CD5 levels determine the extent of proliferation of CD4⁺ cells in non-lymphopenic, Treg-harboring MBP/R⁺ mice. (A) High-proliferating cells display higher CD5 expression levels than low-proliferating cells. 1 × 10⁷ CFSE-labeled splenocytes from Thy1.1 H-2^u/u C57BL/10.PL mice were transferred into Thy1.2 H-2^u/u MBP/R⁺ mice. Twelve days after transfer, Thy1.1⁺-gated lymph node cells were analyzed (n = 5). (B) H-2^d/u OVA- and MBP-specific T cells express low levels of CD5. CD5 expression levels of CD4⁺ T cell–gated lymph node cells from 4- to 7-week-old H-2^d/u OVA/R⁺ (thick line), MBP/R⁺ (thin line), or wild-type mice (filled histogram). Data are representative of 3 mice. (C) The density of self ligands affects CD5 expression levels. Left: overlay of CD5 expression levels of CD4⁺ T cells in 4- to 7-week-old H-2^d/d OVA/R⁺ (filled histogram) or H-2^d/u OVA/R⁺ (open histogram) mice. Right: similar experiment on T cells from H-2^u/u MBP/R⁺ (filled histogram) or H-2^d/u MBP/R⁺ (open histogram) mice. Data are representative of 3 mice. (D) Relationship between CD5 expression levels and homeostatic proliferation in MBP/R⁺ mice. Top row: CD5 staining before sorting (left); sorted CD5^hi cells (middle); and sorted CD5^low cells (right). Middle row: CFSE-labeled sorted CD5^hi and CD5^low cells were injected into Thy1.2 H-2^u/u MBP/R⁺ recipient mice (n = 4 mice/group). Bottom row: CFSE-labeled sorted CD5^hi and CD5^low cells were injected into Thy1.2 H-2^u/u MBP/R⁺.sf recipient mice (n = 4 mice/group). Twelve days after transfer, single-cell suspensions from peripheral lymph nodes were stained with anti-Thy1.1 and anti-CD4 for FACS analysis. MFI, mean fluorescence intensity.