ITGB8 Antibody

What is ITGB8 and what are its key structural and functional properties?

ITGB8 is the beta 8 subunit of integrin, a membrane protein that forms heterodimers with alpha (α) subunits, particularly αv, to create the functional integrin αvβ8. The canonical ITGB8 protein in humans consists of 769 amino acids with a molecular weight of approximately 85.6 kDa and is typically localized to the cell membrane . As a member of the integrin beta chain protein family, ITGB8 plays crucial roles in integrin binding and extracellular matrix protein interactions, contributing significantly to extracellular matrix organization .

One of its most notable functions is as a key activator of transforming growth factor β (TGF-β), which has been implicated in multiple pathological processes including immunosuppression in cancer and fibrotic diseases . The protein undergoes post-translational modifications, with glycosylation being particularly notable . ITGB8 is also known by the synonym "integrin beta-8" and has orthologs in various species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, making it a conserved protein of evolutionary significance .

In which tissues and cell types is ITGB8 predominantly expressed?

Flow cytometric and qPCR analyses of tumor microenvironments have revealed that CD4+CD25+ T cells, particularly regulatory T cells (Tregs) expressing FoxP3, consistently show the highest levels of ITGB8 expression compared to other cell populations . Importantly, ITGB8 expression is 3-5 fold higher in CD4+CD25+ T cells isolated from tumors compared to those from spleen or tumor-draining lymph nodes, suggesting a tumor-specific upregulation mechanism . Some tumor cell lines (such as CCK168 and TRAMPC2) also express αvβ8 in culture, while others (like EMT6 and CT26) show minimal or undetectable expression . This differential expression pattern has significant implications for both experimental design and therapeutic targeting.

What are the most common research applications for ITGB8 antibodies?

ITGB8 antibodies serve multiple research applications in understanding both normal physiological processes and pathological conditions. Based on the available research, the primary applications include:

• Western blot analysis: For detecting and quantifying ITGB8 protein expression in cell and tissue lysates .

• ELISA: For quantitative measurements of ITGB8 in solution .

• Flow cytometry: Critical for analyzing ITGB8 expression on specific cell populations within complex mixtures, particularly in tumor microenvironment studies .

• Immunohistochemistry (IHC): For visualizing ITGB8 expression patterns in tissue sections, particularly important in tumor and inflammation studies .

• Immunofluorescence: For subcellular localization studies and co-localization with interaction partners .

• Functional blocking studies: Using antibodies like ADWA-11 to inhibit αvβ8-mediated TGF-β activation to study downstream effects on immune responses and disease processes .

When selecting ITGB8 antibodies for these applications, researchers should consider factors such as antibody specificity, clone type (monoclonal vs. polyclonal), and validation data for the specific application of interest .

How do researchers verify ITGB8 antibody specificity and functionality?

Verifying antibody specificity and functionality is crucial for obtaining reliable research results. For ITGB8 antibodies, several methodological approaches have been documented:

To validate specificity, researchers have employed transfection models where cell lines that naturally lack αv integrins (e.g., SW480 cells which only express αvβ5) are transfected to express specific integrin heterodimers such as αvβ8, αvβ3, or αvβ6 . Flow cytometry is then performed using the antibody of interest alongside established antibodies for other integrin heterodimers as controls . This approach effectively distinguishes between cross-reactivity and specific binding.

Functional validation is equally important, particularly for blocking antibodies. The ADWA-11 antibody's functionality, for example, was assessed through:

• Cell adhesion assays using U251 cells (which express αvβ8) on plates coated with TGF-β1 latency associated peptide .

• TGF-β activation assays using a reporter cell line system (TMLC luciferase assay) where mink lung epithelial cells express a TGF-β-sensitive portion of the PAI-1 promoter driving firefly luciferase expression . This assay directly measures the antibody's ability to block αvβ8-mediated TGF-β activation.

• In vivo validation through comparison with genetic models, such as demonstrating that an antibody's effects match those seen in conditional knockout models (e.g., CD4-Cre; Itgb8-f/f mice) .

These methodological approaches provide comprehensive validation of both binding specificity and functional efficacy of ITGB8 antibodies.

What methodological considerations are important when detecting ITGB8 in tumor microenvironments?

Detecting ITGB8 in tumor microenvironments presents unique challenges that require specific methodological considerations:

Flow cytometry detection of ITGB8 in cells disaggregated from in vivo tumors has proven difficult, with researchers noting that antibodies that work well on cultured cells often fail to reliably detect the integrin in ex vivo tumor samples . This suggests possible masking of epitopes, conformational changes, or other modifications in the tumor microenvironment.

