Codominant expression of human and mouse CTLA-4 protein by T cells from human CTLA-4 knock-in heterozygotes. (A) Codominant expression of human and mouse CTLA-4 in T cells after antigen stimulation. Spleen cells from human CTLA-4^+/–×P1CTL F1 mice were stimulated for 66 hours in vitro with 0.1 μg/mL P1A peptide. Cells were harvested and stained for cell surface mouse CD3, followed by intracellular mouse and human CTLA-4. The top left panel shows the codominant expression of human and mouse CTLA-4 protein on the same cells as indicated by the diagonal staining pattern. Non–knock-out littermates demonstrated a complete lack of human CTLA-4 expression (bottom left panel). Middle and right panels show isotype controls for each intracellular antibody. All profiles represent cells within the CD3⁺ gate. The same staining pattern has been observed with anti-CD3 mAb–stimulated T cells (K.M., unpublished observations). (B) Expression of mouse and human CTLA-4 molecules in unstimulated spleen CD4 T cells. Spleen cells from WT (CTLA-4 mo/mo), homozygous (human CTLA4^+/+) and heterozygous (human CTLA4^+/–) mice were surfaced-stained with anti-CD4 and anti-CD25 and then stained for intracellular mouse and human CTLA-4 protein. Data shown were gated CD4⁺CD25^– (top panels) and CD4⁺CD25⁺ subsets (bottom panels).