LAYN Human

What is the molecular structure and characterization of human LAYN protein?

Human Layilin (LAYN) is a 55 kDa type I transmembrane protein that belongs to the animal C-type lectin family. The human LAYN cDNA encodes a 374 amino acid protein containing a 19 amino acid signal sequence, a 201 amino acid extracellular region, a 31 amino acid transmembrane segment (containing the distinctive L-A-Y-I-L-I motif for which it is named), and a 123 amino acid cytoplasmic tail . The extracellular domain features a 130 amino acid carbohydrate recognition domain with only one potential Ca²⁺-binding site .
The intracellular region contains three types of LH (layilin homology) repeats: three 16-18 amino acid LH1 repeats, three 5 amino acid (E-G-S-F/W-V) LH2 repeats, and two 4 amino acid (N-D/E-I-Y) LH3 repeats . When expressed in HEK293 systems, the recombinant protein has a predicted molecular weight of 25.4 kDa, but due to glycosylation, it typically migrates to 35-42 kDa as determined by Bis-Tris PAGE analysis .

How is LAYN expressed in different immune cell populations?

LAYN demonstrates distinct expression patterns across immune cell populations, with particularly notable expression in T cells. Research shows that LAYN is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+ T cells in human melanoma . This suggests that LAYN expression may serve as a marker for specific functional states in the T cell compartment.
LAYN expression correlates with immune infiltrating levels of multiple cell types, including CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in various cancers . The correlation is especially pronounced in colon and gastric cancer patients, where LAYN potentially contributes to the regulation of tumor-associated macrophages (TAMs), dendritic cells (DCs), T cell exhaustion, and regulatory T cells (Tregs) .

What methods are used to study LAYN expression in human tissues?

Several complementary methodologies are utilized to evaluate LAYN expression in human tissues:

• Immunofluorescence (IF): Used to visualize LAYN protein localization within cells and tissues.

• Immunohistochemistry (IHC): Applied to evaluate LAYN expression in fixed tissue specimens.

• Enzyme-Linked Immunosorbent Assay (ELISA): Employed for quantitative analysis of LAYN levels in biological samples.

• Flow Cytometry: Used to investigate LAYN expression in different immune cell populations.

• Single-cell RNA Sequencing (scRNA-seq): Provides high-resolution analysis of LAYN expression at the single-cell level .

• Bioinformatics Analysis: Tools such as TIMER and GEPIA databases are used to analyze LAYN expression levels across multiple cancer types and assess correlations with immune infiltration .
  These methodologies provide complementary data on LAYN expression patterns, allowing researchers to comprehensively characterize its distribution and regulation in various physiological and pathological contexts.

How does LAYN modulate CD8+ T cell function in the tumor microenvironment?

LAYN plays a crucial role in modulating CD8+ T cell function within the tumor microenvironment through several distinct mechanisms. Despite being expressed on phenotypically exhausted CD8+ T cells, LAYN paradoxically contributes to maintaining their cytotoxic potential rather than diminishing it .
Mechanistically, LAYN colocalizes with integrin αLβ2 (LFA-1) on T cells, and cross-linking of LAYN promotes the activation of this integrin . This interaction is functionally significant, as LAYN deletion results in attenuated LFA-1-dependent cellular adhesion . The enhanced cellular adhesiveness mediated by LAYN appears to be essential for CD8+ T cells to maintain their cytotoxic potential despite expressing high levels of inhibitory receptors.
Evidence from in vivo models supports this functional role - lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth . Similarly, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing in ex vivo assays . These findings identify LAYN as part of a molecular pathway by which exhausted or "dysfunctional" CD8+ T cells enhance cellular adhesiveness to maintain effective anti-tumor responses.

What is the correlation between LAYN expression and cancer prognosis?

