Recombinant Mouse C-C chemokine receptor type 10 (Ccr10)

What is the basic structure and function of mouse CCR10?

Mouse CCR10 is a G protein-coupled receptor member of the CC chemokine subfamily. The protein is 362 amino acids in length with a predicted molecular weight of 38 kDa. Mouse and rat CCR10 share 88% amino acid sequence identity with the human protein. CCR10 functions primarily through binding to its ligands (notably CCL27 and CCL28), which activates G protein-mediated signaling cascades, including the Akt and NFκB pathways. This activation leads to cytoskeleton rearrangement and chemotactic cell migration, which is critical for proper immune cell homing and positioning .

Where is CCR10 predominantly expressed in mice, and how does this compare to humans?

In mice, CCR10 is predominantly expressed on IgA-producing plasma cells in mucosal tissues, particularly the intestine and lactating mammary gland. It is also found on specific subsets of skin-homing T cells. CCR10 expression is often low or absent on resting peripheral T cells but can be modulated during activation. In comparing mouse and human expression patterns, both species show similar tissue-specific expression profiles, though there are some differences in regulation that researchers should consider when extrapolating from mouse models to human applications .

What are the primary ligands for mouse CCR10 and their expression patterns?

The primary ligands for mouse CCR10 are CCL27 (CTACK) and CCL28 (MEC). CCL27 is predominantly expressed in skin keratinocytes and is involved in the recruitment of CCR10+ cells to the skin. CCL28 is expressed at mucosal sites, particularly in the intestine and lactating mammary gland, where it mediates the recruitment of IgA-secreting plasma cells. Research has shown that CCL28 expression is higher in certain tumor tissues compared to normal tissues, which has implications for cancer immunology research .

What methods are available for detecting CCR10 expression in mouse tissues and cells?

Several methods can be used to detect CCR10 expression:

• Flow cytometry: Using specific anti-CCR10 antibodies such as rat monoclonal antibody (clone 248918) conjugated to fluorophores like PerCP. This approach allows for quantitative assessment of CCR10 expression at the single-cell level .

• RT-PCR: Using specific primers (e.g., forward primer 5′-cattcacaccaaaggggtctt-3′ and reverse primer 5′-agcctctgtggctgtacctc-3′) to amplify CCR10 mRNA. The amplification profile typically involves 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by a 10-min extension at 72°C .

• Reporter mouse models: Transgenic mice in which the CCR10 gene has been replaced with fluorescent reporters like EGFP allow for direct visualization of CCR10 expression without antibodies .

• Western blotting: For protein-level detection in tissue or cell lysates.

When selecting a detection method, researchers should consider that surface CCR10 expression may be affected by enzymatic tissue digestion protocols, which can cleave the extracellular portions of the receptor .

How can I generate and validate CCR10-knockout mice for functional studies?

CCR10-knockout mice can be generated through targeted disruption of the first ATG codon of CCR10 by insertion of reporter/selection markers such as EGFP/NeoR. A detailed approach involves:

• Design of targeting construct: Create a construct where the first ATG codon of CCR10 exon I is mutated into a restriction site (e.g., EcoRV) via PCR-directed mutagenesis.

• Reporter insertion: Insert a reporter gene (such as EGFP) along with a selection marker (such as NeoR) at the mutated ATG site.

• ES cell targeting: Introduce the linearized targeting vector into embryonic stem cells via electroporation (typically ~10^7 cells, 16 μg targeting vector, 400V at 25 μF).

• Selection and verification: Select positive clones using appropriate antibiotics, and confirm homologous recombination by Southern blot analysis.

• Blastocyst injection: Inject confirmed ES cells into blastocysts to generate chimeric mice.

• Breeding: Cross chimeric mice with wild-type mice to achieve germline transmission.

• Genotyping: Confirm knockout by PCR and/or Southern blot analysis of tail genomic DNA .

Validation should include functional assays to confirm the absence of CCR10-mediated responses, such as chemotaxis assays using CCL28 as a chemoattractant.

What recombinant expression systems are most effective for producing functional mouse CCR10 protein?

Several expression systems have been utilized to produce functional mouse CCR10:

• Mammalian cell lines: Transient or stable transfection of HEK293 or CHO cells with CCR10 expression vectors provides properly folded and post-translationally modified receptor, though yields may be lower than other systems.

• Lentiviral expression systems: Using vectors like FUGW lentiviral vector for stable integration and expression in target cells. This approach is particularly useful for engineering T cells to express CCR10 .

• E. coli: While bacterial expression systems can produce high yields of protein, they often require refolding to achieve functionality and lack appropriate post-translational modifications.

For optimal results in functional studies, mammalian expression systems are generally preferred despite lower yields. When designing constructs, consider including purification tags that can be cleaved post-purification to avoid interference with receptor function .

How does CCR10 regulate intestinal IgA responses in mice, and what are the implications of CCR10 deficiency?

