CTLA4 Human, IgG-His, Active

What is the structural composition of CTLA4 Human, IgG-His, Active and how does it differ from native CTLA4?

CTLA4 Human, IgG-His, Active is a recombinant fusion protein comprising the extracellular domain of human CTLA4 joined to a human IgG Fc region with a histidine tag. Unlike native membrane-bound CTLA4, which is primarily expressed on activated T cells and regulatory T cells as a transmembrane protein, this recombinant version is soluble . The IgG fusion creates a dimeric protein that enhances stability and half-life compared to the CTLA4 extracellular domain alone, while the histidine tag facilitates purification and detection in experimental systems . The "Active" designation confirms the protein maintains proper folding and conformation necessary for high-affinity binding to its natural ligands CD80 (B7-1) and CD86 (B7-2) .

How does the expression of CTLA4 change during T cell activation, and what mechanisms regulate this process?

CTLA4 expression follows a tightly regulated temporal pattern during T cell activation. Upon T cell receptor (TCR) engagement, CTLA4 mRNA becomes detectable after approximately 1 hour and reaches peak levels at 24-36 hours post-activation . This upregulation is primarily regulated by the nuclear factor of activated T cells (NF-AT), as inhibiting NF-AT activity significantly reduces CTLA4 transcription . Additionally, cyclic AMP (cAMP) serves as a positive regulator of CTLA4 expression . Intracellularly, CTLA4 undergoes dynamic trafficking between the cell surface and endosomal compartments, with most CTLA4 proteins residing in intracellular vesicles until activation triggers their redistribution to the plasma membrane . This regulated expression ensures that CTLA4's inhibitory function occurs at the appropriate time during immune responses.

What are the primary binding partners of CTLA4 Human, IgG-His, Active and what binding affinities can be expected?

The primary binding partners of CTLA4 Human, IgG-His, Active are:

• CD80 (B7-1) and CD86 (B7-2) - These costimulatory molecules are expressed on antigen-presenting cells and bind CTLA4 with significantly higher affinity than they bind CD28 . CTLA4 binds CD80 with approximately 20-fold higher affinity (KD ~0.2-0.4 nM) than CD28 (KD ~4-8 nM).

• Anti-CTLA4 antibodies - Various research and therapeutic antibodies such as Ipilimumab can bind to the CTLA4 portion of the fusion protein .

The dimeric nature of the CTLA4-IgG-His fusion provides increased avidity through bivalent binding, enhancing its effectiveness as a competitive inhibitor of CD28-CD80/CD86 interactions. When designing binding experiments, researchers should account for the concentration-dependent nature of these interactions, typically using ranges of 0.1-10 μg/mL to observe dose-dependent effects.

What are the optimal experimental conditions for using CTLA4 Human, IgG-His, Active in T cell functional assays?

For optimal results in T cell functional assays using CTLA4 Human, IgG-His, Active:

Reagent preparation:

• Store stock protein at -80°C in aliquots to prevent multiple freeze-thaw cycles

• Working solutions should be prepared in sterile PBS with 0.1% BSA at concentrations of 0.1-10 μg/mL

• Verify protein activity before critical experiments using a simple binding ELISA

Experimental parameters:

• Pre-incubate CTLA4-IgG-His with antigen-presenting cells 30-60 minutes before adding T cells

• For T cell proliferation assays, use freshly isolated T cells labeled with CFSE and stimulate with anti-CD3 (1 μg/mL) plus CD80/CD86-expressing cells

• Optimal culture duration is typically 48-72 hours for proliferation and 24-48 hours for cytokine measurements

• Include dose-response curves (0.1-10 μg/mL) to determine optimal inhibitory concentration

Critical controls:

• Isotype-matched IgG-His fusion protein (negative control)

• Commercial CTLA4-Ig (positive control)

• Anti-CD80/CD86 blocking antibodies (alternative blocking strategy)

For Treg function studies, additionally compare the effects of CTLA4-IgG-His to CTLA4 knockdown approaches, as CTLA4 is crucial for regulatory T cell suppressive function .

