D^d ligation alone stimulates proliferation of CD3^-CD5⁺ cells from D^d-ζ transgenic mice. (A) Freshly isolated splenocytes from homozygous D^d-ζ (130 strain, ▪) or MHC-D^d (▥) mice were incubated with saturating amounts of anti-CD3 or anti-D^d (34-2-12), washed, and cross-linked with plate-bound anti-IgG. After 48 hours the plates were pulsed with ³H-thymidine and harvested 16 hours later. Proliferation was measured by 3H-thymidine uptake and expressed as counts per minute (CPM). (B) Fresh splenocytes (4 million cells) from D^d-ζ mice were CFSE labeled and incubated for 3 days with 10 μg/mL anti-D^d (34-5-8S), irradiated EL4 (0.5 million, 2000 rad), or 2.5 μg/mL concanavalin A (Con A). The cells were stained with Ab to CD3 and CD5, gated as indicated, and evaluated for CFSE loss. EL4-stimulated proliferation of D^d-ζ cells was almost completely blocked by anti–Ly-49A (unpublished observations, March 26, 2001).