Gene targeting strategy for B-hCD3E/h4-1BB/HLA-A2.1 mice.
The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/h4-1BB/HLA-A2.1 mice. The exons 2-7 of mouse Tnfrsf9 gene that encode the extracellular domain were replaced by human TNFRSF9 exons 3-8 in B-hCD3E/h4-1BB/HLA-A2.1 mice. The B2M gene (exon1 to exon3) of mouse were replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains.

Strain specific CD3E expression analysis in homozygous B-hCD3E/h4-1BB/HLA-A2.1 mice by flow cytometry.
Splenocytes were collected from wild-type (WT) mice (+/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice, and analyzed by flow cytometry with species-specific anti-hCD3E antibodies. Mouse CD3E was detectable in WT mice (+/+). Human CD3E was detectable in B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/H) but not in WT mice (+/+).

Strain specific HLA expression analysis in homozygous B-hCD3E/h4-1BB/HLA-A2.1 mice by flow cytometry.
Splenocytes were collected from wild-type (WT) mice (+/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice and analyzed by flow cytometry. Mouse H-2D^b was detectable in WT mice (+/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/+). Human HLA-A2.1 was detectable in B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/H) but not in WT mice (+/+).

Strain specific 4-1BB expression analysis in homozygous B-hCD3E/h4-1BB/HLA-A2.1 mice by flow cytometry.
Splenocytes were collected from wild-type (WT) mice (+/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice stimulated with anti-CD3ε antibody in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibodies. Mouse 4-1BB was detectable in WT mice (+/+). Human 4-1BB was detectable in B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/+) and B-hCD3E/h4-1BB/HLA-A2.1 mice (CD3E^H/H; 4-1BB^H/H; HLA-A2.1^H/H) but not in WT mice (+/+).