TNFR2 Mouse

Basic Research Questions

• What is the physiological role of TNFR2 in mouse regulatory T cells?

TNFR2 plays a critical role in the development, stabilization, and function of regulatory T cells (Tregs) in mice. Research has demonstrated that TNF activation of TNFR2 stabilizes FoxP3 expression in Tregs upon TCR stimulation, which is essential for their suppressive function . The frequency of Tregs in the thymus of mouse strains genetically deficient in TNFR2 or its ligands is significantly reduced, suggesting TNFR2 participation in thymic Treg development .

TNFR2 expression identifies the maximal suppressive and replicating Tregs in mice . Studies have shown that TNF preferentially upregulates TNFR2 expression on Tregs, providing a mechanism for selective enhancement of regulatory versus effector T cell function in inflammatory environments .

• What phenotype do TNFR2 knockout mice display?

TNFR2 knockout (TNFR2−/−) mice do not spontaneously develop apparent autoimmune disorders but show more severe inflammation upon induction of autoimmune disease . The immunological alterations in these mice include:

• Decreased proportion of CD4+FoxP3+ cells in total splenic cells (42% reduction) and lymph node cells (21% reduction) compared to wild-type mice

• Reduced proportion of FoxP3+ cells within CD4+ splenic cells (36% decrease) and CD4+ lymph node cells (22% decrease)

• Approximately 50% reduction in absolute numbers of splenic Tregs

• More severe experimental autoimmune encephalomyelitis (EAE) with delayed remission compared to wild-type mice

These findings suggest that while TNFR2 is not essential for preventing spontaneous autoimmunity, it plays a significant role in limiting induced inflammatory responses through Treg maintenance.

• How does TNF-TNFR2 signaling affect regulatory T cell stability and function?

TNF-TNFR2 signaling affects regulatory T cell stability and function through multiple mechanisms:

• FoxP3 Stabilization: TCR stimulation reduces FoxP3 expression in TNFR2−/− Tregs, and this reduction cannot be reversed by TNF treatment, unlike in wild-type Tregs . This indicates that TNFR2 signaling is crucial for maintaining the stability of the Treg phenotype during activation.

• Expansion and Activation: TNFR2-selective agonistic TNF mutants (R2agoTNF) can expand and activate mouse CD4+CD25+ Tregs ex vivo . This provides a methodological approach for enhancing Treg populations in experimental settings.

• In Vivo Enhancement: Structural optimization of TNFR2 agonists through internal cross-linking or IgG-Fc fusion selectively enhances Treg expansion in vivo , offering potential therapeutic applications.

• Immunosuppressive Function: TNFR2 signaling is critical for the in vivo immunosuppressive function of naturally occurring Tregs . IgG-Fc fusion proteins of TNFR2 agonists have been shown to suppress skin-contact hypersensitivity reactions in mice .

These findings collectively demonstrate that TNFR2 signaling promotes both the quantitative expansion and qualitative functional enhancement of Tregs.

• What experimental models are available for studying TNFR2 function in mice?

Several experimental models have been developed for studying TNFR2 function in mice:

• TNFR2 Knockout Mice: Conventional TNFR2−/− mice with complete deletion of the TNFR2 gene, resulting in reduced Treg numbers and enhanced susceptibility to autoimmune diseases .

• Humanized TNF/TNFR2 Mouse Models:

  • hTNFKI mice: Express human TNF instead of mouse TNF, which cannot signal through mouse TNFR2, resulting in diminished Treg frequencies and exacerbated autoimmune disease .

  • hTNFKI × hTNFR2KI mice: Doubly humanized mice expressing both human TNF and human TNFR2, which normalizes Treg frequencies and autoimmune disease severity compared to hTNFKI mice .

• Conditional TNFR2 Knockout Models: A doubly humanized TNF/TNFR2 mouse line with conditional Cre-mediated deletion of exons 2-6 of the TNFR2 gene allows for cell-specific TNFR2 ablation, such as T reg-restricted TNFR2 deletion .

• In Vitro Systems: mTNFR2/mFas-PA cell line for bioassays to evaluate mTNFR2-mediated effects of TNF mutants .

These models provide valuable tools for investigating TNFR2 function in various contexts, from basic research to therapeutic development.

Intermediate Research Questions

• How can TNFR2-selective agonists be generated for mouse studies?

Generating TNFR2-selective agonists for mouse studies involves sophisticated techniques, primarily phage display:

The phage display approach includes:

• Constructing a phage library displaying TNF mutants with randomization of nine amino acid residues at the predicted receptor-binding site

• Performing competitive panning against mTNFR2 in competition with mTNFR1-Fc to enrich for TNFR2-binding TNF mutants

• Screening selected clones by capture ELISA and bioassays using mTNFR2/mFas-PA and L-M cells to identify candidates with high binding selectivity and bioactivity

One successful example (clone 7) binds strongly to mTNFR2 with an equilibrium dissociation constant (KD) of 7.61 nM while showing greatly reduced affinity for mTNFR1 . This mutant exhibits full bioactivity compared to wild-type TNF.

