ULBP3 Human

What is ULBP3 and what is its role in the immune system?

ULBP3 is a member of a family of cell-surface proteins that function as ligands for human NKG2D, an activating receptor expressed on natural killer (NK) cells, NKT cells, gamma delta T cells, and CD8+ alpha beta T cells. It has also been described under alternative names including RaeT1N (retinoic acid early transcript), NKG2DL3, and ALCAN-gamma . ULBP3 is distantly related to MHC class I proteins but possesses only the alpha 1 and alpha 2 Ig-like domains without the capacity to bind peptide or interact with beta 2-microglobulin .

Unlike many traditional immune markers, ULBP3 is anchored to the cell membrane via a GPI-linkage . Its primary immunological function involves engagement with NKG2D receptors, which results in the activation of cytolytic activity and/or cytokine production by effector cells . This interaction is fundamental to innate immune surveillance mechanisms, particularly in the context of cancer immunology where ULBP3 has been implicated in tumor surveillance .

How can researchers reliably detect ULBP3 expression in experimental systems?

Multiple validated methods exist for ULBP3 detection, each with specific applications:

Flow Cytometry: Human ULBP3 can be detected using specific monoclonal antibodies such as clone 166510 . This methodology is particularly useful for analyzing ULBP3 expression on cell surfaces of intact cells. For optimal results, researchers should include appropriate isotype controls and validate antibody specificity, as approximately 5% cross-reactivity with recombinant human ULBP-2 has been observed in some formats .

Western Blot: This technique allows detection of ULBP3 protein in cell lysates, with antibodies like AF1517 showing good specificity . When performing Western blots, samples can be digested with EndoH to assess glycosylation status, which provides insights into the intracellular processing of ULBP3 .

ELISA: Direct ELISA can be employed to measure ULBP3 levels in various samples . For soluble ULBP3 in serum samples, time-resolved fluoroimmunoassay has been successfully used to distinguish between cancer patients and healthy donors .

Immunohistochemistry: This method allows visualization of ULBP3 expression in tissue sections, providing spatial information about expression patterns .

How does ULBP3 expression influence NK cell-mediated tumor surveillance?

ULBP3 expression on tumor cells significantly impacts NK cell cytotoxicity through complex mechanisms:

Surface ULBP3 Expression: High expression of ULBP3 on the cell surface of tumor cells has been demonstrated to augment NKG2D-mediated NK cell cytotoxicity . This represents the "induced-self" principle of NK cell activation, where stress-induced ligands like ULBP3 trigger NK cell responses against transformed cells .

Soluble ULBP3 (sULBP3): Paradoxically, low levels of soluble ULBP3 (<15 ng/ml) in circulation can weaken NK cell cytotoxicity by decreasing NKG2D expression on NK cells . Research has shown that serum samples from most cancer patients (>70%) contain these low levels of sULBP3, potentially contributing to immune evasion .

Experimental evidence from studies measuring NK cell degranulation provides further insight. When NK cells and CD4+ T cells are activated in vitro and co-cultured with anti-NKG2D monoclonal antibodies, significant changes in NK cell degranulation are observed, demonstrating the functional importance of the NKG2D-ULBP3 interaction pathway .

What is the relationship between ULBP3 and glioblastoma stem-like cells?

Glioblastoma (GBM), the most aggressive brain malignancy in adults with a survival of approximately 14.6 months, has been a focus of ULBP3 research . Studies have investigated how GBM cells respond to NK-mediated cytotoxicity, with particular attention to the role of ULBP3 and other NKG2D ligands.

The culture conditions of GBM cells significantly influence their phenotype and susceptibility to NK cell-mediated killing. Patient-derived GBM samples cultured in different media (NeuroBasal® versus DMEM with FBS) show differential expression of NKG2D ligands including ULBP3 . This methodological consideration is crucial for researchers designing experiments involving GBM and NK cell interactions.

GBM stem-like cells appear to have different ULBP3 expression profiles compared to differentiated GBM cells, potentially affecting their recognition by the immune system . This finding has implications for developing immunotherapeutic approaches that can effectively target the heterogeneous cell populations within GBM tumors.

