Recombinant Guinea pig Cytotoxic T-lymphocyte associated protein 4 (CTLA4), partial (Active)

What is the functional role of CTLA4 in the immune system?

CTLA4 functions as an inhibitory receptor that serves as a key negative regulator of T-cell responses. It binds to the B7 family ligands, CD80 and CD86, with significantly higher affinity than the stimulatory coreceptor CD28, which also interacts with these ligands. This strong binding allows CTLA4 to effectively dampen T-cell activation and prevent excessive immune responses. By inhibiting signals that would normally activate T-cells, CTLA4 helps maintain immune system balance and prevents autoimmunity, ensuring that the immune response remains controlled and targeted, without causing harm to the body's own tissues . This regulatory function positions CTLA4 as a critical checkpoint molecule in immunological research, particularly for studies investigating immune tolerance and autoimmunity.

How does Guinea pig CTLA4 compare structurally to human CTLA4?

Guinea pig CTLA4 shares significant structural homology with human CTLA4, particularly in the extracellular domain that interacts with B7 ligands. The recombinant protein typically encompasses amino acids 37-161, which contains the extracellular domain responsible for ligand binding . The protein sequence (AMHVAQPAVVLASSRGVASFECEYASSHNANEVRVTVLQQVASRTTEICAATYTVERELAFPEDSACAGTSSGTRVNLTIQGLRAADTGLYICKVELMYPPPYFVGTGNGTQIYVIDPEPCPDSD) demonstrates conservation of key binding regions observed in human CTLA4 . While maintaining functional similarity, researchers should note that species-specific differences might affect cross-reactivity in certain experimental applications. These structural similarities make Guinea pig CTLA4 a valuable model for studying immune checkpoint mechanisms, though experimental designs should account for potential species-specific variations in downstream signaling pathways.

What is the significance of using a partial active form of CTLA4 in research?

The partial active form of recombinant Guinea pig CTLA4 typically includes the extracellular domain (amino acids 37-161) responsible for ligand binding but excludes the transmembrane and cytoplasmic regions . This design offers several research advantages: (1) it facilitates solubility and expression in recombinant systems while maintaining binding functionality; (2) it allows for focused study of the B7-ligand interaction without confounding effects from intracellular signaling domains; and (3) it provides a tool for blocking or competitive binding studies without triggering downstream signal transduction. The partial active form has demonstrated biological activity in functional ELISAs with an ED50 of less than 0.4 μg/ml when binding to Mouse B7-1 , making it suitable for multiple experimental applications while offering a simplified system for studying specific protein-protein interactions.

What are the appropriate experimental controls when using recombinant Guinea pig CTLA4 in functional assays?

When designing experiments with recombinant Guinea pig CTLA4, implementing proper controls is essential for result validation. For functional binding assays, include: (1) a negative control using an irrelevant protein with similar size and tag system; (2) a positive control using established CTLA4-B7 interaction partners; (3) a dose-response curve to determine optimal concentration ranges based on the known ED50 (<0.4 μg/ml for B7-1 binding) ; and (4) blocking controls with anti-CTLA4 antibodies to confirm binding specificity. For T-cell suppression assays, include conditions with CD28-only stimulation to demonstrate the inhibitory effect of CTLA4. Additionally, temperature controls are important as protein activity can be temperature-dependent. Finally, include vehicle controls matching the buffer composition (typically 0.2 μm filtered 1xPBS, pH 7.4) to account for potential buffer effects on experimental systems. These comprehensive controls ensure that observed effects are specifically attributable to CTLA4 functionality.

How can recombinant Guinea pig CTLA4 be incorporated into T-cell activation assays?

Incorporating recombinant Guinea pig CTLA4 into T-cell activation assays requires careful experimental design to observe its inhibitory function. The protocol should include: (1) Pre-incubation of antigen-presenting cells expressing B7 ligands with titrated concentrations of recombinant CTLA4 (starting near the ED50 of 0.4 μg/ml) ; (2) Addition of T-cells and appropriate stimulation (antigen or anti-CD3/CD28); (3) Measurement of T-cell activation markers (CD69, CD25), proliferation (CFSE dilution or 3H-thymidine incorporation), and cytokine production (IL-2, IFN-γ) at multiple time points; (4) Parallel experiments with CD28-Ig fusion proteins as comparative controls to demonstrate the competing interactions. Flow cytometry can quantify changes in activation marker expression, while ELISA or multiplex bead arrays can measure secreted cytokines. For more mechanistic studies, analyze intracellular signaling pathways (phosphorylation of ZAP-70, LAT, etc.) in the presence versus absence of CTLA4. This approach allows for comprehensive assessment of CTLA4's inhibitory effects on multiple aspects of T-cell activation.

