ACL5 Antibody

What is CCL5 and what role does it play in neuroinflammatory diseases?

CCL5 (CC Chemokine Ligand 5) is a chemokine that functions primarily to attract inflammatory cells to sites of inflammation. In the context of neuroinflammatory diseases, CCL5 is localized in white matter tracts undergoing demyelination, where it participates in disease pathogenesis by attracting leukocytes into the CNS. This migration of inflammatory cells results in destruction of white matter and subsequent neurological impairment. Mouse models of viral encephalomyelitis using mouse hepatitis virus (a coronavirus) have demonstrated CCL5's crucial role in mediating the inflammatory response that leads to demyelination similar to multiple sclerosis .

How are neutralizing antibodies against CCL5 developed for research applications?

Development of neutralizing antibodies against CCL5 follows a systematic immunization and selection process. Researchers have successfully created these antibodies by:

• Immunizing BALB/c mice with peptides corresponding to specific CCL5 epitopes (e.g., KKWVQEYINYLEMS)

• Extracting spleens from immunized mice and fusing them with SP2/0 myeloma cells using polyethylene glycol

• Selecting hybridoma cell lines producing anti-CCL5 antibodies via ELISA

• Cloning positive candidates twice by limiting dilution

• Testing clones for recognition of full-length CCL5 protein

• Purifying antibodies from culture supernatant using affinity chromatography on protein G-Sepharose columns

This methodology has yielded antibodies with high specificity, such as clone R6G9 (IgG1 isotype, κ L chain), which shows reactivity to recombinant mouse CCL5 at dilutions up to 1/156,000 via ELISA without cross-reactivity to other chemokines .

What in vitro assays confirm the functional activity of anti-CCL5 antibodies?

Chemotaxis assays provide the most direct evidence of anti-CCL5 antibody functionality. These experiments should be designed to:

• Expose target cells (virus-specific T cells or macrophages) to recombinant CCL5 (rCCL5) at appropriate concentrations (e.g., 100 ng/ml)

• Pre-incubate varying concentrations of anti-CCL5 mAb with rCCL5

• Measure cell migration quantitatively

• Include appropriate controls (isotype-matched antibodies)

Research has demonstrated that pre-incubation of rCCL5 with anti-CCL5 mAb results in significant (p ≤ 0.01) decreases in chemotaxis for both virus-specific CD4+ and CD8+ T cells as well as thioglycolate-elicited macrophages .

What controls are essential when evaluating anti-CCL5 antibody specificity in experimental settings?

When evaluating antibody specificity, researchers should implement these controls:

• Test cross-reactivity against other mouse CC chemokines (e.g., CCL2/monocyte chemoattractant protein-1, CCL3/macrophage-inflammatory protein-1α)

• Evaluate binding to CXC chemokines (e.g., CXCL10/IFN-γ-inducible protein-10, CXCL9/monokine induced by interferon-γ)

• Include isotype-matched control antibodies in all functional assays

• Perform dose-response testing with serial dilutions of the antibody

• Confirm specificity through competitive binding assays

Rigorous testing has shown that antibodies like R6G9 demonstrate no cross-reactivity with other mouse chemokines, confirming their CCL5 specificity .

How effective is anti-CCL5 antibody therapy in treating established demyelinating disease in animal models?

Administration of anti-CCL5 antibody has demonstrated remarkable efficacy in treating established demyelinating disease in mouse hepatitis virus (MHV) models. Key findings include:

• Significant improvement in clinical disease severity beginning just 2 days after initial treatment

• Sustained neurological improvement from day 14 through day 21 post-infection (p ≤ 0.05)

• Significant reduction in demyelination severity (p ≤ 0.005)

• Reduced macrophage accumulation within the CNS

• Continued partial protection even after treatment cessation at day 20

These results demonstrate that targeting CCL5 with neutralizing antibodies can effectively reduce CNS disease severity in a viral model of demyelination.

What quantifiable differences in T cell infiltration are observed following anti-CCL5 antibody treatment?

Anti-CCL5 treatment significantly alters T cell infiltration into the CNS in highly specific ways. Research data shows:

This data reveals an approximately 80% reduction (p ≤ 0.0001) in total CD4+ and CD8+ T cell accumulation by day 21 post-infection, with differential effects on virus-specific T cells: a 73% decrease (p ≤ 0.0002) in M133-147-specific CD4+ T cells and a 60% reduction in S510-518-specific CD8+ T cells .

What differential effects does anti-CCL5 antibody treatment have on various antigen-specific T cell populations?

Anti-CCL5 antibody treatment demonstrates selective effects on different T cell populations based on their antigen specificity:

• M133-147-specific CD4+ T cells show greater sensitivity to CCL5 blockade (73% reduction) compared to S510-518-specific CD8+ T cells (60% reduction)

• The frequency of S510-518-specific CD8+ T cells within the total CD8+ population actually increases approximately 1.6-fold (27.9% vs. 17.2%) following anti-CCL5 treatment

• This suggests differential expression of CCL5 receptors (CCR1 and/or CCR5) among virus-specific T cell subsets

These findings indicate that CCL5 regulates T cell migration into the CNS in part based on antigen specificity, providing important insights for targeted immunotherapy development.

How does anti-CCL5 treatment impact chemokine expression networks in the CNS during chronic viral infection?

Anti-CCL5 antibody treatment affects chemokine expression networks in complex ways:

• Treatment reduces CCL5 mRNA expression during the treatment period

• CXCL10 expression is only slightly reduced compared to control-treated mice (not statistically significant)

• The reduction in CCL5 expression likely results from decreased T cell infiltration, as infiltrating T cells are significant producers of this chemokine

• CXCL10 expression remains relatively unchanged because astrocytes (not infiltrating immune cells) are the primary producers of CXCL10 in MHV-infected mice

• Astrocytic CXCL10 production appears regulated primarily by type I IFN rather than by factors from infiltrating T cells

This differential impact on chemokine expression highlights the complexity of chemokine networks in neuroinflammation and has important implications for combination therapy approaches.

What factors determine optimal dosing regimens for anti-CCL5 antibody in neuroinflammatory disease models?

Optimization of anti-CCL5 antibody dosing regimens should consider:

• Dose: 250 μg of anti-CCL5 mAb has been established as effective, with higher doses showing no additional benefit

• Administration route: Intraperitoneal (i.p.) injection has proven effective

• Frequency: Administration every other day provides sustained therapeutic effect

• Treatment window: Beginning treatment during established disease (day 12 post-infection) still yields significant benefits

• Duration: Continuous treatment until day 20 post-infection provides optimal outcomes

• Post-treatment effects: Clinical benefits persist after treatment cessation, though some disease worsening occurs

These parameters provide a foundation for designing effective therapeutic protocols with anti-CCL5 antibodies.

What methodological approaches can resolve contradictory findings in CCL5 antibody research?

When facing contradictory results in CCL5 antibody research, consider these methodological approaches:

• Evaluate antibody specificity through comprehensive cross-reactivity testing

• Compare multiple anti-CCL5 antibody clones recognizing different epitopes

• Assess potential differences in CCL5 receptor expression across experimental models

• Examine time-dependent effects by sampling multiple timepoints during and after treatment

• Analyze both frequency and absolute numbers of cellular populations to avoid misinterpretation

• Consider the impact of the specific disease model (acute vs. chronic, viral vs. autoimmune) on outcomes

These approaches can help reconcile seemingly contradictory findings and advance understanding of CCL5's role in neuroinflammation.