OVA-specific T cells do not proliferate on restimulation after presentation of OVA by DCs in the steady state and show up-regulation of CD25 and CTLA-4. (A) Mice were reconstituted with OVA-specificT cells and were injected with different antibody-OVA conjugates as indicated. Eight days later, KJ1-26⁺ OVA-specific T cells were purified by MACS and cocultured with OVA-pulsed BMDCs. T-cell proliferation was assayed by ³H-thymidine incorporation. (B) To assess survival of the injected DO11.10 T cells, mice were killed after reconstitution and injection of αDEC-OVA conjugates at the time points indicated. Total T-cell suspensions were prepared using magneto-beads and were stained with FITC-labeled clonotypic antibody KJ1-26 and PE-labeled αCD4. Thereafter, FACS was used to analyze cells. Numbers in the diagrams display the percentage of KJ1-26⁺ cells. (C) For phenotypic analysis, 2 or 8 days after injection of the respective antibody-OVA conjugates, CD4⁺ T cells were purified by MACS and double labeled with FITC-conjugated anticlonotypic TCR antibodies (KJ1-26) and PE-labeled anti-CD25 or anti–CTLA-4 antibodies, respectively. Thereafter, total cell suspensions were analyzed using FACS without further gating, and numbers shown are the percentages of cells within the designated quadrant. (D) Mice were treated as in panel C, and T cells were stained with FITC-labeled anti-CD25 and PE-labeled anti–CTLA-4 antibodies and respective isotype controls. Thereafter, cells were analyzed using FACS.