Acute requirement for IL-7 for optimal triggering of T cells. F5 T cells from CD45.1⁺ control F5 and CD45.1⁻ F5 TetIL-7R mice off doxycycline 7 days were CFSE-labeled, mixed at a 1:1 ratio, and transferred (3 × 10⁶ total T cells/mouse) to groups of Rag1^−/− or Il7^−/− Rag1^−/− hosts. Recipient mice were further challenged with flu virus (24 U/mouse). At day 3, mice were culled and F5 T cells populations in spleen analyzed by FACS. (A) Histograms show CFSE profiles of CD8⁺ TCR^hi F5 T cells from the donor mice and in the hosts indicated. (B) Box plot shows the ratio of control F5 to F5 TetIL-7R donor T cells triggered into division in the indicated hosts. Statistics: *P < .016, comparing ratios in Rag1^−/− and Il7^−/− Rag1^−/−. Comparison of frequency of triggered control and F5 TetIL-7R T cells in Rag1^−/− hosts (**P < .001) and in Il7^−/− Rag1^−/− hosts (***P = .66).

IL-7 enhances T cell–DC interactions. (A) Histogram shows class II MHC expression by F5 T cells recovered from spleen 7 days after transfer to Il7^−/− Rag1^−/− (broken line) or Rag1^−/− (solid line) hosts (n = 5 per group) compared with F5 T cells from control F5 mice (gray fill). Graph shows I-A^b staining after transfer to Rag1^−/− (■) or Il7^−/− Rag1^−/− hosts (□) as percentage of expression by F5 T cells from control mice. Data are representative of 3 or more experiments. (B) CD45.1⁺ IL-7R⁺ and CD45.1⁻ IL-7R⁻ F5 T cells were cotransferred to Rag1^−/− hosts (n = 4) and cells analyzed for class II MHC expression 1 day later. Histograms show I-A^b staining by IL-7R⁺ and IL-7R⁻ F5 T cells before (broken line) and after (solid line) transfer to Rag1^−/− hosts. Bar chart is MFI of I-A^b staining by the indicated F5 T cell donor after transfer to Rag1^−/− hosts (*P < .02).

Saturating antigen-presenting capacity overcomes the triggering defect in IL-7R⁻ F5 T cells. (A) Control F5 T cells were CFSE-labeled and transferred to Rag1^−/− hosts (3 × 10⁶ cells/mouse), and groups of mice were challenged with a range of NP68 peptide doses at 0 and 18 hours after transfer. At day 3, mice were culled and the responding T-cell population analyzed by FACS. The scatter plot shows the percentage of control F5 T cells triggered into division in individual mice challenged with different doses of peptide. (B) F5 T cells from CD45.1⁺ control F5 (IL-7R⁺ F5) and CD45.1⁻ F5 TetIL-7R mice off doxycycline 7 days (IL-7R⁻ F5) were CFSE-labeled, mixed at a 1:1 ratio, and transferred (3 × 10⁶ total T cells/mouse) to groups of Rag1^−/− hosts challenged with a range of NP68 peptide doses. At day 3, mice were culled and CFSE profile of IL-7R⁺ F5 and IL-7R⁻ F5T cells analyzed by FACS. Scatter plot shows the percentage of F5 T cells triggered into division for IL-7R⁺ F5 (x-axis) versus IL-7R⁻ F5 (y-axis) T cells in the same recipient (B) and ratio of IL-7R⁻ F5: IL-7R⁺ F5 as a function of peptide dose (C). Data are representative 2 independent experiments.

T-cell triggering is defective over a wide range of antigen doses. (A) Control F5 T cells were CFSE-labeled and transferred to Rag1^−/− hosts (3 × 10⁶/mouse), and groups of mice were challenged with a range of flu doses. At day 3, mice were culled and the responding T-cell population in the spleen analyzed by FACS. The scatter plot shows the percentage of F5 T cells triggered into division in individual mice challenged with different doses of flu virus. (B-D) F5 T cells from CD45.1⁺ control F5 and CD45.1⁻ F5 TetIL-7R mice off doxycycline 7 days were CFSE-labeled, mixed at a 1:1 ratio, and transferred (3 × 10⁶ total T cells/mouse) to groups of Rag1^−/− hosts challenged with 1.6, 6, 24, or 100 U flu virus. At day 3, mice were culled and CFSE profile of CD8⁺ TCR^hi CD45.1⁺ control (IL-7R⁺ F5) and CD45.1⁻ F5 TetIL-7R F5 T cells (IL-7R⁻ F5) analyzed by FACS. Plot shows ratio of IL-7R⁻ F5/IL-7R⁺ F5 cells triggered into division as a function of flu dose (B). Scatter plot shows percentage of F5 T cells triggered into division for IL-7R⁺ F5 (x-axis) versus IL-7R⁻ F5 (y-axis) cells in the same recipient challenged with the flu dose indicated (C). The scatter plot shows the mean division of triggered IL-7R⁺ F5 (x-axis) versus IL-7R⁻ F5 T cells in individual hosts (D). Data are pooled from 3 independent experiments.

