Computational protocol: CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells

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Protocol publication

[…] For the characterization of lymphocytes from CLL patients and healthy donors, fresh blood samples were stained prior to erythrocyte lysis using FACS lysing solution (Becton Dickinson). The following antibodies were used: CD3 FITC, CD19 Brilliant Violet (both Biolegend), CD5 PE-Cy5 (Beckman Coulter), CD8 Pacific Orange (Invitrogen), CD4 PE-Cy7, CD1d PE, CD161 eFluor 450, CD56 PerCP-eFluor 710, CD3 AlexaFluor 700, TCR Vα24 APC (all eBioscience).For determination of TCR-Vβ usage, cells were stained with the IOTest® Beta Mark Kit (Beckman Coulter) where eight sets of three TCR-Vβ - specific antibodies (each set comprises a FITC, PE and FITC/PE conjugated antibody) are combined in a single test. Samples were stained together with antibodies for CD3, CD4, CD8, TCR Vα24, CD56 and CD161.Murine samples (splenocytes of sacrificed mice) were stained using the following antibodies: TCR-Vβ FITC (TCR-Vβ screening panel, BD Pharmingen), CD3 PE, CD1d PE (all BD Pharmingen), CD4 PE-Cy5, CD19 PE-Cy7, CD8 Pacific Blue, CD3 AlexaFluor 700, CD5 PE-Cy5, CD8 PE-Cy7, CD19 Pacific Blue (all Biolegend), NK1.1 PE-Cy7, CD3 FITC (eBioscience).For intracellular detection of cytokines, the cells were stimulated with 100 ng/ml αGalCer (Biovision) for the indicated time and stained using Intracellular Fixation/Permeabilization Buffer Set and monensin (eBioscience) according to the instructions of the manufacturer. The following antibodies were used:mouse: anti-Mouse IFNγ PerCP-Cy5.5, anti-Mouse IL-4 PE, anti-Mouse TNFα APC (all eBioscience).human: anti-human IFNγ FITC, anti-human IL-4 PE, anti-human TNFα APC (all eBioscience).Samples were measured on a Gallios flowcytometer (Beckman Coulter) and analysed using Kaluza 1.2 software (Beckman Coulter). [...] All statistical analyses were performed using GraphPad Prism 5.02 Software or IBM SPSS Statistics 21 software. Overall survival curves were plotted by Kaplan-Meier and the survival distributions of the different cohorts were compared using log-rank (Mantel-Cox) test. Mann-Whitney or Student's t-tests were used as indicated to analyse significance. […]

Pipeline specifications

Software tools Kaluza, SPSS
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Deficiency Diseases, Leukemia, Lymphocytic, Chronic, B-Cell