Recombinant Rat C-C motif chemokine protein (Ccl22), partial (Active)

What is CCL22 and what is its primary immunological role?

CCL22 is a chemokine that plays a critical role in immune regulation by recruiting regulatory T cells (Tregs) to dendritic cells. Dendritic cells in lymph nodes secrete CCL22 to establish cell-cell contacts with CCR4-expressing Tregs, which is essential for immune suppression. CCL22 deficiency results in enhanced T cell immunity, as demonstrated in vaccination, cancer, and inflammatory disease settings .

Which cell types primarily produce CCL22 in rodent models?

Multiple immune cells produce CCL22, with distinct tissue and context dependencies:

• Dendritic cells (DCs) in lymph nodes are major producers, secreting CCL22 to attract Tregs

• M2 macrophages in white adipose tissue prominently express CCL22, particularly during cold exposure

• CCL22 expression is significantly higher in stromal vascular fraction (SVF) cells compared to mature adipocytes in adipose tissue

How does CCL22 signaling pathway regulate immune cell interactions?

CCL22 mediates its effects primarily through the CCR4 receptor expressed on target cells. This signaling axis is particularly important for DC-Treg interactions. In coculture experiments with WT dendritic cells, Tregs outcompete conventional T cells for space on DCs by approximately three-fold. In contrast, when using CCL22-deficient DCs, equal numbers of Tregs and conventional T cells are observed on DC surfaces . The CCL22-CCR4 axis is thus essential for the preferential recruitment of Tregs, influencing downstream immune suppression.

What are effective approaches to study CCL22-mediated Treg recruitment in vitro?

Several validated methods can be employed:

Coculture Assays:

• Mix DCs (either WT or CCL22-deficient) with differentially labeled Tregs and conventional T cells

• Pulse DCs with antigen (e.g., OVA 323-339 peptide) before coculture

• Use fluorescent labeling (e.g., red-labeled Tregs and green-labeled conventional T cells) to visualize competitive interactions

• Quantify the number of each T cell type in contact with DCs

Collagen Gel Migration Assays:

• Use inducible CCL22-expressing cell lines (e.g., DC2.4-CCL22 dox cells)

• Mix cells with T cells in collagen gel with or without CCL22 induction

• Monitor cell-cell contacts through time-lapse microscopy

• Calculate the frequency of DCs in contact with different T cell subsets

Transwell Migration Assays:

• Place CCL22 in the lower chamber at various concentrations (100-1000 ng/ml)

• Add T cell populations in the upper chamber

• Measure migration indices that represent CCL22-induced T cell migration

• Analyze migrated cells by flow cytometry to determine subset enrichment

How can recombinant CCL22 be used effectively in transplantation research?

Recombinant CCL22 has shown promising results in transplantation models through controlled delivery approaches:

Microparticle-Based Delivery System:

• CCL22-releasing microparticles (Recruitment-MP) can be placed at transplantation sites

• In rat hindlimb vascularized composite allotransplantation (VCA) models, this approach:

  • Prolongs allograft survival indefinitely (>200 days)

  • Promotes donor-specific tolerance

  • Enriches Treg populations in allograft skin and draining lymph nodes

  • Enhances Treg function without affecting conventional T cell proliferation

Dosing and Administration:

• Microparticles should release CCL22 in a linear manner over extended periods (~40 days)

• This creates a physiological gradient for effective Treg recruitment

• When scaling to larger animals or humans, careful dose calibration is required (nanogram per kilogram per day range)

What is the protocol for preparing CCL22-releasing microparticles?

The following detailed protocol can be used to formulate PLGA microparticles containing recombinant CCL22:

• Materials Preparation:

  • 200 μl aqueous solution containing:

    • 25 μg recombinant mouse CCL22

    • 2 mg bovine serum albumin

    • 15 mmol NaCl

  • 200 mg PLGA polymer (RG502H) dissolved in 4 ml dichloromethane

• Emulsion Formation:

  • First emulsion: Sonicate the combined solutions for 10 seconds

  • Second emulsion: Homogenize this mixture with 60 ml of 2% polyvinyl alcohol for 60 seconds at 3000 rpm

• Processing:

  • Mix with 1% polyvinyl alcohol and stir for 3 hours to evaporate dichloromethane

  • Collect microparticles and wash four times in deionized water

  • Resuspend in 5 ml deionized water, freeze, and lyophilize for 72 hours

• Characterization:

  • Perform surface analysis using scanning electron microscopy

  • Determine size distribution by volume impedance measurements

  • Verify CCL22 release kinetics by suspending microparticles in PBS at 37°C and quantifying released CCL22 using ELISA

How can researchers quantify functional effects of CCL22 in adipose tissue models?