IL-7 enhances T-cell stimulation by DCs to induce LIP. (A) T cells from F5 Rag1^−/− donors were transferred (2 × 10⁶ /mouse) to Rag1^−/−, β2m^−/− Rag1^−/−, or Il7^−/− Rag1^−/− hosts (n = 5/group). At various days, spleens were taken from recipient mice and stained for CD8, TCR, and CD5. Histograms of CD5 expression 7 days after transfer of F5 T cells to Rag1^−/− hosts (solid line) and Il7^−/− Rag1^−/− (broken line) hosts compared with expression by F5 T cells from control F5 Rag1^−/− mice (gray fill). Histograms of CD8 expression are of F5 T cells 7 days after transfer to Rag1^−/− hosts (solid line), β2m^−/− Rag1^−/− hosts (gray fill), and Il7^−/− Rag1^−/− hosts (broken line). Graph shows CD5 expression after transfer to Rag1^−/− (solid) or Il7^−/− Rag1^−/− hosts (empty) as percentage of expression by F5 T cells from control mice. (B) F5 T cells were CFSE-labeled and transferred (2 × 10⁶ cells/mouse) to Rag1^−/− and Il7^−/− Rag1^−/− hosts. At days 7, 14, and 21, lymphocytes were recovered and stained for expression of CD8 and TCR. Histograms are of representative CFSE profiles at day 14 and indicate gates used to examine cells that had undergone different number of division. Line graphs show TCR expression by F5 T cells that have undergone different numbers of divisions at day 7 (□), day 14 (◪), and day 21 (■) after transfer into the hosts indicated normalized to expression in control F5 Rag1^−/− mice.

Defective triggering of F5 T cells in the absence of IL-7R expression. F5 T cells from CD45.1⁺ control F5 and CD45.1⁻ F5 TetIL-7R mice off doxycycline for 7 days were CFSE-labeled, mixed at a 1:1 ratio, and transferred (3 × 10⁶ total T cells/mouse) to groups of Rag1^−/− hosts. Groups of recipient mice (n = 3) were further challenged with flu virus intravenously (24 U/mouse). At day 3 after transfer, mice were culled and donor populations among host splenocytes analyzed by FACS. (A) Dot plots are of CFSE versus CD45.1 expression by CD8⁺TCR^hi cells from naive or flu-challenged recipients. (B) Histograms are of CFSE profiles for CD45.1⁺ control F5 T cells (IL-7R⁺ F5) and CD45.1⁻ F5 T cells from F5 TetIL-7R donors (IL-7R⁻ F5). Numbers indicate the average percentage of F5 T cells triggered into division plus or minus SD in each case. *P < .008; n = 6. (C) Histograms show cell size as determined by Forward Scatter (FSc) signal for divided and undivided control (solid line) and IL-7R⁻ (gray fill) F5 T cells. Data are representative of 6 independent experiments.

IL-7 does not affect T-cell activation in vitro. (A) Total lymph node cells from F5 Rag1^−/− mice were cultured (10⁶/mL) with 10 nM NP68 in the presence or absence of IL-7 (10 ng/mL). Histograms are of pZap70 and CD69 levels on CD8-gated T cells in unstimulated (gray fill) or NP68-stimulated cultures with (solid line) or without (broken line) IL-7 for the times indicated. pSTAT5 staining is shown for IL-7–stimulated cells cultured with (solid line) or without (broken line) NP68 peptide compared with unstimulated cultures lacking IL-7 and NP68 (gray fill). (B,C) Total F5 lymph node cells were labeled with CFSE and cultured (10⁶/mL) with a range of peptide doses. At day 3, CFSE profile of viable CD8⁺ T cells was analyzed by FACS. Graphs show the percentage cells triggered into division (B) and mean division of triggered cells (C) in response to different doses of agonist NP68 peptide (circles) or the weak agonist NP34 (diamonds) in the absence (empty symbols) or presence (filled symbols) of IL-7 (10 ng/mL). (D) Histograms show expression of IL-7R, TCR, and CD8 by IL-7R⁺ F5 T cells from control (solid line) and IL-7R⁻ F5 T cells from F5 TetIL-7R mice off doxycycline for 7 days (gray fill). Histogram of IL-7R by CD4⁺CD8⁺ DP F5 thymocytes (broken line) is shown as negative control. (E) The graph shows CD69 expression at 18 hours by IL-7R⁺ F5 splenocytes of control mice (●) and CD8⁺ IL-7R⁻ F5 splenocytes from F5 TetIL-7R mice off doxycycline for 7 days (○), stimulated with different doses of NP68 peptide. Data are representative of 3 or more experiments.