CTLA4 Human, igG-His

What is CTLA4 and what are its primary functions in the immune system?

CTLA-4 (CD152) is a protein receptor that functions as a critical immune checkpoint, downregulating immune responses and maintaining immune homeostasis. It is constitutively expressed in regulatory T cells (Tregs) but only upregulated in conventional T cells after activation—a pattern particularly notable in cancer contexts. CTLA-4 acts as an inhibitory "off" switch when bound to CD80 or CD86 on antigen-presenting cells, preventing cell cycle progression and IL-2 production . The protein plays a pivotal role in controlling effector T-cell differentiation, proliferation, and apoptosis while endowing Treg cells with immunosuppressive activity. CTLA-4 inhibitory signals coordinate with phosphatases SHIP2 and PP2A to reduce AKT activity, which is required for the suppressive function of CD4+CD25+Foxp3+ regulatory T cells .

What is the structure and expression pattern of CTLA4 in humans?

CTLA-4 is encoded by the CTLA4 gene in humans (Ctla4 in mice). The protein is predominantly expressed in activated T cells and regulatory T cells. While conventional T cells upregulate CTLA-4 following activation, Tregs constitutively express this molecule . Flow cytometric analysis reveals that CTLA-4 is not detected in B cells but is found in Foxp3+ Tregs, as demonstrated in human CTLA-4 knock-in (Ctla4 h/h) and wild-type mice . The structure consists of an extracellular domain that binds CD80/CD86, a transmembrane domain, and a cytoplasmic tail that mediates signaling. This organization enables CTLA-4 to compete with CD28 for binding to CD80/CD86, thereby inhibiting costimulatory signals necessary for T-cell activation.

What is a CTLA4-Ig fusion protein and how does it differ from native CTLA4?

A CTLA4-Ig fusion protein contains the extracellular domain of CTLA-4 fused to the Fc fragment of human IgG antibody . This construct combines the binding specificity of CTLA-4 with the stability and pharmacokinetic properties of an immunoglobulin. Unlike native membrane-bound CTLA-4, which functions cell-intrinsically, the CTLA4-Ig fusion protein is soluble and can bind to CD80/CD86 on antigen-presenting cells without requiring cell-cell contact. This allows CTLA4-Ig to function as a competitive inhibitor of the CD28-CD80/CD86 interaction, blocking costimulatory signals needed for T-cell activation. The addition of a histidine tag (CTLA4-IgG-His) further facilitates protein purification and detection in research applications through nickel-based affinity chromatography and anti-His antibodies .

How do CTLA4-Ig fusion proteins affect T cell and B cell interactions in experimental models?

CTLA4-Ig fusion proteins exert complex effects on T cell-B cell interactions. Research using human CTLA-4 knock-in mice reveals that CTLA-4 manipulation can significantly impact B cell populations. Administration of Belatacept (a CTLA4-Ig fusion protein) rescues B cells from depletion mediated by anti-CTLA-4 antibody-drug conjugates (Ipi-DM1) and reduces the development of effector memory T cells as well as granzyme B and IFN-γ expression in both CD4 and CD8 T cells .

Studies show that CTLA-4 deficiency or blockade leads to spontaneous differentiation of naïve CD4+ T cells into T follicular helper cells (Tfh), increased germinal center formation, and elevated cytokine production (IL-2, IFN-γ, IL-4, and GM-CSF) . The relationship between CTLA-4 and B cells is bidirectional—patients with CTLA-4 mutations show reduced circulating B cells, associated with hypogammaglobulinemia and lymphopenia . In experimental settings, selective deletion of CTLA-4 in T follicular regulatory cells (Tfr) increases antigen-specific antibody production .

What signaling pathways are affected by CTLA4-Ig binding, and how do they differ from native CTLA4 signaling?

CTLA4-Ig fusion proteins primarily act by competitively inhibiting the CD28-CD80/CD86 interaction, rather than by direct signaling. In contrast, native CTLA-4 engages multiple signaling pathways:

• PI3K/AKT pathway: CTLA-4 regulates the PI3K/AKT signaling pathway, which plays a key role in maintaining the balance of T-cell survival, anergy, and apoptosis, as well as maintaining long-term immune tolerance .

• Phosphatase recruitment: After binding CD80/CD86, CTLA-4 recruits SHIP2 and PP2A to the membrane. SHIP2 inhibits TCR signaling by dephosphorylating CD3 and restrains PI3K/AKT signaling via dephosphorylation of PI3K. PP2A directly dephosphorylates AKT to reduce its activity .

• Downstream effects: These signaling events ultimately reduce the activity of NF-κB, mTOR, Bcl-xl, and the production of IL-2 in T cells .