How can I distinguish between CTLA4 ligand blocking versus signaling effects in my experimental system?

Distinguishing between CTLA4 ligand blocking and signaling effects requires careful experimental design:

Comparative approach strategy:

• Use CTLA4-IgG-His, Active (blocks CD80/CD86 but has no signaling capability)

• Use anti-CTLA4 antibodies that can either:

  • Block CTLA4-CD80/CD86 interaction only

  • Induce CTLA4 downregulation (e.g., Ipilimumab)

  • Potentially engage CTLA4 signaling

Signaling pathway analysis:

• Monitor phosphorylation of AKT and PI3K, as CTLA4 signaling normally activates phosphatases PP2A and SHIP2 that dephosphorylate these proteins

• Assess NF-κB and NF-AT activity, which are downstream of CTLA4 signaling

• Measure IL-2 production and T cell proliferation as functional readouts

Advanced approaches:

• Compare effects in wild-type versus CTLA4 cytoplasmic domain-deleted T cells

• Use fluorescently tagged CTLA4 to track internalization and recycling patterns following different treatments

• Implement intracellular flow cytometry to measure signaling molecule phosphorylation

In interpretating results, effects seen with CTLA4-IgG-His but not with certain anti-CTLA4 antibodies suggest ligand competition rather than signaling, while effects specific to antibodies that trigger internalization may indicate CTLA4 downregulation mechanisms .

What methods can be used to verify the activity and specificity of CTLA4 Human, IgG-His, Active protein preparations?

Multiple complementary methods should be used to verify activity and specificity:

Biochemical verification:

• SDS-PAGE and Western blotting: Confirm expected molecular weight (~90-110 kDa for non-reduced dimer) and reactivity with anti-CTLA4 antibodies

• Size exclusion chromatography: Verify absence of protein aggregation

• Circular dichroism: Confirm proper protein folding

Binding verification:

• Direct ELISA: Coat plates with recombinant CD80/CD86, add CTLA4-IgG-His in serial dilutions, detect with anti-His or anti-IgG

• Surface Plasmon Resonance: Measure binding kinetics to immobilized CD80/CD86

• Flow cytometry: Detect binding to CD80/CD86-expressing cells

Functional verification:

• T cell proliferation inhibition assay: CTLA4-IgG-His should dose-dependently inhibit T cell activation

• Cytokine production assay: Measure reduction in IL-2 and other cytokines

• Competitive binding assay: Set up competition between labeled CD28-Ig and CTLA4-IgG-His for binding to CD80/CD86

Specificity controls:

• Pre-blocking with excess unlabeled CTLA4-Ig should prevent binding

• Effects should be absent on CD80/CD86-deficient cells

• Anti-CTLA4 antibodies like Ipilimumab should block binding to CD80/CD86

A typical verification protocol includes testing at least one binding and one functional assay, with appropriate controls, before proceeding to complex experiments.

How does CTLA4 Human, IgG-His, Active affect different T cell subsets and what are the implications for experimental design?

CTLA4 Human, IgG-His, Active differentially affects various T cell subsets in experimental systems, with important implications for study design:

Effects on conventional T cells:

• Inhibits CD28-mediated costimulation, reducing proliferation and IL-2 production

• Prevents naïve CD4+ T cells from spontaneously differentiating into T follicular helper cells (Tfh)

• Reduces production of multiple cytokines (IL-2, IFN-γ, IL-4, and GM-CSF)

• Impacts PI3K/AKT signaling, affecting the balance between T-cell survival, anergy, and apoptosis

Effects on regulatory T cells (Tregs):

• Mimics some aspects of natural CTLA4 function in Tregs, which is critical for their suppressive activity

• Cannot replicate CTLA4's role in promoting FoxP3 expression induced by TGF-β

• Does not fully recapitulate CTLA4's function in Treg-mediated suppression of dendritic cells

Effects on follicular T cells:

• Impacts both T follicular helper cells (Tfh) and T follicular regulatory cells (Tfr)

• Controls follicular helper T cell activity and downregulates costimulatory molecules on B cells

Experimental design considerations:

• Include separate analyses for different T cell subsets using appropriate surface markers

• When studying Tregs, compare CTLA4-IgG-His effects with CTLA4 knockdown approaches

• For follicular T cell studies, examine germinal center responses and antibody production

• Consider that CTLA4 expression levels differ between T cell subsets, affecting sensitivity to blockade

Understanding these differential effects is essential for correctly interpreting experimental outcomes, particularly when studying complex immune interactions or autoimmune disease models .