How do viral immune evasion strategies interact with ULBP3?

ULBP3 was originally identified based on its binding to human cytomegalovirus (HCMV) glycoprotein UL16, hence the name UL16 binding protein . This interaction represents a viral immune evasion strategy.

Research has demonstrated that viral proteins like Rh159 can interfere with intracellular transport of NKG2D ligands including ULBP3 . Experimental evidence shows that Rh159 co-immunoprecipitates with ULBP3, suggesting a direct interaction that may prevent the ligand from reaching the cell surface . Specifically:

• Viral proteins can associate with ULBP3 and prevent its intracellular transport to the cell surface

• These interactions can reduce steady-state levels of NKG2D ligands

• Both ULBP3 and viral proteins like Rh159 are EndoH sensitive, consistent with ER localization

This mechanism illustrates how viruses have evolved to target the NKG2D-mediated immune surveillance pathway, providing insights for researchers studying viral immunoevasion strategies and their potential impact on tumor immunology.

What recombinant expression systems are effective for producing ULBP3 protein?

Prokaryotic expression systems have been successfully employed to produce recombinant ULBP3 protein for experimental purposes:

The gene encoding human ULBP3 can be cloned into the prokaryotic expression vector pQE30, creating a recombinant plasmid pQE30-ULBP3 . This construct can then be transformed into Escherichia coli M15 to induce the expression of recombinant ULBP3 protein (rec-ULBP3) .

The purified rec-ULBP3 has been effectively used as an antigen for immunizing BALB/c mice to develop monoclonal antibodies against ULBP3 . Through a systematic approach involving cell fusion, sub-cloning, and screening, researchers have successfully generated hybridoma cell clones expressing monoclonal antibodies that specifically recognize both recombinant and native ULBP3 expressed on tumor cell surfaces .

The functionally validated monoclonal antibodies include clones B2-F1-F1 and B4-C5-D11, which have been confirmed to react with both recombinant ULBP3 and naturally occurring ULBP3 on tumor cell surfaces . These reagents provide valuable tools for further investigating ULBP3 function in basic research and potential clinical applications.

What are the optimal conditions for studying ULBP3 in primary cell cultures?

Based on research with patient-derived glioblastoma cells, specific culture conditions have significant effects on ULBP3 expression and experimental outcomes:

NeuroBasal® Medium Conditions:

• Supplement with 1% v/v GlutaMAX®

• Add 2% v/v B-27® (Supplement Minus Vitamin A)

• Include 1% v/v Penicillin/Streptomycin

• Add 20 ng/ml recombinant human epidermal growth factor (EGF)

• Add 20 ng/ml recombinant human basic fibroblast growth factor (FGF)

• Maintain at 37°C in a humidified atmosphere of 5% CO₂

Alternative DMEM Conditions:

• Supplement with 10% fetal bovine serum

• Add non-essential amino acids

• Include 100 U/ml Penicillin/Streptomycin

• Add 400 μM L-glutamine

• Maintain at 37°C in a humidified atmosphere of 5% CO₂

When designing experiments to study ULBP3 expression and function, researchers should be aware that the choice of culture medium can significantly affect cellular phenotype, proliferation, and responses to NK cytotoxicity . This methodological consideration is particularly important for reproducibility and when comparing results across different studies.

How can researchers effectively measure soluble ULBP3 in clinical samples?

For accurate measurement of soluble ULBP3 (sULBP3) in clinical samples, time-resolved fluoroimmunoassay has been validated as an effective methodology . This technique was instrumental in demonstrating that serum from cancer patients, but not from healthy donors, contained elevated levels of sULBP3 .

The threshold of 15 ng/ml appears to be biologically significant, as levels below this concentration have been associated with weakened NK cell cytotoxicity by decreasing NKG2D expression on NK cells . Importantly, research has shown that serum samples from most cancer patients (>70%) contain these low levels of sULBP3 .

