In vitro T lymphocyte function in primary immunodeficiency diseases

Abstract

Diagnosis of primary immunodeficiency diseases (PID), based on laboratory tests and assessment of T lymphocyte function, is crucial in patients who present with lymphopenia. We evaluated T lymphocyte function in healthy children and adults and in patients and with PID using flow cytometry. Whole blood cultures were stimulated with phytohemagglutinin, purified protein derivate (PPD) and candidin, followed by detection of intracellular interferon-gamma (IFN - gamma) and CD25 membrane expression on CD3+ T cells by flow cytometry. Flow cytometry results were compared with 3H-thymidine (3HTdR) lymproliferation after in vitro cell stimulation and with delayed type hypersensitivity reaction (DTH). Patients with PID had lower intracellular IFN - gamma production than healthy children and healthy adults after PHA stimulation for 18 h (p = 0.024 and p < 0.0001, respectively); CD25 expression was also lower in patients with PID than in healthy children and adults after candidin stimulation (p = 0.048 and p < 0.0001, respectively). CD25 expression after PPD and candidin stimulation were also higher in healthy adults when compared with both patients and with healthy children (p < 0.0001 for all comparisons). Lymphoproliferation assay with 3HTdR after candidin stimulation did not show any significant difference between healthy children and patients with PID, but the response was higher in healthy adults (p = 0.029). The DTH for PPD was not different between PID and healthy children (p = 0.281). Intracellular IFN-gamma after PHA stimulation for 18 h and CD25 membrane expression after candidin stimulation for 72 h on CD3+ T cells were most reliable parameters that could discriminate PID patients from healthy children. Our results confirm the high variability in functional cell assays and reinforce the idea that age differences must be taken into consideration during assay evaluation.

Keywords

Cellular immunity - primary immunodeficiency disease - flow cytometry - interferon-gamma - lymphoproliferation assay - delayed type hypersensitivity reaction - candidin - phytohemagglutinin