CD96 Human

What is CD96 and what is its expression pattern in human tissues?

CD96 is a member of the immunoglobulin gene superfamily that functions as an immune checkpoint receptor. Expression analysis using the Human Protein Atlas database shows that CD96 mRNA is group-enriched in blood and lymphoid tissues . At the protein level, CD96 shows low expression across various normal tissues, with localization primarily in the cytoplasm and membrane of immune cells .

Specifically, CD96 shows low expression in non-germinal center cells of normal lymph node tissues and white pulp cells in normal spleen tissues . Flow cytometry analysis reveals that CD96 is expressed on T cells and natural killer (NK) cells, where it plays important roles in regulating immune responses .

How does CD96 expression differ between normal and malignant tissues?

CD96 expression is significantly altered in various cancer types compared to adjacent normal tissues. According to TCGA and GTEx analyses, CD96 expression is significantly increased in multiple cancers including adrenocortical carcinoma (ACC), breast cancer (BRCA), glioblastoma (GBM), lower-grade glioma (LGG), and skin cutaneous melanoma (SKCM), among others .

Conversely, CD96 shows decreased expression in lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and thyroid carcinoma (THCA) compared to normal tissues . Immunohistochemistry analysis reveals moderate CD96 expression in breast cancer and melanoma samples (50-75% positivity) . Importantly, CD96 is highly expressed on CD34+CD38- leukemic stem cells (LSCs) in acute myeloid leukemia (AML), making it a potential LSC-specific marker .

What are the current hypotheses regarding CD96 function in human immune cells?

The function of CD96 in human immune cells remains somewhat controversial. While substantial evidence indicates CD96 acts as an inhibitory immune checkpoint, some studies suggest a costimulatory role:

• Inhibitory role: CD96 has been shown to attenuate T cell cytotoxicity. CRISPR/Cas9-mediated deletion of CD96 in human T cells enhanced their killing of leukemia cells in vitro . Additionally, in NK cells, CD96 functions as an inhibitory receptor that suppresses anti-tumor responses .

• Costimulatory role: Contradictory evidence suggests CD96 may act as a costimulatory receptor that activates CD8+ T cells . Cross-linking of CD96 on mouse and human CD8+ T cells induced T cell activation, proliferation, and cytokine production partly via the MEK-ERK pathway .

This dual and seemingly contradictory functionality makes CD96 a complex target for therapeutic development and necessitates careful experimental design when studying its mechanisms.

What methodologies are most effective for studying CD96 function in human T cells?

Several approaches have proven effective for investigating CD96 function:

• CRISPR/Cas9 gene editing: Researchers have successfully used CRISPR/Cas9 technology to delete CD96 in human T cells. Key methodological considerations include:

  • Targeting shared exons (1, 2, or 3) of the CD96 locus that are common to all annotated CD96 transcripts

  • Verification of knockout efficiency using flow cytometry to assess CD96 protein expression

  • Consideration of different guide RNAs, as efficiency varies (e.g., gRNAs AA, AB, and AC showed nearly complete deletion of CD96, while others were less effective)

• Chimeric Antigen Receptor (CAR) T cell models: Incorporating CD96 endodomain into CAR constructs has allowed researchers to isolate and study CD96 signaling. For example:

  • CAR-T cells incorporating CD96 endodomain (4D5-96z) showed attenuated cytotoxic function compared to those with CD3ζ alone (4D5-z)

  • This approach enables direct assessment of CD96 signaling effects without confounding factors

• Flow cytometry analysis: For expression studies, multi-parameter flow cytometry has been effective for characterizing CD96 expression patterns:

  • Common antibody clones include G8.5 and TH-111

  • Combined with markers like CD34, CD38, CD90, and lineage markers for studying stem cell populations

How does CD96 expression correlate with immune infiltration in different cancer types?