What insights have been gained about CTLA4 trafficking and degradation mechanisms that impact experimental use of CTLA4 Human, IgG-His, Active?

Recent research on CTLA4 trafficking and degradation has revealed critical insights affecting experimental applications:

CTLA4 trafficking dynamics:

• CTLA4 undergoes continuous cycling between the cell surface and endosomal compartments

• Surface CTLA4 has a short half-life due to rapid internalization

• The LRBA-dependent mechanism is essential for CTLA4 recycling to the cell surface after endocytosis

Anti-CTLA4 antibody effects on trafficking:

• Different anti-CTLA4 antibodies exhibit distinct effects on CTLA4 fate:

  • Ipilimumab and TremeIgG1 (irAE-prone antibodies) rapidly direct cell surface CTLA4 to lysosomes for degradation

  • Non-irAE-prone antibodies (HL12, HL32) dissociate from CTLA4 after endocytosis, allowing CTLA4 recycling

  • Disrupting CTLA4 recycling results in robust CTLA4 downregulation by all anti-CTLA4 antibodies

Experimental implications:

• CTLA4-IgG-His serves as a valuable control that blocks CD80/CD86 without affecting cellular CTLA4 levels

• When comparing CTLA4-IgG-His with anti-CTLA4 antibodies, observed differences may reflect:

  • Simple CD80/CD86 blockade (CTLA4-IgG-His effect)

  • CTLA4 downregulation (antibody-specific effect)

• For accurate interpretation of results, researchers should monitor CTLA4 expression levels when using antibodies

Research applications:

• Use CTLA4-IgG-His alongside pH-sensitive anti-CTLA4 antibodies that prevent lysosomal degradation

• Compare trafficking patterns using fluorescently labeled CTLA4 and confocal microscopy

• Investigate how CTLA4 degradation versus simple ligand blocking affects immune responses differently

These findings explain why certain anti-CTLA4 antibodies cause more severe immune-related adverse events and have important implications for both experimental design and therapeutic development .

How can CTLA4 Human, IgG-His, Active be used to investigate the relationship between CTLA4 and B cell responses?

CTLA4 Human, IgG-His, Active provides valuable insights into CTLA4's complex role in regulating B cell responses:

Mechanisms of CTLA4-mediated B cell regulation:

• CTLA4 controls B cell activation through multiple pathways:

  • Direct regulation of follicular helper T cells (Tfh), which are essential for B cell help

  • Downregulation of costimulatory molecules (CD80/CD86) on B cells

  • Modulation of both follicular helper (Tfh) and follicular regulatory (Tfr) T cells

Experimental approaches:

• Germinal center reactions:

  • Use CTLA4-IgG-His to block CD80/CD86 interactions during immunization protocols

  • Analyze germinal center B cell formation, Tfh differentiation, and antibody production

  • Compare with selective CTLA4 deletion in either Tfh or Tfr populations

• B cell survival and proliferation:

  • Investigate how CTLA4-IgG-His affects BCR-induced B cell proliferation

  • Examine B cell survival with/without CTLA4-IgG-His treatment

  • Study impacts on circulating B cell levels and immunoglobulin production

• Antigen-specific antibody responses:

  • Immunize mice with T-dependent antigens with/without CTLA4-IgG-His treatment

  • Measure antigen-specific antibody titers and affinity maturation

  • Analyze plasma cell differentiation and memory B cell formation

Research findings:

• CTLA4 deficiency leads to higher frequency of activated B cells and increased serum immunoglobulin levels

• Selective CTLA4 deletion in both Tfh and Tfr increases B cell responses

• CTLA4 deletion in Tfr alone increases antigen-specific antibody production

• Patients with CTLA4 mutations show complex B cell phenotypes, including reduced circulating B cells and hypogammaglobulinemia

This experimental system allows researchers to distinguish between direct effects on B cells versus indirect effects mediated through T cell subsets, providing crucial insights for autoimmunity and vaccine research.