LAYN has emerged as a promising prognostic biomarker across multiple cancer types. Research indicates that LAYN expression levels correlate with prognosis in several cancers, with particularly strong associations in colon and gastric cancers . In hepatocellular carcinoma (HCC), LAYN has been identified as a potential novel prognostic biomarker predicting poor outcomes .
The prognostic relevance of LAYN appears to be intimately connected to its role in regulating immune infiltration within the tumor microenvironment. Bioinformatic analyses using the TIMER algorithm and GEPIA database have revealed significant correlations between LAYN expression and tumor-infiltrating immune cells, including CD8+ T cells, CD4+ T cells, macrophages, and dendritic cells in HCC .
Notably, LAYN exhibits high expression specifically in HCC specimens with "immune hot" tumor tissues, particularly in infiltrating CD8+ T cells . This suggests that LAYN's prognostic significance may be linked to its impact on the functional state of tumor-infiltrating lymphocytes rather than cancer cell-intrinsic properties.

How do LAYN antagonists affect T cell exhaustion in the context of cancer immunotherapy?

Treatment with LAYN antagonists has demonstrated promising results in addressing T cell exhaustion phenotypes. Studies show that LAYN antagonist treatment can partially restore the exhausted phenotypes and immune function of CD8+ T cells in the tumor microenvironment . This restoration of function suggests potential therapeutic applications for LAYN antagonists in cancer immunotherapy.
The molecular basis for this effect lies in LAYN's role in regulating T cell adhesion and function. By targeting LAYN, antagonists may modulate the integrin signaling pathway, which is critical for T cell activation and function. Specifically, LAYN antagonists might affect the activation state of integrin αLβ2 (LFA-1), altering CD8+ T cell adhesion properties and consequently their anti-tumor activity .
These findings highlight the therapeutic potential of targeting LAYN to recover the antitumor immune response mediated by CD8+ T cells, particularly in cancers like hepatocellular carcinoma where LAYN expression is associated with T cell exhaustion .

What techniques are most effective for evaluating LAYN function in primary human T cells?

Evaluating LAYN function in primary human T cells requires a multi-faceted approach combining molecular, cellular, and functional analyses:

• Gene Editing Approaches: CRISPR-Cas9 technology enables precise genetic manipulation of LAYN in primary human T cells to assess its functional significance . This approach has successfully demonstrated that LAYN deletion reduces direct tumor cell killing by CD8+ T cells in ex vivo assays.

• Functional Assays:

  • Cell Proliferation: CCK8 assays measure how LAYN expression affects T cell proliferative capacity .

  • Killing Activity: CFSE/PI staining protocols evaluate the cytotoxic potential of LAYN-expressing T cells against target cells .

  • Cellular Adhesion Assays: These measure how LAYN modulates LFA-1-dependent adhesion properties .

• Imaging Techniques: Confocal microscopy and proximity ligation assays can visualize the colocalization of LAYN with integrin αLβ2 (LFA-1) and other signaling molecules to elucidate its role in T cell signaling .

• Transcriptomic Analysis: RNA sequencing of LAYN+ versus LAYN- T cells identifies co-expressed genes and characterizes the LAYN+CD8+ T cell exhaustion signature .

• Flow Cytometry: Multi-parameter flow cytometry evaluates how LAYN expression correlates with other exhaustion markers (PD-1, TIM-3, CD39) and activation markers on T cells .
  These complementary approaches provide a comprehensive understanding of LAYN function in primary human T cells, from molecular interactions to functional consequences in tumor immunity.

What are the optimal conditions for reconstitution and storage of recombinant LAYN protein?

The optimal handling of recombinant LAYN protein requires careful attention to reconstitution and storage conditions to maintain its structural integrity and biological activity:
Reconstitution Protocol:

• Recombinant Human LAYN protein is typically supplied in lyophilized form, formulated from a 0.22μm filtered solution in PBS (pH 7.4), often with 8% trehalose added as a protectant before lyophilization .

• Before opening, centrifuge the tube containing the lyophilized protein to ensure all material is at the bottom .

• For optimal reconstitution, dissolve the lyophilized protein in distilled water to a concentration exceeding 100 μg/ml .

• For carrier-free preparations, reconstitute in sterile PBS . For preparations with carriers, reconstitute in sterile PBS containing at least 0.1% human or bovine serum albumin .
  Storage Conditions:

• The lyophilized protein can be stored at -20°C to -80°C for up to 12 months from the date of receipt .