CD96 expression shows significant correlations with immune infiltration across cancer types, though these relationships vary:

CD96 expression strongly correlates with recognized immune checkpoints and various immune infiltrates, including CD8+ T cells, dendritic cells, macrophages, monocytes, NK cells, neutrophils, regulatory T cells (Tregs), and follicular helper T cells (Tfh) in most cancers .

Functional correlation analysis revealed that CD96-related genes were involved in negative regulation of leukocytes in LGG, while in SKCM, they participated in multiple positive immune processes . This suggests context-dependent functions that require careful consideration when designing immunotherapeutic approaches.

What is the prognostic significance of CD96 expression across human cancer types?

CD96's prognostic value varies significantly across cancer types:

Poor prognosis associated with high CD96 expression:

• Glioblastoma (GBM): HR = 1.28, 95% CI = 1.04-1.58, P = 0.020

• Lower-grade glioma (LGG): HR = 2.18, 95% CI = 1.79-2.66, P = 1.5e-14

• Uveal melanoma (UVM): HR = 1.33, 95% CI = 1.08-1.63, P = 0.007

Better prognosis associated with high CD96 expression:

• Bladder cancer (BLCA): HR = 0.96, 95% CI = 0.93-0.98, P = 4.3e-4

• Cervical cancer (CESC): HR = 0.94, 95% CI = 0.89-0.99, P = 0.025

• Head and neck cancer (HNSC): HR = 0.95, 95% CI = 0.91-0.98, P = 0.005

• Skin cutaneous melanoma (SKCM): HR = 0.96, 95% CI = 0.94-0.98, P = 9.3e-4

• Thymoma (THYM): HR = 0.95, 95% CI = 0.92-0.99

These contrasting associations suggest that CD96's impact on cancer progression is highly context-dependent, likely influenced by the specific immune microenvironment of different cancer types.

How can researchers effectively target CD96 in immunotherapy development?

Several strategic approaches for targeting CD96 in immunotherapy have emerged from research:

• Monoclonal antibody development: Anti-CD96 monoclonal antibodies have shown efficacy, particularly in combination therapy approaches:

  • Combination with anti-CTLA-4 or anti-PD-1 antibodies enhances efficacy

  • Effectiveness depends on activation of CD226 signaling in NK cells

  • Consider antibody designs that can induce cytotoxicity, such as antibody-dependent cellular cytotoxicity (ADCC), enhanced macrophage phagocytosis, or complement-dependent cytotoxicity

• CD96-targeted CAR-T cell approaches:

  • Understanding CD96 endodomain signaling is crucial, as it has been shown to attenuate T cell cytotoxic function

  • Careful design of CAR constructs to either block CD96 inhibitory signaling or leverage CD96-specific targeting

• AML stem cell targeting:

  • CD96 is expressed on the majority of CD34+CD38- LSC population in over 60% of human primary AML samples

  • CD96+ cells are highly enriched for LSC activity compared to CD96- AML blasts

  • Consider ex vivo purging or FACS selection strategies using CD96 antibodies for autologous transplantation approaches

• Targeting multiple checkpoints:

  • Evidence supports combination tumor immunotherapy targeting multiple rather than single immune checkpoints

  • Research indicates that CD96 signaling inhibits CAR-mediated CD3ζ activation in human T cells

When developing CD96-targeting therapies, researchers must consider CD96 expression in normal T and NK cells, histiocytes, and some non-hematopoietic cells to minimize off-target effects .

What are the technical challenges in distinguishing CD96+ leukemic stem cells from normal hematopoietic stem cells?