To overcome these limitations, researchers have employed alternative approaches:

• Cell sorting followed by qPCR analysis of Itgb8 mRNA expression has been successful in identifying cell populations expressing ITGB8 within tumors . This approach requires careful isolation of distinct cell populations (e.g., CD4+CD25+ T cells, CD8+ T cells, tumor cells) using flow cytometry followed by RNA extraction and quantitative PCR .

• Comparing expression levels between in vitro cultured tumor cells and those isolated from in vivo tumors has revealed substantial differences, with some tumor cell lines showing high expression in vitro but minimal expression when harvested from in vivo tumors . This highlights the importance of not relying solely on in vitro data when studying ITGB8 biology.

• Analyzing ITGB8 expression in different compartments (tumor vs. draining lymph nodes vs. spleen) provides valuable insights into tissue-specific regulation . The observation that ITGB8 expression is 3-5 fold higher in tumor-infiltrating CD4+CD25+ T cells compared to those in peripheral lymphoid organs suggests tumor-specific regulatory mechanisms .

These methodological considerations underscore the importance of using complementary approaches and appropriate controls when studying ITGB8 in complex tumor microenvironments.

How can researchers effectively use ITGB8 blocking antibodies to study TGF-β activation pathways?

ITGB8 blocking antibodies represent powerful tools for studying TGF-β activation pathways, particularly in cancer and fibrosis research. Effective experimental approaches include:

• In vitro co-culture systems: Using reporter cell lines such as TMLC (mink lung epithelial cells expressing a TGF-β sensitive portion of PAI-1 promoter driving firefly luciferase) co-cultured with αvβ8-expressing cells provides a quantitative readout of TGF-β activation . Researchers can assess the potency of blocking antibodies by measuring the reduction in luciferase activity compared to control antibodies.

• Syngeneic tumor models: Studies with the ADWA-11 antibody demonstrated effective blocking of αvβ8 in multiple syngeneic tumor models including squamous cell carcinoma (CCK168), mammary cancer (EMT-6), colon cancer (CT26), and prostate cancer (TRAMPC2) . These models allow assessment of both tumor growth parameters and changes in immune cell populations and functions.

• Combinatorial treatment approaches: Testing αvβ8 blocking antibodies in combination with other immunomodulators (anti-PD-1, anti-CTLA-4, anti-4-1BB) or treatments like radiotherapy provides insights into potential synergistic pathways and mechanisms .

• Cell depletion studies: Combining αvβ8 blocking with selective depletion of immune cell subsets (e.g., CD8+ T cells) helps define which cells are critical for mediating the effects of αvβ8 blockade .

• Gene expression analysis: Analyzing changes in gene expression profiles in tumor-infiltrating lymphocytes following αvβ8 blockade can reveal downstream pathways affected by TGF-β signaling inhibition. This approach has identified increased expression of genes normally suppressed by TGF-β, including Granzyme B and Interferon-γ in CD8+ T cells .

When designing such experiments, researchers should consider including appropriate controls including isotype control antibodies and effectorless versions of blocking antibodies (e.g., ADWA-11_4mut with E233P, E318A, K320A, and R322A mutations in the Fc region) to distinguish between Fc-mediated effects and those specifically resulting from αvβ8 blockade .

How do genetic models of ITGB8 deletion compare with antibody blocking approaches?

Genetic models of ITGB8 deletion provide complementary insights to antibody blocking approaches, offering distinct advantages and limitations that researchers should consider:

Conditional knockout models such as CD4-Cre; Itgb8-f/f mice, where ITGB8 is specifically deleted in T cells, have proven extremely valuable in defining the cellular source of αvβ8 that contributes to tumor growth . Studies have shown that T cell-specific deletion of Itgb8 significantly suppresses tumor growth and enhances survival in syngeneic tumor models, mimicking the effects of systemic ADWA-11 antibody treatment . Importantly, ADWA-11 administration to CD4-Cre; Itgb8-f/f mice conferred no additional benefit, strongly suggesting that T cell-expressed αvβ8 is the primary target through which these antibodies exert their anti-tumor effects .

In contrast, deletion of Itgb8 from dendritic cells (CD11c-Cre; Itgb8-f/f mice) had no effect on tumor growth or survival, helping to rule out dendritic cells as a significant source of immunosuppressive αvβ8 . This demonstrates the power of cell-type specific genetic models in pinpointing the precise cellular origin of a therapeutic target.

