Biochemical separation of a human B cell mitogenic factor

Area of Science:

• Immunology
• Cell Biology
• Biochemistry

Background:

• Human B lymphocytes can enter cell cycle and synthesize DNA when exposed to factors from lectin-stimulated mononuclear cells.
• Isolation and characterization of these B cell-stimulating factors are crucial for understanding lymphocyte activation.

Purpose of the Study:

• To develop procedures for isolating B cell growth-stimulating cytokines from lectin-stimulated mononuclear cell conditioned media.
• To partially separate B cell and T cell stimulatory factors and characterize their molecular weights.

Main Methods:

• Fractionation of conditioned media using ammonium sulfate precipitation, ion-exchange chromatography (DEAE-Sephadex), and gel filtration chromatography (Bio-Gel P-30 and P-100).
• Assay of proliferation-stimulating activity on purified human T cells, B cells, and murine thymocytes throughout the isolation process.
• Analysis of protein bands using analytic polyacrylamide gel electrophoresis (PAGE) on radiolabeled fractions.

Main Results:

• T cell and B cell stimulatory factors co-purified through initial fractionation steps.
• Partial separation of T and B cell activities was achieved using Bio-Gel P-100 gel filtration.
• PAGE identified distinct protein bands: 14-15 kDa for T cell growth and 12-13 kDa for B cell growth.
• B cell mitogenic fractions induced S-phase entry without detectable IgM secretion.

Conclusions:

• Distinct molecular weight proteins are responsible for T and B cell proliferation stimulation.
• A 12-13 kDa protein fraction was identified as a key B cell mitogenic factor.
• This factor induces B cell cycle progression independent of significant IgM production.