A new simple whole blood flow cytometry-based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation

Abstract

The multiple cellular and soluble elements of the immune system respond in a coordinated way, orchestrated by cytokines, to preserve the integrity of the organism. In this study, we describe a new and unique whole blood method that, with minimal sample manipulation, allows an overall evaluation of immune responses by simultaneously measuring cell activation and cytokine secretion. The identification of cells actively secreting cytokines is based on the stabilization of tumor necrosis factor a (TNFa) at the cell surface through the use of a specific inhibitor of the TNFa-converting enzyme. This inhibitor does not affect the release of cytokines other than TNFa and makes it possible to assess, in the same measurement, the phenotype of TNFa þ -secreting cells and quantify multiple secreted cytokines by using a specific and highly sensitive flow cytometry-based bead immunoassay. Upon stimulation of normal peripheral blood samples with either phorbol 12-myristate 13 acetate (PMA) plus ionomycin or lipopolysaccharide (LPS), both the number of TNFa þ cells and the amount of secreted cytokines progressively increased, the former becoming detectable first. After stimulation for 3 h with PMA plus ionomycin, cellular responses were associated with surface TNFa expression on the majority of CD3 þ T cells and secretion of Th1-associated cytokines: interferon c, interleukin (IL)-2, and to a lesser extent IL4. In turn, stimulation with LPS induced a response mainly by inflammatory cells. After 4 h of LPS-stimulation, the majority of CD14 þ monocytes showed surface TNFa expression; in parallel, high amounts of soluble IL1b, IL6, and IL8 became detectable. Likewise, stimulation of blood samples with cytomegalovirus (CMV) lysates induced viralspecific immune responses detectable in seropositive but not seronegative volunteers; such responses were associated with the detection of increased numbers of TNFa þ monocytes, TNFa þ /CD8 þ T cells and TNFa þ /CD8 À T lymphocytes in association with an increased secretion of IFNc, IL6 and TNFa.

Association of Obesity With Inflammation and Pain After Total Hip Arthroplasty

“…These activators evaluate dynamic response of the immune system and provide mechanistic insight into pathways leading to the release of cytokines and inflammatory response [27]. LPS is used to assess innate immune response to bacterial infection.…”

Expanded cells in monoclonal TCR-αβ+/CD4+/NKa+/CD8−/+dim T-LGL lymphocytosis recognize hCMV antigens

“…Assessment of TNF-␣ ϩ -activated cells as well as quantification of multiple secreted cytokines (IFN-␥, TNF-␣, IL-2, IL-4, IL-6, and IL-10) were performed after stimulation with the peptide on a FACSCalibur flow cytometer, according to procedures that have been previously described in detail. 16 …”

Age-associated Epstein–Barr virus-specific T cell responses in seropositive healthy adults

“…Another PB aliquot was processed under the same conditions but in the absence of the viral lysate (unstimulated sample), and used as a negative control. As a positive control, a third aliquot of PB was cultured with 25 ng/ml phorbol myristate acetate (PMA) (Sigma, St Louis, MO, USA), 1 μg/ml ionomycin (Sigma) and 20 μM of TAPI-2 [49,51]. After stimulation all PB aliquots, including the controls, were incubated at room temperature and labelled with CD3-phycoerythrin cyanin 7 (PE-Cy7) (clone SK7), CD4-peridinin chlorophyll (PerCP) (clone SK3), CD8-allophycocyanin (APC)-Cy7 (SK1 clone), CD45RA-fluorescein isothiocyanate (FITC) (clone HI100), CCR7-PE (clone 3d12) and TNF-α-APC (clone 64,011,111) antibody combination (all purchased from Becton Dickinson Biosciences) or 2 ml/tube of fluorescence activated cell sorter (FACS) lysing solution ×1 (Becton Dickinson Biosciences) for 15 and 10 min, respectively.…”

“…To evaluate T cell responses in vitro, PB stimulation with an EBV lysate was used, as described in detail previously [33,49]. Briefly, two aliquots of PB samples in RPMI-1640 (1:1 vol : vol) adjusted to a cell concentration of 1 × 10 6 leucocytes/μl were incubated for 6 h at 37°C in a 5% CO2-humidified atmosphere with 5 μg/ml of an EBV lysate (strain B95·8; Advanced Biotechnologies, Columbia, MD, USA) together with 20 μM TAPI-2, a TNF-α protease inhibitor that induces TNF-α accumulation on the cell surface (Cytognos SL, Salamanca, Spain), and 1 μg/ml of both anti-CD28 (clone L293; Becton Dickinson Biosciences) and anti-CD49d (clone L25; Becton Dickinson Biosciences) as co-stimulatory monoclonal antibodies (BD Pharmingen, San Diego, CA, USA) [50].…”