Figure 5. D2.1 cells directly induce CD4⁺ Tregs that can target in vivo.

(A) Whole Jedi splenocytes were stained with CellTrace and stimulated with anti-CD28 antibodies and GFP_200–208 peptides in conditioned medium (CM) from D2A1 cells or D2.1 cells for 3 days followed by flow cytometric analysis. (B) Total T cells (CD45⁺CD11b^–CD4⁺ or CD45⁺CD11b^–CD8β⁺) among immune cells (CD45⁺) at the end of culture. (C) Representative flow plots of CD4⁺ and CD8⁺ T cells among CD45⁺ cells and proportion of total CD4⁺ and CD8⁺ cells among CD45⁺ cells. (D and E) Representative flow plots (left) and quantification of divisions (right) of CellTrace-stained CD8⁺ cells (D) or CD4⁺ cells (E) in D2A1 or D2.1 CM. (F) Representative plots (left) and quantification (right) of FoxP3⁺ Tregs in D2A1 and D2.1 CM after 3 days in culture. (G) CD8⁺/Treg ratio in T cells in D2A1 or D2.1 CM. All conditions were performed in triplicate. Statistical comparisons were performed by 2-tailed t test (B, C, F, and G) or Šídák’s 2-way ANOVA (D and E). (H) Growth of D2.1 tumors (1 × 10⁶ cells/mammary fat pad) treated with anti-CTLA4 antibodies (n = 10) or isotype control (n = 9) upon reaching 150 mm³. (I) D2.1 (1 × 10⁶ cells/mammary fat pad) were implanted into female FoxP3-DTR mice and received 0 ng/g DT (Untreated) or 5 doses of 25 ng/g every 4 days (arrows) beginning on day 35 (DT Early) or day 70 (DT late). (J) D2.1 tumor volume comparing all Untreated (n = 14) mice to DT Early (n = 9) at time of DT Early euthanasia (left) or comparing remaining Untreated mice to DT Late (n = 8 each) at end of experiment (right). Statistical comparisons for H and J were performed by Šidák’s 2-way ANOVA and P values shown are at the final time point. Data are presented as mean ± SEM.