CD96 shows promise as a marker to distinguish leukemic stem cells (LSCs) from normal hematopoietic stem cells (HSCs), but several technical challenges must be addressed:

• Expression patterns:

  • Only 4.9 ± 1.6% of cells in the normal HSC-enriched population (Lin-CD34+CD38-CD90+) express CD96

  • In contrast, CD96 is expressed on the majority of CD34+CD38- LSC populations in over 60% of human primary AML samples

  • Expression heterogeneity exists within AML samples, with some showing higher CD96 expression in CD34+ blasts but lower in CD34- blasts

• Flow cytometry considerations:

  • Multiparameter flow cytometry is essential, combining CD96 with stem cell markers (CD34, CD38, CD90) and lineage markers

  • Both antibody clones (G8.5 and TH-111) should be validated for specificity and sensitivity

  • Consider signal amplification methods for detecting low-level expression

• Validation through functional assays:

  • Xenotransplantation experiments have confirmed that CD96+ AML cells are highly enriched for LSC activity compared to CD96- AML blasts

  • Recapitulation of AML heterogeneity after transplantation (CD96+ cells giving rise to both CD96+ and CD96- populations) adds complexity to analysis

• Potential for false negatives:

  • Some AML samples may appear CD96-negative when unsorted leukemia cells are examined, but CD96 positivity may be detected when examining specific subpopulations

These challenges highlight the importance of comprehensive flow cytometry panels and functional validation through xenotransplantation models when studying CD96 as an LSC marker.

How should researchers design experiments to resolve the contradictory roles of CD96 in T cell function?

The apparently contradictory roles of CD96 (inhibitory vs. costimulatory) require careful experimental design:

• Cell type-specific analysis:

  • Separate examination of CD96 function in different T cell subsets (CD4+ vs. CD8+)

  • Consideration of activation state, as CD96 expression increases progressively over six days following T cell activation

  • Analysis of naive vs. memory T cell populations

• Molecular approach diversity:

  • Compare multiple CD96 targeting strategies (gene knockout, antibody blockade, and signaling domain manipulation)

  • CRISPR/Cas9 knockout with multiple guide RNAs targeting different exons

  • Crosslinking experiments to determine if CD96 ligation activates or inhibits T cells

  • Analysis of downstream signaling pathways (e.g., MEK-ERK pathway)

• Functional readouts:

  • Comprehensive assessment of T cell functions:

    • Cytokine production (IFN-γ, TNF-α)

    • Proliferation assays

    • Cytotoxicity against target cells

    • Expression of activation markers (e.g., CD69)

• Context considerations:

  • Evaluation under different cytokine conditions

  • Assessment of the influence of other immune checkpoints (PD-1, CTLA-4)

  • Evaluation of CD96 function in the context of different tumor microenvironments

• In vivo validation:

  • Xenograft models to assess CD96 function in tumor control

  • Analysis of immune infiltration and composition in CD96-manipulated models

What are the most reliable methods for detecting CD96 mutations and their functional impacts?

For comprehensive analysis of CD96 mutations and their functional impacts, researchers should consider:

• Mutation detection approaches:

  • Whole exome or targeted sequencing of CD96 gene

  • Analysis of large cancer genomic databases (TCGA, COSMIC)

  • Special attention to SKCM samples, which demonstrate the highest CD96 mutation frequency among all cancer types

• Functional validation of mutations:

  • Site-directed mutagenesis to introduce specific mutations

  • Expression of mutant CD96 in CD96-knockout cell lines

  • Comparative analysis of wild-type vs. mutant CD96 function:

    • Binding affinity to ligands

    • Effects on immune cell activation

    • Downstream signaling pathway activation

• Structure-function correlation:

  • Analysis of mutation locations in relation to functional domains

  • Prediction of structural impacts using computational modeling

  • Assessment of effects on protein stability, localization, and trafficking

• Clinical correlation:

  • Association of specific CD96 mutations with clinical outcomes

  • Correlation with response to immunotherapy

  • Analysis of mutation co-occurrence with other immune-related genes

By employing these methodological approaches, researchers can better understand how CD96 mutations impact its function and potentially identify mutations that could be exploited therapeutically.

How can CD96 expression be leveraged for improved stratification of leukemia patients?

