A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells

Area of Science:

• Immunology
• Molecular Biology

Background:

• Pathologic rheumatoid factor (RF) levels are characteristic of various human diseases.
• In vitro production of monoclonal RF is crucial for understanding RF antigenic specificities and aberrant RF+ B cell activation mechanisms.

Purpose of the Study:

• To develop an efficient flow cytometry-based method for cloning monoclonal antibodies (mAbs).
• To clone RF-like B cells expressing the immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment.
• To express cloned variable regions as IgM and assess their RF activity.

Main Methods:

• Utilized flow cytometry to isolate and clone B cells expressing specific IgVH gene segments.
• Expressed cloned variable regions as IgM antibodies in vitro.
• Quantified antibody production, achieving concentrations of 5-20 microg/ml.

Main Results:

• Successfully cloned monoclonal antibodies (mAbs) from human B cells containing the IgVH 1-69 gene segment.
• Demonstrated that cloned IgMs from patients with HCV-related mixed cryoglobulinemia exhibit RF activity.
• Achieved consistent IgM production levels between 5 and 20 microg/ml.

Conclusions:

• The developed flow cytometry method efficiently clones monoclonal antibodies (mAbs) with potential RF activity.
• This system is valuable for studying pathogenic, low-affinity IgMs, particularly those encoded by VH1-69.
• Facilitates research into the mechanisms of autoimmune diseases characterized by RF production.