Recombinant Mouse Tumor necrosis factor receptor superfamily member 4 (Tnfrsf4)
Description
Protein Structure
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Tags: Commonly fused with His or Fc tags for purification and detection
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Molecular Weight: 22.1–48.6 kDa (varies by construct and glycosylation)
Production and Quality
| Parameter | Specification | Source |
|---|---|---|
| Purity | >95% (SDS-PAGE) | |
| Endotoxin | <1.0 EU/μg (LAL assay) | |
| Activity | Binds TNFSF4 with ED50 <50 μg/mL | |
| Expression Host | Mammalian cells (e.g., HEK293) |
Functional Roles in Immune Regulation
TNFRSF4/OX40 is a co-stimulatory receptor on activated CD4+ and CD8+ T cells. Key functions include:
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T-Cell Activation: Enhances survival, proliferation, and cytokine production (e.g., IL-2, IFN-γ) .
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Regulatory T-Cell (Treg) Modulation:
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Disease Associations:
Immune Escape in Leukemia
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Mechanism: CML-derived Tregs localize near CTLs in bone marrow and suppress their antileukemic activity .
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Intervention: TNFRSF4 stimulation reduces Treg-mediated protection of leukemic cells, enhancing CTL efficacy .
Atherosclerosis Pathogenesis
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Mouse Models: OX40 deletion reduces atherosclerotic lesions by 40–60% via:
Therapeutic Targeting
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Antibody Studies: Agonistic anti-OX40 antibodies amplify antitumor immunity by expanding effector T cells and destabilizing Tregs .
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Clinical Correlation: Elevated TNFRSF4 mRNA in CML patients correlates with FOXP3+ Treg levels .
Research Applications
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please include them in your order notes, and we will fulfill your request to the best of our ability.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance as additional fees will apply.
Generally, the shelf life of liquid formulations is 6 months at -20°C/-80°C. The shelf life of lyophilized formulations is 12 months at -20°C/-80°C.
Target Background
- The significance of OX40/OX40L signaling in a murine model of asthma has been established. PMID: 29344664
- Combination therapies employing checkpoint inhibitor antibodies (Abs), such as those targeting PD-1 or its ligand (PD-L1), alongside immune stimulatory agonist Abs like anti-OX40, are undergoing clinical trials to enhance antitumor efficacy. PMID: 28848055
- Obese mice induced by a high-fat diet exhibit increased OX40 expression in adipose tissues and splenic T cells. PMID: 28612217
- These findings support the hypothesis that the Ox40/Ox40L pathway drives cellular and humoral autoimmune responses during lupus nephritis in NZB/W F1 mice, highlighting the potential therapeutic value of targeting this pathway in human lupus. PMID: 28696253
- OX40 functions as a death receptor for invariant natural killer T cells and is implicated in pyroptotic cell death. PMID: 28436935
- This study demonstrates that OX40 links a costimulatory receptor to a repressive chromatin remodeling pathway. Notably, OX40 stimulation inhibits IL-17 production and reduces experimental autoimmune encephalomyelitis. PMID: 27317259
- IFNalphaR1 signaling promotes CXCL9 and CXCL10 synthesis, suggesting these chemokines might contribute to the LPS and CD134 costimulation response. PMID: 28432083
- OX40 and ICOS collaborate in a non-redundant manner to maximize and prolong the T follicular helper cell response generated following acute virus infection. PMID: 27895177
- OX40 regulates cardiac remodeling through the modulation of CD4(+) T-lymphocytes. PMID: 27580926
- Bone marrow-derived mast cell (BMMC)-exosomes facilitate the differentiation of naive CD4+ T cells into Th2 cells through ligation of OX40L and OX40 between BMMC-exosomes and CD4+ T cells. This represents a novel mechanism of cell-to-cell communication. PMID: 27066504
- Crystal structures and NMR data reveal that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived alternative decay element (ADE) and in an ADE-like variant present in the Ox40 3'-UTR with identical binding modes. PMID: 27010430
- The OX40-OX40 ligand interaction upregulates intracellular levels of reactive oxygen species in atherogenesis. PMID: 25115246
- OX40 was highly expressed by intratumoral T cells, particularly those of the FoxP3(+) regulatory T-cell (Treg) lineage. PMID: 24732076
