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.

CD25 and IFN-γ expression in the diagnosis of primary immunodeficiencies

“…A positive correlation between age and CD25 or IFN-γ expression in the T cells of healthy donors was demonstrated using several different stimuli [5], emphasizing that T cell-mediated immunity is altered with age even in the absence of disease. Indeed, the authors report several conditions where the measured immune response of both PID patients and healthy children was statistically lower than that of healthy adults, but in these same conditions there were no statistical differences between patients with PID and normal donors when age was considered [5]. This underlines the importance of using the appropriate age-matched controls when assessing immune function in individuals with PID.…”

“…Specifically, the authors discovered that by exposing whole blood to phytohemaglutinin (PHA) for 18 hours, the intracellular levels of interferon-gamma (IFN-γ) produced by activated T cells was statistically lower in pediatric patients with PID than those without. Similarly, expression of CD25 on activated T cells was decreased in patients with PID when compared to children without immune-deficiency after whole blood exposure to candidin for 72 hours [5].…”

“…In addition to highlighting flow cytometry-based assays that may be useful in diagnosing and assessing PID, this work by Almeida et al also emphasizes the importance of using relevant clinical controls when assessing immune function in children [5]. A positive correlation between age and CD25 or IFN-γ expression in the T cells of healthy donors was demonstrated using several different stimuli [5], emphasizing that T cell-mediated immunity is altered with age even in the absence of disease.…”

“…In addition to highlighting flow cytometry-based assays that may be useful in diagnosing and assessing PID, this work by Almeida et al also emphasizes the importance of using relevant clinical controls when assessing immune function in children [5]. A positive correlation between age and CD25 or IFN-γ expression in the T cells of healthy donors was demonstrated using several different stimuli [5], emphasizing that T cell-mediated immunity is altered with age even in the absence of disease. Indeed, the authors report several conditions where the measured immune response of both PID patients and healthy children was statistically lower than that of healthy adults, but in these same conditions there were no statistical differences between patients with PID and normal donors when age was considered [5].…”

“…In this issue of the Journal of Pediatric Biochemistry, Almeida et al demonstrate that the measurement of T cell activation by flow cytometry can be used to distinguish the cellular immune responses of children with PID from those of healthy children in the same age group [5]. Specifically, the authors discovered that by exposing whole blood to phytohemaglutinin (PHA) for 18 hours, the intracellular levels of interferon-gamma (IFN-γ) produced by activated T cells was statistically lower in pediatric patients with PID than those without.…”