Recombinant Mouse Cytotoxic T-lymphocyte protein 4 (Ctla4), partial (Active)

What is the molecular structure of recombinant mouse CTLA-4?

Mouse CTLA-4 is typically expressed as a partial recombinant protein including the extracellular domain (ECD). The amino acid sequence for mouse CTLA-4 ECD spans from approximately residue 36 to 162, with the sequence beginning with "EAIQVTQPSVVLASSHGVASFPCEYS..." and ending with "...PPYFVGMGNGTQIYVIDPEPCPDSD" . The protein contains multiple domains including an IgV-like domain that interacts with B7 ligands. N-glycosylation is important for CTLA-4 dimerization . When expressed as a recombinant protein, mouse CTLA-4 is often fused to an Fc region or includes tags like His-tag to facilitate purification and functional studies.

How does mouse CTLA-4 differ from human CTLA-4 in structure and function?

While mouse and human CTLA-4 share significant homology, there are notable differences:

Despite these differences, both human and mouse CTLA-4 function as inhibitory receptors that negatively regulate T-cell responses . When generating humanized mouse models, special attention must be paid to the exon structure to ensure proper splicing and expression .

Which cell types express CTLA-4 in mice and how is expression regulated?

CTLA-4 is primarily expressed by:

• Activated conventional T cells (upregulated upon activation)

• Regulatory T cells (Tregs) (constitutively expressed)

• Follicular regulatory T cells (Tfr)

Expression patterns vary by anatomical location. In Tfr cells, CTLA-4 expression correlates with high ICOS expression and increased IRF4 expression, suggesting a functional relationship . CTLA-4 expression is relatively universal in Tfr cells regardless of anatomical location (lymph nodes, circulation, Peyer's patches, skin), although the intensity of expression may vary .

Regulation occurs at multiple levels:

• Transcriptionally induced upon T cell activation

• Post-translationally regulated through endocytosis and recycling

• LRBA-dependent mechanism controls CTLA-4 recycling to cell surface

• Lysosomal degradation can be triggered by certain antibodies

How can I monitor CTLA-4 expression and trafficking in real-time experiments?

For real-time monitoring of CTLA-4 expression and trafficking:

• Fluorescent protein tagging: Transfect cells with EGFP-tagged CTLA-4 constructs to visualize trafficking in live cells .

• Surface biotinylation assay:

  • Biotinylate cell surface proteins

  • Stain with fluorophore-labeled streptavidin (e.g., AF549)

  • Monitor internalization and recycling by confocal microscopy

• pH-sensitive fluorescent probes: Use pH-sensitive dyes conjugated to anti-CTLA-4 antibodies to distinguish between surface, endosomal, and lysosomal localization.

• Flow cytometry approach:

  • Stain cells at 4°C (no downregulation) as baseline

  • Compare to cells incubated at 37°C with antibodies

  • Measure surface CTLA-4 using fluorescently labeled secondary antibodies

This approach revealed that CTLA-4 downregulation varies with different antibodies - Ipilimumab and TremeIgG1 caused significant downregulation, while other antibodies (HL12, HL32) allowed recycling .

What are the optimal in vitro assays to evaluate recombinant mouse CTLA-4 functional activity?

Several robust assays can verify CTLA-4 functional activity:

• T cell proliferation/activation inhibition assay:

  • Isolate primary mouse T cells or use Jurkat cell lines

  • Activate with anti-CD3/CD28 or PHA

  • Add recombinant CTLA-4 protein at various concentrations

  • Measure inhibition of proliferation or IL-2 secretion

  • The ED50 for inhibition is typically 0.1-0.4 μg/mL when tested with 1 μg/mL B7-1/CD80

• Binding assays:

  • ELISA: Immobilize B7-1 or B7-2 and detect bound CTLA-4 with specific antibodies

  • Surface Plasmon Resonance (SPR): Capture B7 ligands on chips and measure binding kinetics of CTLA-4

    • Affinity constant of human CTLA-4 to B7-1 is approximately 1.1 nM as determined by SPR

• Competition assays:

  • Test the ability of recombinant CTLA-4 to compete with anti-CTLA-4 antibodies

  • Determine if the protein can block the interaction between CTLA-4 and its ligands

How can recombinant CTLA-4 be used in tumor immunology research?

