Recombinant Pig T-cell surface glycoprotein CD3 epsilon chain (CD3E), partial

What is the structure and function of CD3 epsilon in the porcine T-cell receptor complex?

CD3 epsilon (CD3E) is an essential invariant component of the T-cell receptor (TCR)/CD3 complex in pigs, playing a crucial role in signal transduction following antigen recognition. The CD3 complex consists of CD3 epsilon, gamma, delta, and zeta chains, with CD3E being particularly important for TCR-dependent signal transduction across the lymphocyte membrane .

Structurally, CD3E contains:

• An extracellular domain that associates with CD3 delta or gamma to form heterodimers

• A transmembrane domain involved in complex assembly

• A cytoplasmic domain containing immunoreceptor tyrosine-based activation motifs (ITAMs) that participate in signaling

The cytoplasmic domains of CD3E show a higher degree of conservation across species compared to the extracellular domains, reflecting their critical role in signaling functions . In pigs, CD3E is found on all T lymphocytes and a subpopulation of thymocytes .

How does porcine CD3E compare to CD3E from other species?

Comparative analysis of porcine CD3E with other species reveals both conserved and divergent elements:

The short extracellular domain of the TCR zeta-chain shows 100% conservation between different species, while CD3E's extracellular domains show more variation . Importantly, antibodies directed against the intracytoplasmic domain of human CD3E have been shown to recognize bovine CD3E produced in E. coli, suggesting cross-reactivity in conserved regions . This conservation pattern is significant for developing cross-reactive reagents and understanding evolutionary preservation of T-cell signaling mechanisms.

What expression systems are typically used for producing recombinant pig CD3E?

Several expression systems have been documented for the production of recombinant pig CD3E with varying advantages for different research applications:

• Bacterial systems (E. coli):

  • Commonly used for producing CD3E for structural studies and antibody production

  • Cost-effective and yields high protein amounts

  • Limitations include lack of post-translational modifications and potential folding issues

• Mammalian cell expression systems:

  • HEK293 and CHO cells provide proper folding and post-translational modifications

  • Better for functional studies requiring native protein conformation

  • Lower yield but higher biological relevance

• Yeast expression systems:

  • Intermediate between bacterial and mammalian systems

  • Can perform some post-translational modifications

  • Used for certain pig CD3E recombinant proteins

• Baculovirus expression system:

  • Used for partial pig CD3E expression

  • Provides eukaryotic processing with higher yields than mammalian cells

The choice of expression system should be guided by the intended application, with functional studies typically requiring mammalian expression systems that preserve native conformation and modifications.

How can recombinant pig CD3E be used in the development of T-cell immunotherapies for large animal models?

Recombinant pig CD3E serves as a valuable tool for developing and testing T-cell-based immunotherapies using porcine models, which offer advantages over rodent models due to their physiological similarities to humans. Key applications include:

• CD3E-targeting bispecific antibodies development:

  • Humanized N-terminal epitope models have been developed to express the human epitope of CD3E while maintaining species-specific interactions with CD3 gamma and delta

  • These models facilitate preclinical assessment of T-cell engagers by allowing testing in immunocompetent settings

• CD3E-based CAR-T cell research:

  • The intracellular signaling domains of CD3E can be incorporated into chimeric antigen receptors

  • Studies have shown that CD3E domains can enhance antitumor activity of CAR-T cells while its essential amino acid-rich sequence (BRS) promotes persistence of CAR-T cells by recruiting p85

• CD3E immunotoxins (CD3E-IT) testing:

  • CD3E-IT has demonstrated effectiveness in inducing long-term allograft acceptance in swine models

  • Research shows that differential surface expression of CD3E among T-cell subsets drives regulatory T cell (Treg) enrichment, reshaping organ-specific T-cell composition

This translational research helps bridge the gap between rodent studies and human clinical applications, particularly for transplantation and cancer immunotherapy.

What are the challenges in achieving proper folding and functional activity of recombinant pig CD3E?