What species cross-reactivity considerations apply when using Guinea pig CTLA4 in research?

When working with recombinant Guinea pig CTLA4, species cross-reactivity is a critical experimental consideration. Available data indicates that Guinea pig CTLA4 demonstrates functional binding to mouse B7-1 ligands , but researchers should note that cross-reactivity may vary across different experimental systems. For human systems, preliminary binding validation is essential as sequence differences may affect interaction kinetics. When designing cross-species experiments, consider these approaches: (1) Perform preliminary binding assays using surface plasmon resonance or ELISA to quantify interaction with B7 ligands from different species; (2) If using cell-based assays, include positive controls with species-matched CTLA4-B7 pairs; (3) Validate functional activity in each species system using known biological readouts such as T-cell proliferation inhibition. While certain commercial preparations indicate mouse reactivity and cross-reactivity , independent validation in your specific experimental system is strongly recommended, particularly for complex assays or when precise quantification of inhibitory effects is required.

What are the optimal storage and handling conditions for maintaining recombinant CTLA4 activity?

Maintaining recombinant Guinea pig CTLA4 activity requires careful attention to storage and handling conditions. For long-term storage, the protein should be kept at -20°C to -80°C, with the latter preferred for extended periods . Upon receipt, immediately aliquot the protein to avoid repeated freeze-thaw cycles, which can significantly reduce biological activity. Working aliquots may be stored at 4°C for up to one week , but longer storage at this temperature is not recommended. The lyophilized form (if applicable) demonstrates greater stability than the liquid formulation . When handling the protein, maintain sterile technique to prevent microbial contamination, and avoid vortexing, which can cause protein denaturation—instead, mix by gentle inversion or flicking. If dilution is necessary, use the recommended buffer (typically 0.2 μm filtered 1xPBS, pH 7.4) and prepare fresh dilutions for each experiment. For proteins with His-tags, avoid solutions containing high concentrations of chelating agents (e.g., EDTA) that may strip essential metal ions. Following these handling protocols will maximize protein activity retention and experimental reproducibility.

How should recombinant CTLA4 be reconstituted from lyophilized form to maintain optimal activity?

Reconstitution of lyophilized recombinant Guinea pig CTLA4 requires precise technique to maintain optimal activity. Begin by allowing the vial to equilibrate to room temperature (15-25°C) for approximately 10 minutes before opening to prevent moisture condensation on the protein. For reconstitution, use sterile 0.2 μm filtered 1xPBS (pH 7.4) or the specific buffer recommended in the product documentation. Add the buffer slowly down the side of the vial rather than directly onto the protein cake to prevent foaming, which can denature proteins. Gently rotate or swirl the vial until complete dissolution is achieved; avoid vortexing or vigorous pipetting which can damage protein structure. After reconstitution, allow the solution to sit for 5-10 minutes at room temperature to ensure complete solubilization. The recommended concentration for reconstitution is typically 0.1-1.0 mg/ml, but product-specific guidelines should be followed. Immediately after reconstitution, prepare single-use aliquots in sterile microcentrifuge tubes and flash-freeze in liquid nitrogen before transferring to -80°C storage. Document the reconstitution date, concentration, and buffer composition on each vial for reference.

What quality control parameters should be verified before using recombinant CTLA4 in critical experiments?

