ULBP4 Human

What is ULBP4 and how does it function in the human immune system?

ULBP4 belongs to the UL16-binding protein family of ligands that interact with the NKG2D activating receptor expressed on natural killer (NK) cells and certain T cell subsets. What distinguishes ULBP4 from other NKG2D ligands is its unique dual binding capacity - it functions as a ligand for both the NKG2D receptor and the T cell receptor gamma-delta (TCRγδ), specifically TCRγ9/δ2 .

This dual receptor binding enables ULBP4 to play significant roles in immune surveillance against tumors and viral infections. Research demonstrates that immobilized soluble ULBP4 (rULBP4) induces proliferation of Vδ2+ T cells derived from human carcinomas in vitro, with these T cells secreting Th1 cytokines and displaying strong cytolytic activity against ULBP4-expressing targets . Additionally, ULBP4 expression has been detected on Epstein-Barr virus (EBV)-infected peripheral blood cells, suggesting involvement in antiviral immunity .

What cell types express ULBP4 in humans?

ULBP4 displays a distinctive tissue expression pattern:

• Astrocytes are the predominant cell type expressing ULBP4 in the human brain, particularly in multiple sclerosis (MS) patients. Immunohistochemical studies show significant colocalization of ULBP4 with GFAP (astrocyte marker) but not with MAP2 (neuronal marker) .

• Tumor cells express ULBP4, potentially triggering immune responses by γδT cells and NK cells .

• EBV-infected cells show ULBP4 expression, suggesting a role in viral immunity .

• Neurons express very low levels of ULBP4, even when exposed to cellular stressors that upregulate ULBP4 in astrocytes .

• Peripheral blood immune cells, including monocytes, do not express significant levels of ULBP4 based on transcriptomic analyses from multiple databases showing extremely low to undetectable ULBP4 transcript levels in human blood cell populations .

What methods are appropriate for detecting ULBP4 expression in experimental settings?

Multiple complementary techniques are necessary for reliable ULBP4 detection:

Protein-level detection:

• Western blot: Detects ULBP4 protein in tissue lysates and can distinguish different molecular weight forms (e.g., the 25 kDa soluble form in cerebrospinal fluid) .

• Immunohistochemistry/Immunofluorescence: Visualizes ULBP4 cellular localization through co-staining with cell-specific markers .

• Flow cytometry: Measures ULBP4 surface expression on cells like astrocytes exposed to different stressors .

Transcript-level detection:

• qRT-PCR: Measures ULBP4 mRNA expression using primers with binding sites in specific exons (e.g., exon 3 and exon 4) to detect functional ULBP4 isoforms .

• RNA-Seq: Evaluates ULBP4 expression in large-scale transcriptomic analyses .

Critical methodological considerations:

• Antibody specificity is essential, as demonstrated by the documented cross-reactivity of commercial antibody mAb 709116, which binds to human monocytes despite their lack of ULBP4 expression .

• Validation with multiple methods is recommended to ensure accurate assessment of ULBP4 expression.

• Researchers should use validated antibodies like DUMO1 rather than potentially cross-reactive antibodies that could lead to data misinterpretation .

What is the difference between membrane-bound and soluble forms of ULBP4?

ULBP4 exists in both membrane-bound and soluble forms with distinct biological functions:

Membrane-bound ULBP4:

• Expressed on cell surfaces (astrocytes, tumor cells)

• Functions as a ligand for both NKG2D receptor and TCRγ9/δ2

• Serves as a recognition target for immune surveillance by NK cells and γδT cells

• Upregulated in response to cellular stress signals including inflammation, ER stress, and oxidative stress

Soluble ULBP4:

• Present in cerebrospinal fluid, with a smaller shed/soluble form (25 kDa) significantly elevated in female MS patients

• Previously reported as RAET1E2, a soluble isoform that inhibits NKG2D-mediated NK cytotoxicity

• Enhances production of proinflammatory cytokines (GM-CSF and IFNγ) by CD8+ T lymphocytes

• Increases CD8+ T lymphocyte motility and favors kinapse-like behavior rather than synapse-like behavior

• May participate in modulating immune responses in the CNS, particularly in MS pathology

The mechanisms of ULBP4 shedding from the cell surface and the regulation of this process represent important areas for future investigation.

How does ULBP4 contribute to Multiple Sclerosis pathology?