For studying CCL22's role in adipose tissue biology:

In Vitro Adipocyte Differentiation Assays:

• Isolate stromal vascular fraction (SVF) cells from adipose tissue

• Pretreat cells with recombinant CCL22 (10 ng/ml for 4 days) before differentiation

• Assess beige adipocyte differentiation through:

  • UCP1 mRNA expression analysis

  • Immunofluorescence staining for UCP1 protein

  • Morphological changes (multilocular lipid droplets)

In Vivo Supplementation Studies:

• Administer recombinant CCL22 protein (20 μg/kg per day for 14 days)

• Expose animals to cold conditions to induce beiging

• Analyze adipose tissue using:

  • Histological examination (H&E staining)

  • UCP1 immunofluorescence staining

  • Western blotting for thermogenic proteins

  • qPCR analysis of thermogenic genes

How do researchers distinguish between natural and recombinant CCL22 effects in experimental models?

This requires careful experimental design:

Control Strategies:

• Use CCL22-deficient (Ccl22−/−) models to establish baseline

• Conduct dose-response studies with recombinant protein

• Include vehicle controls in all experiments

• Compare supplementation effects in wild-type versus CCL22-deficient backgrounds

Validation Approaches:

• Confirm that recombinant CCL22 restores phenotypes in CCL22-deficient models

• Verify that effects are abolished in CCR4-deficient models

• Use inducible expression systems (e.g., doxycycline-inducible) to demonstrate direct causality

What factors should be considered when analyzing CCL22-mediated Treg migration?

Several technical considerations affect interpretation:

Concentration-Dependent Effects:

Phenotypic Characterization:

Species Considerations:

• Human and rodent CCL22 may have different potencies

• Both synthetic and recombinant human CCL22 demonstrate comparable T cell-specific chemotactic properties

• Cross-species validation is essential before translational applications

How does CCL22 deficiency impact adaptive immune responses?

CCL22 deficiency has significant effects on immune function:

Enhanced T Cell Immunity:

• Vaccination of CCL22-deficient mice results in stronger T cell responses

• The frequency of antigen-specific and IFN-γ-positive T cells more than doubles in these models

• This suggests CCL22's role as a negative regulator of effector T cell responses

Dendritic Cell Function:

• DCs from CCL22-deficient mice induce substantially stronger T cell immune responses when used for vaccination

• This indicates that CCL22 production by DCs is a key mechanism for limiting excessive T cell activation

• The effect persists even when CCL22-deficient DCs are transferred to wild-type recipients

What are the comparative advantages of synthetic versus recombinant CCL22 for research?

Both forms have distinct characteristics relevant for research applications:

Despite these differences, both synthetic and recombinant human CCL22 exhibit similar T cell-specific chemotactic properties, suggesting either could be used for human applications .

How does CCL22 contribute to adipose tissue thermogenesis?

CCL22 plays an unexpected role in adipose tissue biology:

Cold-Induced Expression:

• Cold exposure markedly increases CCL22 mRNA levels in inguinal white adipose tissue (iWAT)

• This corresponds with increased CCL22 protein secretion throughout cold exposure

• This cold-induced elevation is abolished in lymph node-removed (LNR) mice

Functional Effects:

• Recombinant CCL22 (10 ng/ml) promotes beiging of stromal vascular fraction cells in vitro

• In vivo supplementation (20 μg/kg per day for 14 days) restores cold-induced beiging in LNR mice

• CCL22 supplementation even enhances iWAT beiging in control mice exposed to cold

• These effects appear to be mediated through the CCR4 receptor, as demonstrated in CCR4 knockout models

What are promising translational applications of CCL22-based therapies?

Several potential clinical applications emerge from current research:

Transplantation Tolerance:

• CCL22-releasing microparticles could reduce dependence on systemic immunosuppression

• The approach has shown promise in rat models with implications for scaling to larger animals

• The extremely low doses released locally (nanogram per kilogram per day) suggest favorable safety profiles

Metabolic Disorders:

• CCL22's role in adipose tissue beiging suggests potential applications in metabolic regulation

• Therapeutic manipulation of CCL22 levels could potentially influence energy expenditure and thermogenesis

Autoimmune Diseases:

• Given CCL22's role in promoting Treg function, targeted delivery could help manage autoimmune conditions

• Local administration could potentially avoid systemic immunosuppression complications

What methodological advances would enhance CCL22 research?

Several technical developments would advance the field:

Improved Animal Models:

• Development of cell-type specific CCR4 deletion models (e.g., FoxP3-cre × CCR4-flox mice)

• Creation of reporter systems for tracking CCL22-responsive cells in vivo

• Large animal models with fluorescent or luminescent reporter Tregs for longitudinal imaging

Delivery Systems:

• Optimization of microparticle formulations for specific tissue targeting

• Development of alternative delivery platforms with controllable release profiles

• Integration with existing therapeutic approaches for synergistic effects

Synthetic Chemistry:

• Further refinement of synthetic CCL22 production methods

• Design of CCL22 analogs with enhanced stability or selectivity

• Development of cost-effective production methods suitable for clinical translation