Recombinant CTLA-4 has diverse applications in tumor immunology:

• Blocking tumor-induced immunosuppression:

  • CTLA-4-CD28 chimeras can be used to convert negative CTLA-4 signaling to positive signaling

  • This approach enhances antitumor effects without systemic autoimmunity

  • Gene modification of both CD4+ and CD8+ T cells with CTLA-4-CD28 chimera maximizes antitumor effects

• Studying immune checkpoint timing:

  • Recent research shows CTLA-4 Ig administered after immune checkpoint therapy (ICT) can improve antitumor efficacy

  • This approach helps manage immune-related adverse events (irAEs) without compromising antitumor response

• Visualizing CTLA-4 distribution:

  • Radiolabeled anti-CTLA-4 VHH (heavy chain-only antibody fragment) can be used for whole-animal immuno-PET

  • Studies show surface-accessible CTLA-4 is largely confined to the tumor microenvironment

• Understanding mechanism of action:

  • Studies with monovalent vs. full antibodies demonstrate that CTLA-4-binding antibodies require an Fc domain for antitumor effect

  • Simple CTLA-4 blockade is insufficient for substantial antitumor effects without Fc-dependent activity

What are the key considerations when designing anti-CTLA-4 antibodies for research?

When selecting or designing anti-CTLA-4 antibodies for research:

• Clone selection: Different clones have distinct properties:

  • Clone 9D9: Mouse monoclonal that depletes intra-tumoral Tregs and neutralizes CTLA-4 in vivo

  • Clone 9H10: Syrian hamster IgG that blocks CTLA-4/ligand binding and promotes T cell co-stimulation

  • Clone UC10-4F10-11: Armenian hamster IgG useful for neutralizing CTLA-4 in vitro and in vivo

• Mechanism of action:

  • Blocking antibodies: Prevent CTLA-4 from binding B7 ligands

  • Depleting antibodies: Reduce CTLA-4+ cells through ADCC (antibody-dependent cellular cytotoxicity)

  • pH-sensitive antibodies: Allow CTLA-4 recycling rather than degradation

• Effect on CTLA-4 trafficking:

  • Some antibodies (e.g., Ipilimumab) direct CTLA-4 to lysosomal degradation

  • Others allow CTLA-4 recycling through LRBA-dependent mechanisms

  • pH-sensitive antibodies with tyrosine-to-histidine mutations prevent lysosomal CTLA-4 downregulation

• In vivo effects:

  • Antibodies requiring Fc domains for antitumor effects

  • Potential for immune-related adverse events (irAEs)

  • Different effects in tumor microenvironment vs. periphery

How can I develop and validate CTLA-4 knockdown/knockout models?

Several approaches for CTLA-4 deficiency models with their advantages and limitations:

• Complete knockout models:

  • Conventional CTLA-4 knockout mice die at 2-3 weeks due to massive lymphoproliferation

  • Characterized by lymphocytic infiltration and destruction of major organs

  • T cells show activation markers (CD69, IL-2R), down-regulate CD62L, and proliferate spontaneously

  • Useful for studying fundamental CTLA-4 biology but limited for long-term studies

• Conditional knockout systems:

  • Inducible knockout (iKO) using Cre-loxP systems allows CTLA-4 deletion in adult mice

  • Results in spontaneous lymphoproliferation, hypergammaglobulinemia, and organ-specific autoimmunity

  • Less fatal than congenital deficiency

  • Shows preferential expansion of specific T cell subsets

• Partial knockdown models:

  • RNAi-based knockdown using shRNAs delivered via lentiviral transgenesis

  • Reduces Ctla4 mRNA levels 2-4 fold, more closely mimicking natural variation

  • Can be targeted to specific cell populations

• Human CTLA-4 knockin models:

  • Replace mouse CTLA-4 with human sequence

  • Useful for testing human-specific therapeutics

  • RT-PCR verification confirms proper expression and splicing

How do I troubleshoot inconsistent results when using recombinant CTLA-4 in blocking assays?

