Use of the CD4 in vitro CMI™ platform to measure viral-induced and drug-induced immunosuppression

Abstract

Objective: We have developed a novel platform technology that directly measures the functional role of CD4+ lymphocytes. The assay is based on the ability of CD4+ T-helper cells from either whole blood or peripheral blood mononuclear cells (PBMCs) to respond to stimulation by mitogens, alloantigens, or memory specific antigens. We show that this technology is capable of assessing immune functionality in HIV+ patients and also in organ transplant recipients that are on life-long immunosuppressive medications.Methods: Whole blood or PBMCs were added to wells of a 96-well microtiter plate and stimulated for 4 to 18 hours. After stimulation, CD4+ cells were isolated within the microwells using monoclonal antibody coated magnetic particles. The CD4+ cells were then lysed to release intracellular adenosine triphosphate (ATP). Increases of intracellular ATP precede changes of mRNA and cytokine production and correlated with proliferation of reactive lymphocytes. In vitro CMI™ results were compared to absolute CD4+ cell counts by flow cytometry and to viral-load (for HIV+ patients) or immunosuppressive drug (FK506 or Cyclosporin). To monitor drug-induced or viral-induced immunosuppression, populations of transplant recipients and HIV+ individuals were compared to healthy adults.Results: An initial experiment was conducted to determine the in vitro effect of cyclosporin A (CSA) on mitogen stimulation. It was found that pre-incubation of CSA with whole blood from a healthy volunteer dramatically reduced responses to PHA and concanavalin A in a dose-dependent manner. To monitor the in vivo effects of immunosuppressive therapy, whole blood and PBMCs samples from organ transplant recipients were assessed for responsiveness to mitogens, alloantigens and memory antigens. Activation by PHA was greatly reduced and most of the patients tested were not reactive to memory antigens. A varying degree of immunosuppression was seen in transplant recipients with ATP values ranging from <5 ng/mL to 400 g/mL ATP. Similar to drug-induced immuno-suppression, HIV infected individuals also showed mitogen responses greatly reduced as compared to healthy adults. In a study involving HIV+ drug abusers, the average ATP response to PHA was 140 ng/mL with a range of <5 ng/mL to 300 ng/mL.Conclusions: The CD4 in vitro CMI™ platform provides quantitative functional information on a patient's immune responsiveness. This information provides care-givers a unique opportunity to individualize patient therapy based on monitoring this response.