For generating antibodies against αvβ8, Itgb8 knockout mice have proven valuable. The ADWA-11 antibody was generated by immunizing Itgb8 knockout mice (crossed into the outbred CD1 background to permit post-natal survival) with recombinant αvβ8 integrin . This approach helps overcome immune tolerance to self-proteins and can generate antibodies with higher affinity and specificity.

Researchers should be aware that complete Itgb8 knockout mice often have developmental abnormalities and perinatal lethality, necessitating the use of conditional knockout approaches for most studies . This contrasts with antibody approaches, which can be applied at specific time points and dosages to avoid developmental complications.

What is the current understanding of ITGB8's role in regulatory T cell function in tumor microenvironments?

The role of ITGB8 in regulatory T cell (Treg) function represents a critical area of immunology research with significant implications for cancer immunotherapy. Current research findings indicate:

CD4+CD25+ T cells, which are predominantly Tregs expressing FoxP3, consistently demonstrate the highest levels of Itgb8 expression among all cell populations in various tumor microenvironments . This expression is significantly higher (3-5 fold) in tumor-infiltrating Tregs compared to Tregs isolated from spleen or tumor-draining lymph nodes, suggesting tumor-specific upregulation mechanisms .

Mechanistically, αvβ8 on Tregs functions as a key activator of latent TGF-β, converting it to its active form which subsequently suppresses anti-tumor immune responses . This activation mechanism involves binding of the αvβ8 integrin to the RGD motif in the latency-associated peptide (LAP) that keeps TGF-β in its inactive form, leading to conformational changes that release active TGF-β .

The activated TGF-β impairs CD8+ T cell effector functions by suppressing expression of genes involved in tumor cell killing, including Granzyme B and Interferon-γ . When αvβ8 is blocked using antibodies like ADWA-11 or deleted genetically from T cells, CD8+ T cells show enhanced expression of these effector molecules and improved anti-tumor activity .

The importance of this pathway is underscored by observations that deletion of Itgb8 specifically from T cells is sufficient to inhibit tumor growth in multiple syngeneic models, and that this inhibition is dependent on CD8+ T cells . These findings point to a specialized role for αvβ8 on Tregs in establishing an immunosuppressive tumor microenvironment through local TGF-β activation.

Researchers investigating this pathway should consider examining both ITGB8 expression levels and the activation status of TGF-β within tumor microenvironments, as well as the resulting effects on different immune cell populations, particularly CD8+ T cells.

What molecular mechanisms explain the differential expression of ITGB8 between tumors and peripheral lymphoid tissues?

The observation that ITGB8 expression is significantly higher in tumor-infiltrating CD4+CD25+ T cells compared to those in peripheral lymphoid organs points to tumor-specific regulatory mechanisms that remain incompletely understood. Based on the available data, several potential mechanisms can be proposed:

Tumor-derived factors, including cytokines, chemokines, and metabolites, likely play important roles in upregulating ITGB8 expression specifically within the tumor microenvironment . The identification of these factors represents an important area for future research, as they could provide additional therapeutic targets.

The hypoxic conditions common in solid tumors may contribute to ITGB8 upregulation, as hypoxia-inducible factors (HIFs) have been implicated in regulating various integrin subunits in other contexts. This hypothesis warrants investigation through in vitro studies comparing ITGB8 expression under normoxic versus hypoxic conditions.

Epigenetic reprogramming of Tregs within the tumor microenvironment represents another potential mechanism. Research in other systems has shown that the tumor microenvironment can induce stable epigenetic changes in infiltrating immune cells, altering their phenotype and function. Studies examining the methylation status of the Itgb8 promoter or enhancer regions in tumor-infiltrating versus peripheral Tregs could provide insights into this possibility.

The differential expression also appears to be selective for ITGB8, as the research indicates this is not a general phenomenon affecting all proteins . This suggests specific regulatory mechanisms rather than global alterations in gene expression or protein synthesis.

Understanding these mechanisms could have significant implications for therapeutic approaches aiming to disrupt the ITGB8-TGF-β axis in cancer, potentially allowing for more targeted interventions that specifically affect the tumor microenvironment while sparing peripheral tissues .

How might ITGB8 targeting differ between cancer types that express versus do not express ITGB8 on tumor cells?

The differential expression of ITGB8 on tumor cells across cancer types raises important questions about optimizing therapeutic approaches for different cancers. The research findings provide several key insights:

Studies with the ADWA-11 antibody have demonstrated efficacy across multiple syngeneic tumor models with varying levels of tumor cell ITGB8 expression . Notably, growth suppression or complete regression was observed in models where tumor cells expressed high levels of αvβ8 (CCK168, TRAMPC2) as well as in models with minimal or undetectable expression (EMT6, CT26) . This indicates that tumor cell expression of ITGB8 is not a prerequisite for response to anti-αvβ8 therapy.