Before using recombinant Guinea pig CTLA4 in critical experiments, researchers should verify several key quality control parameters to ensure reliable results. First, assess protein purity through SDS-PAGE analysis, comparing against the manufacturer's specification (typically >90% or >97% as determined by SDS-PAGE). Second, verify protein identity and integrity using Western blot with anti-CTLA4 or anti-His antibodies, depending on the tagged construct. Third, check for endotoxin levels, which should be less than 1.0 EU/μg as determined by LAL method , as endotoxin contamination can significantly alter immune cell responses and confound results. Fourth, confirm biological activity through a functional binding assay with B7-1, comparing to the expected ED50 value (<0.4 μg/ml) . If possible, perform size exclusion chromatography to detect aggregation, which can affect functional activity. Additionally, measure protein concentration using multiple methods (e.g., Bradford assay and UV spectroscopy) to ensure accurate dosing in experiments. For long-stored samples, re-verify activity before use in critical experiments. Documentation of all quality control tests should be maintained for experimental reproducibility and troubleshooting.

How can recombinant Guinea pig CTLA4 be used to study immune checkpoint mechanisms in comparative immunology?

Recombinant Guinea pig CTLA4 offers valuable opportunities for comparative immunology studies of immune checkpoint mechanisms. To leverage this tool effectively, researchers can implement several advanced approaches: (1) Conduct comparative binding kinetics studies using surface plasmon resonance to quantify differences in association and dissociation rates between Guinea pig CTLA4 and B7 ligands from various species (human, mouse, Guinea pig), comparing these to established kinetic parameters of human CTLA4-B7 interactions; (2) Develop cross-species T-cell assays where antigen-presenting cells from one species interact with T-cells from another in the presence of recombinant CTLA4, allowing assessment of evolutionary conservation of this inhibitory pathway; (3) Use mutagenesis studies on the recombinant protein to identify critical residues for B7 binding, comparing these to known human CTLA4 binding sites; (4) Deploy the protein in ex vivo tissue slice models from different species to examine microenvironmental effects on checkpoint activity. These comparative approaches can reveal evolutionary conservation of immune regulation mechanisms and identify species-specific differences that may impact translational research. The partial active construct (aa 37-161) is particularly suitable for these studies as it focuses on the ligand-binding domain.

What approaches can be used to study the interaction between recombinant CTLA4 and B7 family ligands?

Studying the interaction between recombinant Guinea pig CTLA4 and B7 family ligands requires sophisticated biophysical and cellular approaches. For quantitative binding analysis, employ surface plasmon resonance (SPR) by immobilizing the His-tagged CTLA4 on a Ni-NTA sensor chip and flowing B7 ligands at various concentrations to determine association/dissociation constants. Isothermal titration calorimetry (ITC) can complement this by providing thermodynamic parameters of the interaction. For structural insights, circular dichroism spectroscopy assesses secondary structure integrity, while hydrogen-deuterium exchange mass spectrometry can map interaction interfaces. At the cellular level, develop competitive binding assays using flow cytometry where fluorescently labeled CTLA4 competes with CD28 for binding to B7-expressing cells. For in situ visualization, proximity ligation assays or fluorescence resonance energy transfer (FRET) can demonstrate direct interaction in cellular contexts. Functional consequences can be assessed through reporter cell lines where CTLA4-B7 engagement triggers a measurable output such as luciferase expression. Together, these approaches provide a comprehensive understanding of binding parameters (KD < 0.4 μg/ml for Mouse B7-1) , interaction dynamics, and the molecular basis for CTLA4's higher affinity for B7 ligands compared to CD28.

How might recombinant CTLA4 be utilized in developing immunotherapeutic models for autoimmune diseases?

Recombinant Guinea pig CTLA4 can serve as a valuable tool in developing immunotherapeutic models for autoimmune diseases through several advanced research applications. First, create fusion proteins combining the CTLA4 extracellular domain (aa 37-161) with Fc regions to develop CTLA4-Ig analogues, similar to clinical abatacept or belatacept, for testing in Guinea pig autoimmune models. These constructs can be evaluated for their ability to modulate dendritic cell maturation and function through B7 engagement. Second, develop localized delivery systems (such as hydrogels or nanoparticles) containing recombinant CTLA4 to target specific inflammatory tissues, allowing for site-directed immunomodulation while monitoring tissue-specific T-cell responses. Third, combine CTLA4 with other recombinant checkpoint proteins (PD-1, LAG-3) to study combinatorial checkpoint modulation in autoimmune settings, potentially revealing synergistic immunosuppressive effects. Fourth, use the protein in ex vivo organ culture systems from autoimmune disease models to evaluate its potential for restoring immune homeostasis in complex tissue environments. Each approach should incorporate appropriate controls and dose-response analyses based on the known bioactivity (ED50 <0.4 μg/ml) to establish therapeutic windows. These models can provide valuable insights into checkpoint-based interventions for autoimmunity before advancing to more complex in vivo systems.

