Recombinant Human C-C motif chemokine 1 (CCL1), partial  (Active)

What is Recombinant Human C-C Motif Chemokine 1 (CCL1)?

Recombinant Human C-C Motif Chemokine 1 (CCL1) is a chemokine protein typically produced in E. coli expression systems, where the target gene encoding amino acids Lys24-Lys96 is expressed . CCL1 functions as a crucial immunoregulatory molecule that binds to the CCR8 receptor, primarily expressed on regulatory T cells (Tregs) . This chemokine plays a significant role in immune regulation through various mechanisms including potentiation of Treg cell activity, induction of suppressive markers, and regulation of autoimmune responses. The recombinant form allows researchers to study these functions in controlled experimental settings without the variability associated with naturally derived proteins.

How does CCL1 interact with its receptor CCR8?

CCL1 binds specifically to the CCR8 receptor expressed on regulatory T cells, initiating signaling cascades that enhance immune regulatory functions. When CCL1 binds to CCR8, it induces calcium flux through this receptor, which appears to be a unique capability among CCR8 ligands . This interaction initiates downstream signaling that includes ERK1/2 phosphorylation, as demonstrated in studies using the BW5147 CCR8+ thymoma cell line . The interaction between CCL1 and CCR8 establishes an autocrine regulatory loop where CCL1 not only activates the receptor but also upregulates CCR8 expression itself, thereby amplifying the signaling pathway. This positive feedback mechanism enhances the potency of regulatory T cells in suppressing immune responses.

What experimental approaches are used to produce and validate recombinant CCL1?

Recombinant human CCL1 is typically produced using E. coli expression systems where the gene encoding amino acids Lys24-Lys96 is expressed . This approach allows for consistent production of the active protein fragment. Validation of recombinant CCL1 involves multiple methodological steps:

• Structural validation through techniques such as mass spectrometry and circular dichroism

• Functional validation through:

  • Calcium flux assays to confirm receptor activation

  • Chemotaxis assays to verify chemoattractant properties

  • ERK1/2 phosphorylation assays to confirm downstream signaling

  • T cell functional assays to demonstrate biological activity

For long-term stability and in vivo applications, researchers have developed fusion proteins such as CCL1-Ig, which extends the half-life while maintaining biological activity. These constructs retain chemoattraction capabilities and ERK1/2 phosphorylation induction while providing improved pharmacokinetic properties .

How does CCL1 potentiate regulatory T cell function?

CCL1 significantly enhances the suppressive capacity of regulatory T cells through multiple mechanisms. Research demonstrates that CCL1 treatment of human Treg cells (CD4+CD25+CD127low) results in:

• Upregulation of critical regulatory markers:

  • 4-5 fold increase in FOXp3 and CCR8 transcription (p<0.0001)

  • 3.7-fold increase in CD39 transcription

  • 2.5-fold increase in granzyme B and IL-10 transcription (p<0.01)

• Protein level confirmation showed:

  • CCR8 expression increased from 2.65% to 36% of Treg cells

  • FOXp3 expression increased from 89.6% to 95.7% (p<0.01)

  • CD39 expression increased from 4.43% to 16.1% (p<0.0001)

  • Granzyme B expression increased from 3.25% to 18.3% (p<0.0001)

  • IL-10 expression increased from 6.26% to 15.4% (p<0.0001)

This potentiation occurs through direct enhancement of existing Treg cells rather than conversion of non-regulatory T cells, as studies showed no compelling evidence that CCL1 converts FOXp3- T cells into FOXp3+ cells . The multi-faceted enhancement of suppressive markers suggests that CCL1 operates through several complementary pathways to boost Treg function.

What is the role of the CCL1-CCR8 axis in autoimmune disease models?

The CCL1-CCR8 axis plays a crucial role in regulating autoimmune responses, particularly evident in experimental autoimmune encephalomyelitis (EAE) models. Research has revealed:

• CCL1 levels in the central nervous system (CNS) significantly increase after peak disease (up to 14-fold increase on day 22)

• FOXp3+ Treg cells are the principal source of CCL1 in the inflamed CNS, with 13.8-fold higher transcription compared to FOXp3- cells

• CCR8+FOXp3+ T cells at the autoimmune site preferentially express:

  • Granzyme B (32.5% vs. 8.9% in CCR8- cells)

  • CD39 (44% vs. 11.5% in CCR8- cells)

  • IL-10 (11.3% vs. 5.3% in CCR8- cells)

• Administration of CCL1-Ig fusion protein during ongoing EAE:

  • Suppressed disease progression (day 21 mean maximal score of 1±0.13 compared with 2.5±0.23, p<0.01)

  • Reduced histological scores (0.5±0.1 compared with 2.6±0.3 in control groups, p<0.01)

  • Increased the relative number of FOXp3+ Treg cells both in periphery and CNS

  • Enhanced expression of CCR8, CD39, granzyme B, and IL-10 in Treg cells

This evidence suggests an autocrine regulatory loop where Treg cells produce CCL1 that acts back on CCR8 receptors to enhance their own suppressive function at autoimmune sites, providing a potential therapeutic approach for autoimmune conditions.

