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Measurement of CD40 ligand (CD154) expression on resting and in vitro-activated T cells
1Northwestern University Medical School, Children's Memorial Hospital, Chicago, Illinois, USA.
Insights
Measuring CD40 ligand (CD154) expression on activated T cells aids in diagnosing X-linked hyper-IgM syndrome (XHIM). This method also helps investigate T cell costimulatory function abnormalities.
Area of Science:
- Immunology
- Cell Biology
Background:
- CD40 ligand (CD154) is crucial for T cell-mediated immune responses.
- Normal resting lymphocytes lack significant CD154 expression, requiring in vitro activation for measurement.
- X-linked hyper-IgM syndrome (XHIM) is characterized by defects in CD40-CD40L interactions.
Purpose of the Study:
- To detail a method for measuring CD154 expression on activated T lymphocytes.
- To provide a diagnostic tool for X-linked hyper-IgM syndrome (XHIM).
- To facilitate the investigation of T cell costimulatory function abnormalities.
Main Methods:
- In vitro activation of peripheral blood lymphocytes.
- Three-color monoclonal antibody staining for lymphocyte subset identification.
- Flow cytometry for listmode data acquisition.
- Gating and analysis protocols for CD154 expression assessment.
Main Results:
- The described protocol enables reliable measurement of CD154 expression on activated T cells.
- This assay serves as a valuable diagnostic tool for XHIM.
- The method allows for the characterization of T cell costimulatory defects.
Conclusions:
- The in vitro measurement of CD154 expression is essential for diagnosing XHIM.
- This standardized methodology supports the investigation of immune dysfunctions.
- Accurate T cell costimulatory function assessment is critical for immunological research.
Abstract:
Measurement of the ability of in vitro activated T lymphocytes to express CD40 ligand (CD154) is a valuable tool for diagnosis of the X-linked hyper-IgM syndrome (XHIM) and for investigating abnormalities in the costimulatory functions of T lymphocytes. Resting peripheral blood lymphocytes do not normally express any appreciable levels of CD154, necessitating in vitro activation. This unit details the in vitro activation procedure, the three-color monoclonal antibody panels used to label the appropriate lymphocyte subsets, the controls required to interpret the assay, the setup and acquisition of listmode data files, and the gating and analysis protocols used to interpret the data.
