TMIGD2 Antibody

What is TMIGD2 and why is it significant for immunological research?

TMIGD2 (Transmembrane and Immunoglobulin Domain-containing protein 2) is a novel adhesion molecule also known as immunoglobulin-containing and proline-rich receptor 1 (IGPR1). The protein has significant research importance due to its role in cell-cell interactions, cell migration, and angiogenesis. In humans, the canonical protein has 282 amino acid residues with a mass of 30.7 kDa and is primarily localized in the cell membrane. TMIGD2 is notably expressed across multiple tissues, including the colon, stomach, and cerebellum, making it an important target for various immunological investigations .

How does TMIGD2 function at the cellular and molecular level?

At the cellular level, TMIGD2 functions as an adhesion molecule that regulates multiple important processes. It modulates cellular morphology and promotes homophilic cell aggregation, facilitating cell-cell interactions critical for tissue integrity. TMIGD2 activity influences actin stress fiber formation and focal adhesion, suggesting its involvement in cytoskeletal organization and cellular structure maintenance .

On a molecular level, TMIGD2 undergoes post-translational modifications, notably N-glycosylation, which likely affects its function and interaction capabilities. The protein contains transmembrane and immunoglobulin domains, explaining its role in immune cell recognition and communication. Current research suggests that TMIGD2 expression can negatively correlate with angiogenesis, hypoxia, G2/M checkpoint activities, and epithelial-to-mesenchymal transition signaling pathways . This negative association with these processes—many of which are critical in tumor progression—provides insight into why TMIGD2 expression may correlate with improved prognosis in certain cancers.

What are the common synonyms and related proteins in the TMIGD2 family?

When conducting literature searches or designing experiments involving TMIGD2, researchers should be aware of its various synonyms and related proteins to ensure comprehensive coverage:

Protein Aliases:

• CD28 homolog/CD28 homologue

• IGPR-1 (Immunoglobulin and proline-rich receptor 1)

• Immunoglobulin-containing and proline-rich receptor 1

• Transmembrane and immunoglobulin domain-containing protein 2

• Transmembrane and immunoglobulin domain-containing protein 2 variant 2/3

Gene Aliases:

• CD28H

• IGPR-1/IGPR1

• TMIGD2

• UNQ3059/PRO9879

What are the optimal applications for different TMIGD2 antibody formats?

TMIGD2 antibodies are versatile research tools applicable across multiple experimental platforms. The optimal application depends on the specific antibody format and experimental question:

Flow Cytometry: This is the most common application for TMIGD2 antibodies, particularly useful for identifying and quantifying TMIGD2-expressing cells in heterogeneous populations. Unconjugated antibodies require secondary antibody labeling, while directly conjugated antibodies streamline the protocol. Flow cytometry is particularly valuable when identifying Human Group 3 Innate Lymphoid Cells using TMIGD2 as a marker .

Western Blot (WB): Useful for determining TMIGD2 protein expression levels and molecular weight confirmation. This application typically requires unconjugated primary antibodies and is ideal for evaluating protein expression changes in different experimental conditions .

Immunohistochemistry (IHC): Particularly IHC-p (paraffin-embedded samples) allows visualization of TMIGD2 distribution in tissue contexts, providing insights into its localization patterns. This application is crucial for studying TMIGD2 expression in clinical samples and correlating expression with pathological features .

ELISA: Enables quantitative measurement of TMIGD2 in solution, useful for serum or cell culture supernatant analysis. This application provides precise quantification when studying secreted or soluble forms of TMIGD2 .

Researchers should select antibody formats based on their specific experimental needs, considering factors such as sensitivity requirements, sample type, and whether qualitative or quantitative data is needed.

How can researchers validate TMIGD2 antibody specificity for their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For TMIGD2 antibodies, researchers should implement a multi-faceted validation approach:

• Positive and negative control samples: Utilize tissues or cell lines with known TMIGD2 expression profiles. Based on available data, colon, stomach, and cerebellum tissues express TMIGD2 and can serve as positive controls. Comparison with samples from knockout models or siRNA-treated cells provides definitive negative controls.

• Western blot analysis: Verify that the antibody detects a band at the expected molecular weight (approximately 30.7 kDa for the canonical form), while being mindful that post-translational modifications like N-glycosylation may affect observed molecular weight .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide (where available, such as the peptide sequence: CQVDQATAWE RLRVKWTKDG AILCQPYITN GSLSLGVCGP QGRLSWQAPS HLTLQLDPVS LNHSGAYVCW AAVEIPELEE AEGNITRLFV DPDDPTQNRN RIA) to confirm that this abolishes specific staining .

