CRTAM Human

What is CRTAM and where is it expressed in human tissues?

CRTAM is a nectin family member of the immunoglobulin superfamily that functions as an important immune cell receptor. In humans, CRTAM is a 393 amino acid, 80 kDa type I transmembrane glycoprotein with a 17 amino acid signal sequence and a 269 amino acid extracellular domain . It is primarily expressed by activated CD8+ T cells and NK T cells after receptor engagement . Interestingly, CRTAM is also found in spleen, thymus, small intestine, peripheral blood, and has surprisingly high expression in the brain, particularly in Purkinje cells of the cerebellum . Research has shown that approximately 2-5% of activated CD4+ T cells also express CRTAM after TCR stimulation .

How does CRTAM expression differ across T cell subpopulations?

CRTAM expression varies significantly across different T cell subsets:

The CRTAM+ CD4+ T cell population produces high levels of effector cytokines including IFN-γ, IL-17, and IL-22, but not IL-4 . Interestingly, when naive, effector memory, and central memory CD4+ T cells are analyzed separately, each subset contains CRTAM+ cells, with memory cells generating a higher percentage than naive cells . Furthermore, the type of antigen-presenting cell influences CRTAM expression, with dendritic cells inducing more than fourfold higher expression compared to B cells or macrophages .

What functional properties distinguish CRTAM+ CD4+ T cells?

CRTAM+ CD4+ T cells represent a unique hybrid cell population with characteristics of both CD4+ and CD8+ T cells . Comprehensive gene expression analysis has revealed that while CRTAM+ and CRTAM- CD4+ T cells similarly express CD4+ T cell-related genes (including CD4 and ThPOK), CRTAM+ cells also express significant levels of cytotoxic T lymphocyte (CTL)-related genes, such as IFN-γ, CD8α, Granzyme B, and Eomes .

These cells demonstrate cytotoxic functionality after cultivation, suggesting CRTAM identifies or determines a CD4+ cytotoxic T lymphocyte lineage . Approximately 68% of genes are expressed at comparable levels between CRTAM+ CD4+ T cells and CD8+ T cells, with most gene expression profiles being intermediate between typical CD4+ and CD8+ T cells . Importantly, ectopic expression of CRTAM in vivo can induce CD4+ CTL differentiation, indicating a causal role in this developmental pathway .

What are the optimal protocols for detecting and analyzing CRTAM expression?

Detection of CRTAM requires careful attention to timing and methodology due to its transient expression pattern. The following approaches yield reliable results:

Flow Cytometry Protocol:

• Stimulate T cells with anti-CD3/CD28 antibodies or peptide-pulsed APCs

• Harvest cells at multiple timepoints (optimally 24-48 hours post-stimulation)

• Stain with fluorochrome-conjugated anti-CRTAM antibodies

• Include markers to identify T cell subsets (CD3, CD4, CD8)

• Add memory/activation markers (CD44, CD62L) for further characterization

RT-qPCR Analysis:
For transcriptional analysis, researchers should isolate RNA from sorted or unsorted T cell populations and perform RT-qPCR using primers specific for human CRTAM. Expression values should be calculated using the 2-ΔCt method with GAPDH as a reference gene . This approach complements protein-level analysis and can sometimes detect expression changes before surface protein is detectable.

When analyzing experimental data, it's important to note that only 2-5% of activated CD4+ T cells express CRTAM, so analysis strategies must account for this rare population .

How can researchers generate reliable in vitro models for studying CRTAM function?

Several approaches have proven effective for investigating CRTAM function:

Overexpression Systems:
Human CRTAM can be cloned from cell lines such as BT-20 and constructed into lentiviral vectors (e.g., pCDH-CMV-3XFLAG-EF1-PURO) for stable overexpression . The method involves:

• Cloning CRTAM from cDNA and constructing into an appropriate vector

• Producing lentiviruses by transfecting HEK293T cells with the CRTAM vector plus packaging plasmids

• Collecting viral supernatant at 60 hours post-transfection

• Infecting target cells and selecting with puromycin

• Confirming expression by western blot or flow cytometry

Functional Characterization Assays:
After establishing CRTAM-expressing models, researchers can employ multiple functional readouts:

• Phosphorylation status of STAT1 to assess signaling pathway activation

• Expression of interferon-stimulated genes by RT-qPCR

• MHC class I molecule expression analysis

• Cytotoxicity assays against appropriate target cells

• Co-culture experiments with CD8+ T cells to assess immune activation

These approaches allow researchers to dissect the specific contributions of CRTAM to cellular function and immune responses in controlled environments.

