Development of an immune function assay by measuring intracellular adenosine triphosphate (iATP) levels in mitogen-stimulated CD4+ T lymphocytes

Abstract

We developed an immune function assay for monitoring CD4+ T cells activity based on changes in intracellular adenosine triphosphate (iATP) levels after phytohemagglutinin (PHA) stimulation. Blood samples were obtained from 40 healthy subjects and 30 RTRs and incubated with 5 µg/mL of PHA for 15-18 hr at 37°C and 5% CO2. Afterward, the CD4+ T cells were separated by antibody-coated magnetic beads and lysed. Then, iATP content in unstimulated and stimulated conditions was measured by luciferin-luciferase reaction using a log-log standard curve. The iATP levels showed significant increase in CD4+ T cells in both healthy persons (mean: 550 ± 142 ng/mL vs. 109 ± 54 ng/mL) and RTRs (mean: 394 ± 160 ng/mL vs. 52 ± 37 ng/mL) after PHA stimulation (P < 0.001). However, the iATP production in RTRs was significantly lower than that in healthy individuals; both prior to and after stimulation with PHA (P < 0.001). No gender-specific difference in iATP production was observed between women and men subjects. This rapid and low-cost assay reflects the degree of immune cell function through assessment of CD4+ T cells activation. Thus, it can be used for evaluation of immune system status in immunodeficient individuals as well as in immunosuppressed transplant recipients who needs drug adjustment.

Clinical relevance of a CD4+ T cell immune function assay in the diagnosis of infection in pediatric living‑donor liver transplantation

“…It is widely known that administering appropriate doses of immunosuppressive agents is crucial for improving allograft outcome; however, the blood concentration of drugs is not directly correlated with the dose of drug administered due to individual pharmacokinetic differences and variations in the methods used for their detection (19). On the contrary, under conditions of hyper- and hypo-immune suppression, detrimental effects on the graft may occur, and increase the risk of infection and graft rejection (20). The present study, along with other reports, support the hypothesis that monitoring the ATP concentrations of CD4 + T cells may aid in distinguishing between hyper- and hypo immunity for the identification of LDLT patients at risk of infection or graft rejection, and may be applied to increase the efficacy of immunosuppressive therapies (21).…”