Recombinant Human CD160 antigen (CD160) (Active)

What are the structural characteristics of recombinant human CD160 protein?

Recombinant human CD160 protein is typically produced as a full-length mature protein spanning amino acids 27-159 . The protein has a molecular mass of approximately 15.8 kDa and is often tagged with a C-terminal 6His tag to facilitate purification and detection . The amino acid sequence contains specific domains that mediate its binding to MHC class I molecules and TNFRSF14 (HVEM), enabling its various immunoregulatory functions . When produced for research applications, the protein is typically expressed in mammalian cell systems such as HEK 293 cells to ensure proper folding and post-translational modifications, resulting in a highly pure (>90-95%) and bioactive form with minimal endotoxin contamination (<1.0 EU/μg) .

What cell types express CD160 and under what conditions is its expression regulated?

CD160 is primarily expressed on cytotoxic NK cells and specific T-cell subsets under normal conditions . It is not expressed on normal B lymphocytes but is abnormally expressed in B-cell chronic lymphocytic leukemia (CLL) at all disease stages . CD160 expression is typically upregulated upon activation of NK cells and T cells, as demonstrated by in vitro stimulation experiments . In pathological conditions such as chronic viral infections, CD160 expression patterns may change, contributing to altered immune cell function, including T cell exhaustion . Recent studies have also identified CD160 hypomethylation in blood cells correlating with breast cancer in specific populations, suggesting epigenetic regulation of its expression .

What signaling pathways are activated by CD160?

CD160 initiates distinct signaling pathways depending on the cell type and context. In NK cells, CD160 signaling occurs primarily through phosphoinositol 3-kinase (PI3K), while in T cells, it signals via LCK and CD247/CD3 zeta chain . In CLL cells, CD160 expression activates prosurvival signaling through the upregulation of the PI3K/Akt pathway and increases the secretion of cytokines, particularly IL-6 . This interleukin subsequently activates STAT3 and NF-κB, regulating the expression of genes implicated in cell proliferation and survival . Additionally, CD160 decreases apoptosis by downregulating proapoptotic caspases (caspase-3, -9, and -8) and upregulating antiapoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1, thereby blocking both mitochondria-dependent and mitochondria-independent apoptotic pathways .

How should researchers design experiments to study CD160's dual role in stimulatory and inhibitory signaling?

When investigating CD160's dual signaling capabilities, researchers should employ complementary approaches that isolate specific cell types and signaling contexts. To effectively study this dual functionality:

• Cell-specific isolation techniques: Use magnetic or fluorescence-activated cell sorting to obtain pure populations of NK cells, CD8+ T cells, and CD4+ T cells from both healthy donors and patients with relevant pathologies (e.g., chronic viral infections, CLL) .

• Temporal signaling analysis: Implement time-course experiments that monitor signaling events from early (seconds to minutes) to late (hours to days) timepoints following CD160 engagement. This approach helps distinguish between immediate stimulatory signals and delayed inhibitory effects that may occur during chronic stimulation .

• Pathway inhibition studies: Employ specific inhibitors targeting PI3K, LCK, or CD3 zeta chain signaling components to delineate the contribution of each pathway to stimulatory versus inhibitory outcomes .

• Context-dependent stimulation: Compare CD160 signaling under acute versus chronic stimulation conditions, mimicking viral infection scenarios. This can be achieved by using pulsed versus continuous exposure to CD160 ligands or agonistic antibodies .

• Genetic approaches: Utilize CD160-deficient models (CD160−/− mice or CRISPR-edited human cell lines) alongside reconstitution with wild-type or mutant CD160 variants to map specific domains responsible for stimulatory versus inhibitory functions .

This multifaceted experimental design allows researchers to dissect the molecular mechanisms underlying CD160's context-dependent functions while controlling for cell type, activation state, and signaling duration.

What are the best methods for studying CD160's role in NK cell-mediated IFN-γ production?