What factors can lead to inconsistent results when using CTLA4 Human, IgG-His, Active in binding assays?

Several factors can contribute to inconsistent results when using CTLA4 Human, IgG-His, Active in binding assays:

Protein-related factors:

• Protein denaturation due to improper storage or multiple freeze-thaw cycles

• Batch-to-batch variability in protein conformation or activity

• Aggregation of the protein affecting functional binding sites

• His-tag interference with binding in certain experimental contexts

Experimental conditions:

• Variability in pH and buffer composition affecting protein conformation

• Presence of divalent cations influencing His-tag interactions

• Temperature fluctuations altering binding kinetics

• Incubation time variations affecting equilibrium binding

Technical considerations:

• Non-specific binding via the Fc portion to Fc receptors on cells

• Detection antibody cross-reactivity or competition with CD80/CD86 binding sites

• Endotoxin contamination activating cells and confounding functional readouts

• Surface adsorption issues when using plastic labware

Methodological troubleshooting table:

To minimize variability, establish a standardized protocol with appropriate controls, verify protein activity before critical experiments, and consider using commercial CTLA4-Ig as a reference standard for normalization between experiments .

How can I differentiate between effects caused by CTLA4 blockade versus antibody-mediated CTLA4 downregulation in my experimental system?

Differentiating between simple CTLA4 blockade and antibody-induced CTLA4 downregulation requires a systematic comparative approach:

Experimental design strategy:

• Parallel treatment comparison:

  • CTLA4-IgG-His, Active (blocks CD80/CD86 only)

  • Ipilimumab or TremeIgG1 (blocks CD80/CD86 and downregulates CTLA4)

  • pH-sensitive anti-CTLA4 antibodies (block without causing degradation)

  • Anti-CD80/CD86 blocking antibodies (alternative blocking approach)

• CTLA4 expression monitoring:

  • Measure surface CTLA4 by flow cytometry using non-competing antibodies

  • Assess total CTLA4 by Western blot or intracellular staining

  • Track CTLA4 in cellular compartments using confocal microscopy

• Time course analysis:

  • Examine early effects (1-4 hours) when blocking predominates

  • Assess later effects (12-72 hours) when downregulation impacts become apparent

  • Monitor recovery phase after antibody removal

Distinguishing features:

• Effects seen with both CTLA4-IgG-His and anti-CTLA4 antibodies likely reflect CD80/CD86 blockade

• Effects unique to Ipilimumab/TremeIgG1 may indicate CTLA4 downregulation consequences

• Effects that persist after antibody washing may suggest irreversible CTLA4 degradation

Advanced approaches:

• Use lysosomal inhibitors to prevent CTLA4 degradation and isolate blockade effects

• Implement CTLA4-GFP fusion proteins to visualize trafficking in real-time

• Employ LRBA knockdown to enhance degradation effects for clearer differentiation

Research has established that antibody-induced lysosomal degradation of CTLA4 correlates with immune-related adverse events, highlighting the importance of distinguishing these mechanisms when evaluating therapeutic approaches .

What controls and validation steps are essential when studying CTLA4-mediated regulatory T cell functions using CTLA4 Human, IgG-His, Active?

