Recombinant Mouse Tumor necrosis factor receptor superfamily member 8 (Tnfrsf8), partial

What is mouse TNFRSF8 and what are its primary functions in immune signaling?

Mouse TNFRSF8, also known as CD30 or Ki-1, is a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily. It functions as a receptor for CD30 Ligand/TNFSF8 and plays crucial roles in:

• Regulating cellular growth and transformation of activated lymphoblasts

• Modulating gene expression through activation of NF-kappa-B

• Serving as a regulator of apoptosis (can induce either cell death or proliferation depending on cell type)

• Contributing to thymic negative selection by inducing apoptotic cell death of T cells

• Limiting the proliferative potential of autoreactive CD8 effector T cells
  The mouse variant spans amino acids Phe19-Thr281 and has structural differences compared to the human counterpart, most notably containing only three cysteine-rich repeats in its extracellular domain versus six in humans .

How does mouse TNFRSF8 differ structurally from human TNFRSF8, and what implications does this have for experimental design?

The structural differences between mouse and human TNFRSF8 include:

What cell types express TNFRSF8 in mice and how does expression change under different physiological conditions?

In mice, TNFRSF8 is primarily expressed on:

• Activated T cells (not resting T cells)

• Activated B cells (not resting B cells)

• Reed-Sternberg cells in Hodgkin's lymphoma

• Certain lymphoma cells
  Expression is dynamically regulated and increases during:

• Antigen stimulation of lymphocytes

• Inflammatory conditions

• Autoimmune responses

• Lymphoma development
  For experimental detection, immunostaining of mouse splenocytes has been validated using specific antibodies (e.g., 15 μg/mL of goat anti-mouse CD30/TNFRSF8) . Flow cytometry can be used to quantify expression levels across different immune cell populations following activation with mitogens or cytokines .

What are the validated applications for different forms of recombinant mouse TNFRSF8?

Different recombinant forms of mouse TNFRSF8 have been validated for specific applications:

How can recombinant mouse TNFRSF8 be used to study T cell activation and differentiation pathways?

Recombinant mouse TNFRSF8 can be utilized in various experimental approaches to study T cell biology:

• Co-stimulation assays: Immobilize recombinant CD30L (TNFRSF8 ligand) and measure TNFRSF8-dependent co-stimulation of T cell proliferation using:

  • ³H-thymidine incorporation

  • CFSE dilution by flow cytometry

  • Activation marker upregulation (CD25, CD69)

• Signaling pathway analysis:

  • Use recombinant TNFRSF8-Fc to sequester CD30L and block signaling

  • Analyze downstream effects on NF-κB activation using reporter assays

  • Examine phosphorylation of pathway components (JNK, p38, ERK)

• Th1/Th2 differentiation studies:

  • Add soluble recombinant TNFRSF8 during T cell polarization

  • Measure impact on cytokine production (IL-4, IL-5, IFN-γ)

  • Assess transcription factor expression (GATA3, T-bet) by RT-qPCR
    The ED₅₀ for biological effects is typically 1-3 μg/mL when using antibodies against mouse CD30/TNFRSF8 .

What protocols are recommended for using recombinant mouse TNFRSF8 in flow cytometry experiments?

Optimized Protocol for Flow Cytometry with Fluorescent-Labeled Recombinant TNFRSF8:

• Cell Preparation:

  • Harvest cells of interest (primary lymphocytes or cell lines)

  • Wash 2× in cold PBS with 2% FBS

  • Adjust to 1×10⁶ cells/100 μL in staining buffer

• Blocking Step:

  • Incubate cells with 10% normal serum (from species unrelated to antibody source) for 15 minutes at 4°C

  • Add Fc receptor blocking antibody if using primary cells

• Staining with Recombinant TNFRSF8:

  • Add fluorescent-labeled recombinant TNFRSF8 (e.g., Alexa Fluor 647-conjugated)

  • Titrate concentrations (starting recommendation: 2-5 μg/mL)

  • Incubate for 30-45 minutes at 4°C in the dark

• Washing and Analysis:

  • Wash 3× with staining buffer

  • Resuspend in fixation buffer if not analyzing immediately

  • Analyze by flow cytometry with appropriate compensation controls
    Transfected cells have been successfully stained using this approach with recombinant human CD30-Fc Alexa Fluor 647, and a similar approach can be applied with mouse proteins .