• After reconstitution, the protein remains stable at -80°C for approximately 3 months .

• To maximize stability, aliquot the reconstituted protein into smaller volumes for optimal storage and minimize freeze-thaw cycles .

• Use a manual defrost freezer for storage to avoid temperature fluctuations that could compromise protein integrity .
  Adhering to these protocols ensures the maintenance of LAYN protein activity for experimental applications, particularly in functional studies where protein conformation is critical.

How can researchers effectively analyze LAYN expression correlation with immune infiltration in cancer tissues?

Analyzing LAYN expression correlation with immune infiltration in cancer tissues requires a systematic approach combining bioinformatic tools, experimental validation, and computational analyses:
Bioinformatic Analysis Workflow:

• Database Selection: Utilize specialized databases like TIMER (Tumor Immune Estimation Resource) and GEPIA (Gene Expression Profiling Interactive Analysis) to estimate LAYN gene expression levels across cancer types .

• Correlation Analysis: Analyze correlations between LAYN expression and tumor-infiltrating immune cells (CD8+ T cells, CD4+ T cells, macrophages, dendritic cells) using the TIMER database .

• Checkpoint Correlation: Assess relationships between LAYN expression and immune checkpoint receptors such as CD39, PD-1, and TIM-3 .

• Data Normalization: For TCGA cohort analysis, download transcriptomic and clinical data from repositories like UCSC Xena, with normalized expression presented as log₂(TPM+1) .
  Experimental Validation:

• Single-cell RNA Sequencing: Apply scRNA-seq to characterize LAYN expression at the single-cell level within the tumor immune microenvironment .

• Multiplex Immunohistochemistry: Use multiplex IHC to simultaneously visualize LAYN and immune cell markers in tissue sections.

• Flow Cytometry: Quantify LAYN expression on different immune cell populations isolated from tumor tissues.
  This integrated approach allows researchers to establish robust correlations between LAYN expression and immune infiltration patterns in cancer tissues, providing insights into its role in tumor immunology and potential as a prognostic biomarker.

How might targeting LAYN improve current cancer immunotherapy approaches?

Targeting LAYN represents a promising strategy to enhance cancer immunotherapy efficacy through several mechanisms:

• Restoration of Exhausted T Cell Function: LAYN antagonists have demonstrated the ability to partially restore the exhausted phenotypes and immune function of CD8+ T cells in the tumor microenvironment . This restoration could potentially enhance the efficacy of existing immunotherapies such as immune checkpoint inhibitors.

• Combination Therapy Potential: Given LAYN's correlation with immune checkpoint receptors like PD-1 and TIM-3 , combining LAYN antagonists with established checkpoint inhibitors might produce synergistic effects, particularly in patients with high LAYN expression.

• Biomarker-Guided Treatment: LAYN expression could serve as a predictive biomarker to identify patients most likely to benefit from specific immunotherapy approaches. This would enable more personalized treatment strategies, especially in cancers where LAYN is highly expressed, such as colon, gastric, and hepatocellular carcinomas .

• Enhanced T Cell Adhesion Modulation: By targeting LAYN's interaction with integrin αLβ2 (LFA-1), researchers might be able to fine-tune T cell adhesiveness to optimize tumor infiltration and killing capacity . This represents a novel mechanism distinct from current immunotherapeutic approaches.

• Improved CAR-T Cell Efficacy: Engineering CAR-T cells with modified LAYN expression or signaling might enhance their persistence and killing capacity within the tumor microenvironment by optimizing cellular adhesion properties.
  The therapeutic potential of targeting LAYN underscores the importance of further research to develop specific LAYN-targeted agents and evaluate their efficacy in various cancer types.

What experimental models best recapitulate human LAYN function for translational research?

Several experimental models effectively recapitulate human LAYN function for translational research, each with distinct advantages:

• Humanized Mouse Models: Mice engrafted with human immune system components provide a valuable in vivo platform to study LAYN function in a human-relevant context. Lineage-specific deletion of layilin on murine CD8+ T cells has already demonstrated reduced tumor infiltration and increased tumor growth in vivo , suggesting similar approaches with humanized models would be informative.