Additional optimization strategies include:

• Internal cross-linking to increase ligand stability and clustering among ligand-receptor complexes

• IgG-Fc fusion to enhance in vivo activity and half-life

These techniques have successfully produced mTNFR2-selective agonists that serve as valuable analytical tools and potential therapeutic agents.

• What are the advantages of using humanized TNF/TNFR2 mouse models?

Humanized TNF/TNFR2 mouse models offer several distinct advantages for research:

• Translational Relevance: These models bridge the gap between mouse studies and human applications by incorporating human TNF and/or TNFR2 genes .

• Therapeutic Assessment: They enable the evaluation of human-specific antibodies against hTNF or hTNFR2 in various autoimmunity models , providing platforms for preclinical testing of potential therapeutics.

• Overcoming Species Specificity: Human TNF cannot effectively signal through mouse TNFR2 due to species differences. Humanized models overcome this limitation .

• Cell-Specific Studies: Doubly humanized TNF/TNFR2 mice with conditional deletion options allow assessment of cell-specific TNFR2 functions .

• Comparative Analysis: These models facilitate direct comparison between human and mouse TNF-TNFR2 signaling pathways .

As demonstrated in EAE models, hTNFKI mice (expressing human TNF with mouse TNFR2) develop exacerbated disease, while doubly humanized hTNFKI × hTNFR2KI mice show normalized disease progression comparable to wild-type mice . This confirms the importance of species-matched TNF-TNFR2 interactions for proper immune regulation.

• How do TNF binding kinetics differ between wild-type TNF and TNFR2-selective mutants?

Surface plasmon resonance (SPR) analysis reveals significant differences in binding kinetics between wild-type TNF and TNFR2-selective mutants:

Understanding these kinetic differences provides insights into the molecular basis of receptor selectivity and helps optimize TNFR2-targeted therapeutic approaches.

• How does TNFR2 signaling in Tregs influence autoimmune disease progression?

TNFR2 signaling in Tregs significantly influences autoimmune disease progression through multiple mechanisms:

• Disease Severity Modulation:

  • In the EAE model, hTNFKI mice (which lack effective TNF-TNFR2 signaling) show higher disease scores and delayed remission compared to wild-type mice

  • Restoration of TNF-TNFR2 signaling in hTNFKI × hTNFR2KI mice ameliorates disease to levels observed in wild-type mice

• Treg Maintenance During Inflammation:

  • TNFR2 signaling stabilizes FoxP3 expression in Tregs during inflammatory responses

  • This maintains the suppressive capacity of Tregs when they are most needed to control excessive inflammation

• Therapeutic Potential:

  • TNFR2-selective agonists like R2agoTNF can expand and activate Tregs ex vivo

  • IgG-Fc fusion proteins of TNFR2 agonists suppress skin-contact hypersensitivity reactions in mice

• Cell-Type Specific Effects:

  • Conditional deletion of TNFR2 specifically in Tregs can separate its role in regulatory versus effector cells

  • This approach helps identify intrinsic TNFR2 signaling in Tregs as protective in certain autoimmune conditions

These findings highlight the importance of TNFR2 signaling in Tregs for resolving inflammation and suggest therapeutic strategies targeting this pathway for autoimmune disease treatment.

Advanced Research Questions

• What methodological approaches can be used for conditional deletion of TNFR2 in specific cell populations?

Several methodological approaches enable conditional deletion of TNFR2 in specific cell populations:

• Cre-loxP System Implementation:

  • Engineer mice with loxP sites flanking critical exons (2-6) of the TNFR2 gene

  • Cross with mice expressing Cre recombinase under cell-specific promoters

  • For Treg-specific deletion, use FoxP3-Cre mice

  • For temporal control, employ tamoxifen-inducible CreERT2 systems

• Validation Strategies:

  • Flow cytometric analysis with anti-TNFR2 antibodies to confirm deletion in target populations

  • RT-PCR to assess transcript levels of full-length versus truncated TNFR2

  • Functional assays (e.g., TNF-induced signaling, suppression assays for Tregs)

  • Comparison with global knockout controls

• Experimental Design Considerations:

  • Include appropriate Cre-only and floxed-only controls to account for Cre toxicity or leakiness

  • Consider background strain effects and use littermate controls when possible

  • For developmental studies, compare constitutive versus induced deletions

• Advanced Applications:

  • Combine with reporter systems (e.g., YFP) to track cells with deleted TNFR2

  • Implement dual conditional systems for simultaneous manipulation of TNFR2 and other genes

  • Use doubly humanized TNF/TNFR2 conditional models for testing human-specific therapeutics

These approaches allow precise dissection of cell-specific TNFR2 functions in complex biological processes and disease models.

• How can contradictory results regarding TNFR2 function in different disease models be reconciled?

