- Isolate PBMCs and generate T cell clones as previously described (1).
- Purify CD8+ T cells from PBMCs by positive selection with anti-CD8 mAb coupled to magnetic beads (Miltenyi Biotec). In our hands, flow cytometry analysis demonstrated >99% CD8+ cells in the positively purified population and <5% in the CD8-depleted population.
- Derive immature DCs from peripheral monocytes purified by positive selection with anti-CD14 mAb coupled to magnetic beads (Miltenyi Biotec).
- Culture CD14+ cells for 5 days in RPMI 1640 medium containing 5% fetal calf serum (FCS), 2 mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 50 μg/ml kanamycin (Gibco BRL) (complete medium), 50 ng/ml granulocyte macrophage-colony stimulating factor (GM-CFS) (Novartis Pharma), and 1000 U/ml rIL-4.
- Isolate fresh myeloid DCs from PBMCs by two magnetic separation steps, in order to previously deplete B cells from PBMCs with anti-CD19 mAb coupled to magnetic beads, and then positively select CD1c+ DCs using biotin-labeled anti-CD1c mAb and anti-biotin mAb coupled to magnetic beads (Miltenyi Biotec).
- Obtain mature DCs by a 40-h stimulation of iDCs with CD40L-transfected J558L cells (at a DC/J558L ratio of 1:1), as previously described, or with soluble r-CD40L molecules (Alexis Biochemicals, Alexis Corporation). The definition of monocyte-derived or myeloid DCs was based on their different surface phenotype profile by staining with anti-CD14, anti-CD86 (Caltag Laboratories), anti-CD1a, anti-CD1c, anti-CD11c, anti-CD32, anti-CD80 (Becton Dickinson, BD PharMingen) mAbs, Annexin-V (ApoAlert Apoptosis Kit, Clontech Laboratories Inc), propidium iodide (PI) (Sigma-Aldrich), and the appropriate secondary labeled antibodies (BD PharMingen) (Fig. 1).
- After staining, analyse these cells using a FACSCanto flow cytometer (Becton Dickinson) and FACSDiva analysis software (Becton Dickinson).
Apoptotic and control cell preparations.
- Plate cloned CD8+CD95+ T cells (10-100 x 106) in a 24-well plate and incubate for 1 h at 37°C in the presence or absence of 14 μg/ml caspase 3 inhibitor (C3I) (Z-DEVD-FMK), C8I (Z-IETD-FMK), or a negative caspase control (K, Z-FA-FMK) (BD Biosciences PharMingen, San Jose, CA, USA), induced to undergo Fas-mediated apoptosis as described (2).
- Induce apoptosis of T cell clones by the addition of 500 ng/ml anti-Fas (anti-CD95 mAb [clone CH11], Upstate Biotechnology) for at least 6 h (2).
- Measure apoptosis by Annexin-V (ApoAlert Apoptosis Kit, Clontech Laboratories Inc.) and PI (Sigma Aldrich) staining and subsequent analysis by flow cytometry.
- Purify apoptotoc cells by positive selection using Annexin V-conjugated magnetic microbeads (Miltenyi Biotec). As detected by FACS analysis, the positively purified population should consist of >99% Annexin V+ cells. These will be then used as apoptotic cells in all of the appropriate experiments.
- Use as control cells live cloned T cells promptly lysed by repeated freezing and thawing.
- Measure spontaneous apoptosis of CD4+ T cells in the peripheral blood of both HIV-infected and healthy individuals by Annexin-V, PI (Sigma Aldrich), and phycoerythrin (PE)-Cy7-conjugated anti-CD4 mAb (Caltag) staining of fresh PBMCs before and after 18-h incubation in complete medium at 37°C.
- Use live cloned T cells lysed by repeated freezing and thawing as control.
- Measure spontaneous apoptosis of CD4+ T cells in the peripheral blood of both HIV-infected and healthy individuals by Annexin-V, PI (Sigma Aldrich) and phycoerythrin (PE)-Cy7-conjugated anti-CD4 mAb (Caltag) staining of fresh PBMCs before and after 18-h incubation in complete medium at 37°C.
- Treat cloned CD8+CD95+ T cells with 14 μg/ml of caspase 3 inhibitor (Z-DEVD-FMK), caspase 3 inhibitor (Z-IETD-FMK), or a negative caspase control (K, Z-FA-FMK) (BD Biosciences PharMingen), dyed green with PKH27 (Sigma-Aldrich)
- Induce the cells to undergo Fas-mediated apoptosis.
- Co-culture the apoptotic cells at a 1:1 ratio with iDCs, incubated or not with either rabbit anti-αvβ5 polyclonal antibody (Bioline Diagnostici s.r.l.) reacting with a human DC surface integrin implicated in the capture of apoptotic cells (3), or the control rabbit Ig, and dyed red with PKH26 (Sigma-Aldrich).
- After 12 h at 4°C and 37°C, analyze phagocytosis by flow cytometry, as evidenced by the presence of double-positive cells.