• Ex Vivo Human Tumor Explant Cultures: Fresh human tumor samples cultured ex vivo maintain the complex cellular architecture of the tumor microenvironment, allowing for the study of LAYN function in tumor-infiltrating lymphocytes within their native context.

• 3D Organoid Co-culture Systems: Co-culturing cancer organoids with LAYN-manipulated immune cells enables the study of LAYN-dependent interactions in a controlled yet physiologically relevant setting.

• CRISPR-engineered Primary Human T Cells: Gene editing of LAYN in primary human CD8+ T cells has already demonstrated reduced direct tumor cell killing ex vivo . This approach can be expanded to study various aspects of LAYN function in human T cells.

• Patient-Derived Xenograft (PDX) Models: PDX models maintain the heterogeneity of the original tumor and can be used to study how LAYN expression in tumor-infiltrating lymphocytes correlates with response to various immunotherapies.
  Each model system offers unique advantages for studying different aspects of LAYN biology, from molecular mechanisms to functional outcomes in complex tissue environments. The integration of data from multiple model systems will be crucial for translating LAYN-related findings to clinical applications.

What are the most significant unresolved questions regarding LAYN function in human immunology?

Despite recent advances in understanding LAYN biology, several significant questions remain unresolved:

• Ligand Identification: The natural ligand(s) for LAYN's C-type lectin domain remain poorly characterized. Identifying these ligands would provide crucial insights into how LAYN signaling is initiated in different tissue contexts.

• Signaling Mechanisms: While LAYN colocalizes with integrin αLβ2 (LFA-1) and affects its activation , the detailed signaling pathways downstream of LAYN engagement remain incompletely understood. Elucidating these pathways would clarify how LAYN modulates T cell function.

• Expression Regulation: The mechanisms controlling LAYN expression in T cells and other immune cells are not fully defined. Understanding these regulatory pathways could reveal strategies to therapeutically modulate LAYN levels.

• Non-Immune Functions: LAYN may have functions beyond immune regulation that remain to be explored, particularly given its expression pattern across different tissues.

• Therapeutic Targeting Specificity: The optimal strategies for targeting LAYN therapeutically, whether through antibodies, small molecules, or other approaches, need further investigation to ensure efficacy and specificity.
  Addressing these knowledge gaps will be essential for fully harnessing LAYN's potential as both a biomarker and therapeutic target in cancer and potentially other diseases with immune components.

How does LAYN research interface with current trends in immunotherapy development?

LAYN research intersects with several major trends in immunotherapy development:

• Beyond Classical Checkpoints: As the field moves beyond PD-1/PD-L1 and CTLA-4 targeting, LAYN represents a novel immune regulatory axis with distinct functional effects. Its role in maintaining CD8+ T cell cytotoxic potential despite an exhausted phenotype offers a unique angle for therapeutic intervention.

• Biomarker-Guided Therapy: LAYN's correlation with prognosis in multiple cancers positions it as a potential biomarker for patient stratification, aligning with the growing emphasis on precision immunotherapy.

• Microenvironment Modulation: By affecting cellular adhesion and T cell infiltration into tumors , LAYN-targeted interventions could address the challenge of "cold" tumors with limited immune infiltration.

• Combination Therapy Optimization: Understanding LAYN's interaction with other immune pathways can inform rational combination therapies, a central focus in current immunotherapy development.

• Adoptive Cell Therapy Enhancement: Insights from LAYN research could improve CAR-T and TIL therapy by optimizing T cell adhesion, persistence, and function within the tumor microenvironment. LAYN research thus complements and extends current immunotherapy approaches, potentially addressing key limitations such as primary and acquired resistance to existing treatments through novel mechanistic pathways. Human Layilin (LAYN) represents an exciting frontier in immunology and cancer research, with significant implications for understanding T cell biology and developing novel immunotherapeutic strategies. As research in this field progresses, addressing the identified knowledge gaps will likely yield valuable insights for translational applications across multiple cancer types and potentially other immune-mediated diseases.