What in vivo models best capture CRTAM's role in human immune responses?

While fully recapitulating human CRTAM function in animal models presents challenges, several approaches have proven valuable:

Tumor Models:
Immunocompetent mice can be injected with triple-negative breast cancer cells stably overexpressing CRTAM to evaluate its impact on tumor growth and immune infiltration . This approach has demonstrated that CRTAM enhances tumor-associated immune cell infiltration, especially CD8+ T cells . Analysis of these models should include:

• Flow cytometric characterization of tumor-infiltrating lymphocytes

• Immunohistochemistry to assess spatial distribution of immune cells

• Cytokine profiling of the tumor microenvironment

• Assessment of tumor growth kinetics and survival

Transgenic Approaches:
For mechanistic studies, researchers can develop mouse models with T cell-specific CRTAM expression or conditional deletion. These models help distinguish between the role of CRTAM in development versus effector function.

Humanized Mouse Models:
For greater relevance to human biology, immunodeficient mice can be reconstituted with human immune cells, providing a system to study human CRTAM in an in vivo context. This approach is particularly valuable for evaluating therapeutic interventions targeting CRTAM.

How does CRTAM expression impact tumor immune microenvironments?

CRTAM plays a significant role in anti-tumor immunity, particularly in triple-negative breast cancer (TNBC). Research has revealed several critical impacts of CRTAM on tumor immunity:

Enhanced T Cell Infiltration:
CRTAM overexpression promotes infiltration of immune cells into tumors, particularly CD8+ T cells . This infiltration correlates with improved clinical outcomes and may be related to CRTAM's ability to increase expression of MHC class I molecules, facilitating T cell recognition of tumor cells .

Activation of Interferon Signaling:
CRTAM overexpression induces STAT1 phosphorylation and increases expression of interferon-stimulated genes . This interferon response represents a critical antitumor mechanism that enhances immune surveillance and promotes anti-tumor activity.

These findings suggest that CRTAM actively shapes the tumor microenvironment toward an immune-inflamed phenotype that is associated with better clinical outcomes and potentially enhanced responsiveness to immunotherapy.

What methodological approaches best demonstrate CRTAM's functional impact in disease models?

Demonstrating CRTAM's functional impact requires multifaceted approaches that link molecular mechanisms to disease outcomes:

Genetic Manipulation Studies:
Overexpression or knockout studies in relevant cell types provide direct evidence of CRTAM's impact. For example, stable overexpression of CRTAM in triple-negative breast cancer cells demonstrated its ability to induce STAT1 phosphorylation and increase interferon-stimulated gene expression .

Comprehensive Immune Profiling:
Flow cytometry and immunohistochemistry analyses of tumor samples can characterize changes in immune infiltration associated with CRTAM expression . This should include quantification of different immune cell subsets and their functional states.

Transcriptomic Analysis:
RNA sequencing followed by pathway analysis (GO, KEGG, GSEA) can identify biological processes influenced by CRTAM. Studies have shown that CRTAM expression is associated with immune responses and immune system processes .

Survival Analysis:
Kaplan-Meier analysis of patient cohorts stratified by CRTAM expression levels provides clinical relevance . This should be complemented by multivariate Cox regression to adjust for other prognostic factors.

Combined In Vitro and In Vivo Validation:
Findings from cell culture experiments should be validated in animal models. For example, after demonstrating CRTAM's effects on interferon signaling in vitro, researchers confirmed its impact on immune cell infiltration in a mouse tumor model .

This multilayered approach strengthens causal inferences about CRTAM's role in disease processes and provides more robust evidence for potential therapeutic applications.

Can CRTAM expression patterns predict response to immunotherapy?

Emerging evidence suggests CRTAM may serve as a valuable biomarker for immunotherapy response prediction:

Correlation with Established Predictive Factors:
CRTAM expression correlates with features known to predict immunotherapy response, including:

• Enhanced CD8+ T cell infiltration

• Increased MHC class I expression

• Activated interferon signaling pathways

• Immunologically "hot" tumor microenvironment

Predictive Model Development:
LASSO regression analysis identified CRTAM as having the highest predictive value among nine genes associated with the immunomodulatory (IM) subtype of TNBC . The resulting prediction model showed high accuracy with an AUC of 0.80 (95% CI: 0.74-0.86) in the validation cohort . This suggests CRTAM could be incorporated into multiparameter models for patient stratification.

