HAVCR2 Human

What is HAVCR2 and what is its primary function in human cells?

HAVCR2 belongs to the immunoglobulin superfamily and TIM (T-cell immunoglobulin and mucin domain) family of proteins. It functions primarily as an immune regulatory protein expressed on multiple cell types, particularly T cells and NK cells. HAVCR2 regulates macrophage activation, inhibits Th1-mediated auto- and alloimmune responses, and promotes immunological tolerance .

This protein interacts with ligands such as Galectin-9 (LGALS9), potentially resulting in suppression of T-cell responses through mechanisms involving HAVCR2 phosphorylation and disruption of its association with BAG6 . In NK cells, HAVCR2 can enhance IFN-gamma production in response to LGALS9, though its function appears context-dependent, as it has also been shown to suppress NK cell-mediated cytotoxicity in certain settings .

How is HAVCR2 expression regulated in normal human tissues?

HAVCR2 expression is regulated through both epigenetic and genetic mechanisms. Research indicates that HAVCR2 expression shows a negative correlation with DNA methylation, suggesting that hypomethylation of promoter regions may lead to increased expression . Concurrently, HAVCR2 expression positively correlates with copy number variations, indicating that genetic amplification can upregulate its expression .

In immune cells, particularly NK cells, HAVCR2 expression correlates with other functional immune markers. For instance, in bone marrow NK cells from AML patients, HAVCR2 expression positively correlates with NK cell markers like NCAM1 (CD56) and FCGR3A (CD16a), as well as cytotoxic molecules including GNLY, GZMA, GZMB, and PRF1 . This suggests tissue-specific and context-dependent regulation.

What are the common methods to detect HAVCR2 expression in research settings?

Several methodological approaches are employed to detect and analyze HAVCR2 expression:

Transcriptomic Analysis:

• High-throughput transcriptome sequencing for tissue-level expression analysis

• Single-cell RNA sequencing (scRNA-seq) for cell-specific expression patterns

Data Processing and Analysis:

• R packages like "Seurat" for normalization using functions such as "NormalizedData", "FindVariableFeatures", and "ScaleData" with "LogNormalize" methods

• Dimension reduction techniques using "RunUMAP" for visualizing cell populations expressing HAVCR2

Cell Type Annotation:

• "SingleR" package with reference databases like "HumanPrimaryCellAtlasData" for identifying cell types expressing HAVCR2

Differential Expression Analysis:

• "DESeq2" and "DESeqDataSetFromMatrix" functions with statistical thresholds (|log2FC| ≥ 0.5, p < 0.05) to identify genes differentially expressed with HAVCR2

Functional Analysis:

• "clusterProfiler", "enrichGO", and "enrichKEGG" functions for pathway enrichment analysis related to HAVCR2 expression

What are the main signaling pathways associated with HAVCR2 function?

HAVCR2 participates in several critical signaling pathways in immune cells:

Cytokine Signaling Pathways:

• "Cytokine Signaling in Immune system" pathway, suggesting a role in mediating cytokine responses

• "Interleukin-2 family signaling" pathway, crucial for T-cell proliferation and function

T-cell Receptor Signaling:

• HAVCR2-LGALS9 interaction influences T-cell receptor signaling, potentially through pathways involving HAVCR2 phosphorylation

Natural Killer Cell Cytotoxicity Pathways:

• "Natural killer cell mediated cytotoxicity" pathway as revealed by KEGG analysis of HAVCR2-positive NK cells

• Enrichment of cell-killing and leukocyte-mediated cytotoxicity pathways in HAVCR2-positive NK cells

These pathways collectively indicate HAVCR2's involvement in regulating both adaptive and innate immune responses, particularly through its effects on T-cell and NK cell function.

How does HAVCR2 (TIM-3) expression affect natural killer (NK) cell cytotoxicity in acute myeloid leukemia?

The relationship between HAVCR2 expression and NK cell function in AML reveals complex immunoregulatory mechanisms. Single-cell RNA sequencing analysis of NK cells from AML patients demonstrates that HAVCR2-positive NK cells exhibit significantly higher expression of cytotoxic molecules compared to HAVCR2-negative NK cells:

Upregulation of Cytotoxic Machinery:

• Higher expression of cytotoxic granule genes including GNLY (p < 0.0001), GZMA (p = 0.0019), GZMB (p = 0.024), and PRF1 (p = 0.0044)

• Increased transcript levels of NK cell receptors including KLRC1, KLRD1, and JAK1

Pathway Enrichment:

• Significant enrichment of cell-killing and leukocyte-mediated cytotoxicity pathways in HAVCR2-positive NK cells

• Natural killer cell-mediated cytotoxicity pathway identified as the most significantly enriched KEGG pathway

Clinical Correlation:

• Despite correlation with enhanced cytotoxic potential, high TIM-3 levels on NK cells at diagnosis associated with poorer event-free survival (2-year EFS rate: 39.3% vs. 63.4%, p = 0.0074)

This paradoxical relationship suggests that while HAVCR2 expression correlates with enhanced cytotoxic potential, it may also indicate NK cell exhaustion or dysfunction in the AML microenvironment, contributing to less favorable clinical outcomes.