What experimental models are most appropriate for studying CCL1 function?

When designing experiments to study CCL1 function, researchers should consider several model systems based on the specific research question:

• In vitro models:

  • Primary human or mouse Treg cell cultures for studying direct effects on regulatory function

  • Cell lines expressing CCR8 (e.g., BW5147 CCR8+ thymoma) for mechanistic studies of receptor signaling

  • Mixed lymphocyte reaction (MLR) assays to assess suppressive capacity

• In vivo models:

  • Experimental autoimmune encephalomyelitis (EAE) for studying neuroinflammatory conditions

  • Adoptive transfer models using wild-type and knockout mice to assess specific mechanisms

  • BrdU incorporation assays to study proliferative responses of T cell subsets

• Genetic models:

  • CCR8 knockout mice to confirm receptor-specific effects

  • IL-10 knockout mice to dissect cytokine-dependent mechanisms

  • FOXp3-GFP reporter mice for tracking Treg cells in vivo

Each model system offers distinct advantages for addressing different aspects of CCL1 biology. For comprehensive understanding, a combination of in vitro mechanistic studies followed by in vivo validation provides the most robust approach to characterizing CCL1 function in immune regulation.

How should researchers measure CCL1-mediated effects on T cell populations?

To accurately assess CCL1-mediated effects on T cells, researchers should employ a multi-parameter approach:

• Flow cytometric analysis of key markers:

  • Surface markers: CCR8, CD25, CD39, CD127

  • Intracellular markers: FOXp3, granzyme B, IL-10

• Functional assays:

  • Suppression assays to measure Treg inhibitory capacity

  • Calcium flux assays to assess receptor activation

  • BrdU incorporation to measure proliferation

  • Cytokine production profiles using ELISA or intracellular cytokine staining

• Molecular analyses:

  • Real-time PCR for gene expression (normalized to housekeeping genes like β2M)

  • Phosphorylation assays for signaling pathway activation (e.g., ERK1/2)

  • ChIP assays to assess transcription factor binding at relevant gene promoters

• In vivo tracking:

  • Adoptive transfer experiments with labeled cells

  • In situ analysis of tissue infiltration and marker expression

  • Assessment of disease parameters in experimental models

This comprehensive approach allows researchers to correlate phenotypic changes with functional outcomes and mechanistic insights, providing a complete picture of CCL1's effects on T cell populations.

What are the limitations of working with recombinant CCL1 in experimental systems?

Working with recombinant CCL1 presents several technical challenges that researchers should consider:

• Short half-life in vivo:

  • Native chemokines have limited exposure following systemic administration

  • Solution: Development of fusion proteins (e.g., CCL1-Ig) that extend half-life while maintaining biological activity

• Protein stability and storage:

  • Recombinant proteins may lose activity during storage or experimental handling

  • Solution: Appropriate buffer formulations and storage conditions (-80°C for long-term stability)

• Dose-response variability:

  • Different experimental systems may require different optimal concentrations

  • Solution: Careful titration studies to determine effective concentrations for each system

• Specificity confirmation:

  • Ensuring effects are CCR8-dependent rather than off-target

  • Solution: Parallel experiments with CCR8 knockout cells or blocking antibodies

• Translating in vitro findings to in vivo context:

  • In vitro potency may not directly translate to in vivo efficacy

  • Solution: Pharmacokinetic and pharmacodynamic studies to establish appropriate dosing for in vivo models

Understanding these limitations and implementing appropriate solutions ensures more reliable and reproducible research outcomes when working with recombinant CCL1.

How can researchers distinguish CCL1-specific effects from general chemokine activities?

Distinguishing CCL1-specific effects from general chemokine activities requires methodological rigor:

• Receptor specificity controls:

  • Parallel experiments with CCR8 knockout systems

  • Competitive binding assays with known CCR8 ligands

  • Use of receptor-specific blocking antibodies

• Comparative studies with other chemokines:

  • Include chemokines that activate different receptors as controls

  • Compare calcium flux, signaling pathways, and functional outcomes

• Structure-function relationship studies:

  • Use mutated versions of CCL1 that maintain structure but alter function

  • Test CCL1 fragments or domains for specific activities

• Cell type specificity:

  • Compare responses between CCR8+ and CCR8- cell populations

  • Assess effects on different CCR8-expressing cell types (Tregs vs. Th2 cells vs. macrophages)

• Mechanistic dissection:

  • Use pathway inhibitors to block specific downstream signaling events

  • Assess dependency on specific transcription factors or effector molecules

By implementing these controlled experimental approaches, researchers can confidently attribute observed effects specifically to CCL1-CCR8 interactions rather than to general chemokine properties.

How does CCL1 compare to other immune modulators in therapeutic potential?

