4 CARPINTERÍA Y VIDRIOS

Construction of retroviral vectors and production of retroviral supernatant

The construction of retroviral vectors encoding for the TCR chains of the HA-2-specific T-cell clones HA-2.6 and HA-2.5, and the TCR-AV18 chain and TCR-BV13 chain of the CMV pp65- specific T-cell clone have been described previously(5)_{. Briefly, the}

HA-2.6-TCR-AV23, HA-2.5-TCR-AV15 and CMV-TCR-AV18 were cloned into bicistronic retroviral vectors encoding for the marker gene eGFP. The HA-2.6-TCR-BV18, HA-2.5-TCR-BV18, and CMV- TCR-BV13 were cloned in combination with the truncated nerve

growth factor receptor (NGF-R)(54)_{. Retroviral vectors encoding}

eGFP or NGF-R only were used as control vectors. The Moloney murine leukemia virus-based retroviral vector LZRS and packag- ing cells φ-NX-A were used to obtain viral supernatant(55)_. HLA Class I tetrameric complexes and flow cytometric analyses PE- or APC-conjugated tetrameric complexes were constructed as previously described(56) _{with minor modifications. The fol-}

lowing tetramers were used; tetrameric HLA-A2 molecules in complex with CMV-pp65 derived peptide NLVPMVATV (CMV tetramer) or HA-2 peptide YIGEVLVSV (HA-2 tetramer), and tetrameric HLA-B7 molecules in complex with EBV EBNA3A derived RPPIFIRRL (EBV tetramer). For flow cytometric analy- ses cells were labeled with tetramers for 1 h at 4ºC in RPMI without phenol, supplemented with 2% FBS, and washed two times or labeled with monoclonal antibodies (mAb) di- rected against various cell surface molecules for 30 minutes at 4ºC. The mAbs used were anti-BV13, anti-BV7, anti-BV18 and anti-BV2, all PE-conjugated (Beckman Coulter, Mijdrecht, The Netherlands), anti-TCRαβ PE-Cy5-conjugated and CD3 APC- or PE-conjugated (BD Pharmingen, San Diego, CA, USA) and CD8 APC- or PE-conjugated (Invitrogen, Paisley, UK). For detection of ΔNGF-R, anti-human NGF-R mAbs were used either PE- (BD Pharmingen, San Diego, CA, USA) or APC-conjugated (Cedarlane Laboratories, Hornby, Ontario, Canada). For the different adhe- sion and costimulatory molecules PE-conjugated anti-CD54 (CLB, Amsterdam, The Netherlands), anti-CD58 (Southern Biotechnology Associates, Birmingham, AL, USA), anti-CD2 ,

anti-CD11a, and anti-CD50 (BD Pharmingen, San Diego, CA, USA) were used. For intracellular stainings, T-cells were stained with mAbs for 20 minutes at 4ºC and were subsequently fixated for 10 minutes using paraformaldehyde. T-cells were permeabilized by washing 2 times using PBS containing saponine, stained for another 20 minutes at 4ºC, washed and analyzed using FACS. Isolation, transduction and culture of T-cells and LCLs

Purified (>95% pure) HLA-A2-restricted CMV pp65 NLV-specific T-cells (pp65 T-cells) and HLA-B7-restricted EBV EBNA3A RPP- specific T-cells (EBNA3A T-cells) were isolated from peripheral blood of EBV and CMV seropositive healthy individuals using tetramers. After informed consent, peripheral blood mononuclear cells (PBMCs) were harvested and labeled with tetramers for 1 h at 4 ºC in RPMI without phenol supplemented with 2% FBS, washed, and sorted at 4 ºC using the FACS Vantage™ (Becton Dickinson, San Jose, CA, USA). Tetramer-positive T-cells were stim- ulated with PHA (Murex Biotec Limited, Dartford, UK) and irradi- ated feeder cells (30 Gy), and after 2 d of culture, the T-cells were transduced with retroviral supernatant. The transduction proce- dure used for the peripheral blood T-cells has been described pre- viously(57)_{. In brief, 1x10}6 _{T-cells were cultured on CH-296-coated}