ULBP4 appears to be significantly involved in MS pathobiology through multiple mechanisms:

Elevated expression in MS brain tissue:

• ULBP4 protein levels are higher in brain tissues from MS patients compared to controls

• Particularly elevated in active and chronic active lesions and normal-appearing white matter

• Astrocytes are the predominant cell type expressing ULBP4 in MS brains

Soluble ULBP4 in cerebrospinal fluid:

• A smaller shed/soluble form (25 kDa) of ULBP4 is significantly elevated in the CSF of female MS patients compared to controls and male MS patients

• This gender-specific elevation suggests a potential link to the higher prevalence of MS in women

Effects on CD8+ T lymphocyte function and behavior:

• Soluble ULBP4 enhances production of proinflammatory cytokines GM-CSF and IFNγ by CD8+ T lymphocytes

• It increases CD8+ T lymphocyte motility in coculture with human astrocytes

• Promotes kinapse-like behavior (more dynamic interactions) rather than synapse-like behavior

• CD8+ T lymphocytes from MS patients are especially sensitive to these effects

• These changes may facilitate T cell movement within the brain parenchyma and contribute to the inflammatory process

Response to cellular stressors present in MS lesions:

• ULBP4 expression by astrocytes is upregulated in response to inflammation (TNF/IFNγ), ER stress (tunicamycin), and oxidative stress (sodium arsenite)

• These cellular stressors are relevant in the context of MS pathology

These findings highlight ULBP4 as a potential therapeutic target for MS treatment, particularly through strategies that block ULBP4-NKG2D interactions or inhibit ULBP4 shedding.

What cellular stressors induce ULBP4 expression in human astrocytes?

Several cellular stressors relevant to neuroinflammatory conditions can upregulate ULBP4 expression in human astrocytes:

Inflammatory stress:

• Combination of proinflammatory cytokines TNF (tumor necrosis factor) and IFNγ (interferon-gamma) induces the strongest increase in ULBP4 expression

• These cytokines are elevated in MS brain tissues

Endoplasmic reticulum (ER) stress:

• Treatment with tunicamycin, an inducer of ER stress, significantly increases the proportion of astrocytes expressing ULBP4

• ER stress markers are observed in MS lesions

Oxidative stress:

• Exposure to sodium arsenite, which induces oxidative stress, also upregulates ULBP4 expression

• Oxidative stress is a common feature of neuroinflammatory conditions

Notably, these same cellular stressors did not significantly alter the low ULBP4 expression in human neurons isolated from the same CNS tissue, indicating cell type-specific regulation of ULBP4 expression . This differential response suggests distinct regulatory mechanisms across neural cell types, which may be relevant for understanding ULBP4's role in neuroinflammatory diseases.

How does soluble ULBP4 modify CD8+ T lymphocyte function in the context of neuroinflammation?

Soluble ULBP4 modulates CD8+ T lymphocyte function through several mechanisms relevant to neuroinflammation:

Enhanced inflammatory cytokine production:

• Soluble ULBP4 boosts production of two key inflammatory cytokines by activated human CD8+ T lymphocytes :

  • GM-CSF (Granulocyte-macrophage colony-stimulating factor)

  • IFNγ (Interferon-gamma)

• These cytokines are known contributors to neuroinflammation in MS

Altered motility and behavior in the CNS microenvironment:

• In coculture with human astrocytes, CD8+ T lymphocytes exposed to soluble ULBP4 show:

  • Decreased coefficient of arrest, indicating increased motility

  • Shift from synapse-like behaviors (round, poking) to kinapse-like behaviors (dancing, scanning)

  • These changes are more pronounced in CD8+ T lymphocytes from MS patients compared to healthy controls

Mechanism considerations:

• The effects of soluble ULBP4 are not explained by altered NKG2D expression, as both MS patients and controls showed similar NKG2D levels

• Soluble ULBP4 does not appear to have direct chemotactic properties; adding it to a transwell chemotaxis assay had no impact on CD8+ T lymphocyte migration

• The mechanisms enhancing T cell movement within brain parenchyma appear to involve non-chemoattractive pathways

These findings suggest that soluble ULBP4 may contribute to MS pathology by promoting inflammatory cytokine production and facilitating the movement of CD8+ T lymphocytes through the brain parenchyma, particularly in female patients where elevated levels of soluble ULBP4 were detected in CSF.

What are the critical experimental considerations when studying ULBP4?

