Frequency of regulatory CD4⁺ T cells recovered after spontaneous proliferation in a lymphopenic environment. (A) A total of 5 × 10⁶ LN CD4⁺ T cells from CD45.1 C57BL/6 mice was purified, labeled with CFSE, and transferred into C57BL/6 CD3ϵ^−/− hosts. Two and 28 days after transfer, pooled LNs and spleen were analyzed for Foxp3 expression. Fluorescence Foxp3/CFSE dot plots are shown for CD45.1⁺ CD4⁺ T cells 2 and 28 days after transfer. (B) A total of 5 × 10³ to 5 × 10⁶ CFSE-labeled T cells from CD45.1 C57BL/6 mice was transferred into C57BL/6 CD3ϵ^−/− mice and analyzed 1 month after transfer. Proportion of CD45.1⁺ CFSE⁻ CD4⁺ T cells expressing Foxp3 is shown as means ± SEM for 3 independent experiments. The gray area indicates the mean ± SEM proportion of Foxp3-expressing cells among peripheral CD4⁺ T cells from control C57BL/6 mice. ***P < .001; *P < .05; ns, not significant. (C) A total of 10⁶ CD4⁺CD25⁺ T cells from CD45.1 C57BL/6 mice and 10⁶ CD4⁺CD25⁻ T cells from CD45.2 C57BL/6 mice was labeled with CFSE and coinjected into C57BL/6 CD3ϵ^−/− mice. Representative Foxp3 fluorescence histograms of gated CD45.1⁺ or CD45.1⁻ CFSE⁻ CD4⁺ T cells 1 month after transfer. Percentages of Foxp3⁺ cells are indicated.