The reduced AKT activity is particularly important for the suppressive function of CD4+CD25+Foxp3+ regulatory T cells. In Tregs, CTLA-4 signaling prevents FOXO factors from translocating into the nucleus, antagonizing the differentiation and proliferation of natural Treg cells in the thymus .

What is the current understanding of CTLA4-Ig in autoimmune disease models versus tumor immunology?

CTLA4-Ig fusion proteins have divergent roles in autoimmune diseases and cancer immunology:

In Autoimmune Diseases:
CTLA4-Ig functions as an immunosuppressive agent by blocking costimulatory signals required for T-cell activation. This mechanism underlies the therapeutic efficacy of abatacept, a CTLA4-Ig fusion protein used in treating rheumatoid arthritis and other autoimmune conditions . Genetic studies reveal that CTLA-4 deficiency in mice results in severe lymphoproliferative disease, with dramatically increased T-cell blast in lymph nodes and spleens, typically causing death by 3 weeks of age . In humans, CTLA-4 mutations lead to dysfunction of FoxP3+ Treg cells, hyperproliferation of lymphocytes, and activation of effector T cells .

In Tumor Immunology:
Contrary to its use in autoimmunity, blocking CTLA-4 with antibodies is a strategy to enhance anti-tumor immunity. Recent research challenges the view that checkpoint inhibition is the primary mechanism of action for anti-CTLA-4 antibodies in cancer therapy . Rather, evidence suggests that anti-CTLA-4 antibodies may function through Fc-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) against Tregs within the tumor microenvironment. Studies in human CTLA-4 knock-in mice using anti-human CTLA-4 antibody Ipilimumab conjugated to emtansine (Anti-CTLA-4 ADC) demonstrate that targeting CTLA-4-expressing cells can impact both Tregs and promote effector T cell responses against tumors .

How can researchers construct and verify a CTLA4-IgG-His fusion protein expression vector?

The construction of a CTLA4-IgG-His fusion protein expression vector involves several critical steps:

• Primer Design and PCR Amplification: Design specific primers to amplify the extracellular domain of the CTLA-4 gene with appropriate restriction sites. Perform PCR using a high-fidelity polymerase such as Pfu polymerase .

• Cloning Strategy: Ligate the PCR-amplified CTLA-4 fragment into a vector containing the human IgG1 gene. Subsequently, ligate the resulting CTLA4-IgG1 fusion fragment into an expression vector such as pBudCE4.1 .

• Verification Steps:

  • Restriction enzyme digestion pattern analysis

  • DNA sequencing to confirm the correct reading frame and absence of mutations

  • Western blot analysis using anti-CTLA-4 and anti-IgG antibodies to verify protein expression

  • ELISA to confirm binding to CD80/CD86

• Histidine Tag Addition: Incorporate a 6x histidine tag at the C-terminus of the fusion protein for purification purposes through additional PCR steps or site-directed mutagenesis.

The complete verification process should include both genotypic confirmation (sequencing) and phenotypic validation (protein expression and functionality testing) .

What are the optimal expression systems for producing functional CTLA4-IgG-His fusion proteins?

Several expression systems can be employed for producing CTLA4-IgG-His fusion proteins, each with distinct advantages:

CHO-K1 cells are particularly suitable for CTLA4 expression as demonstrated by established stable cell lines . For research purposes where glycosylation is critical for function, mammalian expression systems (CHO or HEK293) are recommended. The choice should be guided by whether the protein is intended for functional studies (where proper folding and PTMs are essential) or structural studies (where high yield might be prioritized) .

What purification strategies yield the highest purity and functionality for CTLA4-IgG-His proteins?

A multi-step purification strategy typically yields the highest purity for CTLA4-IgG-His fusion proteins:

• Initial Capture:

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

  • For IgG-fused constructs: Protein A or Protein G affinity chromatography

• Intermediate Purification:

  • Ion exchange chromatography (typically anion exchange at pH 8.0)

  • Hydroxyapatite chromatography to separate monomeric and aggregated forms

• Polishing Step:

  • Size exclusion chromatography (SEC) to remove aggregates and achieve >95% purity

  • Endotoxin removal using specialized resins if intended for in vivo applications

• Quality Control:

  • SDS-PAGE and western blotting to confirm identity and purity

  • Surface plasmon resonance (SPR) to verify binding to CD80/CD86 with expected affinity (typically low nanomolar KD, similar to ipilimumab at approximately 8.7 nM)

  • Functional assays measuring inhibition of T cell proliferation or IL-2 production

For optimal retention of functionality, purification should be performed at 4°C with appropriate protease inhibitors. The addition of stabilizers (e.g., trehalose or sucrose) in the final formulation buffer can maintain protein stability during storage .

How should researchers design experiments to evaluate CTLA4-IgG-His binding affinity to CD80/CD86?