What are common causes of variability in CTLA4 functional assays and how can they be addressed?

Variability in CTLA4 functional assays can arise from multiple sources that require systematic troubleshooting. First, protein quality issues often contribute to inconsistent results—verify protein activity before each critical experiment using binding assays with B7-1 ligand, ensuring activity remains near the expected ED50 (<0.4 μg/ml) . Temperature fluctuations during handling can affect protein conformation; maintain consistent temperature conditions throughout experiments. Buffer composition differences, particularly in salt concentration or pH, can significantly alter binding kinetics; standardize buffer systems across experiments and prepare fresh working solutions. For cell-based assays, variation in B7 ligand expression levels on antigen-presenting cells can dramatically affect outcomes; quantify B7 expression by flow cytometry before each experiment. The activation state of T-cells prior to assay setup introduces another variable; standardize T-cell isolation and resting protocols. To address these issues: (1) implement detailed standard operating procedures; (2) include internal calibration standards in each experiment; (3) perform technical replicates across multiple protein lots; (4) maintain detailed records of cell passage numbers and activation states; and (5) consider automation of pipetting steps to reduce handling variation. Statistical approaches should include power calculations to determine appropriate replicate numbers based on observed variability.

How should researchers interpret discrepancies between in vitro CTLA4 binding data and functional T-cell assay results?

When confronted with discrepancies between in vitro binding data and functional T-cell assay results for recombinant Guinea pig CTLA4, researchers should employ a systematic analytical approach. First, examine the assay formats—pure protein-protein binding assays (such as ELISA or SPR) measure direct interactions under controlled conditions, while T-cell assays involve complex cellular environments with multiple competing interactions. The protein concentration achieving 50% inhibition in cellular assays may differ substantially from the ED50 in binding assays (<0.4 μg/ml) due to these contextual differences. Second, evaluate buffer and environmental conditions—factors such as divalent cations, serum proteins, or pH differences between binding and cellular assays can affect interaction kinetics. Third, consider the multimolecular nature of immune synapses—in cellular contexts, CD28-B7 interactions occur simultaneously with TCR-MHC engagement, potentially altering CTLA4 binding dynamics. Fourth, assess the temporal aspects—binding assays provide equilibrium measurements, while T-cell assays involve dynamic processes occurring over hours to days. To resolve these discrepancies: (1) perform dose-response experiments across broader concentration ranges; (2) develop competition assays where CTLA4 competes with CD28 for B7 binding; (3) use microscopy to visualize CTLA4-B7 interactions in cellular contexts; and (4) employ computational modeling to integrate binding and functional data into a comprehensive mechanistic framework.

What factors should be considered when comparing results between different commercial preparations of recombinant CTLA4?

Comparing results between different commercial preparations of recombinant Guinea pig CTLA4 requires careful consideration of several critical factors. First, expression systems significantly impact protein folding and post-translational modifications—preparations expressed in yeast versus E. coli may exhibit different glycosylation patterns or tertiary structures, affecting functionality. Second, construct design variations, particularly in the expressed amino acid ranges, can alter activity—while most preparations include the extracellular domain (aa 37-161) , minor boundary differences can affect binding regions. Third, tag location and composition (typically C-terminal 6xHis tags) may differentially impact protein function or introduce steric hindrance in certain assay formats. Fourth, purity levels vary between preparations (from >90% to >97% by SDS-PAGE), potentially introducing different levels of contaminants or truncated products. Fifth, stability and formulation differences—liquid versus lyophilized forms and buffer composition variations—affect long-term activity retention. For valid cross-preparation comparisons: (1) perform side-by-side functional testing using the same assay conditions; (2) normalize activity based on functional rather than absolute concentration; (3) verify molecular weights empirically, as theoretical calculations (14.1 kDa vs. 7.9 kDa ) may reflect different constructs or calculation methods; (4) document lot numbers and acquisition dates; and (5) maintain consistent storage conditions (-20°C/-80°C) to minimize preparation-independent variables.

