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Updated: Aug 8, 2026
Generation of Human CD40-activated B cells
Published on: October 17, 2009
Expression of a 32-kDa ligand for the CD40 antigen on activated human T lymphocytes
P Hermann1, D Blanchard, B de Saint-Vis
1Schering-Plough, Laboratory for Immunological Research, Dardilly, France.
Insights
Researchers identified the CD40 ligand on activated T cells using a CD40-Fc fusion protein. This binding protein is approximately 32 kDa and its detection is enhanced by depleting B cells.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- The CD40 antigen is a crucial co-stimulatory molecule involved in immune responses.
- Identifying the specific ligand for CD40 is essential for understanding T cell activation and B cell interactions.
Purpose of the Study:
- To identify the ligand(s) that bind to the human CD40 antigen.
- To characterize the binding properties and molecular weight of the CD40 ligand.
- To investigate the expression of the CD40 ligand on activated T lymphocytes.
Main Methods:
- Construction of a CD40-Fc fusion protein by combining the extracellular domain of CD40 with the Fc region of human IgG1.
- Binding assays using the CD40-Fc fusion protein to detect interactions with activated T cell clones (CD4+ and CD8+).
- Radioiodination of the CD40-Fc fusion protein for quantitative binding studies and determination of equilibrium dissociation constant (Kd).
- Analysis of protein size using SDS-PAGE and Western blotting.
- Investigation of CD40 ligand expression on primary T cells with and without B cell depletion.
Main Results:
- The CD40-Fc fusion protein specifically bound to activated CD4+ and CD8+ T cell clones.
- The equilibrium dissociation constant (Kd) for the binding of labeled CD40-Fc to an activated CD4+ T cell clone (MT9) was determined to be 10-20 nM.
- The human CD40-binding protein on activated T lymphocytes was identified as a monomeric protein of approximately 32 kDa, with minor components of 29 kDa and 17 kDa.
- A small population of activated CD4+ and CD8+ blood mononuclear T cells expressed the CD40 ligand, with detection being optimal after B cell depletion.
- The presence of B cells inhibited the binding of CD40-Fc to anti-CD3 activated T cells.
Conclusions:
- The study successfully identified and characterized a CD40-binding protein on activated T lymphocytes, representing the CD40 ligand.
- The CD40 ligand is a monomeric protein of approximately 32 kDa expressed on a subset of activated T cells.
- B cells appear to play an inhibitory role in the detection or expression of the CD40 ligand on T cells.
Abstract:
To identify the ligand(s) of the human CD40 antigen, a cDNA encoding the extracellular domain of the CD40 antigen was fused to a cDNA encoding the constant region (Fc) of human IgG1. The CD40-Fc fusion protein was able to specifically bind to CD4+ and various CD8+ T cell clones activated with immobilized anti-CD3. The 125I-labeled CD40-Fc fusion protein bound anti-CD3 activated CD4+ T cell clone (MT9) with an equilibrium dissociation constant (Kd) of 10-20 nM. The human CD40-binding protein expressed on the cell surface of activated T lymphocytes is a monomeric protein of approximately 32 kDa. Minor components of 29 kDa and 17 kDa were also detected. A small proportion of CD4+ and CD8+ blood mononuclear T cells activated by anti-CD3 expressed the CD40 ligand but its detection was best observed following depletion of B cells. Addition of B cells to purified T cells abolished the binding of CD40-Fc obtained after anti-CD3 activation.