To evaluate CTLA4-IgG-His binding affinity to CD80/CD86, researchers should implement a comprehensive experimental approach:

• Surface Plasmon Resonance (SPR):

  • Immobilize recombinant CD80 or CD86 on sensor chips

  • Flow CTLA4-IgG-His at multiple concentrations (typically 0.1-100 nM)

  • Determine association (kon) and dissociation (koff) rates

  • Calculate equilibrium dissociation constant (KD)

  • Expected values: low nanomolar range (reference: ipilimumab KD ≈ 8.7 nM)

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Coat plates with recombinant CD80 or CD86

  • Incubate with serial dilutions of CTLA4-IgG-His

  • Detect binding using anti-His or anti-IgG antibodies

  • Generate binding curves to determine EC50 values

• Cell-Based Binding Assays:

  • Use CD80/CD86-expressing cells (e.g., activated dendritic cells)

  • Incubate with fluorescently labeled CTLA4-IgG-His

  • Analyze binding by flow cytometry

  • Perform competitive binding assays with known ligands

• Bio-Layer Interferometry:

  • Alternative to SPR with similar principles

  • Allows real-time monitoring of protein-protein interactions

  • Provides kon, koff, and KD values

Results should be compared to commercial CTLA4-Ig products (e.g., abatacept) as reference standards to establish relative binding efficiency and specificity .

What cellular and animal models are most appropriate for testing CTLA4-IgG-His immunomodulatory functions?

Selection of appropriate models for evaluating CTLA4-IgG-His functions should consider disease relevance and translational potential:

In Vitro Cellular Models:

• Mixed Lymphocyte Reaction (MLR):

  • Measures inhibition of T-cell proliferation in response to allogeneic stimulation

  • Quantify using tritiated thymidine incorporation or CFSE dilution

  • CTLA4-IgG-His should dose-dependently inhibit proliferation

• T-Cell Activation Assays:

  • CD3/CD28-stimulated primary human T cells

  • Measure IL-2 production, CD25 expression, and proliferation

  • CTLA4-IgG-His should suppress these activation markers

• Treg Functional Assays:

  • Assess impact on Treg suppressive capacity

  • Co-culture Tregs with effector T cells in the presence of CTLA4-IgG-His

  • Evaluate changes in T-cell proliferation and cytokine production

In Vivo Animal Models:

• Human CTLA-4 Knock-in Mice:

  • Express human instead of murine CTLA-4

  • Allows testing of human-specific CTLA4-IgG-His

  • Enables assessment of effects on T-cell subsets and B-cell populations

• Autoimmune Disease Models:

  • Collagen-induced arthritis (CIA)

  • Experimental autoimmune encephalomyelitis (EAE)

  • CTLA4-IgG-His should reduce disease severity and inflammatory markers

• Transplantation Models:

  • Skin, heart, or islet allografts

  • Measure graft survival time and rejection biomarkers

  • CTLA4-IgG-His should prolong graft survival

• Humanized Mouse Models:

  • NOD-scid-gamma (NSG) mice reconstituted with human immune cells

  • Provides translational insights into human immune responses

  • Particularly valuable for evaluating specificity for human CD80/CD86

What considerations are important when analyzing contradictory data in CTLA4-IgG-His research?

When encountering contradictory data in CTLA4-IgG-His research, several methodological considerations are essential:

• Context-Dependent Effects:

  • CTLA-4 functions differently in various cell types and microenvironments

  • Studies report that CTLA-4's effects on PI3K/AKT signaling vary depending on experimental conditions, cell types, organs, and disease states

  • Example: SHIP2 inhibits PI3K/AKT in some contexts but enhances PI3K activity in others

• Genetic Background Considerations:

  • Different mouse strains may yield contradictory results

  • Human versus mouse CTLA-4 may have subtle functional differences

  • Use human CTLA-4 knock-in models for translational research

• Protein Quality Variables:

  • Fusion protein design (linker length, orientation, tag position)

  • Glycosylation differences between expression systems

  • Aggregation or degradation during purification or storage

  • Always verify protein integrity before functional experiments

• Experimental Design Factors:

  • Dose-dependency: Use a wide concentration range (0.1-100 μg/ml)

  • Time-course analyses: Acute versus chronic exposure effects

  • Cell activation status: Resting versus pre-activated cells respond differently

• Reconciliation Strategies:

  • Reproduction with consistent protocols across laboratories

  • Meta-analysis of published data with attention to methodological details

  • Combined in vitro and in vivo validation

  • Molecular mechanism studies to identify context-specific signaling differences

How does CTLA4-IgG-His compare with antibody-based CTLA4 modulators in research applications?

