• The mouse Trav2 that is driven by mouse H2-K promoter was inserted into Hipp11 (H11) locus in OT-II mice. The mouse Trbv5 gene is driven by mouse Trav17 promoter was inserted into TIGRE locusin OT-II mice.
• OT-II mice exhibit a significant shift in their T cell population, with increasing in the CD4+ to CD8+ T cell ratio compared to wild-type mice.
• The mouse TCR Vα2+TCR Vβ5+ cells were exclusively detectable in homozygous OT-II mice.
• Antigen-specific activation increases surface CD25 and CD69 expression, IL-2 expression of OT-II CD4+ T cells.
• OT-II mice have normal T cell immunogenic function induced by OVA_323-339.
• Application: this product is used for tracking antigen-specific T-cell responses in vivo and in vitro, investigating thymic selection, and modeling autoimmune or infectious diseases.

Gene targeting strategy for OT-II mice. The mouse Trav2 that is driven by mouse H2-K promoter was inserted into Hipp11 (H11) locus in OT-II mice. The mouse Trbv5 gene is driven by mouse Trav17 promoter was inserted into TIGRE locus in OT-II mice.

The CD4+/CD8 ratio between wild-type C57BL/6JNifdc mice and homozygous OT-II mice. Splenocytes, blood and lymph nodes were collected from wild-type C57BL/6JNifdc mice and homozygous OT-II mice (Male, 7-week old). OT-II mice exhibit a significant shift in their T cell population, with increasing in the CD4+/CD8+ T cell ratio compared to wild-type mice.

Strain specific mouse TCR Vα2 and TCR Vβ5 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-OT-II mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous OT-II mice (H/H), and analyzed by flow cytometry with species-specific anti-mTCR Vα2 antibody (Biolegend, 127807) and anti-mTCR Vβ5.1, 5.2  antibody (Biolegend, 139505). The mouse TCR Vα2+TCR Vβ5+ cells were exclusively detectable in homozygous OT-II mice.

Antigen-specific activation increases surface CD25 and CD69 expression of OT-II CD4+ T cells. Splenocytes from wild-type C57BL/6JNifdc mice and homozygous OT-II mice were exposed for 24h to OVA_323-339. In parallel, medium and OVA_257-264 (10 µg/mL) were included as negative controls. And anti-mCD3e mAb were included positive controls. Percentage of CD25+ (A) and CD69+ (B) gated CD4+ T-cells were assessed by flow cytometry. Flow cytometry analyses of OVA_323–339 challenged CD4+ T cells from OT-II mice showed higher surface expression levels of the early T cell activation markers CD69 and CD25. Data are mean ± SEM values.

Antigen-specific activation increases IL-2 but not IFN-γ expression of OT-II splenocytes. (A) Culture supernatants from wild-type C57BL/6JNifdc mice, homozygous OT-II mice (Biocytogen splenocytes stimulated for 24h with medium, OVA_257-264 (10 µg/mL), anti-mCD3e mAb (0.5 mg/mL) or OVA_323-339 (10 µg/mL) were assayed for IL-2 and IFN-γ levels by ELISA. ELISA analysis of culture supernatants from OVA_323-339-challenged (10 µg/mL) OT-II mice splenocytes for 24h revealed increased levels of IL-2. Data are mean ± SEM values.

Detection of OVA-induced immune responses in OT-II mice by IFN-γ ELISpot assay. Female OT-II mice from the Biocytogen were immunized with intraperitoneal injection of 0.5 mg of OVA protein (Simga, A5503-25MG) and 50 μg poly (I:C) (InvivoGen, tlrl-pic). Three weeks after the last immunization, mice were sacrificed. Mice were immunized with OVA two times at 1-week interval. One week after the last immunization, mice were sacrificed. The splenocytes were extracted, stimulated with OVA_323-339, or no peptide as negative control (NC), or Cell Activation Cocktail (without Brefeldin A), (Biolegend, 42330) as positive control, and then measured for IFN-γ secretion. No significant difference in body weight among groups (Data was not shown). (A) Representative results showing stimulation of splenocytes harvested from immunized mice with negative control, or OVA_323-339, or positive control in duplicates. (B) Summary of results. These data indicate that OT-II mice have specific response to the OVA_323-339.