How should researchers design experiments to study the relationship between CTLA4 and other immune checkpoint molecules?

Designing experiments to study interactions between CTLA4 and other immune checkpoint molecules requires sophisticated approaches that capture both independent and interdependent functions. Begin with comprehensive expression profiling of multiple checkpoint receptors (CTLA4, PD-1, LAG-3, TIM-3, etc.) on various T-cell subsets under different activation conditions using multiparameter flow cytometry. Design co-expression analyses using labeled antibodies against multiple checkpoints simultaneously, enabling identification of receptor co-localization patterns. For functional studies, develop factorial experimental designs where recombinant Guinea pig CTLA4 is combined with other checkpoint proteins at varying concentrations, allowing detection of additive, synergistic, or antagonistic effects on T-cell activation. Implement CRISPR-Cas9 gene editing to create T-cell lines with selective checkpoint receptor deletions, then reconstitute with wild-type or mutant forms to assess functional interdependencies. For mechanistic insights, analyze intracellular signaling pathway convergence using phospho-flow cytometry or mass cytometry (CyTOF) after stimulation with various checkpoint ligand combinations. Time-course experiments are crucial, as different checkpoint receptors may function at distinct phases of T-cell activation. Include appropriate statistical designs, such as two-way ANOVA with interaction terms, to formally test for synergistic effects between CTLA4 and other checkpoint molecules, with post-hoc analyses to characterize specific interaction patterns.

What considerations apply when incorporating recombinant CTLA4 into cancer immunotherapy research models?

Incorporating recombinant Guinea pig CTLA4 into cancer immunotherapy research models requires careful experimental design that addresses both fundamental immunology and translational considerations. First, establish baseline CTLA4-B7 interaction dynamics in your specific tumor model using binding assays with the recombinant protein (ED50 <0.4 μg/ml for B7-1) . For in vitro cancer models, develop co-culture systems with tumor cells, T-cells, and antigen-presenting cells where CTLA4 blockade or supplementation can be manipulated while monitoring tumor-specific T-cell responses. When designing in vivo approaches, consider developing CTLA4-Fc fusion constructs based on the recombinant protein sequence that can modulate systemic immunity in tumor-bearing animals. For mechanistic studies, implement spatially-resolved techniques such as multiplexed immunohistochemistry to visualize CTLA4 expression and function within the tumor microenvironment before and after experimental interventions. The Cancer Center Support Grant (CCSG) guidelines for clinical research studies provide valuable frameworks for designing translational experiments, particularly regarding protocol standardization and data collection methodologies. When comparing CTLA4-targeted approaches with other checkpoint inhibitors, design factorial experiments that allow assessment of potential synergistic effects. Additionally, incorporate relevant biomarkers into experimental designs to identify predictors of response to CTLA4-modulating interventions, enhancing the translational relevance of preclinical findings.

How can researchers effectively integrate recombinant CTLA4 studies with in silico modeling approaches?

Integrating recombinant Guinea pig CTLA4 experimental data with in silico modeling creates powerful research synergies that enhance mechanistic understanding and predictive capabilities. Begin by generating comprehensive binding kinetics data using surface plasmon resonance with the recombinant protein and its B7 ligands across concentration ranges centered around the known ED50 (<0.4 μg/ml) . These quantitative parameters provide essential inputs for molecular dynamics simulations that can model CTLA4-B7 interactions at the atomic level, identifying key binding residues within the recombinant protein's sequence . Develop agent-based models of T-cell activation that incorporate CTLA4's inhibitory functions, calibrated with dose-response data from in vitro T-cell assays using the recombinant protein. For systems biology approaches, generate time-course transcriptomic and proteomic data from T-cells exposed to varying CTLA4 concentrations, then apply network analysis algorithms to identify regulatory modules affected by CTLA4 signaling. Machine learning methods can identify patterns in these multi-omic datasets that predict functional outcomes of CTLA4 engagement under different conditions. To validate in silico predictions, design targeted experiments that test model-generated hypotheses about specific amino acid residues or concentration thresholds that affect CTLA4 function. This iterative cycle between wet-lab experiments with the recombinant protein and computational modeling creates a powerful research framework that accelerates discovery while reducing experimental resources.