- Collect apoptotic and control cells, wash them twice with phosphate-buffered saline (PBS), centrifuge the cells at 1600 rpm for 10 min.
- Dissolve the resulting pellets in 300 µl of two-dimensional sample buffer containing 8M urea (Sigma-Aldrich), 2% 3-[3-(cholamidopropyl)-dimethyl-ammonio1-1propanesulphonate] (CHAPS; Amersham Biosciences, Uppsala, Sweden), 100mM 1,4-dithio-DL-threitol (DTT; Sigma-Aldrich), 0.2% Ampholine pH 3-10 (BioRad Laboratories), 1 mM sodium orthovanadate, 1 μg/ml protease inhibitor cocktail, and a trace of bromophenol blue (Sigma-Aldrich), that will be subsequently rehydrated into 11-cm nonlinear immobilized pH gradient (IPG) strips (3-10 NL IPG 11 cm [Biorad]).
- Perform active sample rehydration into IPG strips at 50 V overnight.
- Perform focusing in a Bio-Rad Protean Isoelectric Point Focusing Cell under the following conditions: (i) 30 min at 500 V, (ii) 30 min at 1000 V, (iii) 15 min at 2000 V, (iv) 30 min at 3000 V, and (v) 7 h at 5000 V.
- After isoelectric point focusing, reduce the IPG strips and alkylate them with 2% w/v DTT and 2.5% w/v iodoacetamide (Sigma-Aldrich), respectively, in 10-min incubations in an equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS [Sigma-Aldrich]).
- To perform the second dimension, mount each strip on an SDS-PAGE gel containing 12% T and 2.5% C (w/v) acrylamide/bis-acrylamide (Amersham) and then overlayer it with a solution containing 0.5% w/v agarose and 0.5 M Tris-HCl pH 8.8. The SDS-PAGE gel was electrophoresed at 15 mA/gel for 15 min and at 30 mA/gel for 5 h at 10°C.
- Fix the two-dimensional gels with a solution containing 50% ethanol, 2% phosphoric acid for 2 h, wash them in deionizated water for 30 min, and transfer to a solution containing 34% methanol, 17% (w/v) ammonium sulphate, and 2% phosphoric acid. 8. After 1 h, stain gels with Coomassie Brilliant Blue G-250 (0.06% (w/v) (Fluka; Sigma-Aldrich Group).
MALDI-TOF-MS and database searching
- Excise each 2DE spot of interest and in-gel digest with trypsin, as described (4,5).
- Carry out MALDI-TOF-MS of tryptic protein hydrolysates and identify proteins from the peptide mass spectra produced by MALDI-TOF-MS using the peptide mass fingerprinting analysis software PeptIdent, accessed via ExPASy (http://us.expasy.org). 3. Use the Swiss-Prot database to conduct the searches, allowing for up to one missed cleavage, considering methionine in the oxidized form and cysteine treated with iodoacetamide to form Cys_CAM.
Purification of proteins from 2DE gels
- To extract protein fragments, transfer 2DE gels onto nitrocellulose membranes electrophoretically (Schleicher & Schuell Bioscience, Inc.).
- Stain the membranes with Ponceau S solution (P7170; Sigma), and cut out the spots of interest
- Elute the proteins derived from these spots in a electron-elution solution (50 mM Tris-Cl pH 9.5, 1% [v/v] Triton X-100, 2% [w/v] SDS [Sigma-Aldrich]) (6).
- After centrifugation at 5000 rpm for 5 min, remove the supernatant, and precipitate the proteins with 25% trichloroacetic acid (TCA, Sigma-Aldrich).
- Wash the resulting protein pellet twice with cold acetone and quantify the protein content (6) before to conduct the cross-presentation experiments.
Immunoblot of cellular vimentin
- Collect apoptotic cells, CI-treated apoptotic cells, and control cells, wash twice with PBS, and then centrifuge them at 1600 rpm for 10 min.
- Mix the resulting pellets and dissolved them in 50 µl of lysis buffer solution (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholic acid, 150 mM sodium chloride, 1 mM ethylene-glycol-bis[2-amino-ethylether]-N,N,N’,N’-tetra-acetic acid [EGTA], 1 mM phenylmethanesulfonyl fluoride [PMSF], 1 mM sodium orthovanadate, 1 μg/ml protease inhibitor cocktail [Sigma-Aldrich]).
- Quantify the protein contents and separate proteins by SDS-PAGE.
- Transfer each gel to a nitrocellulose membrane (Schleicher & Schuell), and immunoblot the proteins for vimentin with a relevant guinea pig polyclonal antibody (Research Diagnostics Inc.), which recognize both the entire and fragmented vimentin forms (see Fig. 2c), and with the secondary horseradish peroxidase (HRP)-conjugated donkey anti-guinea pig IgG antibody (Research Diagnostics Inc.).
- Perform chemiluminescent detection (SuperSignal West PicoChemiluminescent substrate [Pierce]) to visualize the resulting proteins.
Caspase 3 digestion and SDS-PAGE of recombinant vimentin.