CCR10 plays a critical role in regulating intestinal IgA responses through several mechanisms:

• Plasma cell positioning: CCR10 is essential for positioning IgA-producing plasma cells in the intestinal lamina propria, allowing for efficient secretion of IgA into the intestinal lumen.

• IgA memory maintenance: CCR10 is required for the long-term maintenance of IgA-producing plasma cells and IgA+ memory B cells in the intestine following pathogen exposure.

Studies with CCR10-knockout mice have revealed complex consequences:

These findings highlight CCR10's essential role in intestinal immune memory maintenance rather than in primary IgA responses .

What is the significance of CCR10 in lactating mammary gland IgA production?

The lactating mammary gland model has been particularly valuable for studying CCR10 function because:

• IgA antibody-secreting cells (ASCs) in the mammary gland lack expression of CCR3 (another receptor for CCL28), making CCR10 the sole receptor mediating their recruitment via CCL28.

• Studies using CCR10-deficient mice have demonstrated that CCR10-mediated interactions are indispensable for efficient accumulation of high levels of IgA ASCs in the lactating mammary gland.

• This model provides a clean system to study CCR10-specific effects without confounding factors from other chemokine receptors.

The significance extends to understanding maternal antibody transfer to offspring, as IgA in breast milk provides critical passive immunity to neonates. CCR10's role in this process makes it a potential target for strategies to enhance passive immunity transfer through breast milk .

How do CCR10 expression dynamics change during inflammation and immune cell activation?

CCR10 expression is dynamically regulated during inflammation and immune cell activation:

• T cell activation: In activated human T cells, CCR10 expression shows a biphasic pattern, with an initial increase on day 4 post-activation, followed by a subsequent decrease. This temporal regulation is important when designing experiments with activated T cells .

• Monocyte-to-macrophage differentiation: As monocytes differentiate into macrophages or dendritic cells, their chemokine receptor expression profile changes. While CCR2 expression predominates in Ly6C^hi monocytes, differentiation toward macrophages is accompanied by upregulation of CCR1 and CCR5 with concurrent downregulation of CCR2. This pattern has been elegantly demonstrated using inflammatory chemokine receptor reporter (iCCR-REP) mice .

• Tissue inflammation: During inflammation, tissue-specific patterns of chemokine upregulation influence the recruitment of CCR10+ cells. For example, in inflammation-driven hepatocarcinogenesis, there is significant upregulation of the CCR10 ligand CCL28, which can recruit CCR10+ cells to the liver .

Understanding these dynamics is crucial for designing experiments targeting appropriate time points and for interpreting results in inflammatory disease models .

How can CCR10 be exploited to enhance T cell trafficking in adoptive cell therapy?

CCR10 has emerged as a promising candidate for improving T cell trafficking in adoptive cell therapy, particularly for targeting solid tumors. The strategy involves:

• Identifying suitable targets: Analysis of chemokine expression in tumor tissues compared to normal tissues has identified CCL28 (the CCR10 ligand) as highly expressed in several cancer types, including breast cancer and lung carcinomas, while being minimally expressed in normal tissues.

• Engineering approach: Since activated T cells typically express low levels of CCR10, genetic engineering using viral vectors (particularly lentiviral vectors) can be employed to overexpress CCR10 in therapeutic T cells. This approach provides more stable, long-term expression compared to transient methods like mRNA electroporation.

• Functional improvements: CCR10-engineered T cells demonstrate significantly enhanced migration toward CCL28 gradients. In experimental models, even 25% CCR10 receptor expression resulted in approximately 3.8-4.4 times higher chemotaxis index when exposed to human recombinant CCL28.

• Integration with other therapeutic modalities: CCR10 can be co-expressed with tumor-targeting receptors (such as TCRs or CARs) to create dual-function therapeutic cells that both recognize tumor antigens and migrate efficiently to tumor sites.

This approach addresses one of the major challenges in solid tumor immunotherapy: insufficient trafficking of therapeutic cells to tumor sites .

What role does CCR10 play in inflammation-driven hepatocarcinogenesis, and how might this inform cancer research?

Research has uncovered significant connections between CCR10 and inflammation-driven hepatocellular carcinoma (HCC):

• Upregulation in inflammatory contexts: CCR10 is significantly upregulated in hepatocytes isolated from inflammation-driven human HCC tumors and matching paracancerous tissues. This upregulation is also observed in experimental models of inflammatory hepatocarcinogenesis using tetrachloromethane (CCl4) or diethylnitrosamine (DEN).

• Signaling pathway: TNF stimulation increases CCR10 expression in hepatocytes, leading to enhanced Akt phosphorylation, PCNA expression, and hepatocellular proliferation. Importantly, TNF also increases secretion of CCL28, the natural CCR10 ligand-agonist, creating a potential autocrine/paracrine signaling loop.