• Cross-reactivity testing: When working with non-human samples, consider the limited ortholog sequence identity (23% for mouse and rat) which may affect antibody recognition .

• Comparison of multiple antibody clones: Using antibodies recognizing different epitopes of TMIGD2 helps confirm specific detection of the same protein.

These validation steps should be documented in experimental methods to establish confidence in the specificity of observed TMIGD2 signals.

What are the key considerations for optimizing immunohistochemical detection of TMIGD2?

Optimizing immunohistochemical detection of TMIGD2 requires careful attention to several technical aspects:

• Fixation and antigen retrieval: TMIGD2 is membrane-localized, so fixation methods that preserve membrane integrity while allowing antibody access are critical. Test both formaldehyde-fixed, paraffin-embedded (FFPE) and frozen section protocols to determine optimal preservation of TMIGD2 epitopes. For FFPE samples, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is often a good starting point.

• Antibody concentration and incubation conditions: Titrate antibody concentrations to determine optimal signal-to-noise ratio. Extended incubation periods (overnight at 4°C) may improve detection of lower-expression samples.

• Detection system selection: Amplification systems (such as polymer-based detection or tyramide signal amplification) may be necessary for detecting low-abundance TMIGD2, particularly in clinical samples with variable expression levels.

• Counterstaining considerations: Given TMIGD2's membrane localization, select counterstains that allow clear visualization of cellular boundaries and don't obscure membrane staining patterns.

• Multiplex staining optimization: When co-staining for TMIGD2 alongside other immune markers, carefully select antibody combinations that don't cross-react and detection systems that can be clearly distinguished .

When studying glioma samples specifically, consider the heterogeneity of TMIGD2 expression observed in different glioma subtypes, with higher expression reported in astrocytoma and IDH-1 mutated tumors .

How does TMIGD2 expression correlate with clinical outcomes in gliomas?

Recent research on TMIGD2 in gliomas has revealed significant correlations between expression levels and clinical outcomes:

These findings highlight TMIGD2's potential value as both a prognostic biomarker and possible therapeutic target in glioma management, though these observations contrast with findings in some other cancer types where TMIGD2 expression has been associated with poorer outcomes.

What is the relationship between TMIGD2 expression and immune cell infiltration in tumor microenvironments?

The relationship between TMIGD2 expression and immune cell infiltration in tumor microenvironments reveals complex immunological interactions:

These observations suggest that TMIGD2 may play a role in shaping the immune landscape within tumors, potentially promoting anti-tumor immune responses in gliomas. Understanding these relationships could inform immunotherapeutic strategies targeting or leveraging TMIGD2.

What are the proposed mechanisms by which TMIGD2 affects tumor progression in gliomas?

Based on current research, several mechanisms have been proposed for how TMIGD2 may influence tumor progression in gliomas:

• Negative regulation of angiogenesis: TMIGD2 expression negatively correlates with angiogenesis signaling pathways. This relationship is significant as angiogenesis is a critical process for tumor growth and progression. The anti-angiogenic effect may contribute to limiting tumor expansion and invasiveness .

• Inhibition of epithelial-to-mesenchymal transition (EMT): High TMIGD2 expression negatively correlates with EMT signaling pathways. As EMT is associated with increased invasiveness and metastatic potential, TMIGD2's inhibitory effect on this process may reduce tumor aggressiveness .

• Modulation of hypoxia response: TMIGD2 expression inversely correlates with hypoxia pathways. Since hypoxia drives numerous processes favoring tumor survival and progression, this negative regulation may impair tumor adaptation to low oxygen environments .

• Cell cycle regulation: TMIGD2 expression negatively correlates with G2/M checkpoint pathways, potentially influencing cell proliferation rates .

• Immune microenvironment modulation: As detailed in the previous question, TMIGD2 expression associates with increased infiltration of anti-tumor immune cells and decreased presence of immunosuppressive cells. This immune profile modification could enhance host anti-tumor responses .

• Cell adhesion effects: As an adhesion molecule, TMIGD2 regulates cellular morphology and cell-cell interactions, potentially affecting tumor cell migration and invasion capabilities .

How can TMIGD2 antibodies be utilized in therapeutic development strategies?

The potential therapeutic applications of TMIGD2 antibodies emerge from recent research findings and present several strategic approaches:

• Combination therapy development: Research suggests that combining anti-VEGF treatment with selective activation of naive T cells in patients with high TMIGD2 expression could enhance anti-tumor responses and improve prognosis in glioma patients. This approach addresses both angiogenesis (which standard bevacizumab treatment alone failed to adequately control) and immune activation pathways .

• Patient stratification biomarker: TMIGD2 expression levels could serve as a biomarker for patient selection in clinical trials, identifying those most likely to benefit from immunotherapy approaches. The significant correlation between TMIGD2 expression and survival suggests its utility in creating more homogeneous treatment groups .