Researchers face several challenges when producing functional recombinant pig CD3E:

• Heterodimer formation requirements:

  • CD3E naturally forms heterodimers with CD3 delta or gamma in the TCR complex

  • Refolding CD3E/δ heterodimers from E. coli-expressed inclusion bodies has proven difficult, unlike CD3E/γ

  • Stabilization methods include refolding in the presence of antibody fragments (e.g., single-chain variable domain fragments like UCHT1-scFv)

• Disulfide bond formation:

  • The CxxC motif in CD3E's stem region requires proper oxidizing conditions for disulfide formation

  • Weak or no density was observed for the CxxC motif in some refolded ectodomains, suggesting challenges in reproducing this structural feature

• Conformational validation methods:

  • Verification techniques include SEC-HPLC (showing >90% purity) and functional binding assays

  • Surface staining of cells expressing CD3E with specific monoclonal antibodies confirms proper conformation

• Post-translational modifications:

  • Glycosylation patterns affect antibody recognition and protein stability

  • Bacterial systems lack glycosylation capabilities, potentially affecting conformation and function

These challenges highlight the importance of selecting appropriate expression systems and validation methods based on the intended application of the recombinant protein.

What methodologies are most effective for studying CD3E-mediated signal transduction in porcine T-cells?

Investigating CD3E-mediated signaling in porcine T-cells requires multiple complementary approaches:

• Monoclonal antibody-based activation studies:

  • Antibodies like PPT3 and BB23-8E6 recognize porcine CD3E and can activate T-cells

  • Activation can be demonstrated through calcium mobilization, increases in protein tyrosine phosphorylation, and proliferation assays

  • Flow cytometry with double-color analysis confirms binding specificity to swine lymphocyte populations

• Phosphorylation analysis of signaling components:

  • Quantitative mass spectrometry to analyze ITAM phosphorylation levels after TCR activation

  • Western blot analysis to characterize downstream signaling events

• Fluorescence resonance energy transfer (FRET) approaches:

  • FRET has been used to demonstrate that TCRs can contain both human and mouse CD3E chains in transgenic models

  • This technique helps determine CD3E subunit stoichiometry and organization in the receptor complex

• Genetic modification studies:

  • CRISPR/Cas9 engineering of porcine T-cells to introduce mutations or tags in CD3E

  • Allows for tracking of signaling dynamics in real-time when combined with fluorescent reporters

• Reconstitution experiments:

  • Expression of recombinant pig CD3E in cell lines lacking endogenous CD3E

  • Enables structure-function analysis of specific domains or mutations

These methods collectively provide insights into how CD3E contributes to T-cell receptor signaling pathways in porcine systems, with implications for both veterinary medicine and translational research.

How is CD3E expressed in porcine models of primary immunodeficiencies?

CD3E expression exhibits notable patterns in porcine immunodeficiency models, particularly in SCID (Severe Combined Immunodeficiency) pigs:

• ARTEMIS-deficient SCID pigs:

  • Despite severe lymphopenia, ART16/16, ART12/12, and ART12/16 SCID pigs unexpectedly maintain small populations of CD3ε+ cells in circulation and lymph nodes

  • CD3ε+ cells are detectable in newborn pigs (0 days of age) within lymph nodes prior to environmental exposure

  • These CD3ε+ cells show a skewed CD4α+CD8α+CD8β− T helper memory phenotype

• Functional evidence of residual T-cell development:

  • PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRβ V20 and J1.1, has detected rearranged VDJ joints in lymph node cells

  • TCRα and TCRδ constant region transcripts are expressed in thymic and lymph node tissues, though with abnormal patterns compared to carrier animals

• Organ-specific distribution:

  • Flow cytometry analysis shows variable frequencies of CD3ε+ cells in different tissues

  • Some SCID pigs maintain thymic tissue predominantly containing CD3ε+ cells

This "leaky" CD3E phenotype in porcine SCID models has important implications for using these animals in biomedical research, as it represents a variation from the classical SCID phenotype that may affect experimental outcomes.

How does CD3E surface expression vary across different porcine T-cell subsets?

