Recombinant Human Lymphotoxin-alpha protein (LTA) (Active)

What is the molecular structure of recombinant human LTA protein?

Recombinant human Lymphotoxin-alpha/TNF-beta is a 22 kDa protein derived from E. coli expression systems. The protein consists of amino acids Leu35-Leu205 with an N-terminal methionine. Biologically active LTA forms homotrimers that bind to and activate specific receptors including TNF RI/TNFRSF1A, TNF RII/TNFRSF1B, and HVEM/TNFRSF14. The protein shares approximately 73% amino acid sequence identity with mouse and rat LTA/TNF-beta, which is important to consider when designing cross-species experiments .

What cell types express LTA and under what conditions?

LTA is primarily expressed by activated T and B lymphocytes. Expression is tightly regulated and typically occurs during immune activation. In research contexts, understanding the activation conditions that promote LTA expression is crucial for experimental design. While constitutive expression is limited, various activation protocols using cytokines, mitogens, or receptor-mediated signaling can induce LTA expression in lymphocytes .

What is the optimal reconstitution protocol for recombinant human LTA?

For carrier-free formulations (211-TBB/CF), reconstitute the lyophilized protein at 100 μg/mL in PBS. For formulations containing carrier protein (211-TBB), reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin. After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles that can compromise activity. Store reconstituted protein at -20°C to -80°C, and use a manual defrost freezer to maintain protein integrity .

How should I determine the appropriate dose of LTA for in vitro cytotoxicity assays?

The effective dose for LTA-induced cytotoxicity varies by application and cell type. For the L-929 mouse fibroblast cell line, the ED50 for cytotoxicity in the presence of actinomycin D is 4-20 pg/mL for the newer formulation (211-TBB) and 0.1-0.4 ng/mL for the older formulation (211-TB). When designing cytotoxicity assays, establish a dose-response curve (0.1 pg/mL to 10 ng/mL) to determine optimal concentrations for your specific cell system. Include actinomycin D (typically 1 μg/mL) as a metabolic inhibitor to enhance cytotoxic effects, and implement appropriate controls including TNF-alpha as a comparative standard .

What experimental controls should be included when studying LTA-mediated effects?

When designing experiments with LTA, include the following controls:

These controls help differentiate specific LTA-mediated effects from non-specific or assay-related outcomes .

How can I design experiments to distinguish between LTA homotrimer and LTA/LT-beta heterotrimer signaling?

Distinguishing between signaling pathways activated by LTA homotrimers versus LTA/LT-beta heterotrimers requires careful experimental design. Use receptor-specific blocking antibodies to differentiate between pathways: LTA homotrimers signal through TNF RI/II and HVEM, while LTA/LT-beta heterotrimers signal through LT-beta R.

For advanced studies, implement the following approaches:

• Use receptor-specific reporter cell lines expressing individual receptors

• Employ CRISPR/Cas9 to knock out specific receptors in your cell system

• Use recombinant soluble receptors as competitive inhibitors

• Compare effects of recombinant LTA alone versus co-culture with LT-beta-expressing cells

Verification can be performed using receptor-specific phosphorylation assays and downstream signaling analysis by Western blot or phospho-flow cytometry .

What methodologies are optimal for studying LTA's role in lymphoid organ development?

Investigating LTA's role in lymphoid organ development requires integrating in vitro and in vivo approaches:

• Organoid culture systems: Establish lymphoid organoids incorporating stromal cells, lymphocytes, and controlled LTA exposure.

• 3D tissue engineering: Create artificial lymphoid structures with controlled gradients of LTA.

• Conditional knockout models: Use Cre-lox systems for cell type-specific and temporally controlled LTA deletion.

• Adoptive transfer experiments: Transfer LTA-sufficient or LTA-deficient lymphocytes to appropriate recipient models.

• High-resolution imaging: Employ two-photon microscopy to visualize LTA-dependent cellular interactions in developing lymphoid tissues.

Analysis should incorporate comprehensive immune phenotyping, spatial transcriptomics, and functional evaluation of the developing structures .

How does human LTA cross-reactivity with mouse receptors impact translational research models?

• Validate receptor binding: Confirm binding affinity of human LTA to mouse TNF-RI, TNF-RII, and LT-beta R using surface plasmon resonance or cellular binding assays.

• Compare signaling potency: Perform parallel dose-response analyses in human and mouse cells to establish equivalent functional doses.

• Account for species-specific differences: Some downstream pathways may show divergent responses due to species-specific adapter proteins or signaling intermediates.