24-well non-tissue culture-treated plates (Falcon) together with 1 ml thawed retroviral supernatant overnight at 37ºC, washed, and transferred to 24-well tissue culture plates. TCR transduced (td) virus-specific T-cells were sorted on bases of marker gene expression and cultured in IMDM supplemented with 5% FBS, 5% human serum and IL-2 (100 IU/ml) (Chiron, Amsterdam, The

Netherlands). T-cells were nonspecifically stimulated every 2 wk with feeder cell mixtures containing 1x106_{/ml irradiated allogeneic}

PBMCs (30Gy), 1x105_{/ml irradiated EBV-transformed B cells (LCLs;}

50 Gy), and PHA (800 ng/ml).

For TCR internalization and re-expression assays differ- ent LCLs were used. LCL-Z and LCL-RZ originate from an HLA- identical sibling pair with HA-2 disparity. LCL-Z is HLA-A2 positive but HA-2 negative, while LCL-RZ is HLA-A2 and HA-2 positive. Both LCLs were transduced with HLA-B7, and pulsed with the EBNA3A peptide in particular experiments. To obtain LCLs presenting endogenously processed pp65, LCL-Z was transduced with the lower matrix protein pp65 of HCMV AD169 (Zpp65). LCLs were maintained in IMDM supplemented with 10% FBS. This study was approved by the Leiden University Medical Center institutional review board.

TCR internalization assay

2x104 _{TCR td and mock td T-cells were plated in 96-well}

U-bottomed microtiter plates. LCL-Z was used as stimulator cell, either unpulsed, or pulsed with 1 µM of pp65 or HA-2 peptide for 1 h at 37ºC. In some assays, HLA-B7 td LCL-Z was pulsed with 1 µM of EBNA3A peptide. At different time points, 2x104 _{LCLs were}

added to the T-cells in a final volume of 200 µl in medium contain- ing 30 IU/ml of IL-2. After a stimulation period of maximal 72 h at 37ºC and 5% CO2, 96-well U-bottomed microtiter plates were spinned down, and T-cells were either tested functionally in a chromium release assay or cells were stained with different mAbs and analyzed for cell surface expression by flow cytometry. Cell

surface expression was calculated as follows: [MFI of T-cells with stimulator / MFI of T-cells without stimulator] * 100.

Cytotoxicity assay

1x104 _{TCR td and mock td T-cells were plated in 96-well}

U-bottomed microtiter plates and stimulated using LCL-Z either unpulsed or pulsed with 1 µM of pp65 or HA-2 peptide. Target cells were labeled with 70 µCi Na251_{CrO4 for 1 h at 37ºC, washed}

three times, and added at different time points after antigen- specific stimulation to the effector cells in a final volume of 150 µl IMDM supplemented with 10% FBS in 96-well U-bottomed microtiter plates, resulting in a 10:1 effector-to-target-ratio. Target cells were pulsed with EBNA3A, pp65 or HA-2 peptide (1 µM) during Na251_{CrO4 labeling. Targets incubated in medium or}

1% Triton X-100 were used for determination of the spontane- ous and maximum release, respectively. The tests were done in triplicate. After 4 h of incubation at 37ºC and 5% CO2, 25 µl of the supernatant was harvested and measured in a luminescence counter (Topcount-NXT, Packard Instrument Company, Meriden, CT, USA). The percentage of specific lysis was defined as [(ex- perimental release – spontaneous release) / (maximum release – spontaneous release)] * 100.

Statistics

Cell surface expression of the TCR chains and complexes, CD3 and CD8 of stimulated TCR and mock transduced virus-specific T-cells were evaluated in a paired fashion by use of the students’

T-test at each time interval (4h, 24h and 48h). Reported P values are 2-sided and were considered statistically different if <0.01.