The spontaneous proliferation of regulatory T cells in a lymphopenic environment requires help from conventional CD4⁺ T cells. (A) A total of 10² LN CD4⁺ T cells from CD45.1 C57BL/6 mice was injected into CD45.2 C57BL/6 CD3ϵ^−/− mice. Fluorescence dot plots show TCR, CD44, CD25, and Foxp3 expression on gated CD4⁺CD8⁻ T cells from a control C57BL/6 mouse and a representative recipient mouse 1 month after transfer. (B) A total of 10² CD4⁺CD25⁻ or CD4⁺CD25⁺ T cells from CD45.1 C57BL/6 mice was injected into CD45.2 C57BL/6 CD3ϵ^−/− mice, alone or together with 5 × 10⁴ CD45.2⁺CD4⁺ T cells. One month later, the presence of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells in the periphery of recipient mice was individually tested. Left: absolute numbers of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells; right: frequency of positive mice and absolute numbers of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells in positive mice. Data show means ± SEM of mice for 3 independent experiments. (C) A total of 10² CD4⁺CD25⁺ T cells from CD45.1 C57BL/6 mice and 5 × 10⁴ CD8⁺ T cells from CD45.2 C57BL/6 mice was cotransferred into C57BL/6 CD3ϵ^−/− IIKO or C57BL/6 CD3ϵ^−/− mice. The absolute numbers of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells recovered from recipient mice 1 month after transfer are shown. (D) A total of 10² CD45.1⁺CD4⁺CD25⁺ T cells from C57BL/6 mice was coinjected with 5 × 10⁴ CD4⁺ T cells from C57BL/6 IL-2^−/− mice or C57BL/6 control littermates into C57BL/6 CD3ϵ^−/− mice. Absolute numbers of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells recovered from recipient mice 1 month after transfer are shown. Each point represents an individual mouse, and horizontal lines represent the mean number of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells in positive mice (B left; C-D). ns, not significant; NA, not applicable.