Thymic CD4⁺ single-positive thymocytes require MHCII contact to proliferate during IL-7 therapy. Enriched CD45.1⁺CD4⁺_SPT cells from thymuses were labeled with CFSE prior to their transfer into CD45.2⁺C57BL/6-MHC^+/+ or CD45.2⁺C57BL/6-MHC^−/− hosts. (A) Representative flow cytometric analysis of transferred CD4⁺_SPT into MHC^+/+ or MHC^−/− hosts after 6 days of IL-7 therapy. (B) Histogram analysis showing the absolute number of CD45.1⁺CD4⁺_SPT cells. (C) Experiment in (A) was repeated and cells were parked for 3 days in their appropriate recipient prior to initiating IL-7 therapy. (D) Histogram analysis showing the absolute number of CD45.1⁺CD4⁺_SPT recovered in MHCII^+/+ and MHCII^−/− hosts after parking and 6 days of IL-7 therapy. (E) Flow cytometric analysis showing STAT5-phosphorylation in CD4⁺_PERI exposed to varying concentrations of IL-7 ^+/− anti-CD3 stimulation. Data are representative of 2 mice per group. (F) Schematic representation of the experimental design where mice were conditioned with FLT3 ligand for 14 days and peripheral FL-CD4⁺_PERI or PBS-CD4⁺_PERI isolated and transferred into wild-type mice treated with IL-7 for 6 days. (G) Flow cytometric analysis showing CFSE profile of FL-CD4⁺_PERI or PBS-CD4⁺_PERI after 6 days of IL-7 treatment. (H) Graphic representation of the percentage of FL-CD4⁺_PERI or PBS-CD4⁺_PERI proliferating cells. Data show 6 mice pooled from 3 independent experiments. (I) Graphic summary of the relative expression of miR181a transcript in peripheral CD4⁺GFP⁻, CD4⁺_RTE GFP⁺, and CD4⁺_SPT GFP⁺. Data show 3 mice per group pooled from 3 independent experiments.