0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
free from immune-modulating therapy for at least 3 months preceding entry
in the study. Local Ethical Committee approval and written informed consent
from all the subjects will be obtained before study initiation. Comparisons
between pairs of data-sets will be performed by means of Student’s t-tests for
unrelated samples.
Flow cytometry and cell preparation. Flow Cytometric analysis will be carried
out on a FACSCalibur, on a FACSCanto (BD Bioscience) or CyAn (Coulter).
High Speed Cell sorting will be performed by a 4 laser MoFlo (Coulter).
Human mononuclear cells will be isolated by Ficoll gradient centrifugation
(Pharmacia, Uppsala, Sweden). Dead cells will be excluded by propidium
iodide. Data will be analyzed using Flowjo software (Treestar Inc., Ashland,
OR).
Antibodies and reagents. The following antibodies will be used: Foxp3 from
eBioscience, San Diego, CA; 5-(and-6)-carboxyfluorescein diacetate, succinimidyl
ester (CSFE) will be purchased from Invitrogen (Carlsbad, CA, USA).
CD2, CD3, CD4, CD5, CD8, CD25, CD26, CD27, CD28, CD30, CD38, CD40L,
CD44, CD45RO, CD45RA, CD45RB, CD49d, CD50, CD54, CD56, CD58,
CD62L, CD69, CD70, CD95, CD122, CD134, CD162, CTLA4, HLA-DR, CCR4,
CCR6, CCR7, CXCR2, CXCR3, CXCR5 are from BD Pharmingen; GITR,
CCR1, CCR2, CCR3, CCR5, CCR8, CCR9, CXCR4, CXCR6 are from R&D.
These antibodies are conjugated with the following fluorochromes: FITC, PE,
Cy5-PE, APC, PE-Cy7, PE-TexasRed, APC-Cy5.5, APC-Cy7, APC-Alexa 750,
PE-Alexa 610, Cascade Yellow, Pacific Blue. IFN-g, IL-10, IL-4, TNF-a are
from Becton Dickinson; IFN alpha is from Miltenyi Biotech.
Isolation and immunostaining of blood cells. PBMC will be isolated from blood
samples by discontinuous density centrifugation over Ficoll-hypaque according
to standard procedures. Cells will be resuspended in PBS with 1% human
serum. For each staining 5 x 10 6 PBLs will be used, in a final volume of 100 ml.
Monoclonal antibodies (conjugated with the appropriate fluorochrome) will
be added to predetermined optimal concentrations and cells will be incubated
for 10 minutes at 4 °C in the dark. Cells will be then washed twice, resuspended
in 250 ml of PBS contaning 0,5% FCS, and analyzed at the flow
cytometer.
Isolation of regulatory T cells from PBMC. In order to achieve this goal we will
use a combination of several methodologies: 1) Polychromatic Flow Cytometer
Analysis (3 Laser MoFlo, Coulter); 2) MACS Cytokine Secretion Assays -
detection and isolation of viable antigen-specific T cells; 3) Accurate high
speed cell sorting. The different subsets of regulatory T cells identified in the
first part of the project with polychromatic flow cytometry will be sorted
either directly in 96 well plates with a high speed cell sorter (Coulter) for cell
cloning, or in tubes in the case that the cells will be used in a suppression
assay.
PBLs will be stimulated with EBV pentamers (Pro5 ® MHC Class I Pentamers
Proimmune), or with plate-bound anti-CD3 and soluble anti-CD28
(1 mg/ml, both from Pharmingen) as a positive control. Production of inflam-
646 2009