TNFR2 Human, His

What is TNFR2 and what distinguishes it structurally from other TNF receptors?

TNFR2 (also known as TNFRSF1B or CD120b) is a type I transmembrane protein and member of the TNF receptor superfamily (TNFRSF). It belongs to the TRAF (TNF-receptor-associated factor)-interacting subgroup of TNFRSF . Human TNFR2 protein typically comprises amino acids 23-257 of the sequence encoded by GenBank Accession No. NM_001066 . Unlike TNFR1, TNFR2 lacks an intracellular death domain, which results in fundamentally different signaling outcomes. His-tagged versions often include a C-terminal histidine tag to facilitate purification and detection, with recombinant forms frequently designed as fusion proteins with molecular weights around 55 kDa .

How does TNFR2 signaling differ from TNFR1 signaling pathways?

While TNFR1 is the dominant receptor for pro-inflammatory responses, TNFR2 has more complex and context-dependent signaling outcomes . TNFR2 activates both canonical and non-canonical NF-κB pathways. In the canonical pathway, TNFR2 ligation leads to IκBα degradation . Additionally, TNFR2 induces transcription of NFKB2 and RELB genes, which encode proteins forming the non-canonical NF-κB transcription factor . TNFR2 signaling in regulatory T cells (Tregs) enhances their proliferation and stability through the PI3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway . Unlike TNFR1, TNFR2 is preferentially activated by membrane-bound TNF rather than soluble TNF .

What are the expression patterns of TNFR2 across different cell types?

TNFR2 expression is typically high in myeloid cells but is also found in certain T and B cell subsets and some non-immune cells such as endothelial cells, glial cells, and cardiomyocytes . Regulatory T cells (Tregs) constitutively express high levels of TNFR2 . In unstimulated conditions, TNFR2 is primarily expressed by CD4+Foxp3+ Treg cells, but not by other evaluated lymphocyte populations . Following stimulation with PHA-P/IL-2, TNFR2 is additionally expressed by CD4+Foxp3− and CD8+ effector T cells, and NK cells . Antigen-specific CD8+ T cells, such as those responding to CMV pp65, can display particularly high intensity TNFR2 expression . Mouse NK cell TNFR2 expression differs from human patterns, highlighting important species differences in TNFR2 biology .

What are the optimal methods for purifying and validating His-tagged TNFR2 proteins?

His-tagged human TNFR2 can be efficiently purified using immobilized metal affinity chromatography (IMAC). For optimal purification:

• Express the protein in an appropriate system (HEK293 cells are commonly used for TNFR2)

• Use a buffer system containing 8 mM phosphate, pH 7.4, with 110 mM NaCl for stability

• Verify purity using SDS-PAGE (TNFR2-Fc-His fusion proteins typically appear around 55 kDa)

• Validate functionality through binding assays with TNF-alpha

Essential validation steps include functional assessment using chemiluminescent binding assays with biotinylated TNF-alpha to generate inhibition curves, confirming proper folding and biological activity of the purified protein .

How can researchers effectively detect and characterize TNFR2 expression in experimental and clinical samples?

Multiple complementary approaches can be used to detect TNFR2 expression in various sample types:

• Flow cytometry:

  • Utilize fluorescently-labeled anti-TNFR2 antibodies

  • Combine with lineage markers (CD3, CD4, CD8, CD25, CD56, Foxp3)

  • Essential for distinguishing TNFR2 expression on different cell populations

• Immunohistochemistry:

  • Use validated anti-TNFR2 antibodies

  • Consider dual staining with cell-type specific markers

  • Particularly valuable for tissue localization studies

• Western blotting:

  • Use denaturing conditions that maintain epitope recognition

  • Include recombinant TNFR2-His as positive control

  • Expected molecular weight is approximately 55 kDa

For cerebrospinal fluid (CSF) analysis, measuring soluble TNFR2 (sTNFR2) can provide insights into TNFR2-related processes in neurological conditions like Alzheimer's disease .

What controls are essential when using His-tagged TNFR2 in binding and functional assays?