• Immune checkpoint modulation: The negative correlation between TMIGD2 and inhibitory immune checkpoints (PDL-1, B7-H3, B7-H6) suggests potential complementary or synergistic effects when combining TMIGD2-targeted therapies with established checkpoint inhibitors .

• Antibody-drug conjugates (ADCs): Given TMIGD2's expression on tumor cells in glioma patients, it could potentially serve as a target for ADCs that would deliver cytotoxic payloads specifically to TMIGD2-expressing cells.

• NK cell activation: Research using B7H7+ tumor cells has demonstrated that TMIGD2 can function as an activator of NK cells, suggesting the potential development of TMIGD2 agonist antibodies to enhance anti-tumor NK cell responses .

These approaches represent promising directions for therapeutic development, though each would require rigorous preclinical validation before clinical translation.

What methodological challenges exist when studying TMIGD2 across different species models?

Researchers investigating TMIGD2 across species models face several significant methodological challenges:

• Limited sequence homology: The relatively low sequence identity between human TMIGD2 and rodent orthologs (only 23% identity to mouse and rat) presents a major challenge for antibody cross-reactivity. Antibodies raised against human TMIGD2 may not recognize mouse or rat orthologs with sufficient specificity or sensitivity .

• Expression pattern differences: While TMIGD2 orthologs have been reported in bovine, chimpanzee, and chicken species, the expression patterns and functional conservation across species remain incompletely characterized. This knowledge gap complicates the translation of findings between species .

• Functional conservation uncertainty: Given the limited sequence homology, the degree to which TMIGD2 functions are conserved across species is uncertain. Processes like cell adhesion, migration, and immune cell interactions may vary between humans and common laboratory animal models.

• Model selection challenges: For researchers studying TMIGD2 in the context of specific diseases like glioma, developing appropriate animal models that recapitulate the relevant TMIGD2 biology becomes difficult due to these interspecies differences.

• Antibody validation requirements: Researchers must perform extensive validation when using TMIGD2 antibodies across species, including positive and negative controls specific to each species and comparison of staining/detection patterns with known expression data.

To address these challenges, researchers might consider species-specific antibodies, humanized mouse models, or in vitro systems using cells of the relevant species expressing either native TMIGD2 or engineered constructs with tags to facilitate detection.

How can conflicting data on TMIGD2's role in different cancer types be reconciled?

The seemingly contradictory findings regarding TMIGD2's role across different cancer types present an intriguing research question requiring careful consideration:

• Context-dependent functions: TMIGD2 appears to have divergent roles depending on the cancer type. In gliomas, high TMIGD2 expression correlates with better prognosis, while in gastric cancer and oral squamous cell carcinoma, its expression has been associated with poorer outcomes. This suggests that TMIGD2's function is highly context-dependent and influenced by the specific tumor microenvironment .

• Methodological reconciliation approaches:

  • Comprehensive pathway analysis: Comparing the signaling pathways and molecular interactions of TMIGD2 across different cancer types may reveal how the same molecule produces opposite effects in different contexts.

  • Isoform-specific studies: Investigating whether different cancer types express distinct TMIGD2 isoforms that may have different or even opposing functions.

  • Ligand-dependent effects: Examining whether TMIGD2 interacts with different binding partners in different cancer types, potentially activating distinct downstream pathways.

  • Immune context analysis: Given TMIGD2's relationship with immune cell infiltration, differences in baseline immune environments between cancer types may explain the divergent outcomes.

• Integrated multi-omics approach: Combining transcriptomic, proteomic, and immunophenotyping data across cancer types could help identify the molecular basis for these seemingly contradictory roles. This approach might reveal cancer-specific co-expression patterns or regulatory mechanisms that modify TMIGD2's function.

• Temporal considerations: Investigating whether TMIGD2's role changes during cancer progression might explain some contradictions, as studies often represent different disease stages.

Understanding these context-dependent functions of TMIGD2 is crucial for developing targeted therapeutic approaches and predicting potential side effects in different tissues.

What criteria should guide the selection of TMIGD2 antibodies for specific research applications?

Selecting appropriate TMIGD2 antibodies requires evaluation of several key criteria based on the intended application:

• Application-specific validation: Choose antibodies specifically validated for your intended application (Flow Cytometry, Western Blot, IHC, or ELISA). For instance, flow cytometry applications may benefit from directly conjugated antibodies, while Western blot applications require antibodies that perform well under denaturing conditions .

• Epitope considerations:

  • For detection of specific isoforms (up to 2 different isoforms have been reported for TMIGD2), select antibodies targeting epitopes unique to those isoforms.