- OX40 and IL-7 play synergistic but distinct roles in the homeostatic proliferation of CD4(+) effector memory T cells. PMID: 25103720
- OX40-OX40L interaction regulates the expression of NFATc1, which might play a critical role in atherosclerotic plaque formation and may therefore have implications for the pathophysiology of atherosclerosis. PMID: 24045961
- OX40 stimulation of virus-specific CD4 T cells promoted expression of the transcriptional repressor Blimp-1 and diverted the majority of cells away from follicular Th cell differentiation. PMID: 24101548
- Interruption of the OX40-OX40L signaling pathway, with decreases in dietary cholesterol, induces regression of atherosclerosis by inducing IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reducing Th2 and mast cells. PMID: 24068673
- OX40-OX40L interaction promotes proliferation and activation of lymphocytes via NFATc1 in ApoE-deficient mice. PMID: 23593329
- These data demonstrate that OX40-OX40L signaling contributes to the evolution of the adaptive immune response to an allograft through the differential control of alloreactive effector and regulatory T cell survival. PMID: 23817421
- These results suggest that OX40 costimulation crucially engages survivin during antigen-mediated Th2 responses. PMID: 23616302
- Mediates responsiveness to respiratory syncytial virus in neonates. PMID: 23036746
- OX40 facilitates the control of persistent virus infections. PMID: 22969431
- Mechanistically, OX40 activates the ubiquitin ligase TRAF6, which triggers induction of the kinase NIK in CD4( ) T cells and the noncanonical transcription factor NF-kappaB pathway. This subsequently leads to the generation of T(H)9 cells. PMID: 22842344
- Both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8+ T cell proliferation following both primary and secondary antigen challenge. PMID: 22956587
- Upon ligation by OX40L, OX40 (CD134) assembles a unique complex that not only contains TRAF2, RIP, and IKKalpha/beta/gamma but also CARMA1, MALT1, BCL10, and PKC – molecules previously shown to regulate NF-kappaB activation through the T-cell receptor (TCR). PMID: 21282629
- OX40-OX40L interaction can regulate the mRNA and protein expressions of NFATc1 in lymphocytes of ApoE-/- mice. PMID: 21924079
- Development of skin lesions was more likely attributable to deletion of Ikbk2 in skin keratinocytes in OX40(Cre) mice. PMID: 22363815
- Following OX40 stimulation, blockade of Treg-cell suppression and enhancement of the Tem-cell adjuvant effect both contribute to freeing DCs from immunosuppression and activating the immune response against the tumor. PMID: 22229156
- OX40L and PD-L2 expressed on dendritic cells differentially regulate cytokine production during recall responses in the lung. PMID: 22266281
- In vivo OX40 engagement in naive mice induces initial expansion of Foxp3+ regulatory T (Treg) cells. However, the expanded Tregs exhibit poor suppressive function and display features of exhaustion, which can be prevented by exogenous interleukin (IL)-2. PMID: 22147766
- sOX40 inhibits MC degranulation, potentially providing an in vivo therapeutic tool for allergic diseases. PMID: 21653238
- Mxd4 and Mnt upregulation following OX40 engagement likely increases T-cell survival. PMID: 21400495
- The vascular OX40/OX40L system plays a significant role in the formation of vasa vasorum and subsequent atherosclerosis. PMID: 20584752
- Activation of OX40 augments Th17 cell function, contributing to ocular inflammation. PMID: 20952591
- OX40 is a key factor in shaping Treg sensitivity to IL-2 and promoting cell proliferation and survival. PMID: 20806292
- OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. PMID: 20019337
- OX40 plays a crucial role in the homeostasis of intestinal Foxp3+ T regulatory cells and in the suppression of colitis. PMID: 20368580
- Findings indicate that OX40, a marker of both T cell activation and memory, is selectively upregulated in the brain during ECM. Its distribution among CD4(+) and CD8(+) T cells accumulated in the brain vasculature is approximately equal. PMID: 19710907
- Plays a role in the activation of CD8(+) intraepithelial lymphocytes. PMID: 11739485
- Engagement of OX40 enhances antigen-specific CD4(+) T cell mobilization/memory development and humoral immunity. PMID: 11739496
- Dendritic cell-induced CD4+ and CD8+ T cell responses in vivo are significantly amplified when Ox40 signaling is provided through an anti-Ox40 monoclonal antibody. PMID: 11777959
- Constitutive interaction of OX40 with its ligand provides a system that plays a potential key role in the immune regulation of various autoimmune diseases. PMID: 12370402