When designing experiments with His-tagged TNFR2, several critical controls should be included:

• Binding specificity controls:

  • Irrelevant His-tagged protein of similar size

  • Unlabeled TNF for competitive binding

  • TNFR1-specific controls to distinguish receptor-specific effects

• Functional assay controls:

  • Pathway inhibitor controls (NF-κB, PI3K-Akt-mTOR)

  • Cell-type controls (TNFR2-expressing vs. non-expressing cells)

  • Multiple timepoints to capture both early and late signaling events

• Technical controls:

  • Endotoxin testing to prevent confounding inflammatory effects

  • Concentration gradient to establish dose-response relationships

  • Lot-to-lot consistency validation

TNFR2: TNF-alpha binding inhibition assays using biotinylated TNF-alpha with chemiluminescent detection provide a robust readout for functional validation .

How does TNFR2 regulate regulatory T cell (Treg) biology?

TNFR2 plays multiple critical roles in Treg biology through several mechanisms:

• NF-κB pathway activation:

  • Canonical pathway: TNFR2 ligation activates canonical NF-κB signaling via IκBα degradation

  • Non-canonical pathway: TNFR2 induces transcription of NFKB2 and RELB genes

  • TNFR2 ligation in combination with IL-2 stabilizes NF-κB-inducing kinase (NIK) protein

  • RelB translocation from cytosol to nucleus occurs following TNFR2 stimulation

• Epigenetic regulation:

  • TNFR2 agonism induces expression of histone methyltransferase EZH2

  • EZH2 forms a complex with Foxp3 in activated Tregs

  • This complex is important for Treg suppressive activity

• Proliferative effects:

  • TNFR2 activation enhances IL-2-induced cell cycle entry in Tregs

  • This leads to dramatic Treg expansion (>300% increase 5 days after treatment)

  • The expanded Tregs maintain their suppressive function

Inhibition of RelB nuclear translocation blocks the proliferative response, demonstrating that the non-canonical NF-κB pathway is crucial for TNFR2-mediated Treg expansion .

How can researchers design and validate TNFR2-specific agonists for experimental applications?

Developing effective TNFR2-specific agonists involves several strategic approaches:

• Fusion protein design:

  • Create single-chain TNF80 trimers (sc(mu)TNF80) fused to modified IgG1

  • Example: NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) with FcγR-binding defective IgG1

  • This architecture provides superior serum retention via FcRn interaction

• Structure-guided design:

  • Modify TNF to enhance TNFR2 selectivity over TNFR1

  • Alternative approaches include DARPin technology (e.g., DARPin 18/D18)

• Validation methodology:

  • Confirm receptor specificity using TNFR2-deficient cells

  • Assess canonical NF-κB activation through IκBα degradation

  • Measure non-canonical NF-κB pathway activation (RelB translocation)

  • Evaluate functional outcomes like Treg expansion in vitro and in vivo

• In vivo validation:

  • Assess pharmacokinetics and serum retention

  • Measure biological effects such as Treg expansion (sustained at 200% for ~10 days)

  • Evaluate efficacy in disease models (e.g., protection against graft-versus-host disease)

The design of NewSTAR2 exemplifies this approach, demonstrating how a single injection can dramatically enhance Treg numbers and function in vivo .

What are the key differences in TNFR2 signaling between regulatory T cells and effector T cells?

TNFR2 signaling exhibits significant cell type-specific differences:

• Expression patterns:

  • Tregs constitutively express high levels of TNFR2

  • Effector T cells express minimal TNFR2 at baseline but upregulate it after activation

  • Expression intensity is generally higher in Tregs than activated effector T cells

• Functional outcomes:

  • In Tregs: TNFR2 activation enhances suppressive function and proliferation

  • In CD8+ T cells: TNFR2 can mediate activation-induced cell death (AICD) through reactive oxygen species production

  • In activated CD8+ effector T cells: TNFR2 activation increases memTNF expression, which can in turn promote Treg proliferation via TNFR2

• Molecular mechanisms:

  • In Tregs: TNFR2 robustly activates both canonical and non-canonical NF-κB pathways

  • The PI3K-Akt-mTOR signaling axis is particularly important in Tregs

  • TNFR2-stimulated Tregs demonstrate enhanced suppressive capacity against conventional T cell proliferation and activation marker expression

These differences create a complex regulatory network where TNFR2 can simultaneously enhance Treg-mediated suppression and modulate effector T cell responses in a context-dependent manner.