To effectively investigate CD160's role in NK cell IFN-γ production, researchers should implement a comprehensive methodological approach:

• In vivo and in vitro comparative studies: Utilize both in vivo tumor challenge models (such as B16 melanoma or RMA-S lymphoma) and in vitro NK cell stimulation assays to assess IFN-γ production. This dual approach allows for the validation of findings across different experimental contexts .

• Genetic manipulation: Compare NK cells from wild-type, CD160−/−, and CD160/RAG−/− mice to isolate NK cell-specific effects from those potentially mediated by T cells. This approach has been successfully used to demonstrate CD160's essential role in NK cell-mediated IFN-γ production .

• Ex vivo stimulation protocols: Harvest splenocytes or purified NK cells from tumor-bearing or poly(I:C)-treated mice for ex vivo stimulation with PMA/ionomycin, plate-bound antibodies, or cytokines. This technique allows for the assessment of how in vivo priming affects subsequent NK cell responses .

• Functional targeting: Use soluble CD160-Ig to block CD160 signaling, which has been shown to impair tumor control and IFN-γ production. This provides a complementary approach to genetic deletion studies .

• Molecular analysis: Implement real-time PCR to determine IFN-γ expression without requiring ex vivo stimulation, thereby capturing the in vivo state of NK cells. This approach has revealed that CD160 is required for cytokine production specifically in activated NK cells but not in naive NK cells .

• Flow cytometry: Use intracellular cytokine staining to quantify IFN-γ production at the single-cell level, allowing for the identification of specific NK cell subsets that depend on CD160 for optimal cytokine production .

By combining these methodologies, researchers can comprehensively analyze how CD160 regulates NK cell effector functions, particularly IFN-γ production, in various physiological and pathological contexts.

How can CD160 expression be targeted in chronic lymphocytic leukemia research?

CD160 presents a unique research target in chronic lymphocytic leukemia (CLL) due to its aberrant expression on malignant B cells but not on normal B lymphocytes. Researchers studying CD160 in CLL context should consider these methodological approaches:

• Therapeutic antibody development: Engineer anti-CD160-GPI monoclonal antibodies (mAbs) that can simultaneously engage CD160-GPI receptors on both NK cells and CLL-B cells along with FcγRs on NK cells. This approach aims to reactivate exhausted NK cells in the CLL microenvironment while potentially triggering antibody-dependent cellular cytotoxicity (ADCC) against CD160-expressing leukemic cells .

• Functional studies on dual signaling: Investigate how CD160 engagement differentially affects NK cells versus CLL B cells. While CD160 stimulation enhances NK cell activity, it may also promote CLL B cell proliferation and resistance to apoptosis, creating a complex therapeutic challenge that requires careful experimental design .

• Minimal residual disease (MRD) detection: Develop flow cytometry or molecular assays that leverage CD160's specific expression pattern for detecting minimal residual disease in CLL patients. This could potentially improve clinical management and prevent disease relapse .

• Microenvironment modeling: Create in vitro co-culture systems that recapitulate the CLL microenvironment, including T cells, NK cells, and stromal components, to study how CD160-expressing cells interact within this complex milieu .

• Epigenetic regulation studies: Investigate the mechanisms regulating CD160 expression in CLL B cells, focusing on whether genetic or epigenetic alterations drive its aberrant expression. Recent evidence of CD160 hypomethylation in other cancer contexts suggests epigenetic dysregulation may be involved .

These approaches facilitate the exploration of CD160 as both a prognostic marker and a potential therapeutic target in CLL, addressing the urgent need for novel treatment strategies for this malignancy.

What is the functional significance of CD160's binding to HVEM versus MHC class I molecules?

The differential binding of CD160 to HVEM (herpesvirus entry mediator, a TNF family member) versus MHC class I molecules represents a critical area of investigation with important experimental considerations:

• Binding affinity comparisons: Implement surface plasmon resonance or bio-layer interferometry to quantitatively compare the binding affinities of CD160 for HVEM versus various MHC class I molecules. Previous research has suggested that human CD160 binds to HVEM with significantly higher affinity than to MHC class I .

