right panel). Either irradiated allogeneic (B6 solid lines) or syngeneic splenocytes (B10.A broken lines) were used as stimulators. For the cytotoxicity assay ConA blasts of the respective strain were used as targets. Values are shown plus or minus SE, n = 3, P < .05. (C) Differences in the TCR Vβ repertoire of CD8 T cells primed on pulsed or nonpulsed DCs. CDR3 length profile of certain Vβ populations was determined by TCR spectratype analysis. cDNA of naive (top panels), R-DCspulsed, or R-DCsnonpulsed ETCs (bottom panels) were amplified by PCR with 21 Vβ-specific primers (Vβ1, Vβ2, Vβ3, Vβ4, Vβ5.1, Vβ5.2, Vβ6, Vβ7, Vβ8.1, Vβ8.2, Vβ8.3, Vβ9, Vβ10, Vβ11, Vβ12, Vβ13, Vβ14, Vβ15, Vβ16, Vβ17, Vβ18) and 1 Cβ1-Cβ2 primer. FAM-labeled PCR products were analyzed by capillary electrophoresis. The Vβ1 is shown exemplarily for a nonaffected CDR3-profile (left panels), the Vβ5.1 and Vβ5.2 for skewed CDR3-profiles (middle and right panels). Representative results of 3 independent experiments are shown.