- Examines the relative importance of CD134 (OX40) and CD137 (4-1BB) in the costimulation of CD4+ and CD8+ T cells under comparable conditions of antigenic stimulation. PMID: 12516549
- The OX40/OX40L pathway has a broad impact on graft-versus-host disease induction. PMID: 12521997
- T-cell stimulation via OX40 engagement during cryptococcosis infection using OX40L fusion protein (OX40L:Ig) promotes cell-mediated immunity and IFN-gamma production by CD4+ T cells, reducing pathogen burden in the lung and associated eosinophilia. PMID: 12794142
- Expressed on memory CD4 cells. OX40-OX40L interactions are pivotal to the efficiency of recall responses regulated by memory Th2 cells. PMID: 12860930
- Interactions mediated by CD134 affect the onset and development of experimental allergic encephalomyelitis. Cd134-/- mice exhibit less severe clinical signs of disease and markedly reduced inflammatory infiltrates within the central nervous system. PMID: 14644025
- OX40 signals may play a crucial role in mediating skin allograft rejection in CD28/CD154 double knockout mice. PMID: 14734751
- Important roles are revealed for OX40 signals in regulatory T (Treg) cell development, homeostasis, and suppressive activity, showing how OX40 signals can oppose Treg-mediated suppression when delivered directly to antigen-engaged naive T cells. PMID: 15004159
- OX40 is one of the costimulatory molecules that can contribute signals to regulate the accumulation of antigen-reactive CD8 cells during immune responses. PMID: 15067059
Q&A
What is the structure of mouse TNFRSF4 protein and how does it compare to human TNFRSF4?
Mouse OX40/TNFRSF4 is a 454 amino acid type I transmembrane glycoprotein that contains several distinct domains. The protein structure includes:
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A 19-21 amino acid signal sequence
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A 191-192 amino acid extracellular domain (ECD) with four cysteine-rich domains (CRDs)
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A pre-ligand assembly domain (PLAD) that mediates constitutive trimer formation
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A 23-25 amino acid transmembrane domain
The ECD of mouse TNFRSF4 shares approximately 63% sequence identity with human TNFRSF4 and about 90% with rat TNFRSF4 . This notable difference in sequence homology between species is important to consider when designing cross-species experiments.
What are the primary cellular sources of TNFRSF4 expression and what stimulates its upregulation?
TNFRSF4 is primarily expressed on:
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Activated CD4+ and CD8+ T cells following TCR engagement
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Regulatory T cells (Tregs) where it is constitutively expressed
Expression is upregulated by:
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Engagement of the T cell receptor (TCR) by antigen presenting cells
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Co-stimulation through CD40-CD40 Ligand interactions
This expression pattern makes TNFRSF4 an important therapeutic target for modulating T cell responses in autoimmunity, cancer, and vaccine development.
How should recombinant mouse TNFRSF4 protein be reconstituted and stored to maintain optimal activity?
| Storage Parameter | Recommendation |
|---|---|
| Lyophilized form | Stable for up to 12 months at -20°C to -80°C |
| Reconstitution | Reconstitute at 100-250 μg/mL in sterile PBS |
| Reconstituted solution | Store at 4-8°C for 2-7 days |
| Aliquoted samples | Stable at < -20°C for 3 months |
| Freeze/thaw cycles | Avoid repeated cycles |
For carrier-free versions:
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Reconstitute in PBS without BSA when the presence of BSA could interfere with your application
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For cell or tissue culture applications, the version with BSA is generally recommended for enhanced stability
Always centrifuge tubes before opening, and aliquot reconstituted protein to minimize freeze-thaw cycles which can significantly reduce biological activity .
What are the validated methods for detecting binding activity of recombinant mouse TNFRSF4?
Several validated methods have been reported for assessing recombinant mouse TNFRSF4 binding activity:
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Functional ELISA: When mouse OX40L is immobilized at 1.0 μg/mL, mouse TNFRSF4-Fc chimera typically binds with an EC50 of 25-100 ng/mL .