  • For detection across species, evaluate the conservation of the epitope sequence in target species (noting the limited 23% homology between human and rodent orthologs) .

  • Consider whether the epitope might be masked by post-translational modifications, particularly N-glycosylation which has been reported for TMIGD2 .

• Clonality selection:

  • Monoclonal antibodies offer high specificity and reproducibility for well-defined epitopes.

  • Polyclonal antibodies (such as the rabbit polyclonal antibodies available from several suppliers) may provide greater sensitivity by recognizing multiple epitopes, especially useful when protein expression is low .

• Reactivity profile: Verify the antibody has been validated against human TMIGD2 if studying human samples, and be cautious about cross-reactivity claims for other species given the limited sequence homology .

• Format compatibility: Consider whether unconjugated formats (requiring secondary detection) or directly conjugated antibodies better suit your experimental workflow and multiplexing needs .

Thorough evaluation of these criteria will help ensure selection of antibodies that provide reliable, reproducible results for specific TMIGD2 research applications.

How do post-translational modifications affect TMIGD2 antibody binding and detection?

Post-translational modifications (PTMs) of TMIGD2, particularly N-glycosylation, can significantly impact antibody binding and detection in multiple ways:

• Epitope masking: N-glycosylation can physically block antibody access to protein epitopes, particularly if glycosylation sites are located within or adjacent to the antibody recognition sequence. This may result in false-negative results despite TMIGD2 being present in the sample .

• Altered molecular weight: Glycosylation adds significant mass to the protein, causing TMIGD2 to migrate at an apparent molecular weight higher than the predicted 30.7 kDa in gel electrophoresis. Researchers should be aware of this when interpreting Western blot results, as the observed band may appear larger than expected .

• Sample preparation considerations: Deglycosylation treatments (such as PNGase F for N-linked glycans) prior to analysis may be necessary for certain applications, particularly when using antibodies whose epitopes might be masked by glycosylation.

• Tissue-specific glycosylation patterns: The extent and pattern of TMIGD2 glycosylation may vary between tissues or disease states, potentially resulting in variable antibody detection efficiency across different sample types.

• Other potential PTMs: While N-glycosylation is specifically mentioned in the literature, other potential modifications like phosphorylation (particularly given TMIGD2's role in signaling) could also affect antibody recognition and should be considered when unexpected results occur .

When selecting antibodies for TMIGD2 detection, researchers should review available information about the epitope location relative to known or predicted PTM sites, and consider validation using both native and deglycosylated samples to fully understand detection capabilities.

What advanced multiplexing strategies can be employed for studying TMIGD2 in complex immune cell populations?

Advanced multiplexing approaches offer powerful tools for studying TMIGD2 in heterogeneous immune populations, providing contextual information about expression patterns and cellular interactions:

• Multiparameter flow cytometry strategies:

  • Combine TMIGD2 antibodies with lineage markers for detailed immune cell phenotyping

  • Use fluorochrome combinations optimized for minimal spectral overlap

  • Include functional markers (activation, exhaustion, proliferation) alongside TMIGD2 to correlate expression with cellular states

  • Consider spectral cytometry for expanded parameter detection beyond conventional flow cytometry limitations

• Mass cytometry (CyTOF) applications:

  • Metal-labeled TMIGD2 antibodies can be integrated into CyTOF panels allowing simultaneous detection of 40+ parameters

  • This approach is particularly valuable for comprehensive immune profiling in limited clinical samples

  • Can reveal rare TMIGD2-expressing cell populations within complex immune landscapes

• Multiplex immunohistochemistry/immunofluorescence:

  • Sequential multiplex IHC allows co-staining of TMIGD2 with multiple markers on the same tissue section

  • Spectral imaging and unmixing technologies can distinguish closely overlapping fluorophores

  • Spatial analysis of TMIGD2-expressing cells relative to other immune populations provides valuable microenvironmental context

  • Particularly relevant given TMIGD2's differential association with various immune cell infiltrates in gliomas

• Single-cell sequencing integration:

  • Index sorting approaches can link TMIGD2 protein expression data from flow cytometry with subsequent single-cell RNA sequencing

  • This allows correlation of TMIGD2 protein levels with comprehensive transcriptional profiles

  • Can help identify novel markers co-expressed with TMIGD2 for improved phenotyping

• Computational analysis workflows:

  • Dimensionality reduction techniques (tSNE, UMAP) and clustering algorithms help visualize TMIGD2 expression patterns across immune populations

  • Trajectory analysis can reveal relationships between TMIGD2 expression and cellular differentiation states

These advanced approaches enable comprehensive characterization of TMIGD2's expression and function across diverse immune populations in both healthy and disease contexts.