Panel A shows a schematic diagram of the genetic strategy, with the Lag3 gene containing CreERT2 inserted at the translational start site and linked through a 2A peptide, and the Rosa26 locus containing a LoxP-STOP-LoxP-tdTomato cassette that recombines after tamoxifen induction to express tdTomato. Panel B shows the experimental design in which B16-F10 tumors are implanted into Lag3iCreERT2 Rosa26LSL-tdTomato mice, followed by tamoxifen administration on days 0 to 2, 3 to 5, 8 to 10, 13 to 15, or 16 to 18, with tissue collection on day 19, while a control group receives vehicle only. Panel C shows flow cytometry plots and a bar graph of tdTomato expression in Thy1.2 positive T cells from tumors, identifying the most effective labeling schedule for capturing tdTomato positive cells. Panel D shows flow cytometry plots of tdTomato expression in CD8 positive, TCONV (CD4 positive FOXP3 negative), and TREG (CD4 positive FOXP3 positive) cells gated on Thy1.2 positive T cells. Panel E shows flow cytometry plots and pie charts of LAG3 and tdTomato expression in Thy1.2 positive CD8 positive T cells, with the pie charts displaying the proportions of LAG3 positive tdTomato positive, LAG3 positive tdTomato negative, LAG3 negative tdTomato positive, and LAG3 negative tdTomato negative cells recovered from tumors on day 19.
Generation of a tamoxifen-inducible LAG3 + cell–specific fate-mapping Cre line. (A) Schematic of the Lag3iCreERT2Rosa26LSL-tdTomato mouse. (B) B16-F10 tumor implanted in Lag3iCreERT2Rosa26LSL-tdT mice, treated with three daily tamoxifen injections (2 mg in 5% EtOH/sunflower oil) in a time-course–dependent manner (d0–2, d3–5, d8–10, d13–15, and d16–18), and harvested on d19. A subset of mice was given 5% EtOH/sunflower oil as vehicle (Veh) control. (C) Lymphocytes were isolated from tumors, and tdT expression was assessed on the T cell compartment to identify the best labeling scheme to capture tdT+ cells, gated on Thy1.2+ T cells. (D) tdT expression was assessed on CD8+, Tconv (CD4+ FOXP3−), and Treg (CD4+ FOXP3+) cells in tumors treated with tamoxifen, gated on Thy1.2+ T cells. (E) LAG3 and tdT expression was assessed on CD8+ T cells isolated from B, gated on Thy1.2+ CD8+ T cells. Proportion of respective LAG3−tdT−, LAG3+tdT−, LAG3+tdT+, and LAG3−tdT+ CD8+ T cells recovered from B16-F10 tumor at d19. Data in C–E are from n = 5–8 mice for each group, pooled from three independent experiments. *P < 0.05; **P < 0.01; ns, not significant by unpaired t test (C and D). Error bars represent mean ± SEM.