What are the optimal storage conditions for maintaining the activity of recombinant mouse TNFRSF8?

Proper storage is critical for maintaining recombinant TNFRSF8 activity. Follow these guidelines based on protein formulation:
For Lyophilized Protein:

• Store at -20°C for up to 12 months

• After reconstitution, store at 4°C for 2-7 days for immediate use

• For longer storage, aliquot and keep at -80°C for 3-6 months

• Avoid repeated freeze-thaw cycles

• Use within one month after reconstitution
  For Solutions (e.g., Fluorescent-Labeled Proteins):

• Protect from light

• Use a manual defrost freezer

• Avoid repeated freeze-thaw cycles

• Consider adding carrier protein (0.1% HSA or BSA) for long-term storage

• For proteins in glycerol (e.g., 10% glycerol in PBS), store at 4°C if entire vial will be used within 2-4 weeks
  Stability Testing:
  Before conducting critical experiments, verify protein activity using functional assays such as ELISA or cell-based assays to ensure the stored protein has retained its biological activity.

How can I validate the biological activity of recombinant mouse TNFRSF8 before using it in my experiments?

Validation of biological activity is essential for ensuring experimental reproducibility. These methods can be used:

• Binding Assays:

  • ELISA: Measure binding to immobilized anti-CD30 antibody or CD30 ligand

  • Surface Plasmon Resonance: Determine binding kinetics and affinity constants

  • Example: For biotinylated human CD30, the EC₅₀ was 11.5 ng/mL when measured by ELISA at 0.5 μg/mL antibody concentration

• Cell-Based Functional Assays:

  • Co-stimulation of T cell proliferation

  • NF-κB reporter assay in appropriate cell lines

  • Apoptosis assays in CD30-responsive cell lines

• Structural Validation:

  • SDS-PAGE: Confirm expected molecular weight and purity

  • Western blot: Verify immunoreactivity

  • Size exclusion chromatography: Assess oligomeric state

  • Typical results: >95% purity by Tris-Bis PAGE and SEC-HPLC for quality recombinant proteins

• Endotoxin Testing:

  • Use LAL method to ensure endotoxin levels are below 0.1 EU per 1 μg protein

What are common technical challenges when working with recombinant mouse TNFRSF8 and how can they be addressed?

How does mouse TNFRSF8 contribute to negative selection in the thymus and what experimental approaches can be used to study this process?

TNFRSF8 plays a critical role in thymic negative selection by inducing apoptotic cell death of CD4+CD8+ T cells. This process is essential for eliminating self-reactive T cells and preventing autoimmunity .
Experimental Approaches to Study TNFRSF8 in Thymic Selection:

• Ex vivo Thymic Organ Culture (FTOC):

  • Isolate fetal thymic lobes and culture with recombinant CD30L

  • Add recombinant TNFRSF8-Fc to block CD30-CD30L interactions

  • Analyze T cell development by flow cytometry

  • Assess deletion of specific T cell receptor (TCR) specificities

• Bone Marrow Chimeras:

  • Generate mixed chimeras with TNFRSF8-deficient and wild-type bone marrow

  • Analyze development of specific T cell populations

  • Assess competitive fitness of TNFRSF8-deficient vs. wild-type thymocytes

• TCR Transgenic Models:

  • Cross TNFRSF8-knockout mice with TCR transgenic mice

  • Analyze negative selection of self-reactive T cells

  • Assess autoimmune phenotypes

• Single-Cell Transcriptomics:

  • Analyze transcriptional profiles of developing thymocytes

  • Identify TNFRSF8-dependent gene expression programs

  • Map the "effectorness gradient" that shapes T cell responses to cytokines
    These approaches can reveal how TNFRSF8 signaling interfaces with other pathways of thymocyte selection and contributes to central tolerance .

What are the molecular mechanisms by which TNFRSF8 regulates the balance between cell survival and apoptosis in mouse T cells?