Surface expression of CD3E shows significant variation across porcine T-cell subpopulations, with important functional implications:

• Expression level differences between T-cell subsets:

  • CD4+Foxp3- T cells typically show higher CD3E surface expression compared to CD4+Foxp3+ regulatory T cells (Tregs)

  • CD62Lhi Tregs (mostly CD44lo) exhibit higher CD3E expression than CD62Llo Tregs (mostly CD44hi)

  • This differential expression pattern affects susceptibility to CD3E-targeting therapies

• Quantitative representation in different tissues:

  • CD3E antigen is present on 68-82% of normal peripheral blood lymphocytes and 65-85% of thymocytes

  • The fraction of Foxp3+ Tregs varies among secondary lymphoid and non-lymphoid organs, ranging from 5.2% to 28.3% of CD4+ T cells

• Response to CD3E-targeting agents:

  • CD3E-immunotoxin (CD3E-IT) treatment preferentially depletes T cells with high CD3E expression (CD3Ehi)

  • This selective depletion results in enrichment of CD3Edim cells, particularly CD62Llo Tregs

  • The CD4/CD8 ratios typically increase following CD3E-IT treatment, indicating preferential depletion of CD8+ T cells over CD4+ T cells

This heterogeneity in CD3E expression creates opportunities for selective targeting of specific T-cell populations in therapeutic applications, particularly for transplantation tolerance induction.

What are the methods for validating the specificity and functionality of recombinant pig CD3E preparations?

Comprehensive validation of recombinant pig CD3E requires multiple complementary approaches:

• Biochemical and biophysical characterization:

  • SDS-PAGE analysis under reducing conditions with 5% enrichment gel and 15% separation gel

  • Size-exclusion high-performance liquid chromatography (SEC-HPLC) to confirm purity >90%

  • Mass spectrometry to verify sequence integrity and post-translational modifications

• Structural validation:

  • Circular dichroism spectroscopy to assess secondary structure content

  • X-ray crystallography has been used to determine three-dimensional structures of CD3 heterodimers at resolutions as high as 1.9 Å

  • Molecular replacement techniques to compare with known structures

• Functional binding assays:

  • Flow cytometry to demonstrate binding to porcine lymphocytes

  • Overlay peak curve analysis showing cell surface staining compared to isotype controls

  • Acquisition of >10,000 events to ensure statistical reliability

• Signaling capacity assessment:

  • Calcium mobilization assays following stimulation with anti-CD3E antibodies

  • Protein tyrosine phosphorylation analysis by Western blotting

  • T-cell proliferation assays to confirm functional activity

• Heterodimer formation:

  • Co-expression with CD3 delta or gamma to validate heterodimer formation capacity

  • Co-immunoprecipitation studies to confirm interactions with other TCR components

These validation methods ensure that recombinant pig CD3E preparations maintain both structural integrity and functional activity for reliable research applications.

What are the key challenges in developing cross-reactive antibodies for porcine CD3E research?

Developing antibodies that effectively target porcine CD3E presents several specific challenges:

• Domain conservation variations:

  • The cytoplasmic domains of CD3E show higher conservation across species than extracellular domains

  • Antibodies targeting conserved cytoplasmic epitopes may offer better cross-reactivity but require cell permeabilization for binding

• Epitope accessibility issues:

  • Some CD3E epitopes may be partially masked by glycosylation or interactions with other TCR/CD3 components

  • The bulky carbohydrate moieties of CD3δ and CD3γ can cover protein surfaces, contributing to poor antigenicity

• Validation methodologies:

  • Double-color flow cytometry analysis is essential to confirm specific binding to swine lymphocyte populations

  • Competitive binding studies with known anti-pig CD3 antibodies (e.g., STH164) can verify binding to shared epitopes

• Clone selection considerations:

  • Clones like BB23-8E6-2B3C (an immunoglobulin isotype switch variant of BB23-8E6) have demonstrated utility in immunoprecipitation, immunocytochemistry, and costimulation

  • The PPT3 monoclonal antibody has been validated for activation capabilities through calcium mobilization, protein tyrosine phosphorylation, and proliferation assays

For researchers seeking to develop new antibodies, targeting the conserved regions of the CD3E molecule while considering species-specific variations in the extracellular domain is crucial for achieving both specificity and potential cross-reactivity.