Reconciling contradictory results regarding TNFR2 function requires methodological examination of several key factors:

• Context-Dependent Signaling Analysis:

  • Systematically compare TNFR2 signaling pathways activated in different cell types

  • Examine temporal dynamics of signaling in acute versus chronic inflammation

  • Investigate potential co-receptor interactions that may modify TNFR2 function

• Cell Type-Specific Contributions:

  • Utilize conditional knockout models targeting TNFR2 in specific cell populations

  • Compare phenotypes between global versus cell-specific deletions

  • Analyze relative contributions of TNFR2+ Tregs versus TNFR2+ effector cells in each model

• Species-Specific Considerations:

  • Compare results between conventional and humanized mouse models

  • The observation that hTNFKI mice develop exacerbated EAE while doubly humanized mice show normalized disease progression highlights species-specific interactions

• Experimental Protocol Standardization:

  • Standardize disease induction protocols across laboratories

  • Control for mouse genetic background, age, sex, and microbiome

  • Establish consensus phenotyping parameters for each disease model

• Integrated Analysis Framework:

  • Develop mathematical models that incorporate cell-specific and temporal aspects of TNFR2 signaling

  • Use systems biology approaches to predict net outcomes in complex disease environments

  • Implement multi-parameter analysis to identify key variables that determine protective versus pathogenic roles

This methodological framework helps resolve apparent contradictions by revealing the nuanced, context-dependent functions of TNFR2 across different experimental settings.

• What are the technical challenges in developing selective TNFR2 agonists with full bioactivity?

Developing selective TNFR2 agonists with full bioactivity presents several technical challenges requiring specific methodological solutions:

• Receptor Selectivity Engineering:

  • Traditional site-directed mutagenesis yields suboptimal results; human TNFR2 binding affinity of the double mutant D143N-A145R was ~5–10 fold less than wild-type TNF

  • Phage display with randomized receptor-binding sites provides superior selectivity

  • Competitive panning against mTNFR2 in the presence of mTNFR1-Fc enriches for TNFR2-selective variants

• Bioactivity Optimization:

  • Selective mutants often show altered binding kinetics compared to wild-type TNF

  • Surface plasmon resonance analysis reveals that while wild-type TNF shows slow association/dissociation with mTNFR2, selective mutants exhibit quicker kinetics

  • Different binding modes may affect downstream signaling quality and duration

• Species Specificity Barriers:

  • Human TNFR2-selective TNF mutants do not bind mouse TNFR2

  • Species-specific agonists must be developed separately for preclinical testing

  • Humanized mouse models partially address this limitation

• Structural Optimization Approaches:

  • Internal cross-linking increases stability and enhances receptor clustering

  • IgG-Fc fusion improves pharmacokinetics and in vivo efficacy

  • These modifications must preserve receptor selectivity while enhancing activity

• In Vivo Testing Complexities:

  • Biodistribution and tissue penetration must be optimized

  • Potential immunogenicity of mutant proteins requires assessment

  • Cell-specific targeting strategies may improve therapeutic window

The successful development of clone 7 (a mouse TNFR2-selective agonist) demonstrates that these challenges can be overcome through sophisticated protein engineering approaches .

• What methodological approaches can expand regulatory T cells via TNFR2 for therapeutic applications?

Expanding regulatory T cells via TNFR2 for therapeutic applications involves several methodological approaches:

• TNFR2-Selective Agonist Development:

  • R2agoTNF expands and activates mouse CD4+CD25+ Tregs ex vivo

  • Structural optimization through internal cross-linking enhances efficacy

  • IgG-Fc fusion further improves in vivo expansion capability

• Ex Vivo Expansion Protocol Optimization:

  • Cell isolation: Magnetic bead or flow cytometry-based purification of CD4+CD25+/FOXP3+ Tregs

  • Culture conditions: Determine optimal TNFR2 agonist concentration, timing, and duration

  • Supplementary factors: Test combinations with IL-2, rapamycin, or vitamin D3

  • Quality assessment: Monitor FOXP3 stability, suppressive function, and phenotypic markers

• In Vivo Expansion Strategies:

  • Direct administration of optimized TNFR2 agonists (e.g., IgG-Fc fusion proteins)

  • Combined approaches with low-dose IL-2 or other Treg-promoting agents

  • Targeted delivery systems to enhance Treg selectivity

  • Dosing regimens optimized for sustained Treg expansion without effector T cell activation

• Therapeutic Efficacy Assessment:

  • Preclinical testing in relevant disease models (e.g., skin-contact hypersensitivity)

  • Monitoring both quantitative (Treg numbers) and qualitative (suppressive function) parameters

  • Long-term stability evaluation of expanded Treg populations

  • Safety assessment focusing on potential off-target effects

• Translational Considerations:

  • Humanized models to test human-specific TNFR2 agonists

  • GMP-compatible manufacturing processes for clinical application

  • Biomarkers for patient stratification and response monitoring

  • Combination with existing immunosuppressive therapies

These methodological approaches provide a framework for developing TNFR2-based strategies to expand regulatory T cells for treating autoimmune diseases, transplant rejection, and other inflammatory conditions.