TNFR2 encodes TNF receptor 2, a member of the TNF receptor superfamily. TNF receptor 2 and TNF receptor 1 form a heterocomplex that recruits two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. While the role of IAPs in TNF receptor signaling remains unclear, c-IAP1 is believed to enhance TNF-induced apoptosis by promoting the ubiquitination and degradation of TNF receptor-associated factor 2 (TRAF2), a mediator of anti-apoptotic signaling. Knockout studies in mice further indicate that this protein helps protect neurons from apoptosis through the activation of antioxidant pathways.
In TFNR2 humanized mice (B-hTFNR2), exons 2-6 of mouse Tfnr2 gene that encodes the extracellular domain was replaced by human TFNR2 exons 2-6. Human TNFR2 expression was detectable exclusively in homozygous B-hTNFR2 mice and not in wild-type mice. Humanization of TNFR2 does not alter immune cell frequency or distribution in the spleen or lymph nodes.
Key Advantages
Validation
Applications
Human exons 2–6 of TNFR2 replace the mouse Tnfr2 exons encoding the extracellular domain in TNFR2 humanized mice (B-hTNFR2).
Strain specific analysis of TNFR2 gene expression in wild-type C57BL/6 mice and homozygous TNFR2 humanized mice (B-hTNFR2) by RT-PCR. The splenocytes RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous TNFR2 humanized mice (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TNFR2 primers.
Strain specific analysis of TNFR2 gene expression in wild-type C57BL/6 mice and homozygous TNFR2 humanized mice by RT-PCR. The splenocytes RNA was isolated from wild-type C57BL/6 mice (+/+) and homozygous TNFR2 humanized mice (H/H), and then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TNFR2 primers.
Strain specific TNFR2 protein expression analysis in wild-type C57BL/6 mice and homozygous TNFR2 humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous TNFR2 humanized mice (H/H), and analyzed by flow cytometry with species-specific anti-TNFR2 antibody (anti-mouse CD120b (TNF R Type II/p75) Antibody, Biolegend, 113405; anti-human CD120b, Biolegend, 358403).
Strain specific TNFR2 expression analysis in wild-type C57BL/6 mice and homozygous TNFR2 humanized mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous TNFR2 humanized mice (H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-TNFR2 antibody (anti-mouse CD120b (TNF R Type II/p75) Antibody, Biolegend, 113405; anti-human CD120b, Biolegend, 358403).
Analysis of leukocyte subpopulations by flow cytometry in immune organs of TNFR2 humanized mice. Splenocytes and lymph nodes were isolated from female C57BL/6 and TNFR2 humanized mice (female, 6-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.
Analysis of splenocytes of TNFR2 humanized mice by flow cytometry. Splenocytes were isolated from TNFR2 humanized mice (female, 6-week-old, n=3) treated with anti-mouse CD3ε (0.2 or 1 μg/mL) and anti-mouse CD28 (1 μg/mL) in vitro. Single live cells were gated for CD45 population and used for further analysis as indicated here. Human TNFR2 expression was detectable on CD3+ T cells in TNFR2 humanized mice as evidenced by hTNFR2 Ab2 binding vs isotype control (hIgG). mTNFα enhanced anti-human TNFR2 antibody Ab2 binding under mild anti-mouse CD3ε (0.2 μg/mL) stimulation, suggesting that mTNFα/hTNFR2 signaling pathway also works well in TNFR2 humanized mice.
Establishment of a MC38 model and in vivo efficacy study of an anti-human TNFR2 antibodies in TNFR2 humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous TNFR2 humanized mice (male, 9-10-week-old, n=8).
Antitumor activity of anti-human TNFR2 antibodies in TNFR2 humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. These results demonstrate that TNFR2 humanized mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibodies.
The overage of this tumor model is 40%.
Antitumor activity of anti-human TNFR2 antibodies in TNFR2 humanized mice. Tumor cells growth of individual mice. These results demonstrate that TNFR2 humanized mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibodies.
Establishment of a MC38 model and in vivo efficacy study of an anti-human TNFR2 antibodies in TNFR2 humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous TNFR2 humanized mice (male, 8-9-week-old, n=8).
Antitumor activity of anti-human TNFR2 antibodies in TNFR2 humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. (C) Tumor cells growth of individual mice. These results demonstrate that TNFR2 humanized mice provide a powerful preclinical model for in vivo evaluation of anti-human TNFR2 antibodies. The overage of this tumor model is 40%.
Q1: What are TNFR2 humanized mice?
B-hTNFR2 mice are genetically engineered models in which exons 2-6 of the mouse Tnfr2 gene, encoding the extracellular domain, are replaced by the corresponding human TNFR2 exons. This allows for the expression of the human TNFR2 protein in a mouse model.
Q2: Why are TNFR2 humanized mice important for immuno-oncology research?
TNFR2 is a key immunomodulator expressed on T cells and Tregs. This model enables the in vivo evaluation of anti-human TNFR2 therapeutics, which can inhibit tumor growth by modulating the tumor microenvironment, such as depleting Tregs and activating effector T cells.
Q3: How is human TNFR2 expression validated in these mice?
Human TNFR2 expression is exclusively detectable at both the mRNA and protein levels in homozygous B-hTNFR2 mice, as confirmed by RT-PCR, RT-qPCR, and flow cytometry using species-specific antibodies, while being absent in wild-type mice.
Q4: Does the humanization of TNFR2 affect normal immune development?
No. Comprehensive immune profiling demonstrates that the development, differentiation, and distribution of major immune cell populations (including T, B, and NK cells) in the spleen and lymph nodes of B-hTNFR2 mice are comparable to wild-type controls.
Q5: What is the evidence that this model is predictive for drug efficacy?
Multiple in vivo efficacy studies show that anti-human TNFR2 antibodies can dose-dependently inhibit tumor growth in B-hTNFR2 mice, validating it as a powerful preclinical model for evaluating anti-TNFR2 therapies.