How is TNFR2 involved in cancer biology and immunotherapy approaches?

TNFR2 plays complex roles in cancer biology with therapeutic implications:

• Expression patterns in tumors:

  • TNFR2 is highly expressed by tumor-infiltrating Tregs

  • Lower expression is found on tumor-infiltrating effector T cells

  • This pattern has been observed in lung cancer patients and mouse tumor models

• Functional effects:

  • TNFR2 affects tumor development and metastasis through multiple mechanisms

  • TNFR2 can promote tumor immune escape by stimulating immunosuppressive cells

  • TNFR2-specific agonists can inhibit tumor growth in experimental models

• Immunotherapy approaches:

  • TNFR2-specific agonists enhance tumor infiltration by CD8+ T cells

  • TNFR2 stimulation increases IFN-γ synthesis by CD8+ T cells

  • Target-agnostic phenotypic screening has identified TNFR2 as a promising target for cancer immunotherapy

Researchers have found that TNFR2 emerged as a key target from unbiased screens, suggesting its fundamental importance in tumor immunology and potential as a therapeutic target .

What is the evidence for TNFR2's role in neurodegenerative diseases?

TNFR2 shows emerging significance in neurodegenerative diseases, particularly Alzheimer's disease:

• Genetic associations:

  • A genetic variant of TNFR2 (rs976881 in the TNFRSF1B gene) correlates with modulation of Alzheimer's disease severity

  • This variant is associated with a slower rate of functional decline over one year

  • It may serve as a marker of cognitive resilience

• Mechanistic insights:

  • TNF plays important roles in synaptic plasticity and responses to neural injury

  • TNFR2 signaling may contribute to neuroprotection and cognitive resilience

  • The TNFR2 pathway potentially explains why some patients show better cognitive performance despite significant pathology

• Clinical implications:

  • Understanding TNFR2's role could contribute to developing precision medicine interventions

  • The TNFR2 pathway represents a potential novel target for Alzheimer's therapies

  • Genetic TNFR2 variants could serve as biomarkers for predicting disease progression

These findings highlight TNFR2 as an important focus for understanding and potentially treating neurodegenerative diseases through novel mechanisms beyond traditional amyloid and tau pathways .

How can TNFR2-targeted approaches be applied in autoimmune and inflammatory conditions?

TNFR2-targeted therapies show promise for autoimmune and inflammatory conditions:

• Graft-versus-host disease (GvHD):

  • TNFR2 agonism with NewSTAR2 shows protection against acute GvHD

  • A single preemptive administration five days before allogeneic hematopoietic cell transplantation protected mice from acute GvHD

  • This effect correlates with Treg expansion and enhanced suppressive function

• Inflammatory modulation:

  • TNFR2 activation upregulates CD39 (adenosine-regulating ectoenzyme) on Tregs

  • This enhances the Treg-mediated suppression of inflammatory responses

  • TNFR2-stimulated Tregs show enhanced ability to suppress conventional T cell activation

• Cell therapy applications:

  • TNFR2 agonists can be used to expand Tregs ex vivo for adoptive transfer

  • Enhanced longevity of TNFR2 agonists (like NewSTAR2) allows for more effective in vivo administration

  • The improved serum retention of Fc-fusion TNFR2 agonists enables treatment with a single dose

• Other immune-regulatory cell types:

  • TNFR2 signaling is important in mesenchymal stem cells (MSCs), enabling them to inhibit T cell proliferation and activation

  • TNFR2 mediates survival and function of group 2 innate lymphoid cells (ILC2s)

  • TNFR2 is critical for endothelial progenitor cells' (EPCs) immunosuppressive capabilities

These findings suggest that targeted TNFR2 activation could provide novel therapeutic avenues for treating inflammatory and autoimmune conditions through multiple cellular mechanisms.