When facing inconsistent results:

• Protein quality assessment:

  • Verify protein integrity by SDS-PAGE (expect 25-30 kDa band under reducing conditions due to glycosylation)

  • Confirm activity by binding assays with known ligands

  • Check N-glycosylation status as it's critical for dimerization and function

• Experimental variables to control:

  • Temperature: CTLA-4 trafficking is temperature-dependent; maintain consistent conditions

  • Concentration: Establish dose-response curves (typical ED50: 0.1-0.4 μg/mL)

  • Incubation time: CTLA-4 internalization kinetics affect results

  • Cell activation status: CTLA-4 function depends on activation state

• Cell source considerations:

  • Primary cells vs. cell lines respond differently

  • Cell activation state affects CTLA-4 expression

  • Different T cell subsets (conventional T cells vs. Tregs) have different baseline CTLA-4 levels

• Assay-specific troubleshooting:

  • For binding assays: Use proper controls including isotype controls and known CTLA-4 ligands

  • For functional assays: Include positive controls (anti-CD28) and negative controls

What strategies exist for studying CTLA-4 function while avoiding lethal autoimmunity?

Several approaches can be used to study CTLA-4 function without triggering lethal autoimmunity:

• Timing-controlled CTLA-4 deletion:

  • Inducible knockout systems allow CTLA-4 deletion in adult mice

  • Adult deletion causes autoimmunity but is less fatal than congenital deficiency

• Partial CTLA-4 inhibition:

  • RNAi-based knockdown reduces CTLA-4 levels 2-4 fold

  • More closely mimics natural genetic variation

• Blocking B7-CTLA-4 interactions:

  • Treatment with CTLA-4 Ig (fusion protein) blocks B7-CD28 interactions

  • Can delay onset of lymphoproliferative disease in CTLA-4-deficient mice

  • Treatment starting at day 12 after birth delays disease onset

• Cell-specific CTLA-4 manipulation:

  • Express CTLA-4 mutants in specific T cell populations

  • CTLA-4-CD28 chimera gene modification enhances antitumor effect without systemic autoimmunity

• Combination approaches:

  • Post-immunotherapy CTLA-4 Ig treatment

  • Timing is critical: Starting CTLA-4 Ig after ICT improved antitumor response, while concomitant administration diminished efficacy

What are the best methods for producing high-quality recombinant mouse CTLA-4 protein?

For optimal recombinant mouse CTLA-4 production:

• Expression systems:

  • HEK293 cells are preferred for mammalian expression

  • Ensures proper folding and post-translational modifications, especially glycosylation

• Construct design:

  • Include extracellular domain (typically aa 36-162 for mouse CTLA-4)

  • Consider fusion partners:

    • Fc tag for dimerization and purification

    • His-tag for affinity purification

    • Fluorescent protein tags for tracking studies

• Purification strategy:

  • Affinity chromatography using protein A/G for Fc-tagged proteins

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Size exclusion chromatography for final polishing

  • Target purity: >95% as determined by SDS-PAGE and HPLC

• Quality control:

  • SDS-PAGE analysis (expect 25-30 kDa under reducing conditions due to glycosylation)

  • Western blot verification

  • ELISA binding assays with B7 ligands

  • Endotoxin testing (<1 EU/μg protein using LAL method)

  • Functional testing through T cell assays

What techniques are most effective for studying CTLA-4-mediated signaling pathways?

For investigating CTLA-4 signaling:

• Phosphorylation studies:

  • Western blot analysis of key CTLA-4 downstream signals

  • Phospho-specific antibodies to detect activation of pathways

  • Immunoprecipitation to isolate CTLA-4 complexes

• Genetic approaches:

  • CRISPR/Cas9-mediated mutation of key signaling residues

  • Domain swapping (e.g., CTLA-4-CD28 chimeras)

  • Cytoplasmic tail truncations or point mutations

• Biochemical techniques:

  • Pull-down assays to identify interaction partners

  • Mass spectrometry to identify novel binding partners

  • In vitro kinase assays to assess phosphorylation events

• Imaging approaches:

  • Confocal microscopy to visualize CTLA-4 clustering with signaling components

  • FRET/BRET to detect protein-protein interactions

  • Live cell imaging using fluorescently tagged proteins

• Functional readouts:

  • Multiplex cytokine analysis

  • Transcriptomic analysis following CTLA-4 engagement

  • Cell proliferation and survival assays

  • Calcium flux measurements