CTLA4-IgG-His fusion proteins and anti-CTLA-4 antibodies represent fundamentally different approaches to modulating CTLA-4 biology:

Research indicates that anti-CTLA-4 antibodies may function through mechanisms beyond simple checkpoint inhibition. For instance, anti-CTLA-4 antibody-drug conjugates (ADCs) like Ipi-DM1 can target CTLA-4-expressing cells, impair Treg function, and drive T-cell hyperproliferation with increases in effector memory T cells . In contrast, CTLA4-Ig fusion proteins like Belatacept can rescue B cells from depletion mediated by anti-CTLA-4 ADCs and reduce effector T-cell activation .

These different modalities allow researchers to select the appropriate tool based on their specific research question—whether investigating immunosuppression (CTLA4-Ig) or immune activation (anti-CTLA-4 antibodies) .

What are the methodological approaches to studying CTLA4-IgG-His in models of autoimmunity?

Investigating CTLA4-IgG-His in autoimmunity models requires methodological rigor across multiple experimental systems:

• Disease Model Selection:

  • Rheumatoid arthritis: Collagen-induced arthritis (CIA) or K/BxN serum transfer

  • Multiple sclerosis: Experimental autoimmune encephalomyelitis (EAE)

  • Type 1 diabetes: NOD mice or streptozotocin-induced diabetes

  • Systemic lupus erythematosus: MRL/lpr or NZB/W F1 mice

• Treatment Protocols:

  • Preventive (before disease onset) versus therapeutic (after disease manifestation)

  • Dose-finding studies (typically 1-25 mg/kg)

  • Frequency determination (daily, weekly, biweekly administration)

  • Combination with other immunomodulatory agents

• Disease Assessment Methods:

  • Clinical scoring systems specific to each model

  • Histopathological analysis of affected tissues

  • Autoantibody measurements by ELISA

  • Inflammatory cytokine profiling in serum and tissues

• Immunological Evaluations:

  • Flow cytometric analysis of T-cell subpopulations (Th1, Th17, Treg)

  • Assessment of CD80/CD86 expression on APCs

  • Antigen-specific T-cell recall responses

  • Transcriptomic analysis of target tissues

• Molecular Mechanistic Studies:

  • Phospho-flow analysis of signaling pathways (PI3K/AKT)

  • RNA-seq of sorted immune cell populations

  • Chromatin immunoprecipitation to assess transcription factor binding

  • In situ methods to visualize immune cell interactions

When studying CTLA-4-deficient states, researchers should note that genetic mutations in CTLA-4 lead to severe autoimmune phenotypes, including dysregulated T-cell responses, lymphocyte hyperproliferation, and early mortality in mice . These models provide valuable insights into CTLA-4 biology that complement studies using CTLA4-IgG-His as a therapeutic agent.

How can researchers optimize CTLA4-IgG-His half-life and tissue distribution for experimental applications?

Optimizing the pharmacokinetic properties of CTLA4-IgG-His involves several strategic approaches:

• Structural Modifications:

  • Fc engineering: Introduce mutations (e.g., M252Y/S254T/T256E) to enhance FcRn binding and extend half-life

  • Glycoengineering: Modify glycosylation patterns to reduce clearance

  • PEGylation: Site-specific addition of polyethylene glycol moieties

  • Alternative fusion partners: Consider albumin fusion instead of or in addition to IgG

• Formulation Strategies:

  • Buffer optimization: Test various pH conditions (typically pH 6.0-7.5)

  • Excipient screening: Include stabilizers like trehalose, sucrose, or polysorbates

  • Concentration adjustments: Higher concentrations may form aggregates that alter PK

  • Lyophilization: Develop freeze-dried formulations for improved stability

• Administration Route Optimization:

  • Subcutaneous vs. intravenous: SC administration typically provides more sustained levels

  • Local delivery: Site-specific administration for targeted effects (e.g., intra-articular)

  • Sustained release formulations: Encapsulation in biodegradable polymers

  • Osmotic pumps for continuous delivery in experimental animals

• Monitoring Methods:

  • ELISA: Detect CTLA4-IgG-His in serum samples over time

  • Functional bioassays: Measure CD80/CD86 occupancy on APCs

  • Tissue distribution: Immunohistochemistry with anti-His or anti-CTLA-4 antibodies

  • Pharmacokinetic modeling: Calculate clearance, volume of distribution, and half-life

• Special Considerations:

  • Anti-drug antibody monitoring: Screen for immune responses against the fusion protein

  • Target-mediated drug disposition: Account for binding to CD80/CD86 in PK analyses

  • Species differences: Human CTLA4-IgG-His may have different PK in mice versus humans

  • Disease state effects: Inflammation can alter distribution and clearance

These optimization strategies should be implemented systematically, with careful attention to how modifications might affect the biological activity of the CTLA4-IgG-His fusion protein in the specific experimental context.