Mechanistic Rationale:
CRTAM's biological functions provide a mechanistic basis for its predictive value. CRTAM promotes interferon responses and CD8+ T cell infiltration - both essential for effective immunotherapy. Research has indicated that CRTAM can be used as a predictive marker for immune therapy responses, potentially benefiting TNBC patients .

To advance CRTAM as a clinical biomarker, prospective studies specifically correlating CRTAM expression with response to various immunotherapeutic agents (anti-PD-1/PD-L1, anti-CTLA-4, etc.) are needed, ideally with standardized assessment methods and clearly defined response criteria.

How does CRTAM interact with established immune checkpoint pathways?

While the provided search results don't explicitly detail CRTAM's interaction with established checkpoint molecules, several hypotheses can be formulated based on available data:

Potential Complementary Roles:
CRTAM+ CD4+ T cells exhibit enhanced cytotoxic functions and cytokine production , suggesting CRTAM may function as a co-stimulatory rather than inhibitory checkpoint. This raises the possibility that CRTAM activation could complement blocking of inhibitory checkpoints like PD-1 or CTLA-4.

Biomarker Relationships:
CRTAM's potential as a biomarker for immunotherapy response suggests functional relationships with pathways targeted by current immunotherapies. Determining whether CRTAM expression correlates with expression of PD-1, PD-L1, or other checkpoint molecules could provide insights into these relationships.

Signaling Pathway Interactions:
CRTAM induces STAT1 phosphorylation and interferon-stimulated gene expression , potentially intersecting with signaling pathways affected by other checkpoint molecules. Understanding these signaling interactions could inform combination therapy approaches.

Future research should investigate:

• Co-expression patterns between CRTAM and established checkpoint molecules

• Functional consequences of combined CRTAM activation and checkpoint blockade

• Signaling pathway cross-talk between CRTAM and other immune regulators

• Potential for CRTAM-targeted therapies to enhance current immunotherapeutic approaches

What mechanisms drive CRTAM's role in determining CD4+ cytotoxic T lymphocyte differentiation?

CRTAM appears to play a critical role in the differentiation of CD4+ T cells toward a cytotoxic phenotype through several potential mechanisms:

Transcriptional Reprogramming:
CRTAM+ CD4+ T cells express CTL-related genes including Granzyme B, IFN-γ, and Eomesodermin (Eomes) . This suggests CRTAM signaling activates transcriptional programs associated with cytotoxic function, potentially through induction or activation of key transcription factors like Eomes.

Hybrid Identity Development:
Gene expression analysis reveals that CRTAM+ CD4+ T cells maintain CD4+ T cell-related genes while acquiring CD8+ T cell-related genes . Approximately 68% of genes are expressed at comparable levels between CRTAM+ CD4+ T cells and CD8+ T cells . This hybrid transcriptional profile suggests CRTAM may function at a developmental branch point that allows CD4+ T cells to acquire cytotoxic properties while maintaining helper functions.

Causative Role:
Critically, ectopic expression of CRTAM in vivo can induce CD4+ CTL differentiation , providing strong evidence that CRTAM actively drives this developmental pathway rather than simply marking cells with pre-existing cytotoxic potential.

Future research should explore:

• The temporal relationship between CRTAM expression and acquisition of cytotoxic function

• The signaling pathways linking CRTAM engagement to transcriptional changes

• Epigenetic modifications associated with CRTAM-induced cytotoxic programming

• Potential therapeutic approaches to manipulate this pathway in disease contexts

What are the methodological challenges in studying CRTAM's role in human clinical samples?

Investigating CRTAM in clinical contexts presents several methodological challenges that require specialized approaches:

Transient Expression Dynamics:
CRTAM is transiently expressed following T cell activation , making its detection time-sensitive. Researchers must implement:

• Time-course experiments capturing multiple post-activation timepoints

• Preservation techniques that maintain CRTAM detection during sample processing

• Analysis methods that account for transient expression patterns

Low Frequency Population Analysis:
Only 2-5% of activated CD4+ T cells express CRTAM , requiring:

• High-sensitivity detection methods

• Enrichment strategies before analysis

• Single-cell approaches to characterize this rare population

• Sufficient sample sizes to obtain reliable data

Clinical Sample Variability:
Human samples exhibit significant donor-to-donor variability, necessitating:

• Larger cohorts to establish statistically significant patterns

• Stratification based on relevant clinical parameters

• Paired analyses (e.g., tumor vs. adjacent tissue) when possible

• Robust normalization methods for comparative analyses

Functional Assessment Challenges:
Determining CRTAM's functional impact in complex disease environments requires:

• Multiparameter analysis linking CRTAM expression to functional readouts

• Ex vivo functional assays that preserve physiological relevance

• Spatial analysis techniques to understand CRTAM+ cell localization and interactions

• Correlation with clinical outcomes to establish relevance

These methodological considerations are essential for generating reliable, clinically relevant data about CRTAM's role in human disease and its potential as a therapeutic target or biomarker.