How can recombinant pig CD3E contribute to developing improved xenotransplantation models?

Recombinant pig CD3E has significant potential for advancing xenotransplantation research:

• T-cell depletion strategies optimization:

  • CD3E-immunotoxins (CD3E-IT) have demonstrated effectiveness in inducing long-term allograft acceptance in swine models

  • Understanding the differential depletion of T-cell subsets based on CD3E expression levels can improve transplant protocols

  • CD3E-IT treatment enriches Foxp3+ regulatory T cells (Tregs) in tissue-resident pools, potentially contributing to tolerance induction

• Chimeric receptor engineering:

  • Transgenic expression of human CD3E in pigs could create more humanized immune systems

  • Studies in mice have shown that transgenic human CD3E can incorporate into T-cell receptor complexes, allowing normal T-cell development and selection

• Tolerance induction mechanisms:

  • CD3E-IT treatment alone or in combination with short-term immunosuppressive chemotherapy has shown promise in transplant models

  • Recent CD3E-IT-mediated donor hematopoietic stem cell transplant studies have demonstrated promising results through mixed chimerism in monkey and swine models

• T-cell monitoring tools:

  • Recombinant CD3E can be used to develop improved flow cytometry reagents for monitoring T-cell responses post-transplantation

  • Multiparameter analysis can track the emergence of donor-reactive T cells or regulatory T cells

These applications could collectively advance our understanding of the porcine immune system's role in xenograft rejection and facilitate the development of more effective strategies for inducing transplantation tolerance.

What are the emerging techniques for studying CD3E structure-function relationships in porcine T-cells?

Several cutting-edge methodologies are advancing our understanding of CD3E biology in porcine systems:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of complete TCR/CD3 complexes without crystallization

  • Can reveal the spatial arrangement of CD3E in relation to other components

  • Particularly valuable for understanding the "paddle-shaped" CD3E/γ and ε/δ ectodomains that may lie close to the membrane

• Single-cell transcriptomics and proteomics:

  • Reveals heterogeneity in CD3E expression and associated signaling components across T-cell populations

  • Can identify novel interaction partners and signaling networks

  • RNA sequencing of the repertoire provides insights into T-cell development and selection

• CRISPR/Cas9 genome editing in primary porcine T-cells:

  • Enables precise modification of CD3E domains to assess functional consequences

  • Can introduce reporter tags for live-cell imaging of CD3E dynamics

  • Facilitates structure-function studies by creating domain deletions or point mutations

• Advanced imaging techniques:

  • Super-resolution microscopy reveals nanoscale organization of CD3E within TCR clusters

  • Fluorescence resonance energy transfer (FRET) approaches determine CD3E subunit stoichiometry

  • Live-cell imaging captures dynamic reorganization during T-cell activation

• Molecular dynamics simulations:

  • Computational modeling of CD3E interactions with other TCR components

  • Insights into how specific domains contribute to complex stability and signaling

  • Prediction of critical residues for targeted mutagenesis studies

These methodologies collectively provide unprecedented resolution for understanding how CD3E structure relates to its function in porcine T-cells, with implications for both basic immunology and translational applications.

What strategies can overcome poor expression or folding of recombinant pig CD3E in bacterial systems?

Researchers can employ several strategies to improve the production of correctly folded recombinant pig CD3E in bacterial expression systems:

• Co-expression with chaperones:

  • Co-expression with molecular chaperones like GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor

  • Reduces inclusion body formation and enhances proper folding

• Optimization of induction conditions:

  • Lower growth temperatures (16-25°C) to slow protein synthesis and improve folding

  • Reduced IPTG concentrations (0.1-0.5 mM) for more gradual induction

  • Use of auto-induction media for gradual protein expression

• Fusion tag selection:

  • Solubility-enhancing tags like glutathione S-transferase (GST), maltose-binding protein (MBP), or SUMO

  • TCR zeta-chain has been successfully produced as a GST fusion protein in E. coli

• Refolding with stabilizing partners:

  • Refolding in the presence of binding partners like antibody fragments

  • CD3-ε/δ heterodimers have been successfully refolded with UCHT1-scFv, producing stable 1:1:1 complexes

• Oxidative refolding optimization:

  • Careful control of redox conditions with optimal glutathione ratios

  • Step-wise dialysis with decreasing denaturant concentrations

  • Addition of stabilizing agents like L-arginine or glycerol

• Construct design considerations:

  • Expression of functional domains rather than full-length protein

  • Removal of hydrophobic transmembrane regions

  • Codon optimization for E. coli expression

These approaches have been successfully applied to various CD3E expression challenges, with the choice of method depending on the specific experimental requirements and downstream applications.

How can researchers distinguish CD3E expression in tissue-resident T-cells from circulating T-cells in porcine models?

Accurately differentiating tissue-resident from circulating T-cells based on CD3E expression requires specialized techniques:

• Intravascular staining approach:

  • Intravenous injection of fluorochrome-conjugated anti-CD3E antibodies (IVS+) prior to tissue collection

  • Cells in circulation will be labeled, while tissue-resident cells remain unlabeled

  • Research has used this approach to identify tissue-resident (IVS−) pools of CD3E+ cells in various organs

• Multi-parameter flow cytometry panels:

  • Combining CD3E with tissue-residency markers:

    • CD69 (early activation marker retained on tissue-resident cells)

    • CD103 (αE integrin, expressed on many tissue-resident T cells)

    • CD62L (L-selectin, low on tissue-resident effector memory cells)

  • CD3E+ cells in SCID pigs were found to be primarily CD8α+CD8β+CD4α− and CD8α+CD8β−CD4α+ phenotypes

• Immunohistochemistry with spatial analysis:

  • CD3E staining of tissue sections reveals localization patterns

  • Tissue-resident cells often show distinct distribution in tissue parenchyma versus vasculature

  • Has been used to identify CD3E+ cells in neonatal SCID pig lymph nodes

• Transcriptional profiling:

  • Tissue-resident T cells have characteristic transcriptional signatures

  • Single-cell RNA sequencing can classify cells based on residence markers

  • TCR sequencing can identify clonal relationships between populations

• Parabiosis experiments:

  • Surgical joining of circulation between animals

  • Allows distinction between cells that equilibrate between partners (circulating) versus those that remain host-derived (resident)

These approaches collectively provide robust methods for distinguishing CD3E expression in tissue-resident versus circulating T-cells, critical for understanding T-cell biology in porcine models.

What controls should be included when using recombinant pig CD3E in binding and functional assays?

Rigorous experimental design for recombinant pig CD3E studies requires comprehensive controls:

• Positive and negative expression controls:

  • Positive: Known CD3E-expressing porcine T-cell lines or primary T-cells

  • Negative: Non-T cells (B cells, NK cells) that never express CD3E

  • Background control: Secondary antibody only to assess non-specific binding

• Isotype controls for antibody experiments:

  • Matched isotype control antibodies (e.g., mouse IgG1 or IgG2b for anti-CD3E antibodies)

  • Used at the same concentration as the experimental antibody (e.g., 1μg/1×10^6 cells)

  • Critical for flow cytometry to establish gating boundaries

• Specificity controls:

  • Competitive binding with known anti-CD3E antibodies

  • Binding to CD3E-knockout or CD3E-depleted cells

  • Cross-species reactivity assessment with human and mouse T-cells

• Functional validation controls:

  • Positive control for T-cell activation: PMA/ionomycin or concanavalin A

  • Inhibition control: Cyclosporin A to block TCR-mediated activation

  • Dose-response curves to establish optimal concentrations

• Reagent quality controls:

  • Endotoxin testing to ensure preparations are free from bacterial contaminants

  • Protein concentration verification through multiple methods (BCA, Bradford)

  • Storage stability testing with repeat analysis after freeze-thaw cycles

• Technical replicates and biological replicates:

  • Minimum of three technical replicates per experiment

  • Independent biological samples from different animals

  • Statistical analysis appropriate for sample size and distribution