• Functional consequence analysis: Design experiments that selectively block CD160-HVEM versus CD160-MHC class I interactions using specific antibodies or mutated ligands to determine the downstream functional consequences on NK and T cell activation, cytokine production, and cytotoxicity .

• Context-dependent expression studies: Analyze the expression patterns of HVEM versus MHC class I molecules in different tissues and disease states to understand when each interaction might predominate physiologically .

• Species-specific variations: Compare human and mouse CD160 binding preferences, as there may be important species-specific differences in ligand affinity that affect experimental interpretation and translational applications .

• Signaling pathway dissection: Determine whether CD160-HVEM and CD160-MHC class I interactions activate distinct signaling pathways by performing phosphoproteomic analysis following selective ligand engagement .

Understanding these binding preferences and their functional outcomes is essential for interpreting seemingly contradictory data from different experimental systems and for developing targeted therapeutic approaches that modulate specific CD160 interactions.

What are the optimal storage and handling protocols for recombinant CD160 protein to maintain bioactivity?

Proper handling and storage of recombinant human CD160 protein is critical for maintaining its structural integrity and biological activity in research applications. Based on manufacturer recommendations and research protocols, the following guidelines should be implemented:

• Storage temperature: Store lyophilized recombinant CD160 powder at -20°C/-80°C for long-term stability, where it can remain stable for up to 12 months. For reconstituted protein in liquid form, storage at -20°C/-80°C maintains stability for up to 6 months .

• Aliquoting strategy: Upon receipt, prepare small single-use aliquots of reconstituted protein to avoid repeated freeze-thaw cycles, which can significantly degrade protein quality. Working aliquots can be stored at 4°C for up to one week .

• Reconstitution buffer: Reconstitute lyophilized CD160 protein in a buffer matching its formulation buffer, typically a 0.2 μm filtered 20 mM phosphate buffer with 150 mM NaCl at pH 7.4. This maintains optimal protein folding and activity .

• Freeze-thaw management: Limit freeze-thaw cycles to preserve bioactivity. Research indicates that protein activity can decrease by 10-30% with each freeze-thaw cycle, particularly for complex proteins with multiple domains .

• Functional validation: Verify bioactivity before experimental use through functional ELISA, testing the protein's ability to bind its known ligands such as Mouse TNFRSF14. The ED50 for bioactivity should be less than 50 μg/ml .

• Quality control: Confirm protein purity (>90% by SDS-PAGE) and endotoxin levels (<1.0 EU/μg) prior to use in sensitive cellular assays to prevent experimental artifacts from contaminants .

Following these optimized protocols ensures that experiments utilizing recombinant CD160 protein yield reliable and reproducible results, particularly in complex functional assays measuring immune cell activation or cytokine production.

How can CD160 knockout models advance our understanding of immune regulation?

CD160 knockout models, particularly CD160−/− mice, have provided valuable insights into the functional significance of this receptor in immune regulation. Researchers considering the use of these models should be aware of the following findings and experimental approaches:

• Normal lymphocyte development: CD160−/− mice show no abnormalities in lymphocyte development, indicating that CD160 is not essential for basic immune cell development but rather for functional responses .

• Tumor control deficiencies: Despite normal development, CD160−/− mice exhibit severely compromised control of NK-sensitive tumors, demonstrating CD160's critical role in antitumor immunity .

• Selective functional deficits: Surprisingly, the cytotoxicity of NK cells remains intact in CD160−/− mice, while IFN-γ secretion is markedly reduced. This selective functional deficit highlights CD160's specific role in cytokine production rather than all NK effector functions .

• Compound knockout approaches: Creating compound knockouts, such as CD160/RAG−/− mice (lacking both CD160 and the adaptive immune system), allows researchers to isolate the role of CD160 specifically on NK cells in the absence of adaptive immunity .

• Tumor challenge models: B16 melanoma and RMA-S lymphoma models in CD160-deficient backgrounds provide robust systems for studying NK cell-mediated tumor responses. The specificity of this effect is confirmed by the absence of difference in growth of MHC class I-expressing RMA tumors, which do not activate NK cells .

