Gene expression in CD4⁺ T cells in response to transient or persistent antigen presented by steady-state or activated DCs. (A) Experimental outline. Host mice were treated as indicated, transferred with unlabeled CD90.1⁺AND T cells. These were sorted 60 h later and their RNA analyzed with microarrays. (B) Gene expression in AND T cells primed as indicated in A, compared with untreated control animal 1. Genes up- or down-regulated relative to control by >2-fold are depicted in black, and their numbers are indicated. (C) The kinetics of antigen presentation regulates gene expression in CD4⁺ effector cells. (Upper) The FC/FC plot compares expression values of treatments 3 and 5 relative to the control condition 1. (Lower) Magnified view of the Inset in Upper on activation response transcripts (5/1 FC >3). Color-coding distinguishes three sets of activation-induced transcripts with different response modes and is detailed in SI Methods.

Deletion versus memory-cell development depends on both antigen persistence and additional features of DC activation. (A) CD45.1⁺ AND LN cells were transferred into dtg mice. At 3, 10, and 24 days later, s.c. LNs were surgically removed and analyzed by flow cytometry. On day 25, all animals were challenged by antigen reexpression (together with anti-CD40 to maximize responses), and LN cells were analyzed 3 d later. Numbers indicate the proportion of donor CD4⁺ T cells. Splenocytes were also restimulated in vitro and stained for IFNγ (gray histograms) or a control mAb (open histograpms). (B) Compilation of several experiments. Each point depicts the percentage of CD4⁺CD45.1⁺ T cells in s.c. LNs; each symbol represents an independent experiment.

Comparison of CD4⁺ T cell responses in animals with transient and persistent TIM expression and activated DCs. (A) Lymph node (LN) and spleen (SPL) cells of dtg recipients of CFSE-labeled CD90.1⁺ AND T cells were analyzed 60 h after transfer. Dtg mice were treated with dox for 1 or 3.5 d (gray boxes), and treated with a stimulatory αCD40 mAb (thick black arrow). Numbers indicate the percentages of donor cells among CD4⁺ cells. Representative of three experiments. (B) For IFNγ analysis, lymph node cells were restimulated for 6 h and stained intracellularly for IFNγ (open histogram) or with an isotype control-Ig (filled histogram). Results are representative of two experiments. Histograms are gated on CD4⁺CD90.1⁺ cells.

Effector CD4⁺ T cell gene expression is shaped by the kinetics of antigen presentation. FC/FC plots compare changes elicited by transient antigen without (Left) and with DC activation via CD40 (Center) and by continuous antigen without DC activation (Right) relative to responses to persistent antigen presentation by activated DCs (x axes). Correlation coefficients of r² = 0.34, 0.84, 0.94 (Left, Center, and Right, respectively); the slopes of the linear regression trend lines are shown. Data and conditions are as in Fig. 2.

In vivo DC activation extends MHC class II-restricted antigen presentation in dtg mice, but not after mAb-mediated antigen targeting. (A) Dtg mice were treated as depicted before the transfer of CFSE-labeled cells from CD90.1⁺ AND TCR-tg mice. CFSE profiles of CD4⁺CD90.1⁺ LN and SPL cells were analyzed 60 h later. Control mice received PBS or irrelevant rat IgG2a mAb. Data are representative of three experiments. (B) MCC/E^k complexes disappear from activated DCs ≈6 d after the turn-off. Experiment is as in A, with the time between 24-h dox feeding and the AND cell transfer varied from 0 to 9 d. Recipients were injected with PBS (open circles) or αCD40 (filled squares) at the initiation of dox exposure. (C) The effect of αCD40 vanishes within 3 days. Experiment performed as in A, except that dtg animals were treated with dox at different times relative to αCD40. Representative of three experiments. (D) Extended antigen presentation with mAb-mediated antigen targeting to DCs is not detectable. B10.BR animals were injected with hen egg lysozyme coupled to 33D1 or an isotype control and 6 h later with αCD40. Spleen CD11c⁺CD8⁻ DCs were analyzed 1 and 4 d later with Aw3.18 (filled histograms) or a control mAb (open histograms). Data are representative of two experiments.