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Surface Plasmon Resonance (SPR): SPR analysis reveals that mouse TNFRSF4 binds to mouse OX40L with equilibrium dissociation constants (KD) in the range of 7.61×10⁻⁹ to 20.5×10⁻⁹ M, depending on the exact construct used .
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Bio-Layer Interferometry (BLI): When loaded on AR2G Biosensor, mouse OX40L-His can bind mouse OX40-His with an affinity constant of approximately 0.16 μM .
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Flow Cytometry: Detection of cell surface expression can be achieved using specific anti-TNFRSF4 antibodies followed by fluorophore-conjugated secondary antibodies, as demonstrated in L-929 mouse cell line studies .
What experimental controls should be included when testing TNFRSF4 agonistic activity?
When evaluating TNFRSF4 agonistic activity, the following controls are essential:
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Isotype control antibodies: Use control antibodies (e.g., AB-108-C) alongside test antibodies to confirm binding specificity .
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Competitive inhibition controls: Include soluble TNFRSF4 ligand to demonstrate competitive binding.
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TNFR1/TNFR2 controls: When studying TNFRSF4-specific effects, include controls for other TNF receptor family members (particularly TNFR1/TNFR2) to exclude off-target effects .
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Signaling pathway inhibitors: Include inhibitors like Nec-1s or Sorafenib when assessing downstream signaling to determine pathway specificity .
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Cell viability controls: Essential for cytotoxicity assays to distinguish between specific TNFRSF4-mediated effects and general cellular toxicity.
Research has shown that experiments lacking these controls may yield misleading results due to the complex interplay between different TNF receptor family members and their shared downstream signaling pathways .
How do TNFRSF4 and TNFRSF25 differ in their effects on T cell subpopulations?
Research comparing TNFRSF4 and TNFRSF25 agonistic antibodies has revealed important functional differences in their costimulatory effects:
| Parameter | TNFRSF4 (OX40) | TNFRSF25 |
|---|---|---|
| CD8+ T cell proliferation | Strong costimulator (primary and secondary challenge) | Strong costimulator (primary and secondary challenge) |
| CD4+ Tconv proliferation | Potent costimulator | Weak costimulator |
| Treg proliferation | Weak costimulator | Strong costimulator |
| IgG1 production | Moderate | Strong |
| IgG2a production | Weak | Strong |
| IgG2b production | Minimal | Strong |
These differences highlight the non-redundant activities of these receptors in T cell immunity. When used in combination, TNFRSF4 and TNFRSF25 agonists showed additive effects on CD8+ T cell proliferation, suggesting potential synergistic applications in vaccine development and cancer immunotherapy .
What is the significance of TNFRSF4 expression in acute myeloid leukemia (AML) and its correlation with genetic mutations?
Analysis of TCGA datasets has revealed critical correlations between TNFRSF4 expression and genetic mutations in AML:
TNFRSF4 expression was also significantly associated with risk stratification, being higher in intermediate (p = 0.0004) and poor (p = 0.0011) risk groups compared to good prognosis patients. Additionally, relapsed AML samples showed significantly higher TNFRSF4 expression (p = 0.0099) than de novo samples .
These findings suggest TNFRSF4 as a potential prognostic biomarker and therapeutic target in AML, warranting further functional and mechanistic studies.
How does TNFRSF4 signaling in regulatory T cells influence leukemic stem cell immune escape?
Recent research has identified TNFRSF4-expressing regulatory T cells (Tregs) as key mediators of immune escape in leukemic stem cells (LSCs). In chronic myeloid leukemia (CML) models:
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Increased frequency of Tregs was observed in the bone marrow of CML mice compared to naive mice
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Close proximity between Tregs and leukemic Gr-1+ cells was demonstrated
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A positive correlation existed between Treg frequency and leukemic cell burden
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TNFRSF4-expressing Tregs showed enhanced suppressive function against cytotoxic T lymphocytes (CTLs)
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Activation of TNFRSF4 signaling on Tregs inhibited anti-leukemic immunity
These findings suggest that targeting TNFRSF4 on Tregs could potentially reduce their immunosuppressive function and enhance anti-leukemic immunity, offering a novel therapeutic approach for leukemia treatment.
What strategies have been successful in generating TNFRSF4-selective agonists with high receptor specificity?