Researchers investigating ULBP4 should be aware of several critical experimental considerations:

Antibody validation:

• Careful validation of antibodies is essential due to documented cross-reactivity issues

• The commercial antibody mAb 709116 has been shown to bind to a non-ULBP4 surface molecule on human monocytes, leading to potential misinterpretation

• Use of validated antibodies like DUMO1 is recommended for reliable ULBP4 detection

Multi-method approach for expression studies:

• Combine protein-level detection (Western blot, flow cytometry, immunohistochemistry) with transcript analysis (qRT-PCR, RNA-Seq)

• For qRT-PCR, design primers that can detect all functional cell-bound ULBP4 isoforms

• Include multiple tissue or cell sources to establish expression patterns

Soluble vs. membrane-bound ULBP4 distinction:

• Distinguish between membrane-bound and soluble forms, which may have different molecular weights and functional properties

• Use appropriate methods to detect soluble ULBP4 in biological fluids

• Consider how sample processing might affect detection of soluble factors

Cellular stress protocols:

• When studying stress-induced ULBP4 expression, optimize stressor concentrations and exposure times to avoid significant cell death while inducing stress responses

• Include appropriate positive controls for stress induction

• Consider physiologically relevant stressors for the biological context under investigation

Sex-specific effects:

• Consider potential sex differences in ULBP4 expression and function, given the elevated levels of soluble ULBP4 in CSF from female MS patients compared to males

• Account for disease state when comparing samples, as conditions like MS can significantly alter ULBP4 expression patterns

What contradictions exist in the literature regarding ULBP4 expression in human immune cells?

Several notable contradictions exist in the scientific literature regarding ULBP4 expression in immune cells:

Transcriptomic evidence:

• Multiple independent transcriptomic datasets show extremely low to undetectable levels of ULBP4 mRNA in human monocytes and other peripheral blood immune cells :

  • The database of immune cell expression (DICE) contains RNASeq data from 13 immune cell subsets derived from 91 healthy donors, showing no ULBP4 transcripts in any blood cell subset except extremely low levels (0.1 TPM) in memory CD4 T cells

  • Data from Monaco et al. covering 29 different immune cell types showed extremely low levels of ULBP4 transcripts (0.2 to 1.1 pTPM) across analyzed immune cell subsets

  • The Human Protein Atlas project reported ULBP4 transcripts at extremely low levels (0.1 to 0.4 pTPM) in various immune cells

The current consensus, based on multiple lines of evidence, suggests that ULBP4 is not expressed by monocytes and likely not by other peripheral blood immune cells . This restricted expression pattern distinguishes ULBP4 from some other NKG2D ligands with broader expression profiles.

What therapeutic approaches might target ULBP4 in inflammatory or autoimmune diseases?

Given ULBP4's emerging role in conditions like Multiple Sclerosis, several therapeutic strategies could be considered:

Blocking ULBP4-receptor interactions:

• Development of monoclonal antibodies that specifically bind to ULBP4 and prevent its interaction with NKG2D and/or TCRγ9/δ2

• Creation of soluble receptor decoys that can bind ULBP4 and prevent it from engaging cell-surface receptors

• Small molecule inhibitors designed to disrupt ULBP4-receptor binding interfaces

Inhibiting ULBP4 shedding:

• Targeting the proteases responsible for generating the soluble form of ULBP4, which enhances CD8+ T cell inflammatory functions in MS

• This approach might be particularly beneficial for female MS patients, who show elevated levels of the shed/soluble form of ULBP4 in CSF

Reducing ULBP4 expression:

• Targeting the cellular stress pathways that upregulate ULBP4 expression in astrocytes, such as inflammation (TNF/IFNγ signaling), ER stress, or oxidative stress

• Development of gene silencing approaches to reduce ULBP4 production

Cell type-specific approaches:

• Since astrocytes are the predominant cells expressing ULBP4 in MS brains, developing astrocyte-targeted delivery systems for ULBP4-modulating therapeutics could increase efficacy while reducing off-target effects

Therapeutic applications:

• Multiple Sclerosis: Targeting ULBP4 could reduce CD8+ T cell activation and motility in the CNS, potentially limiting inflammatory damage

• Tumor immunotherapy: Given ULBP4's role in activating γδT cells against tumor targets, approaches to enhance ULBP4 expression on tumor cells might boost anti-tumor immunity

• Viral infections: As ULBP4 is expressed on EBV-infected cells, strategies to modulate ULBP4-mediated immune responses could be explored for viral diseases