Integrated measurements by flow cytometry of the cytokines IL-2, IFN-γ, IL-12, TNF-α and functional evaluation of their receptors in human blood

Immunodeficiencies might be caused not only by the lack of cytokine production, but also by defective expression and/or function of the cytokine receptors. We have measured by flow cytometry, within 2 days, not only the production of IL-2, IFN-gamma, IL-12 and TNF-alpha, but also the functional expression of the receptors for these cytokines in blood samples obtained from 15 healthy donors and 13 patients suffering from tuberculosis. Cytoplasmic and surface staining with monoclonal antibodies (mAbs) was used to assess the production of cytokines and their receptors, respectively, after polyclonal stimulation. To evaluate receptor activity, peripheral blood mononuclear cells (PBMC) were first incubated with the corresponding recombinant human (rh) cytokine. CD69 was detected on lymphocytes after incubation with rhIL-2; IFN-gamma was detected in lymphocytes after co-stimulation with rhIL-12 plus PHA; iNOS induction and upregulation of major histocompatibility complex (MHC) II and MHC I was detected on monocytes after recombinant human interferon-gamma (rhIFN-gamma) stimulation; finally, COX-2 expression and MHC II upregulation were detected on monocytes after rhTNF-alpha stimulation. The assay that was developed can be used clinically to assess the activities of components of the cytokine signaling pathways of patients with immunodeficiencies or those with chronic intracellular infections such as tuberculosis.