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Molecular and biological characterization of human 4-1BB and its ligand
M R Alderson1, C A Smith, T W Tough
1Department of Cellular Immunology, Immunex Research and Development Corporation, Seattle, WA 98101.
Abstract:
4-1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4-1BB (mu4-1BB-L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4-1BB in the immune response we undertook to clone the human homologue of 4-1BB-L. However, attempts to isolate a cDNA encoding the human 4-1BB-L by cross-hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein consisting of the extracellular portion of hu4-1BB coupled to the Fc region of human immunoglobulin G1 (hu4-1BB.Fc) was then used to identify and clone a gene for human 4-1BB-L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4-1BB-L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high-affinity binding of hu4-1BB.Fc to either native or recombinant human 4-1BB-L. Both monoclonal antibody to hu4-1BB and cells transfected with hu4-1BB-L induced a strong proliferative response in mitogen co-stimulated primary T cells. In contrast, ligation of 4-1BB on T cell clones enhanced activation-induced cell death when triggered by engagement of the TCR/CD3 complex.
Insights
Researchers identified and cloned the human 4-1BB ligand (4-1BBL), crucial for T cell immune responses. This discovery advances understanding of T cell activation and regulation, impacting immune system research.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- 4-1BB is a T cell receptor protein involved in immune responses.
- The murine ligand for 4-1BB (4-1BBL) was previously identified.
- The role of 4-1BB in the immune response necessitates understanding its human counterpart.
Purpose of the Study:
- To clone the human homologue of 4-1BB ligand (4-1BBL).
- To characterize the interaction between human 4-1BB (hu4-1BB) and hu4-1BBL.
- To investigate the functional consequences of 4-1BB/4-1BBL interaction on T cells.
Main Methods:
- Cross-species hybridization was used to isolate the human 4-1BB (hu4-1BB) cDNA.
- A fusion protein (hu4-1BB.Fc) was generated for ligand identification.
- Direct expression cloning identified the human 4-1BB ligand (hu4-1BBL) from activated T cells.
- Scatchard analysis assessed binding affinity.
- Functional assays evaluated T cell proliferation and activation-induced cell death.
Main Results:
- Human 4-1BB ligand (hu4-1BBL) was successfully cloned and shares 36% amino acid identity with its murine counterpart.
- High-affinity binding was confirmed between hu4-1BB and hu4-1BBL.
- Engagement of 4-1BB with 4-1BBL stimulated T cell proliferation.
- Ligation of 4-1BB enhanced T cell death upon TCR/CD3 engagement.
Conclusions:
- The human 4-1BB/4-1BBL pathway plays a significant role in modulating T cell responses.
- This pathway influences both T cell activation and programmed cell death.
- Understanding this interaction is key for developing immunotherapies.