When investigating CTLA4's role in regulatory T cell (Treg) functions using CTLA4 Human, IgG-His, Active, comprehensive controls and validation steps are essential:

Essential controls:

• Cellular controls:

  • CTLA4-sufficient vs. CTLA4-deficient Tregs

  • CD4+CD25- conventional T cells (negative control)

  • Foxp3 reporter systems to ensure Treg purity

• Reagent controls:

  • Irrelevant IgG-His fusion protein (negative control)

  • Commercial CTLA4-Ig (positive control)

  • Anti-CTLA4 antibodies with known Treg effects

  • Dose titration (0.1-10 μg/mL) to establish dose-response

• Functional controls:

  • CD28 costimulation blockade (alternative approach)

  • IL-2 supplementation (bypasses some CTLA4 effects)

  • CTLA4 blockade in Treg-depleted cultures

Validation steps:

• Phenotypic validation:

  • Verify Treg markers (CD25high, FOXP3+, CD127low)

  • Confirm CTLA4 expression levels before experiments

  • Check CD80/CD86 expression on target APCs

• Functional validation:

  • Suppression assays with varied Treg:Teffector ratios

  • Cytokine production analysis (IL-10, TGF-β, IL-35)

  • Measurement of Treg stability (FOXP3 maintenance)

• Mechanistic validation:

  • CD80/CD86 trans-endocytosis assessment

  • DC phenotype analysis (costimulatory molecule expression)

  • PI3K/AKT signaling pathway evaluation

Interpretation considerations:

• CTLA4-IgG-His blocks CD80/CD86 but cannot recapitulate membrane CTLA4 functions like trans-endocytosis

• Studies have shown that CTLA4-depleted Tregs may still have suppressive activity through upregulation of other inhibitory molecules (IL-10, LAG-3, PD-1)

• Both cell-intrinsic and extrinsic mechanisms contribute to CTLA4's role in Treg function

These controls and validation steps ensure that observed effects are specifically attributable to CTLA4's role in Treg biology rather than experimental artifacts or alternative pathways.

How is CTLA4 Human, IgG-His, Active being used to develop improved cancer immunotherapies with reduced adverse events?

CTLA4 Human, IgG-His, Active is playing a crucial role in developing next-generation cancer immunotherapies with improved safety profiles:

Mechanism-based therapeutic innovation:

• Research comparing CTLA4-IgG-His with anti-CTLA4 antibodies has revealed that antibody-induced CTLA4 lysosomal degradation strongly correlates with immune-related adverse events (irAEs)

• This insight has led to the development of pH-sensitive anti-CTLA4 antibodies that dissociate from CTLA4 in endosomes, preventing lysosomal targeting while maintaining anti-tumor efficacy

Experimental approaches:

• Antibody engineering:

  • Introduction of tyrosine-to-histidine mutations creates pH-sensitive antibodies that prevent CTLA4 downregulation

  • CTLA4-IgG-His serves as a reference standard for ligand blocking without degradation

  • Comparative assays measuring CTLA4 surface expression, total levels, and functional outcomes guide optimization

• Tumor microenvironment targeting:

  • Investigation of how CTLA4 blockade without degradation affects intratumoral Tregs versus peripheral Tregs

  • Development of bispecific antibodies targeting CTLA4 and tumor-specific antigens

  • Localized delivery approaches to restrict CTLA4 modulation to the tumor site

• Combination therapy optimization:

  • Studies show that pH-sensitive anti-CTLA4 antibodies have increased bioavailability and enhanced efficacy in intratumor Treg depletion

  • CTLA4-IgG-His helps distinguish synergistic versus redundant mechanisms in combination strategies

Key research findings:

• pH-sensitive anti-CTLA4 antibodies that avoid CTLA4 downregulation demonstrate dramatically attenuated irAEs

• Surprisingly, by avoiding CTLA4 downregulation and due to increased bioavailability, these pH-sensitive antibodies show improved efficacy in rejecting established tumors

• This establishes a new paradigm for cancer immunotherapy that simultaneously reduces toxicity while enhancing therapeutic effects

This research direction represents a significant advance in tumor immunotherapy, moving beyond simple CTLA4 blockade to more sophisticated approaches that selectively modulate CTLA4 function in specific cellular contexts.