CCL1 demonstrates distinct properties that differentiate it from other immune modulators:

• Cell-type specificity:

  • CCL1 primarily acts on CCR8+ cells, which includes Tregs and Th2 cells

  • Unlike broad immunosuppressants, this specificity may reduce off-target effects

• Multi-faceted enhancement of regulatory function:

  • Simultaneously increases multiple suppressive mechanisms (FOXp3, CD39, granzyme B, IL-10)

  • This multi-pronged approach may provide more robust regulation than single-pathway modulators

• Autocrine regulation:

  • Creates a positive feedback loop where Tregs produce CCL1 to enhance their own function

  • This self-sustaining regulation may provide more durable effects than exogenous modulators

• Comparison with other approaches:

• Context-dependency:

  • Studies show CCL1 can effectively suppress ongoing autoimmunity in EAE models

  • Effects may be independent of IL-10, suggesting multiple mechanisms of action

The unique properties of CCL1 suggest potential therapeutic applications particularly in autoimmune conditions where targeted enhancement of regulatory mechanisms is desired rather than global immunosuppression.

What are the most pressing research questions regarding CCL1 in disease contexts?

Several critical research questions about CCL1 require further investigation:

• Tissue-specific regulation:

  • How does CCL1 function differ across tissue environments?

  • Is CCL1 production regulated differently in various inflammatory contexts?

• Disease relevance beyond EAE:

  • What is the role of the CCL1-CCR8 axis in other autoimmune conditions?

  • How does CCL1 contribute to immune dysregulation in allergic diseases and DRESS syndrome?

• Integration with other regulatory pathways:

  • How does CCL1 signaling interact with other Treg-enhancing pathways?

  • Can CCL1 restore regulatory function in dysfunctional Tregs?

• Therapeutic translation:

  • What formulations could overcome the pharmacokinetic limitations of CCL1?

  • How can CCL1-based therapies be targeted to specific tissues?

• Biomarker potential:

  • Can CCL1 or CCR8 expression serve as biomarkers for disease activity or treatment response?

  • Does serum CCL1 correlate with regulatory T cell function in patients?

• Mechanistic details:

  • What transcriptional networks are activated by CCL1-CCR8 signaling?

  • How does CCL1 differentially regulate distinct Treg subpopulations?

Addressing these questions will provide deeper insights into CCL1 biology and potentially lead to novel therapeutic approaches for immune-mediated diseases.

What are optimal protocols for assessing CCL1 activity in vitro?

To effectively assess CCL1 activity in vitro, researchers should implement the following optimized protocols:

• CCR8 receptor activation assays:

  • Calcium flux measurement: Load CCR8+ cells with Fluo-4 AM calcium indicator, establish baseline, add CCL1, and monitor fluorescence changes over time

  • ERK1/2 phosphorylation: Treat CCR8+ cells with CCL1 for 5-15 minutes, lyse cells, and detect phosphorylated ERK1/2 by Western blot or flow cytometry

• Treg potentiation assays:

  • Isolate CD4+CD25+CD127low Treg cells from peripheral blood using magnetic sorting

  • Culture with anti-CD3/CD28 stimulation ± CCL1 for 36-72 hours

  • Assess marker expression (FOXp3, CCR8, CD39, granzyme B, IL-10) by flow cytometry and qPCR

• Functional suppression assay:

  • Isolate Tregs and responder T cells (CD4+CD25-)

  • Pretreat Tregs with CCL1 for 24-48 hours

  • Co-culture at various Treg:responder ratios with stimulation

  • Measure responder proliferation using CFSE dilution or 3H-thymidine incorporation

• Chemotaxis assay:

  • Use transwell chambers with CCL1 in the lower chamber

  • Add CCR8+ cells to upper chamber and incubate for 2-4 hours

  • Count migrated cells to determine chemotactic response

These protocols should include appropriate controls, including CCR8- cells, isotype controls for flow cytometry, and vehicle controls for treatments to ensure reliable interpretation of results.

How can researchers effectively quantify CCL1 and CCR8 expression in experimental samples?

Accurate quantification of CCL1 and CCR8 expression is critical for experimental interpretation. Researchers should consider these methodological approaches:

• mRNA quantification:

  • Real-time quantitative PCR using gene-specific primers

  • Normalize to stable reference genes (e.g., β2M, GAPDH)

  • Express as fold-change relative to appropriate controls

  • Consider digital PCR for absolute quantification in low-abundance samples

• Protein quantification:

  • ELISA for soluble CCL1 in supernatants or biological fluids

  • Flow cytometry for CCR8 cell surface expression

  • Western blot for total protein levels in cell lysates

  • Immunohistochemistry for tissue localization

• Single-cell approaches:

  • Flow cytometry with fluorochrome-conjugated antibodies for population-level analysis

  • RNA-seq for transcriptomic profiling

  • Mass cytometry for simultaneous assessment of multiple parameters

• In situ detection:

  • Immunofluorescence microscopy for tissue localization

  • RNA in situ hybridization for mRNA detection in tissue sections

  • Multiplexed imaging for co-localization studies

• Reporting guidelines for quantification:

Following these guidelines ensures reproducible and comparable quantification across different experimental contexts.