• Bone marrow chimera experiments: Reciprocal bone marrow transfers between wild-type and CD160-deficient mice help distinguish between intrinsic and extrinsic effects of CD160 on immune cell function .

These experimental approaches using CD160 knockout models have established CD160 as an essential regulator of NK cell cytokine production and demonstrated its importance in antitumor immunity, providing a foundation for therapeutic strategies targeting this pathway.

What technical considerations are important when measuring CD160 expression in clinical samples?

Accurate assessment of CD160 expression in clinical samples requires careful attention to technical details to ensure reliable and reproducible results:

• Sample preservation: Fresh clinical samples are optimal, but if storage is necessary, cryopreservation protocols must be standardized to prevent selective loss of CD160 expression. The GPI-anchored nature of one CD160 isoform makes it particularly susceptible to degradation during improper sample handling .

• Detection antibody selection: Choose antibodies that recognize both GPI-anchored and transmembrane forms of CD160, as these may have distinct expression patterns and functional significance in different pathological conditions .

• Multi-parameter analysis: Implement multi-color flow cytometry panels that allow simultaneous assessment of CD160 along with lineage markers, activation markers, and other functional molecules to contextualize CD160 expression within specific cell subsets .

• Quantitative standardization: Use appropriate quantitative controls such as antibody capture beads to standardize mean fluorescence intensity values across different experiments and clinical centers, enabling reliable comparison of expression levels .

• Molecular verification: Complement protein-level detection with mRNA assessment using RT-qPCR to validate expression patterns and potentially identify splice variants of CD160 that may not be detected by all antibodies .

• Epigenetic analysis: Consider analyzing CD160 promoter methylation status, particularly in cancer contexts where hypomethylation has been associated with aberrant expression .

These technical considerations are particularly important when using CD160 as a biomarker for conditions such as chronic lymphocytic leukemia, where its expression serves as a potential indicator for minimal residual disease and may guide clinical management decisions.

What are the most promising therapeutic applications targeting CD160?

Based on current research findings, several promising therapeutic strategies targeting CD160 are emerging:

• Engineered anti-CD160-GPI monoclonal antibodies: These antibodies could simultaneously engage CD160 on NK cells and CLL B cells while also binding FcγRs on NK cells, potentially reactivating exhausted NK cells in the tumor microenvironment while triggering ADCC against CD160-expressing malignant cells .

• NK cell-based immunotherapies: Given CD160's essential role in NK cell IFN-γ production, strategies that enhance CD160 signaling specifically in NK cells could boost antitumor immune responses, particularly in contexts where T cell-based approaches have limited efficacy .

• Minimal residual disease detection: Leveraging CD160's aberrant expression on CLL cells for sensitive detection of minimal residual disease could improve clinical management and prevent relapse in leukemia patients .

• Differential targeting of CD160 interactions: Developing therapeutic agents that selectively block or enhance specific CD160 interactions (either with HVEM or MHC class I) could provide more precise modulation of immune responses in various disease contexts .

What emerging technologies will advance CD160 research in the coming years?

Several cutting-edge technologies are poised to significantly advance our understanding of CD160 biology and its therapeutic applications:

• Single-cell multi-omics: Integration of single-cell RNA sequencing, ATAC-seq, and proteomics will provide unprecedented resolution of CD160's expression patterns and functional states across immune cell populations in health and disease .

• Advanced protein engineering: Structure-guided protein engineering will enable the development of modified CD160 variants or decoy receptors with altered binding preferences, allowing more precise modulation of specific CD160 interactions .

• CRISPR-based screening: Genome-wide CRISPR screens in CD160-expressing cells will help identify novel regulators of CD160 expression and signaling, potentially revealing new therapeutic targets .

• Improved in vitro disease models: Advanced organoid and microphysiological systems that better recapitulate the complexity of the tumor microenvironment will enable more accurate assessment of CD160-targeting therapies before clinical translation .

• Real-time imaging technologies: Intravital microscopy and other advanced imaging approaches will allow visualization of CD160-mediated interactions in living tissues, providing insights into the spatiotemporal dynamics of CD160 signaling in immune responses .