How can researchers address the contradictory data on TNFR2 function in different experimental systems?

Reconciling contradictory findings on TNFR2 requires systematic consideration of experimental variables:

• Cell type considerations:

  • TNFR2 functions differently across cell types (Tregs vs. effector T cells vs. non-immune cells)

  • Expression levels vary dramatically between populations

  • Cell purity is critical - minor contaminating populations can skew results

• Experimental conditions:

  • TNF concentration affects TNFR1 vs. TNFR2 activation balance

  • Soluble vs. membrane-bound TNF preferentially activates different receptors

  • Culture conditions (serum, cytokines) modulate TNFR2 expression and signaling

• Species differences:

  • Mouse and human TNFR2 have different expression patterns and functions

  • For example, NK cell TNFR2 expression differs between mice and humans

  • Species-specific reagents are essential for accurate interpretation

• Methodological approach to reconciliation:

  • Direct side-by-side comparisons under identical conditions

  • Detailed reporting of experimental conditions and reagents

  • Use of highly selective TNFR2 agonists (like DARPin 18 or NewSTAR2)

  • Systems biology approaches to model context-dependent signaling networks

These considerations help explain why TNF has been variably reported to promote, inhibit, or have no effect on Treg function in different experimental systems .

What are the major technical challenges in studying TNFR2 signaling pathway cross-talk?

Investigating TNFR2 signaling pathway cross-talk presents several technical challenges:

• Receptor specificity:

  • TNF can bind both TNFR1 and TNFR2, complicating pathway-specific analysis

  • Need for highly selective TNFR2 agonists (e.g., DARPin 18, NewSTAR2)

  • Requirement for receptor-specific knockout models

• Temporal dynamics:

  • TNFR2 activates multiple pathways with different kinetics

  • Canonical NF-κB signaling occurs rapidly

  • Non-canonical NF-κB and other pathways develop more slowly

  • Comprehensive temporal analysis is required

• Pathway interconnections:

  • TNFR2 connects to multiple pathways (NF-κB, PI3K-Akt-mTOR)

  • Challenge in isolating pathway-specific effects

  • Example: The precise mechanism linking TNFR2 to the PI3K-Akt-mTOR axis in Tregs remains to be fully clarified

• In vivo translation:

  • Limited serum half-life of many TNFR2-targeting molecules

  • Newer approaches like NewSTAR2 with improved FcRn binding address this challenge

  • Engineering TNFR2 agonists with extended half-life enables more effective in vivo studies

Addressing these challenges requires innovative approaches combining genetic models, highly selective pharmacological tools, and advanced analytical techniques.

What are emerging directions for TNFR2 research in immune modulation?

Several promising research directions are emerging for TNFR2:

• Enhanced therapeutic design:

  • Development of TNFR2 agonists with improved pharmacokinetics

  • Examples include NewSTAR2, which demonstrates significantly improved serum retention compared to earlier TNFR2 agonists

  • Potential for context-dependent TNFR2 modulation in different disease settings

• Cell-specific targeting:

  • Exploring differential effects on Tregs vs. effector cells

  • Developing strategies to selectively target TNFR2 on specific cell populations

  • Understanding cell type-specific signaling networks

• Combinatorial approaches:

  • TNFR2 agonism combined with checkpoint inhibitors in cancer

  • TNFR2 modulation with current standard-of-care therapies in autoimmune conditions

  • Potential synergy with IL-2 pathway modulation, as TNFR2 and IL-2 show cooperative effects

• Novel disease applications:

  • Beyond cancer and autoimmunity to neurodegenerative diseases

  • Investigation of TNFR2's role in cognitive resilience in Alzheimer's disease

  • Potential applications in ILC2-dependent asthma, where the TNF-TNFR2 axis is crucial for ILC2 survival and function

These emerging directions highlight the continued importance of TNFR2 as a research focus with significant therapeutic potential across multiple disease areas.