TNFRSF8 exhibits context-dependent effects on cell survival and death through complex signaling mechanisms:
Survival Signaling Pathway:

• CD30L binding triggers recruitment of TRAF2 and TRAF5 to the cytoplasmic domain of TNFRSF8

• TRAF proteins activate the canonical NF-κB pathway

• NF-κB induces expression of anti-apoptotic proteins (Bcl-2, Bcl-xL)

• This pathway predominates in certain activated T cell subsets
  Apoptotic Signaling Pathway:

• In specific cellular contexts, TNFRSF8 can activate death signaling pathways

• This involves recruitment of different adapter proteins

• Leads to activation of caspase cascades

• Results in programmed cell death
  Experimental Approaches to Dissect These Pathways:

• Protein Interaction Studies:

  • Immunoprecipitation with tagged recombinant TNFRSF8

  • Mass spectrometry analysis of binding partners

  • Domain mapping using truncated TNFRSF8 constructs

• Signaling Analysis:

  • Phospho-specific antibodies to detect activated pathway components

  • Inhibitor studies to block specific signaling nodes

  • Genetic approaches (CRISPR/Cas9) to delete pathway components

• Functional Readouts:

  • Flow cytometry for apoptosis markers (Annexin V, caspase activation)

  • Live-cell imaging with fluorescent reporters

  • Transcriptional profiling of survival/apoptosis genes
    The dual nature of TNFRSF8 signaling makes it an important regulator of immune homeostasis .

How can recombinant mouse TNFRSF8 be used to study its role in lymphoma development and progression?

TNFRSF8 is implicated in various lymphomas, making it an important target for cancer research . Recombinant TNFRSF8 can be utilized in multiple experimental approaches:

• Functional Studies in Lymphoma Models:

  • Treat lymphoma cell lines with recombinant CD30L to activate TNFRSF8 signaling

  • Use TNFRSF8-Fc chimeras to block endogenous CD30L-CD30 interactions

  • Assess effects on proliferation, survival, and gene expression

  • Measure changes in cancer-related signaling pathways

• In Vivo Models:

  • Inject recombinant TNFRSF8-Fc to block CD30 signaling in lymphoma xenograft models

  • Develop bi-specific antibodies incorporating anti-TNFRSF8 domains

  • Evaluate effects on tumor growth, metastasis, and survival

  • Analyze tumor microenvironment changes

• Diagnostic Applications:

  • Develop TNFRSF8-based imaging probes using fluorescent-labeled recombinant proteins

  • Optimize detection of TNFRSF8-expressing cells in tissue samples

  • Correlate TNFRSF8 expression with clinical outcomes

• Therapeutic Target Validation:

  • Screen for compounds that modulate TNFRSF8 signaling

  • Test combinations with established lymphoma therapies

  • Develop TNFRSF8-targeted chimeric antigen receptor (CAR) T cells
    Cancer types associated with TNFRSF8 expression include Non-Hodgkin Lymphoma, Hodgkin Lymphoma, Cutaneous T-cell lymphoma, and Diffuse Large B-Cell Lymphoma .

What experimental approaches can be used to investigate the interaction between TNFRSF8 and its ligand (CD30L/TNFSF8) in mouse models?

The TNFRSF8-CD30L interaction can be studied using various sophisticated techniques:

• Biophysical Interaction Analysis:

  • Surface Plasmon Resonance (SPR) with recombinant proteins

  • Isothermal Titration Calorimetry (ITC) to determine binding thermodynamics

  • Bio-Layer Interferometry to measure association/dissociation kinetics

  • Employ both full-length and domain-specific constructs to map interaction sites

• Structural Biology Approaches:

  • X-ray crystallography of the TNFRSF8-CD30L complex

  • Cryo-electron microscopy for larger assemblies

  • Hydrogen-deuterium exchange mass spectrometry to map interfaces

  • Molecular dynamics simulations based on experimental structures

• Cell-Based Interaction Studies:

  • Bioluminescence Resonance Energy Transfer (BRET) in live cells

  • Proximity Ligation Assay (PLA) to visualize interactions in situ

  • Flow cytometry with fluorescent-labeled recombinant proteins

  • Time-lapse imaging of receptor-ligand trafficking

• In Vivo Imaging:

  • Two-photon microscopy of labeled proteins in lymphoid tissues

  • Positron Emission Tomography (PET) with radiolabeled TNFRSF8

  • Intravital microscopy to track cellular interactions
    An ELISA system has been established using immobilized Human CD30 Ligand, His Tag at 5 μg/mL (100 μL/well), with dose response curves for interacting proteins providing an EC₅₀ of 27.1 ng/mL . Similar approaches can be adapted for mouse proteins to quantitatively assess interactions under different experimental conditions.