• Consider humanized models: For highly translational studies, use mouse models with humanized TNF receptors.

Quantify these differences by comparing ED50 values between species and adjust dosing accordingly to ensure translational relevance .

What strategies can resolve inconsistent results in LTA-induced cytotoxicity assays?

Inconsistent cytotoxicity results can stem from multiple factors:

• Protein activity loss: Recombinant LTA can lose activity due to improper storage or handling. Ensure proper aliquoting to avoid freeze-thaw cycles and verify protein integrity by SDS-PAGE before critical experiments.

• Cell line variability: L-929 and other reporter cell lines may change sensitivity over passages. Maintain low-passage cells and periodically verify sensitivity using standard curves.

• Metabolic inhibitor effectiveness: The potency of actinomycin D is critical for observing cytotoxic effects. Ensure fresh preparation and appropriate concentration (typically 1 μg/mL).

• Media components interference: Serum factors can neutralize LTA activity. Consider titrating serum concentration or using serum-free conditions during treatment periods.

• Cell density effects: Cytotoxicity is influenced by cell density at treatment time. Standardize seeding density and treatment timepoint relative to plating.

Implementing a standard operating procedure with these considerations can significantly reduce variability in cytotoxicity assays .

What analytical methods should be used to confirm the integrity and activity of recombinant human LTA?

Multiple complementary methods should be employed to confirm LTA integrity:

• Structural analysis:

  • SDS-PAGE under reducing conditions (should show a band at 19 kDa)

  • Size exclusion chromatography to confirm trimeric assembly

  • Circular dichroism spectroscopy to verify secondary structure

• Functional assessment:

  • L-929 cytotoxicity assay with actinomycin D (ED50 4-20 pg/mL)

  • Reporter cell lines expressing TNF receptors coupled to luciferase or other reporters

  • Binding assays using surface plasmon resonance with purified receptors

• Immunological confirmation:

  • Western blot with specific anti-LTA antibodies

  • ELISA to quantify protein concentration

For critical experiments, performing both structural and functional assessments is recommended to ensure reproducible results .

How can researchers distinguish between direct LTA effects and secondary cytokine responses?

Distinguishing primary LTA effects from secondary cytokine responses requires temporal and mechanistic controls:

• Temporal analysis: Perform detailed time-course studies capturing both early (minutes to hours) and late (hours to days) responses.

• Transcriptional inhibition: Use actinomycin D at sub-cytotoxic doses or other transcriptional inhibitors to block secondary cytokine production.

• Translational blockade: Apply cycloheximide to inhibit protein synthesis and prevent secondary cytokine effects.

• Receptor occupancy analysis: Use competitive binding assays with labeled LTA to determine receptor occupancy kinetics.

• Cytokine neutralization: Implement comprehensive cytokine neutralization using antibody cocktails to eliminate secondary effects.

• Single-cell analysis: Employ single-cell transcriptomics or proteomics to identify directly responding cells versus those responding to secondary signals.

These approaches can be combined for robust differentiation between direct LTA signaling and downstream cytokine cascades .

How can multiparametric analysis be optimized to study LTA-induced inflammatory pathways?

Optimizing multiparametric analysis requires integration of complementary technologies:

• Phospho-flow cytometry: Simultaneously evaluate multiple phosphorylation events (p38 MAPK, JNK, ERK, NF-κB) in heterogeneous cell populations following LTA stimulation.

• Multiplex cytokine analysis: Employ bead-based multiplex assays to quantify up to 50 cytokines simultaneously from culture supernatants.

• Transcriptomic profiling: Use RNA-seq or targeted NanoString panels to identify transcriptional networks activated by LTA.

• Pathway inhibitor panels: Systematically apply specific kinase inhibitors in a matrix format to delineate signaling dependencies.

• CRISPR screens: Implement focused CRISPR libraries targeting inflammatory pathway components to identify essential mediators of LTA responses.

Data integration across these platforms using computational approaches such as principal component analysis or pathway enrichment can reveal the multidimensional nature of LTA-induced inflammatory responses .

What methodologies enable investigation of LTA's role in TNF superfamily cross-talk?

Investigating cross-talk between LTA and other TNF superfamily members requires specialized approaches:

• Sequential and simultaneous stimulation protocols: Apply LTA before, simultaneously with, or after other TNF family ligands to identify synergistic, additive, or antagonistic effects.

• Receptor complex immunoprecipitation: Use proximity ligation assays or co-immunoprecipitation to identify receptor complex formation and composition.