When implementing this assay:

• Collect serum samples using standardized protocols to minimize pre-analytical variables

• Include appropriate calibration standards and quality controls

• Validate the assay's specificity for ULBP3 versus other NKG2D ligands

• Consider potential interfering factors in cancer patient samples

For correlation with functional outcomes, these measurements can be paired with NK cell cytotoxicity assays, such as the lactate dehydrogenase release assay, to determine the relationship between sULBP3 levels and NK cell function in individual patients .

How does ULBP3 genetic structure influence expression and function?

ULBP3 is encoded by a gene located on chromosome 6 (NC_000006.12 in the GRCh38.p14 Primary Assembly) . It exists within a cluster of ten related genes, six of which encode potentially functional glycoproteins . The amino acid sequence identity within this family ranges from a moderate 30% to a substantial 60% .

The protein structure of ULBP3 includes only the alpha 1 and alpha 2 Ig-like domains characteristic of MHC class I proteins, but notably lacks the capacity to bind peptide or interact with beta 2-microglobulin . ULBP3 anchors to the membrane via a GPI-linkage, in contrast to some other family members that possess transmembrane domains .

The mature ULBP3 protein spans from Gly27 to Pro216 , with critical regions for receptor binding and immune recognition. Researchers investigating ULBP3 genetic variants and expression should consider accessing genomic sequence information through resources such as:

• NC_000006.12 (Chromosome 6 Reference GRCh38.p14 Primary Assembly)

• NC_060930.1 (Chromosome 6 Alternate T2T-CHM13v2.0)

• NC_000006.11 (Chromosome 6 Reference GRCh37.p13 Primary Assembly)

Understanding these genetic elements is crucial for designing effective gene manipulation experiments, interpreting polymorphism data, and evaluating evolutionary conservation of ULBP3 across species.

What is the relationship between ULBP3 expression and clinical outcomes in cancer patients?

Research investigating ULBP3 expression in cancer contexts has revealed complex relationships with clinical outcomes. The dual nature of ULBP3's effects on immune surveillance presents a nuanced picture:

ULBP3 overexpression has been observed on various tumor cell lines and tumor tissues . This surface expression theoretically enhances tumor recognition by NK cells through the NKG2D receptor pathway, potentially contributing to better immune surveillance.

This complex relationship suggests that researchers should consider both membrane-bound and soluble forms when evaluating ULBP3 as a potential biomarker or therapeutic target. The finding that serum samples from most cancer patients (>70%) contain low levels of sULBP3 suggests this may be an important immune evasion mechanism worthy of further investigation .

For comprehensive clinical correlation studies, researchers should consider:

What approaches can target the ULBP3-NKG2D axis for cancer immunotherapy?

ULBP3 represents a promising therapeutic target for enhancing immune responses against cancer based on its role in regulating NK cell activity . Several potential approaches emerge from the research:

Blocking sULBP3: Developing antibodies or other biologics that neutralize soluble ULBP3 could potentially prevent the downregulation of NKG2D on NK cells, thereby maintaining their cytotoxic potential . This approach might be particularly relevant since over 70% of cancer patients exhibit low levels of sULBP3 that weaken NK cell responses .

Enhancing membrane ULBP3 expression: Strategies to upregulate ULBP3 expression on tumor cell surfaces could increase their susceptibility to NK cell-mediated killing . Screening for small molecules or other agents that enhance ULBP3 transcription or prevent its shedding from the cell surface represents a promising research direction.

Bispecific engagers: Engineering molecules that simultaneously bind ULBP3 on tumor cells and activating receptors on NK cells could enhance targeted immune responses against ULBP3-expressing tumors.

Adoptive NK cell therapy: Ex vivo expansion and activation of NK cells, potentially combined with genetic engineering to enhance NKG2D expression or prevent its downregulation, could overcome the immunosuppressive effects of soluble ULBP3.

Research using monoclonal antibodies against ULBP3 has already provided valuable tools for investigating these approaches . Hybridoma clones such as B2-F1-F1 and B4-C5-D11 have demonstrated specific reactivity with both recombinant and naturally occurring ULBP3 , potentially forming the basis for therapeutic antibody development.

The observation that ULBP3 can regulate NK cell activity in both positive and negative ways highlights the need for careful therapeutic design that enhances the immune-stimulatory aspects while mitigating immune-suppressive effects.