The common denominator across all responsive tumor models appears to be high expression of ITGB8 on CD4+CD25+ T cells within the tumor microenvironment, suggesting that targeting T cell-expressed ITGB8 may be sufficient for therapeutic efficacy regardless of tumor cell expression status . This is further supported by the observation that T cell-specific deletion of Itgb8 recapitulates the effects of systemic antibody treatment .

From a practical standpoint, these findings suggest that patient selection for αvβ8-targeting therapies should not be limited to those with ITGB8-positive tumors. Instead, assessment of ITGB8 expression on tumor-infiltrating lymphocytes, particularly CD4+CD25+ Tregs, might be a more relevant biomarker for predicting response to therapy .

What methodological approaches are most effective for studying ITGB8's role in fibroinflammatory diseases?

Research into ITGB8's role in fibroinflammatory diseases, particularly chronic obstructive pulmonary disease (COPD) and other airway remodeling conditions, requires specialized methodological approaches:

Engineered antibodies optimized for specific targeting of human αvβ8, such as the B5 antibody, have been developed for studying TGF-β activation in fibroinflammatory disease models . These tools are particularly valuable when used in transgenic mouse models expressing human rather than mouse ITGB8, allowing for assessment of human-specific therapeutic approaches .

For studying the role of ITGB8 in airway remodeling, researchers have employed models where airway fibroblasts from COPD patients are compared with those from healthy controls for ITGB8 expression levels and TGF-β activation capacity . This approach has revealed increased expression of αvβ8 in airway fibroblasts in COPD, identifying it as a potential therapeutic target .

Assessment of TGF-β activation in the context of airway inflammation requires specialized assays that can distinguish between latent and active forms of TGF-β. Reporter cell systems, such as the TMLC luciferase assay mentioned earlier, provide quantitative readouts of TGF-β activation that can be correlated with disease severity or treatment effects .

Imaging approaches, including immunohistochemistry and immunofluorescence, are valuable for localizing ITGB8 expression within fibrotic tissues and determining its relationship to areas of active fibrosis and inflammation . These techniques can help establish spatial relationships between ITGB8-expressing cells and pathological features of disease.

To establish causality, intervention studies using either genetic approaches (conditional knockout models) or pharmacological approaches (blocking antibodies) combined with assessments of disease progression provide the strongest evidence for ITGB8's role in pathogenesis . Measurement of fibrosis markers, inflammatory mediators, and lung function parameters before and after intervention helps quantify the impact of ITGB8 blockade on disease outcomes.

What are the most promising combination strategies involving ITGB8 antibodies for cancer immunotherapy?

Research with ITGB8 blocking antibodies has identified several promising combination strategies that enhance therapeutic efficacy in cancer models:

Combination with immune checkpoint inhibitors has shown particular promise. ADWA-11 significantly enhanced the anti-tumor effects of both anti-PD-1 and anti-CTLA-4 blocking antibodies across multiple syngeneic tumor models . This synergy likely stems from complementary mechanisms of action: while checkpoint inhibitors primarily release T cells from inhibitory signals, αvβ8 blockade prevents TGF-β-mediated suppression of T cell effector functions .

Combination with immune activators represents another effective approach. The research demonstrates that ADWA-11 enhances responses to anti-4-1BB agonist antibodies, which directly stimulate T cell activation and expansion . This combination potentially addresses both removal of immunosuppressive signals (via αvβ8 blockade) and provision of positive costimulatory signals (via 4-1BB activation).

Radiotherapy combined with αvβ8 blockade has also shown enhanced efficacy compared to either treatment alone . Radiotherapy can increase tumor antigen release and presentation, potentially creating a more immunogenic tumor microenvironment that, when coupled with relief from TGF-β-mediated immunosuppression through αvβ8 blockade, allows for more effective anti-tumor immune responses.

The mechanistic basis for these synergistic effects appears to involve enhanced CD8+ T cell activity, as depletion of CD8+ T cells abrogated the beneficial effects of combined treatments . Gene expression analysis revealed that αvβ8 blockade increases expression of genes involved in tumor cell killing, including Granzyme B and Interferon-γ, in tumor-infiltrating CD8+ T cells .

When designing combination studies, researchers should consider factors such as timing and sequencing of treatments, potential overlapping toxicities, and mechanistic analyses to understand the cellular and molecular basis of observed synergies. The development of effectorless versions of blocking antibodies (such as ADWA-11_4mut) can help distinguish between Fc-mediated effects and those specifically resulting from αvβ8 blockade in combination settings .