What is the prognostic significance of HAVCR2 upregulation in testicular germ cell tumors?

HAVCR2 upregulation has emerged as a significant prognostic factor in testicular germ cell tumors (TGCTs):

Correlation with Outcome:

• Significant upregulation in TGCT tissue correlating with poor prognosis

• Identified as a high-potential biomarker for diagnosis, prognosis, and treatment guidance

Immune Microenvironment Impact:

• Positive correlation with infiltration of six immune cell types: B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and myeloid dendritic cells

• Higher HAVCR2 expression associated with increased immune cell abundance in tumor microenvironment

Treatment Response Prediction:

• Tumor Immune Dysfunction and Exclusion (TIDE) scoring analysis showed HAVCR2 related to immunotherapy responsiveness in TGCTs

• Drug sensitivity analysis revealed HAVCR2 correlation with sensitivity to multiple antitumor drugs

These findings establish HAVCR2 as a multifaceted biomarker in TGCTs, potentially guiding clinical decision-making for diagnosis, prognosis assessment, and precision treatment selection.

How does HAVCR2 expression correlate with immune cell infiltration in cancer tissues?

Analysis of HAVCR2 expression in cancer tissues reveals significant associations with immune cell infiltration patterns:

Multi-lineage Correlation:

• Significant positive correlation between HAVCR2 expression and infiltration of six immune cell types in TGCTs

• Associated immune cells include B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and myeloid dendritic cells

Quantitative Relationship:

• Higher HAVCR2 expression levels correlate with increased immune cell abundance in the tumor microenvironment

• This suggests HAVCR2 may influence immune cell recruitment or retention within tumor tissues

Therapeutic Implications:

• The correlation between HAVCR2 and immune infiltration has implications for immunotherapy approaches

• TIDE scoring analysis indicates HAVCR2 expression relates to immunotherapy responsiveness

These correlations suggest HAVCR2 plays a role in shaping the tumor immune microenvironment, potentially affecting both anti-tumor immune responses and treatment outcomes.

What are the experimental considerations when studying HAVCR2 methylation patterns in relation to its expression?

Research demonstrates that HAVCR2 expression negatively correlates with its DNA methylation , indicating epigenetic regulation. When investigating this relationship, researchers should consider:

Comprehensive Methylation Analysis:

• Utilize techniques such as bisulfite sequencing, methylation-specific PCR, or methylation arrays

• Focus on promoter and enhancer regions that likely have strongest regulatory impact

Multi-omics Integration:

• Design experiments for simultaneous analysis of methylation patterns and expression levels

• Include copy number variation analysis, as HAVCR2 expression positively correlates with copy number levels

• Assess relative contributions of genetic and epigenetic factors to expression regulation

Tissue and Cell Type Considerations:

• Compare methylation patterns across different tissues and cell types

• Employ single-cell methylation analysis when possible to account for cellular heterogeneity

• Consider tumor microenvironment influences on HAVCR2 methylation in cancer tissues

Functional Validation:

• Implement targeted methylation/demethylation using CRISPR-based epigenome editing tools

• Analyze transcription factor binding sites potentially affected by methylation

• Investigate functional consequences of altered HAVCR2 expression due to methylation changes

This multi-faceted approach would enable more comprehensive understanding of how methylation patterns regulate HAVCR2 expression across different cellular contexts.

How can researchers resolve conflicting results regarding HAVCR2's role in immune suppression versus activation?

The literature contains apparently conflicting observations regarding HAVCR2 function, particularly in NK cells where it has been reported to both enhance IFN-gamma production and suppress cytotoxicity . To resolve these conflicts, researchers should consider:

Context-Dependent Experimental Design:

• Analyze HAVCR2 function across multiple cell types under identical conditions

• Compare function in different disease models and activation states

• Systematically vary experimental conditions to identify factors that influence HAVCR2 function

Single-Cell Resolution Analysis:

• Employ single-cell technologies to identify heterogeneity within HAVCR2-expressing populations

• Correlate HAVCR2 expression with functional markers at single-cell level

• Identify cell subsets that might exhibit different HAVCR2-mediated functions

Temporal Dynamics:

• Implement time-course analyses to track HAVCR2 function over the course of immune responses

• Use inducible expression systems to control timing of HAVCR2 activity

• Monitor real-time changes in HAVCR2-expressing cells during activation/inhibition

Signaling Network Analysis:

• Analyze HAVCR2 function with or without specific ligands (e.g., LGALS9)

• Investigate co-receptors that might modulate HAVCR2 signaling

• Map interconnected signaling networks using systems biology approaches

Methodological Standardization:

• Employ multiple complementary functional assays

• Validate key findings using independent experimental approaches

• Include appropriate controls for antibody specificity and genetic models

This systematic approach would help reconcile conflicting observations and develop a more nuanced understanding of HAVCR2's context-dependent functions.