Flow-Cytometric Assessment ofin VivoCytokine-Producing Monocytes in HIV-Infected Patients

Major peptide autoepitopes for nucleosome-specific T cells of human lupus

“…We wanted to confirm that the peptide autoepitopes ( Figure 6) were also relevant to unmanipulated CD4 T cells in PBMCs of patients with lupus. Flow cytometry for newly synthesized, intracellular cytokines is ideal for rapidly detecting antigen-specific responses and determining the frequency of CD4 T cells response to a particular epitope (19)(20)(21). Because autoimmune T cells are preactivated in vivo in patients with active disease, we found that activation markers, such as CD69, CD25, or CD79, were not as reliable.…”

“…An equal number of events from every sample were analyzed. After doing preliminary studies with test antigens, such as tetanus toxoid, mumps, and candida, and taking into account previous experience in other systems (19)(20)(21), we considered a response to the nucleosomal antigens to be unequivocally positive when the frequency of positive cells was greater than 0.2% and the values were at least 2-fold higher than respective background values ("medium"; Figures 8 and 9). Repeat assays with…”

“…Measurements of antigen-specific, intracellular cytokine responses of T cells were done as described with slight modifications (19)(20)(21). Purified PBMCs (10 6 ) were placed in polystyrene tissue culture tubes (12 × 75 mm; Becton Dickinson and Co., Franklin Lakes, New Jersey, USA) containing 0.5 mL of HL-1 serum-free medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (GIBCO BRL; Grand Island, New York, USA), appropriately titered peptide antigen preparations, and 1 µg/mL anti-CD28 mAb, L293 (Becton-Dickinson and Co.).…”

“…Staining of PBMCs for intracellular cytokines and surface markers was done as described (19)(20)(21). Five parameter analyses were performed on FACScalibur (Becton Dickinson Immunocytometry Systems) using FITC, PE, and PerCP as the fluorescent parameters.…”

“…Five parameter analyses were performed on FACScalibur (Becton Dickinson Immunocytometry Systems) using FITC, PE, and PerCP as the fluorescent parameters. Methods of cytometer setup and data acquisition have been described previously (1,17,(19)(20)(21). For each analysis, 10,000 events were acquired and gated on CD4 expression and a light scatter gate designed to include only viable lymphocytes (1,17,19).…”