- Filter human r-vimentin (Research Diagnostic Inc.) by using a Centricon Vivaspin 500 (10,000 MWCO PES [Sartorius Group]) to remove urea.
- Add 1 ml of caspase 3 buffer (50 mM Tris-Cl pH 7.4, 100 mM NaCl, 100 mM 1,4-dithioerythritol [DTE], 1 mM ethylenediaminetetracetic acid [EDTA], 0.1% CHAPS [Sigma-Aldrich]) to the Centricon tubes and centrifuge twice at 4000 rpm for 30 min.
- Collect 100 μg of vimentin and incubate with 150 ng of caspase 3 (C1224, Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37°C.
- Separate the complete vimentin and the digested vimentin proteins on the basis of molecular weight under SDS-PAGE conditions (Fig. 2b) (7).
- Stimulate highly purified CD8+ T cells (1 x 10>5) from PBMCs with the different peptides (5 μg/ml per single peptide) and irradiated autologous CD8-depleted PBMCs, used as antigen-presenting cells (APCs).
- Perform this stimulation directly in 96-well ELISPOT nitrocellulose-backed plates (MAHA S4510, Millipore) that had been coated with capture anti-IFN-γ mAb (BD PharMingen) (ELISPOT plate (8).
- Reveal the IFN-γ spot formation after 6 to 8 h at 37°C by using the AID ELISPOT Reader (AID GmbH).
- Determine resting CD8+ T cell responses by counting IFN-γ-spots formed by short-term CD8+ T-cell lines obtained after one or two rounds of in vitro stimulation with peptides and irradiated autologous CD8-depleted PBMCs.
Pentamer and intracellular cytokine staining
- Stain PBMCs with allophycocyanin [APC]-labeled-HLA-A*0201 pentamers (complexed to vimentin78-87 [LLQDSVDFSL], non-muscle myosin478-486 [QLFNHTMFI], non-muscle myosin741-749 [VLMIKALEL], or actin266-274 [FLGMESCGI] peptide) (Proimmune Limited), APC-Cy7-labeled anti-CD8 mAb (BD, PharMingen), fluorescein isothiocyanate (FITC)-labeled perforin mAb (BD, PharMingen), and PE-labeled granzyme B mAb (PeliCluster (9).
- Obtain negative controls by staining cells with an irrelevant isotype-matched mAb.
- Analyse samples using a FACSCanto flow cytometer and FACSDiva analysis software (Becton Dickinson).
- Stimulate part of the pentamer/anti-CD8-stained PBMCs with peptide (10 μg/ml) plus anti-CD28 mAb (4 μg/ml) (BD PharMingen) for 6 h at 37° C.
- At the second h, add 10 μg/ml Brefeldin-A (Sigma-Aldrich), wash cells with Perm/Wash buffer (BD PharMingen and stain intracellularly with phycoerythrin (PE)-labeled anti-IFN-γ mAb (BD PharMingen) for 30 min at 4°C.
- Fix and permeabilize cells using Cytofix/Cytoperm solution (BD PharMingen) at 4°C for 20 min, and analyze them by flow-cytometry.
- In some experiments, perform pentamer staining after stimulation with peptide (50 μg/ml) overnight, in order to determine the degree of TCR down-regulation (9).
- Pulse immature monocyte-derived DCs or myeloid DCs (3 x 10>4), as APCs, with increasing concentrations of CI-treated, caspase-treated, untreated, r-vimentin-loaded apoptotic cells, control lysed cells, soluble antigens or peptides, in the presence or absence of 80 mM lactacystin (Sigma-Aldrich) in U-bottom 96-well plates for 18 h.
- Wash APCs and coculture them with antigen-specific CD8+ T cells (2-3 x 10>4)
- Reveal IFN-γ spot formation after 6–8 h at 37°C by an ELISPOT assay(8).
- Pulse iDCs with apoptotic or control cells (derived from HLA-A2- Epstein-Barr virus [EBV]-transformed B cells) previously infected by either WT-VV or NS3Ag-expressing VV (5 plaque-forming units/cell) and then cocultured with NS31406-1415-specific CD8+ T cell clones.
- After 6 to 8 h at 37°C, reveal IFN-γ spot formation by CD8+ T cells by ELISPOT assay.
Cross-presentation experiments ex vivo
- Double-stain PBMCs from patients or HDs with pentamers and anti-CD8 mAb)
- Culture PBMC (in the presence of soluble r-CD40L molecules) with autologous iDCs that have been pulsed or not with apoptotic cloned T cells.
- After 6–8 h, test PBMC for their capacity to produce IFN-γ by ICSS assay (9).
Peptide synthesis and class I binding
- Identify peptides containing HLA binding motifs.
- Synthesize these peptides and test them for class I binding capacity, as described in detail in (10).
- Binding data should be presented as 50% inhibitory concentration (IC50) nanomolar values. Peptides with binding affinity ≤50 nM may be classified as "good" binders, and peptides in the 50 to 500 nM range as "intermediate" binders.