Domain-specific CD6 monoclonal antibodies identify CD6 isoforms generated by alternative-splicing

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• CD6 glycoprotein is a transmembrane receptor crucial for T-cell function.
• Characterizing CD6 structure and interactions is vital for understanding T-cell regulation.
• Monoclonal antibodies (mAbs) have aided in studying CD6's scavenger receptor cysteine-rich (SRCR) domains.

Purpose of the Study:

• To definitively characterize the CD6 glycoprotein's ectodomain architecture and extracellular interactions.
• To identify specific binding sites for CD6 mAbs within its SRCR domains.
• To investigate the functional consequences of alternative splicing on CD6 isoforms and CD166 binding.

Main Methods:

• Utilized engineered CD6 isoforms, both membrane-bound and soluble, lacking specific SRCR domains.
• Employed CD6 mAbs (OX125, OX126, OX124) to probe domain recognition.
• Analyzed CD6 isoform expression at mRNA and protein levels following T-cell activation.

Main Results:

• Conclusively demonstrated that CD6 mAbs OX125 and OX126 recognize CD6 domain 2.
• Confirmed that CD6 mAb OX124 targets CD6 domain 3 and blocks CD166 interaction.
• Identified and confirmed the expression of the CD6Δd3 isoform, lacking the CD166-binding domain, after T-cell activation.

Conclusions:

• Established precise mapping of CD6 mAbs to specific SRCR domains, enhancing CD6 characterization.
• Provided definitive evidence for CD6 domain 2 recognition and CD6 domain 3's role in CD166 binding.
• Confirmed the prevalence of the CD6Δd3 isoform post-T-cell activation, impacting CD166 interactions.