How might therapeutic targeting of CRTAM impact anti-tumor immunity?

Given CRTAM's role in enhancing anti-tumor immune responses, several therapeutic strategies merit investigation:

CRTAM Agonism:
Development of agonistic antibodies or recombinant ligands could potentially enhance cytotoxic function in CD4+ T cells and promote anti-tumor immunity. This approach could be particularly valuable in "cold" tumors with limited T cell infiltration.

Expansion of CRTAM+ T Cells:
Ex vivo expansion of CRTAM+ T cells followed by adoptive transfer represents a potential cellular therapy approach. This would leverage the enhanced cytotoxic and cytokine-producing capabilities of these cells .

Combination Approaches:
CRTAM-targeted therapies might synergize with existing immunotherapies:

• Combining CRTAM agonism with immune checkpoint blockade

• Using CRTAM status to guide selection of appropriate immunotherapy

• Sequential approaches that first induce CRTAM expression then target other pathways

Predictive Biomarker Application:
Incorporating CRTAM expression analysis into patient stratification algorithms could improve selection for immunotherapy, particularly in triple-negative breast cancer where CRTAM has shown predictive value .

Research in these areas should carefully assess both efficacy and safety, as enhancing cytotoxic T cell function could potentially exacerbate autoimmune or inflammatory conditions.

What technological innovations would advance CRTAM research?

Several emerging technologies could significantly advance our understanding of CRTAM biology:

Single-Cell Multi-Omics:
Integrating single-cell transcriptomics, proteomics, and epigenomics would provide comprehensive characterization of CRTAM+ cells and their developmental trajectories. This approach could identify regulatory networks controlling CRTAM expression and function.

Advanced Imaging Technologies:
Multiplexed imaging techniques (e.g., Imaging Mass Cytometry, CODEX, Multiplex Immunofluorescence) would enable spatial analysis of CRTAM+ cells within complex tissues, revealing their interactions with other immune and non-immune cells.

CRISPR-Based Functional Genomics:
High-throughput CRISPR screens could identify genes regulating CRTAM expression or mediating its downstream effects. This approach could uncover new therapeutic targets within the CRTAM pathway.

Engineered Organoid Systems:
Development of immune-competent organoid models would provide more physiologically relevant systems for studying CRTAM function in tissue-specific contexts, bridging the gap between simple cell culture and complex in vivo models.

AI-Driven Data Integration:
Machine learning approaches could integrate diverse datasets (genomic, transcriptomic, clinical) to build predictive models of CRTAM function and its relevance to disease outcomes. This could accelerate biomarker development and therapeutic targeting.

These technological advances would address current limitations in CRTAM research and potentially accelerate translation to clinical applications.

How might CRTAM biology inform our understanding of CD4+ T cell plasticity?

The discovery that CRTAM identifies a unique CD4+ T cell population with hybrid CD4+/CD8+ characteristics offers valuable insights into T cell plasticity:

Lineage Reprogramming:
CRTAM+ CD4+ T cells challenge traditional views of helper and cytotoxic T cell lineages as strictly separate entities . Understanding how these cells acquire cytotoxic function while maintaining helper characteristics could reveal fundamental mechanisms of lineage plasticity.

Developmental Branch Points:
The ability of CRTAM expression to induce CD4+ CTL differentiation suggests it marks or creates a developmental branch point. Characterizing the molecular determinants of this branch point could identify other contexts where lineage reprogramming occurs.

Tissue-Specific Adaptation:
CRTAM+ CD4+ T cells are enriched in mucosal tissues and inflammatory sites , suggesting this hybrid phenotype may represent adaptation to specific tissue environments. This could inform broader understanding of how tissue niches shape T cell function.

Therapeutic Implications:
Understanding the plasticity of CD4+ T cells through CRTAM biology could inform development of cellular therapies with enhanced functionality. For example, engineering T cells to express CRTAM might generate more effective anti-tumor responses by combining helper and cytotoxic functions.

Further research into CRTAM-mediated CD4+ T cell reprogramming could fundamentally change our understanding of T cell differentiation and reveal new opportunities for therapeutic manipulation of immune responses.