Ag-experienced T-cell proliferation is preferentially inhibited during on ongoing immune response. (A) Two-cohort experimental design. A first cohort of 10⁶ naive CD45.2 CFSE-labeled Marilyn cells was injected intravenously into CD45.2 B6 mice that were immunized by injection of 10⁶ H-Y peptide–loaded LPS-matured DCs into the footpad the next day. After a variable (n) number of days, a second cohort of either 10⁶ naive, 5 × 10⁶ effector, or 2 × 10⁶ memory CD45.1 CFSE-labeled Marilyn cells was injected. CFSE profiles of gated TCR⁺CD45.1⁺ cells from the popliteal and inguinal lymph nodes draining the immunization site (DLN) were assessed 6 days after transfer of the second cohort. (B) Kinetics of the response of transferred Marilyn TCR Tg T cells. Naive (10⁶) or memory (2 × 10⁶) CD45.1 CFSE-labeled Marilyn cells were injected intravenously into B6 mice that were primed the next day by injection of 10⁶ H-Y peptide–loaded LPS-matured DCs into the footpad. The percentage of transferred T cells (CD45.1⁺TCR⁺) in the draining lymph node was measured at the indicated time points and compared with the percentage measured at day 1 in the absence of antigen. Each dot represents one mouse. Pooled data from 2 independent experiments. (C) Stronger inhibition of Ag-experienced versus naive T-cell proliferation. Naive (left panels), effector (middle panels), or memory (right panels) Marilyn T cells were injected into primed B6 hosts in the absence (top panels) or the presence (bottom panels) of a previous cohort of responding T cells injected 6 days earlier, as in panel A. Dot plots of gated TCR⁺CD45.1⁺ cells from the DLN are representative of at least 3 experiments with 2 mice each per group. The percentage of undivided cells, among total Tg Marilyn T cells, is indicated. (D) Antigen persists in vivo at least 18 days. H-Y peptide–loaded LPS-matured DCs were injected into the footpad of female B6 mice, previously injected (right panels) or not (left panels) with naive CD45.2 CFSE-labeled Marilyn LN cells. At the indicated time points, naive CD45.1 CFSE-labeled Marilyn LN cells were transferred intravenously. Dot plots of gated TCR⁺CD45.1⁺ cells from the DLN injected at the indicated time after DC priming and analyzed 6 days later. Representative of 2 independent experiments with 2 mice per group for each one. (E) Functional avidity measurement in vivo. Naive (10⁶) or memory (2 × 10⁶) CD45.1 CFSE-labeled Marilyn cells were injected intravenously into B6 mice, which were then primed by injection into the footpad of 10⁶ LPS-matured DCs loaded with the indicated amount of H-Y peptide. Five days later, the number of naive and memory T cells recovered in the DLN was measured.