T-cell subsets and their intracellular cytokine production were identified by multiparametric flow cytometry.11- 13 Peripheral blood mononuclear cells were isolated from 20 mL of anticoagulated (EDTA) venous blood by density centrifugation and incubated at 37°C in a 5% carbon dioxide incubator for 4 hours in RPMI (Roswell Park Memorial Institute; Sigma-Aldrich Corp, St Louis, Mo) medium containing 10-µg/mL brefeldin A (Epicentre Technologies, Madison, Wis) to dissaggregate the Golgi complex and enable the intracellular accumulation of proteins.14 During the incubation period, half the cells were separately stimulated with 20-ng/mL phorbolmystrateacetate (Sigma-Aldrich Corp) and 1-µg/mL ionomycin (Sigma-Aldrich Corp) to assess submaximal cytokine production, while the other half remained unstimulated. After incubation, cell suspensions were divided into 18 tubes with approximately 1×106 cells per tube corresponding to cell type (CD4+, CD8+, or γδ+), cytokine combination (isotype control, IFN-γ/IL-4, or IL-2/IL-10), and stimulation category (unstimulated or stimulated). Cells were resuspended in buffer (phosphate-buffered saline plus 0.1% sodium azide plus 1% bovine serum albumin plus 5-mmol EDTA) and incubated for 15 minutes at 25°C with a 1:5 dilution of Tricolor-conjugated (a tandem conjugate of phycoerythrin and Cy-5; Caltag Laboratories, San Francisco, Calif) anti-γδ T-cell receptor, anti-CD4, or anti-CD8 monoclonal antibodies for phenotypic identification. After washing in phosphate-buffered saline, cells were fixed in 100 µL of solution A (cell permeabilization fixation; Caltag Laboratories) for 15 minutes at 25°C and washed again in phosphate-buffered saline. Intracellular cytokines were identified by resuspension in 50 µL of the permeabilization solution B (Caltag Laboratories) containing a 1:500 dilution of dinitrophenyl-conjugated anti–IFN-γ and a 1:100 dilution of biotin-conjugated anti–IL-4; or dinitrophenyl-conjugated anti–IL-10 and biotin-conjugated anti–IL-2; or dinitrophenyl-conjugated anti-mouse IgG1 and biotin-conjugated anti-mouse IgG2 for 15 minutes at 25°C. Specific intracellular cytokine identification was achieved by subsequent incubation (Strepavidin Red 670; Pharmingen Inc, San Diego, Calif) and fluorescein isothiocyanate–conjugated anti-dinitrophenyl antibody (Molecular Probes Inc, Eugene, Ore) for 15 minutes at 25°C. Finally, cells were resuspended in 0.5 mL of cytoflow buffer for analysis. Surface and intracellular fluorescence was measured using a flow cytometer (FACScan; Becton Dickinson Immunocytometry Systems, San Jose, Calif) and fluorescence data were analyzed with software (CellQuest; Becton Dickinson Immunocytometry Systems). After standard calibration of the flow cytometer, data were recorded until 5000 cells were acquired or the cell suspension volume of 0.5 mL was exhausted. The minimum number of cells was lowered to 2000 cells for γδ+ T cells since these cells are much scarcer in the peripheral blood. Intracellular cytokine detection was performed by double gating each cell surface marker and a lymphocyte gate and measuring fluorescence channel 1 (fluorescein isothiocyanate) and fluorescence channel 3 (Strepavidin Red 670) fluorescence intensity. The data recorded included the total number of acquired cells, the percentage of total cells gated for each subset, and the number of cells positive for fluorescence channel 1 and fluorescence channel 3 internal fluorescence. Background fluorescence was measured using isotype-matched fluorochrome-conjugated IgG and arithmetically subtracted from the measured intracellular cytokine fluorescence to obtain the net detectable intracellular fluorescence. The average background fluorescence measured was less than 1%. Reference assays using fixed, but not permeabilized, cells demonstrated a reduction in the intracellular detection of IL-2 and IFN-γ by 99% and 98%, respectively. Preincubation with recombinant human cytokines before intracellular staining reduced intracellular detection of IL-2 and IFN-γ by 99% and 98%, respectively. Serum IL-6 levels were measured by enzyme-linked immunosorbent assay by the Clinical Investigation Facility at David Grant Medical Center.