Traditional site-directed mutagenesis approaches for creating TNFRSF4-selective agonists have shown limited success, with resulting mutants often demonstrating reduced binding affinity. More successful strategies include:
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Phage display technique: This approach has yielded TNFRSF4-selective TNF mutants with both high receptor specificity and full bioactivity. For example, clone 7 demonstrated strong binding to mouse TNFRSF4 (KD = 7.61 nM) with minimal binding to TNFR1 .
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Competitive panning methods: This refinement to phage display has produced optimized agonists with superior selectivity profiles.
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Structure-guided design: Based on the three-dimensional structure of the TNF-TNFR complex to create mutations at key receptor-binding interfaces.
Notably, there are species differences between human and mouse TNFRSF4, requiring species-specific optimization. For example, human TNFRSF4-selective TNF mutants (like R2-7) do not bind mouse TNFRSF4, necessitating separate development programs for preclinical and clinical applications .
How can recombinant TNFRSF4 be modified to enhance its stability and activity in experimental systems?
Several approaches have been validated for enhancing the stability and activity of recombinant TNFRSF4:
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Fc fusion proteins: Incorporating an Fc domain (typically human IgG1 or mouse IgG2a) significantly increases serum half-life and enhances receptor clustering through FcγR binding. Recombinant TNFRSF4-Fc chimeras demonstrate improved stability while maintaining binding specificity .
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His-tagging strategies: Addition of 6-His tags facilitates purification while minimally impacting biological activity. His-tagged TNFRSF4 constructs maintain binding to OX40L with affinity constants in the sub-micromolar range .
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Oligomerization enhancement: Strategies to increase ligand stability and clustering among ligand-receptor complexes include:
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Carrier protein addition: Addition of BSA as a carrier protein enhances stability, increases shelf-life, and allows for storage at more dilute concentrations .
The choice of modification strategy should be guided by the specific experimental application, with carrier-free versions preferred for applications where BSA might interfere with results.
What are the key differences in binding kinetics between wild-type TNF and TNFRSF4-selective mutants?
Surface plasmon resonance (SPR) analysis has revealed important differences in the binding kinetics between wild-type TNF and TNFRSF4-selective mutants:
| Parameter | Wild-type TNF | TNFRSF4-selective mutants (e.g., Clone 7) |
|---|---|---|
| Association (kon) | Slow association rate | Quick association rate |
| Dissociation (koff) | Slow dissociation rate | Quick dissociation rate |
| Maximum binding response (Rmax) | Higher | Lower |
| Equilibrium dissociation constant (KD) | ~10.4×10⁻⁹ M | ~7.61×10⁻⁹ M (for Clone 7) |
| EC50 value | 3.01 ng/ml | 0.48 ng/ml (for Clone 7) |
This kinetic information is crucial for researchers designing therapeutic approaches targeting TNFRSF4, as it helps predict in vivo behavior and potential off-target effects.
How does TNFRSF4 agonism affect different T cell subsets in vaccine development?
TNFRSF4 agonism has distinct effects on T cell subsets that are relevant for vaccine development:
| T Cell Subset | Effect of TNFRSF4 Agonism | Mechanism |
|---|---|---|
| CD8+ T cells | Enhanced proliferation and survival | Increased cytokine production; prevention of activation-induced cell death |
| CD4+ Tconv cells | Potent costimulation of proliferation | Enhanced division and cytokine production |
| Tregs | Weak to moderate proliferation | Increased sensitivity to IL-2; potential decrease in suppressive function |
| Memory T cells | Enhanced maintenance and recall response | Promotion of memory cell formation and survival |
These differential effects position TNFRSF4 agonists as potential vaccine adjuvants that can:
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Enhance CD8+ T cell responses against intracellular pathogens and tumors
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Boost CD4+ helper T cell responses to support antibody production
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Potentially limit Treg-mediated suppression of vaccine responses
Studies have demonstrated that TNFRSF4 agonism can provide strong costimulatory signals in both primary and secondary antigen challenges, making it valuable for both prime and boost vaccination strategies .
What is the significance of TNFRSF4 in regulatory T cell function in disease models?
TNFRSF4 plays complex and sometimes paradoxical roles in regulatory T cell (Treg) function across different disease models:
