Increased frequency of peripheral CD4⁺ T cells undergoing spontaneous proliferation in NOD mice compared with C57BL/6 mice. (A) A total of 10 to 5 × 10⁴ LN CD4⁺ T cells from CD45.2 NOD mice was transferred into CD45.1 NOD Cα^−/− mice. A total of 25 to 5 × 10⁴ CD4⁺ T cells from CD45.1 C57BL/6 mice was injected into CD45.2 C57BL/6 CD3ϵ^−/− mice. Absolute numbers of CD45.2⁺CD4⁺CD8⁻ TCR⁺ cells in NOD Cα^−/− recipient mice (circle) and of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells in C57BL/6 CD3ϵ^−/− recipient mice (diamond) are shown. (B) Absolute numbers of CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells in C57BL/6 CD3ϵ^−/− mice or C57BL/6 Cα^−/− mice receiving 5 × 10³ CD4⁺ T cells. (C) CD25, CD44, CD45RB, and CD69 expression as a function of Foxp3 expression on gated LN CD4⁺CD8⁻ T cells was compared between C57BL/6 and NOD mice. The dot plots shown were generated from the data for 1 mouse, but are representative of 3 individual experiments with at least 2 mice per group. (D) LN CD4⁺ T cells from C57BL/6 and NOD mice were purified, and naive CD4⁺ T cells were electronically sorted on the basis of their nonexpression of CD25 and their low or absent expression of CD44. A total of 5 × 10³ purified naive CD4⁺ T cells (CD4⁺CD25⁻CD44^−/low) from CD45.1 C57BL/6 mice and CD45.2 NOD mice was injected into CD45.2 C57BL/6 CD3ϵ^−/− mice and CD45.1 NOD Cα^−/− mice, respectively. Absolute numbers of recovered CD45.1⁺CD4⁺CD8⁻ TCR⁺ cells in C57BL/6 CD3ϵ^−/− recipient mice and of CD45.2⁺CD4⁺CD8⁻ TCR⁺ cells in NOD Cα^−/− recipient mice are shown 1 month after transfer. ***P < .001; **P < .01; ns, not significant. Each point represents an individual mouse, and horizontal lines represent the mean number of CD4⁺ T cells recovered from positive mice (A-B, D).