• FRET/BRET analysis: Employ fluorescence or bioluminescence resonance energy transfer to study receptor clustering and heterotypic interactions.

• Competitive binding assays: Quantify displacement of labeled ligands to determine binding competition at shared receptors.

• Chimeric receptors: Engineer domain-swapped TNF receptors to identify regions mediating cross-talk.

• Bispecific reagents: Develop bispecific antibodies or fusion proteins targeting multiple TNF receptors to modulate cross-talk experimentally.

These approaches can reveal how LTA signaling is influenced by and influences other TNF superfamily members in complex immune responses .

How can researchers accurately model the role of LTA in autoimmune disease pathogenesis?

Modeling LTA's role in autoimmune pathogenesis requires multilevel approaches:

• Patient-derived materials: Analyze LTA expression and polymorphisms in samples from autoimmune disease patients, correlating with clinical parameters.

• Humanized mouse models: Develop models with human immune system components to better reflect human LTA biology.

• Tissue-specific conditional expression: Use inducible promoters to express LTA in relevant tissues mimicking autoimmune conditions.

• Ex vivo tissue models: Culture affected tissues (synovium for rheumatoid arthritis, CNS for multiple sclerosis) with controlled LTA exposure.

• Systems biology integration: Combine genomic, transcriptomic, and proteomic data to create predictive models of LTA-dependent disease progression.

• Therapeutic intervention models: Test LTA-pathway inhibitors at different disease stages to identify optimal intervention points.

These approaches can help define LTA's precise contributions to specific autoimmune conditions and identify targeted therapeutic strategies .

How should researchers interpret differences in potency between older (211-TB) and newer (211-TBB) recombinant human LTA formulations?

The significant potency difference between older (211-TB, ED50: 0.1-0.4 ng/mL) and newer (211-TBB, ED50: 4-20 pg/mL) formulations represents an approximately 25-fold increase in specific activity. This difference should be interpreted considering:

• Protein folding and trimeric assembly: The newer formulation likely maintains more native trimeric structure.

• Experimental design implications: Protocols developed for the older formulation require dose adjustment when transitioning to the newer product.

• Historical data comparison: When comparing new results with published literature using older formulations, normalize to biological activity rather than protein mass.

• Receptor binding kinetics: Perform comparative receptor binding assays to determine if higher potency correlates with increased receptor affinity.

• Data reproducibility: Higher specific activity may improve signal-to-noise ratio and data consistency in functional assays.

When publishing results, always specify the exact formulation used and include standardized bioactivity measurements to facilitate cross-study comparisons .

What analytical frameworks help distinguish between LTA's direct cytotoxic effects and its immunomodulatory functions?

Distinguishing between direct cytotoxicity and immunomodulation requires systematic analytical frameworks:

• Cell type-specific response patterns:

• Temporal discrimination: Direct cytotoxicity typically manifests within 6-24 hours, while immunomodulatory effects may develop over days.

• Concentration-dependent effects: Plot complete dose-response curves, as cytotoxic effects often require higher concentrations than immunomodulatory functions.

• Pathway-specific inhibitors: Apply selective inhibitors of death receptor signaling (caspase inhibitors) versus immunomodulatory pathways (NF-κB inhibitors).

• Genetic manipulation: Compare effects in cells with genetic deficiencies in apoptotic versus immunomodulatory signaling components.

This analytical framework provides a structured approach to differentiate LTA's diverse functional effects across experimental systems .

How can researchers effectively compare and integrate LTA data across different experimental models and species?

Cross-model and cross-species data integration requires standardized frameworks:

• Activity normalization: Convert all doses to biological activity units rather than mass concentrations to account for potency differences.

• Receptor expression profiling: Quantify TNF receptor expression levels across models to normalize for receptor availability.

• Pathway activation mapping: Develop standardized readouts of key signaling nodes (e.g., NF-κB, MAPK, caspase activation) across models.

• Temporal alignment: Adjust timepoints based on model-specific kinetics of receptor expression and signaling.

• Comparative transcriptomics: Identify conserved gene modules responsive to LTA across species and models.

• Meta-analysis frameworks: Apply statistical methods designed for heterogeneous data integration with appropriate weighting of model relevance.

• Visualization tools: Implement dimensionality reduction techniques like PCA or t-SNE to visualize relationships between datasets from different models.

These approaches facilitate meaningful comparisons across diverse experimental systems while acknowledging model-specific contexts and constraints .