OT-II mice: Biological Roles and Therapeutic Strategies

• Gene Information: The OT-II mouse carries co-injected transgenes encoding the mouse TCR α-chain (Vα2) and β-chain (Vβ5) under the control of an endogenous TCR promoter, targeting the locus to enforce expression of a single TCR rearrangement.
• Protein Expression: The transgenic Vα2/Vβ5 TCR complex pairs exclusively with the CD4 coreceptor, leading to a marked four-fold increase in the peripheral CD4+ to CD8+ T-cell ratio, where virtually all CD4+ T cells express this specific receptor.
• Signaling Pathway: Upon specific recognition of the chicken ovalbumin residues 323–339 peptide presented by MHC class II (I-A^b) molecules, the transgenic TCR triggers CD3 phosphorylation cascades, driving robust, dose-dependent CD4+ T-cell activation, IL-2 secretion, and proliferation.
• Therapeutic Inhibition: This model serves as a standardized in vivo and in vitro platform to track antigen-specific CD4+ T-cell responses, investigate CD4-mediated helper T-cell (Th1/Th2/Th17) differentiation, and study immunological tolerance or autoimmune disease mechanisms.

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.

• 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.

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).

• The mouse TCR Vα2+TCR Vβ5+ cells were exclusively detectable in homozygous OT-II 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).

• 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.

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. Data are mean ± SEM values.

• 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.

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. Data are mean ± SEM values.

• OT-II mice have specific response to the OVA_323-339.

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.