CD96 expression shows promise for stratifying leukemia patients, particularly those with AML:

• Diagnostic applications:

  • Flow cytometry panels incorporating CD96 alongside established markers (CD34, CD38) can identify LSC-enriched populations

  • CD96 is expressed on the majority of CD34+CD38- LSC population in over 60% of human primary AML samples

• Risk stratification approaches:

  • Quantification of CD96+ LSC frequency at diagnosis may correlate with treatment response

  • Assessment of minimal residual disease (MRD) using CD96 as a marker for persistent LSCs

  • Analysis of CD96 expression patterns in relation to cytogenetic and molecular risk groups

• Treatment selection considerations:

  • Patients with high CD96+ LSC burden might benefit from more intensive consolidation or transplantation

  • Potential for CD96-targeting therapies in patients with high CD96 expression

  • Consideration of CD96 in combination with other LSC markers for improved precision

• Monitoring protocols:

  • Serial assessment of CD96+ LSCs during and after therapy

  • Analysis of CD96 expression changes in response to treatment

  • Correlation of persistent CD96+ LSCs with relapse risk

A key advantage of CD96 as a stratification marker is that it allows distinction between LSCs and normal HSCs, potentially enabling more precise targeting of leukemic cells while sparing normal hematopoiesis .

What approaches should be considered when developing CD96-targeting antibodies for clinical applications?

Development of CD96-targeting antibodies requires careful consideration of multiple factors:

• Antibody design specifications:

  • Selection of antibody format (full IgG, F(ab')2, scFv)

  • Isotype selection to optimize effector functions

  • Engineering for enhanced effector functions (ADCC, CDC, ADCP)

  • Consideration of bispecific formats targeting CD96 and another immune checkpoint

• Epitope selection strategy:

  • Target epitopes that block interaction with ligands

  • Avoid epitopes that might trigger inhibitory signaling

  • Consider epitopes that are preferentially exposed on tumor-infiltrating immune cells or cancer cells

  • Evaluate epitope conservation across species for preclinical model relevance

• Efficacy assessment protocols:

  • In vitro functional assays (T cell activation, NK cell cytotoxicity)

  • Ex vivo testing on primary patient samples

  • Combination testing with established checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Dependency on CD226 signaling should be evaluated

• Safety considerations:

  • Expression profiling across normal tissues to predict off-target effects

  • Attention to CD96 expression on normal T cells, NK cells, and histiocytes

  • Dose-escalation strategy for first-in-human studies

  • Biomarker strategy to identify patients most likely to benefit

Combination approaches targeting multiple immune checkpoints rather than CD96 alone are supported by research data and may provide superior clinical outcomes .

What are the most promising areas for future CD96 research based on current knowledge gaps?

Several promising research directions emerge from current understanding of CD96:

• Resolving functional duality:

  • Molecular mechanisms underlying CD96's seemingly contradictory roles in different contexts

  • Identification of cofactors that determine whether CD96 functions as inhibitory or costimulatory

  • Characterization of CD96 isoforms and their functional differences

• Therapeutic targeting optimization:

  • Development of context-specific CD96 targeting approaches

  • Identification of optimal combination partners for CD96-targeting therapies

  • Discovery of small molecule modulators of CD96 signaling

• Biomarker development:

  • Validation of CD96 as a prognostic marker across cancer types

  • Development of CD96-based companion diagnostics for immunotherapy

  • Integration of CD96 expression data with other immune markers for improved prediction models

• Expanded cancer applications:

  • Investigation of CD96 in additional cancer types beyond those currently studied

  • Analysis of CD96's role in metastasis and therapy resistance

  • Exploration of CD96 in cancer stem cells beyond AML

• Basic biology exploration:

  • Complete characterization of CD96 signaling pathways

  • Identification of additional CD96 ligands beyond known partners

  • Understanding of CD96's role in normal immune development and homeostasis

Addressing these knowledge gaps will enhance understanding of CD96 biology and accelerate development of effective CD96-targeting therapeutic approaches.