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

green, louisa;

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 beadbased 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.