mRNA expression analysis: Mouse Icos mRNA was detected in thymic tissue isolated from wild-type and heterozygous B-hICOS (H/+) mice. Human ICOS mRNA was detected only in heterozygous B-hICOS (H/+) mice. The positive band was confirmed by sequencing. Protein expression analysis: Human ICOS was exclusively detected in homozygous B-hICOS (H/H) mice. Proliferation analysis: CD4+ and CD8+ T cell activation in heterozygous B-hICOS mice (H/+) and homozygous B-hICOS mice (H/H) was upregulated by anti-mCD3ε and human ICOSL recombinant protein, similar to the activation by anti-mCD3ε and anti-mCD28. This demonstrates that the introduction of human ICOS in place of its mouse counterpart does not affect T cell activation. Functional assay: The basal serum levels of IgG1 in B-hICOS mice were significantly higher than those in wild-type mice, which may be caused by the humanization of ICOS. OVA-specific IgG1 and IgE levels in B-hICOS mice were similar to those in wild-type mice.