• Functional impact: CCR10 knockout in DEN-treated mice significantly increases hepatocellular apoptosis and reduces compensatory proliferation, resulting in lower liver tumor incidence and smaller tumors. Mechanistically, this occurs through modulation of Akt phosphorylation pathways.

These findings suggest CCR10 as a potential therapeutic target in inflammation-associated liver cancers, with CCR10 antagonists potentially serving as adjuvant treatments. The research also highlights the value of studying chemokine receptors in non-immune cells within the tumor microenvironment .

How can reporter mouse models advance our understanding of chemokine receptor expression dynamics?

Innovative reporter mouse models, such as the inflammatory chemokine receptor reporter (iCCR-REP) mice, represent a transformational advance in studying chemokine receptor biology:

• Simultaneous tracking of multiple receptors: These models express spectrally distinct fluorescent reporters for multiple chemokine receptors (e.g., CCR1, CCR2, CCR3, and CCR5) within a single animal. The reporters used (mTagBFP2, Clover, mRuby2, and iRFP682) have discrete excitation and emission spectra, allowing clear discrimination.

• Advantages over antibody-based detection:

  • Reporter expression remains detectable after enzymatic tissue digestion, which often cleaves receptor extracellular domains

  • Eliminates background non-specific staining issues seen with antibodies due to homology between different chemokine receptors

  • Enables detection of receptors for which reliable antibodies are unavailable

• Application to complex biological questions: These models have revealed that chemokine receptor expression is highly specific and more selective than previously anticipated, challenging the notion of redundancy in the chemokine system.

• Experimental design opportunities: Researchers can now analyze individual and combinatorial receptor expression patterns on myeloid cells in both resting and inflamed conditions, offering unprecedented insights into the spatial and temporal dynamics of receptor expression.

These models can address fundamental questions about how chemokines orchestrate inflammatory responses and may help identify more precise therapeutic targets in inflammatory diseases .

What are the common challenges in detecting native CCR10 expression, and how can they be overcome?

Researchers frequently encounter several challenges when detecting native CCR10 expression:

• Low baseline expression: CCR10 is often expressed at low levels in resting cells, making detection difficult.

  • Solution: Use more sensitive detection methods such as qRT-PCR or RNAscope for mRNA detection, or high-sensitivity flow cytometry with signal amplification.

• Receptor internalization: Chemokine receptors rapidly internalize upon ligand binding, potentially giving false negative results.

  • Solution: Ensure cells are not exposed to ligands before staining, and consider using intracellular staining protocols to detect internalized receptors.

• Poor antibody specificity: Commercial antibodies may show cross-reactivity with other chemokine receptors due to homology.

  • Solution: Always include appropriate negative controls (ideally cells from CCR10-knockout mice) to establish specific staining. Consider using reporter mice or reporter-tagged constructs when available .

• Enzymatic digestion artifacts: Tissue digestion protocols commonly cleave the extracellular portions of chemokine receptors.

  • Solution: Optimize tissue digestion protocols to minimize receptor damage, or utilize genetic reporter systems that are unaffected by extracellular proteolysis .

• Dynamic regulation: CCR10 expression varies with activation state and differentiation.

  • Solution: Carefully time experiments and perform kinetic analyses rather than single time-point measurements .

What are the best practices for reconstituting and handling recombinant mouse CCR10 protein?

Optimal handling of recombinant mouse CCR10 protein requires attention to several factors:

• Reconstitution protocols:

  • For lyophilized preparations with carrier protein (BSA): Reconstitute at 25 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin.

  • For carrier-free (CF) preparations: Reconstitute at 100 μg/mL in sterile PBS.

• Storage conditions:

  • Store reconstituted protein at -20°C to -80°C in single-use aliquots.

  • Use a manual defrost freezer and avoid repeated freeze-thaw cycles, which significantly decrease protein activity.

  • For short-term use (less than 1 week), store at 2-8°C.

• Working solution preparation:

  • Dilute in buffers containing carrier protein (0.1-1% BSA or serum) to prevent adhesion to tubes and loss of activity.

  • For cell culture applications, ensure sterile filtration after dilution.

• Stability considerations:

  • CCR10 is relatively unstable compared to many other proteins; activity may decline even with proper storage.

  • Consider including protease inhibitors in working solutions to prevent degradation.

  • Always perform functional validation (e.g., chemotaxis assays) before critical experiments .

How can contradictory findings in CCR10 research be reconciled and addressed methodologically?

Contradictory findings in CCR10 research can arise from multiple sources and require systematic approaches to resolve:

When addressing contradictions specifically for CCR10, it's important to recognize that its functions are often context-dependent. For example, the apparently normal intestinal IgA levels in CCR10-knockout mice under homeostatic conditions (which contradicted expected results) were later explained by identifying compensatory mechanisms involving enhanced generation of IgA+ cells in isolated lymphoid follicles .