What insights has CTLA4 Human, IgG-His, Active provided about the relationships between CTLA4 polymorphisms and autoimmune disorders?

CTLA4 Human, IgG-His, Active has been instrumental in elucidating connections between CTLA4 genetic variations and autoimmune disease mechanisms:

CTLA4 genetic variations and functional consequences:

• Studies using CTLA4-IgG-His as a reference standard have helped characterize how various CTLA4 polymorphisms affect:

  • Protein expression levels and stability

  • Binding affinity to CD80/CD86

  • Trafficking dynamics and cell surface expression

  • Resistance to degradation by certain antibodies

Experimental approaches:

• Functional comparison studies:

  • Side-by-side binding assays comparing wild-type CTLA4-IgG-His with variant CTLA4-IgG-His constructs

  • Surface expression recovery assays following antibody-induced internalization

  • Assessment of variant CTLA4 sensitivity to lysosomal degradation pathways

• Disease model applications:

  • Investigation of how CTLA4 variants affect susceptibility to experimental autoimmune conditions

  • Comparative efficacy of CTLA4-IgG-His treatment in models with different CTLA4 genetic backgrounds

  • Analysis of variant-specific responses to CTLA4-targeting therapeutic approaches

Clinical correlations:

• Patients with CTLA4 mutations exhibit dysfunction of FoxP3+ Treg cells, hyper-proliferation of lymphocytes, and activated effector T cells

• CTLA4 deficiency results in a large number of lymphocytes in circulation and lymphoid organs

• Some CTLA4 mutations are associated with hypogammaglobulinemia and lymphopenia

• CTLA4-IgG-His has helped distinguish between variants causing signaling defects versus trafficking abnormalities

This research has significant implications for personalized medicine approaches to autoimmune disorders, potentially guiding variant-specific therapeutic strategies and explaining differential responses to CTLA4-based therapies among patients with various CTLA4 polymorphisms.

How are researchers utilizing CTLA4 Human, IgG-His, Active to investigate the role of CTLA4 in novel immune cell populations beyond conventional T cells?

Researchers are leveraging CTLA4 Human, IgG-His, Active to explore CTLA4's functions in diverse immune cell populations:

B cell biology applications:

• CTLA4-IgG-His helps investigate how CTLA4 controls follicular helper T cell activity and downregulates costimulatory molecules on B cells

• Studies reveal CTLA4's role in suppressing B cell activation and antibody production

• Research shows that CTLA4 deficiency leads to higher frequency of activated B cells and increased immunoglobulin levels

• CTLA4 antibody-drug conjugates have revealed mechanisms of autologous B cell destruction

Dendritic cell (DC) interactions:

• CTLA4-IgG-His facilitates studies of how CTLA4 regulates DC maturation and function

• Research demonstrates that CTLA4 deficiency in Treg cells increases CD80 and CD86 expression on DCs

• Investigations into how CTLA4 engagement affects DC cytokine production and T cell stimulatory capacity

Innate lymphoid cells (ILCs):

• Emerging research using CTLA4-IgG-His examines potential CTLA4 expression and function in ILC subsets

• Studies explore whether ILCs express CD80/CD86 and can be regulated by CTLA4-mediated mechanisms

• Investigation of how CTLA4 blockade affects ILC activation in various tissue microenvironments

Myeloid cell populations:

• Research into whether myeloid-derived suppressor cells (MDSCs) utilize CTLA4 for their suppressive functions

• Studies of how macrophage polarization and function is affected by CTLA4-CD80/CD86 interactions

• Investigation of potential CTLA4 expression on tumor-associated myeloid populations

Experimental approaches:

• Flow cytometry panels to identify CTLA4 expression across diverse immune lineages

• Functional assays comparing CTLA4-IgG-His effects on conventional versus unconventional cell types

• Single-cell RNA sequencing to identify CTLA4-responsive gene signatures in various immune populations

These expanding research directions are revealing CTLA4's unexpectedly broad influence across the immune system, extending well beyond its classical role in conventional T cells to encompass regulatory functions in multiple cellular lineages.