Research Article

Development of an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay

Louisa Green

Oxford University Hospitals NHS Trust, Churchill Hospital, Old Road, Headington, Oxford OX3 7LJ, UK


13 Jun 2014


6 Nov 2014


17 Nov 2014






PHA, anti-CD2/CD3/CD28 beads, T cell, proliferation, diagnosis, immunodeficiency


Severe combined immunodeficiency (SCID) has many phenotypes, including those which result in the formation of T cells but render them incapable of an optimal level of proliferation following stimulation by an antigen. Phytohaemagglutinin (PHA) is a mitogen commonly used to assess T-cell proliferation, although anti-CD2/CD3/CD28-coated beads may provide a more physiological trigger. Some patients and healthy controls display poor T-cell proliferation (<45%) in response to PHA, but may be capable of an increased percentage proliferation when stimulated by another mitogen. This study aimed to develop an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay and to compare T-cell proliferation generated by two mitogens both in healthy controls and in poor responders to PHA. A bead-based T-cell proliferation assay was optimized and validated. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood samples of 10 healthy controls and 3 poor PHA responders. PBMCs were incubated with carboxyfluorescin diacetate succinimidyl ester and three separate stimulants: PHA, Beads only and Beads plus IL-2. Flow cytometry was performed on Day 6, following fluorochrome staining with CD3 PerCP and CD4 APC, to identify CD4+ T cells. No significant difference was found between the results generated by PHA- or Bead stimulation in 10 healthy controls. A strong positive correlation was observed between the use of the Beads alone and the addition of IL-2. A varied response to the Beads was produced among the poor PHA responders. It has been concluded that the bead-based assay will be run alongside every PHA test in the Oxford Immunology diagnostic laboratory. The step to add IL-2 on Day 3 will not be included as this did not significantly impact T-cell proliferation. The Bead assay provides a more physiological addition to the current PHA test and may be useful in assessing T-cell proliferation in those patients who respond poorly to PHA. However, some individuals who respond poorly to PHA may also respond poorly to the anti-CD2/CD3/CD28 Beads.

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