CFSE^- CD4⁺ T-cell generation depends strictly on interactions with MHC molecules. (A) Lymph node CD4⁺ T cells (5 × 10⁶) were injected intravenously into CD3ϵ^-/-mice and CD3ϵ^-/-MHC II^Δ/Δ mice. Two or 56 days after transfer, ζ protein levels (ζ) and the extent of ζ chain phosphorylation (Pζ) in recovered CD4⁺ T cells were determined by immunoblotting, and the Pζ/ζ ratio was calculated. Purified CD4⁺ T cells from C57BL/6 mice were used as controls. (B-C) CFSE-labeled lymph node CD4⁺ T cells (5 × 10⁶) from C57BL/6 mice were injected intravenously into CD3ϵ^-/- mice and CD3ϵ^-/-MHC II^Δ/Δ mice. CFSE-labeled lymph node CD4⁺β ^-/- T cells (5 × 10⁶) from C57BL/6 β_2m^-/- mice were injected intravenously into CD3ϵ^-/- β_2m^-/- MHC II^Δ/Δ mice. CFSE fluorescence histograms of recovered CD4⁺ TCR^hicells are shown 28 days after transfer (B). Twenty-eight days after transfer the absolute numbers of CFSE⁺ and CFSE^- CD4⁺ TCR^hi cells were calculated (C); data represent mean ± SD.