HLA-DP4 expression and immunity to NY-ESO-1: correlation and characterization of cytotoxic CD4+ CD25- CD8- T cell clones

Patients

We typed 102 patients with metastatic NY-ESO-1-expressing cancers of different kinds for the presence of the HLA-DP4 allele and tested them for detectable NY-ESO-1 serum antibody using Western blot assays. Of these, four HLA-A2+ DP4+ patients were selected for the evaluation of NY-ESO-1-specific CD4+ and CD8+ T cell responses. At the time lymphocytes were collected for analysis, two patients (melanoma patient NW1454 and non-small cell lung cancer patient NW1662) had detectable NY-ESO-1 serum antibody and NY-ESO-1-specific CD8+ T cell reactivity, and two patients (melanoma patient NW1045 and ovarian cancer patient NW1691) were NY-ESO-1 antibody negative.

Expression of NY-ESO-1 in tumor tissues

Expression of NY-ESO-1 mRNA in tumor specimens frozen immediately after biopsy at -80°C was assessed by RT-PCR, using PCR conditions and primers as previously described (1).

NY-ESO-1 serum antibody

NY-ESO-1 serum antibody was assessed by standard Western blot, using NY-ESO-1 recombinant protein purified from Escherichia coli as described (7, 13).

Cell lines

EBV-B cells were derived from PBMCs obtained from individuals of different HLA types using culture supernatant from the EBV-producing B95-8 cell line. EBV-B cell lines and the mutant TAP-deficient lymphoblastoid cell line T2 (CEMx721.174.T2) were maintained in RPMI 1640 medium supplemented with 10% FCS, L-asparagine (50 mg/l), L-arginine (24 mg/l), and L-glutamine (300 mg/l) as described (13). The EBV-B cell lines MZ070782 and DUCAF were a kind gift from Dr. Ghislaine Sterkers, Hopital Robert Debre, Paris, France.

The NY-ESO-1-expressing melanoma cell lines NW-MEL-38, NW-MEL-450, NW-MEL-634, SK-MEL-29, Mel624, and SK-MEL-37, and the NY-ESO-1-negative melanoma cell line NW-MEL-8, were established from surgically resected melanoma metastases and cultured in DMEM (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% FCS, L-asparagine (50 mg/l), L-arginine (24 mg/l), and L-glutamine (300 mg/l) as described (13). For the cytotoxicity assay, the tumor cells were pretreated with IFN-gamma (100 U/ml, Bioferon, Laupheim, Germany) for 24 h.

Peptides

The HLA-DP4-restricted NY-ESO-1 peptide p157-170 (SLLMWITQCFLPVF) was selected to analyze the CD4+ T cell response to NY-ESO-1 in HLA-DP4+ patients (31). Two HLA-A2-restricted NY-ESO-1 peptides, p157-167 (SLLMWITQCFL) and p157-165 (SLLMWITQC), were selected to analyze the CD8+ T cell response to NY-ESO-1 (13). The HLA-A2-binding Melan A peptide p29-37 (GILTVILGV) was included for control purposes. All peptides were synthesized by Multiple Peptide Systems (San Diego, CA, USA) with a purity of >90%, as determined by reverse-phase HPLC.

Viral constructs

Adenovirus encoding the full-length NY-ESO-1 cDNA (Ad2/ESO) was provided by Genzyme Corporation (Framington, MA, USA), and v.v. WT and v.v. ESO were provided by Therion Biologics (Cambridge, MA, USA). Viral constructs were used for the preparation of NY-ESO-1-expressing APCs as described (14).

Presensitization of CD4+ and CD8+ effector T cells

CD4+ and CD8+ T lymphocytes were separated from PBMCs by antibody-coated magnetic beads (MiniMACS, Miltenyi Biotec, Auburn, CA, USA) and seeded into 24-well plates (Greiner) at 5 x 10⁵ cells/well in RPMI 1640 medium supplemented with 10% human serum and L-glutamine (300 mg/l). PBMCs depleted of CD4+ and CD8+ T cells were either pulsed with peptide (10 µg/ml) or infected with recombinant Ad2/ESO at 1000 infection units/cell and used as APCs. These APCs were then irradiated and added to plates containing CD4+ or CD8+ T cells at a concentration of 1 x 10⁶ cells/well. IL-2 (2.5 ng/ml, Biotest, Dreieich, Germany) and IL-4 (10 U/ml, Strathmann Biotech, Hannover, Germany) were added to CD4+ T cell cultures, and IL-2 (2.5 ng/ml) was added to CD8+ T cell cultures (16).

Generation of NY-ESO-1-specific CD4+ T cell clones

NY-ESO-1-specific CD4+ T cell clones were obtained from limiting dilution cultures. Selected CD4+ T cells were stimulated with irradiated autologous APCs pulsed with the HLA-DP4-restricted NY-ESO-1 peptide p157-170 (10 µg/ml) in the presence of irradiated HLA-DP4+ EBV-B cells as feeder cells, and recombinant interleukin-2 (rIL-2) at 10 ng/ml and rIL-4 at 10 U/ml. Clones were maintained by weekly stimulation with irradiated HLA-DP4+ EBV-B cells pulsed with NY-ESO-1 peptide p157-170 (10 µg/ml) and supplemented with rIL-2 and rIL-4 (10 ng/ml and 10 U/ml, respectively).

Antigen-presenting target cells

DCs were generated from plastic-adherent peripheral blood monocytes that were cultured for 6 d in the presence of IL-4 (1000 U/ml) and GM-CSF (1000 U/ml, Leukomax, Sandoz, Nurnberg, Germany). On day 6 of in vitro culture, APCs were treated with IL-4 (1000 U/ml), IL-6 (1000 U/ml), IL-1-beta (10 ng/ml), TNF-alpha (10 ng/ml, all obtained from Pharma Biotechnologie, Hannover, Germany), GM-CSF (1000 U/ml), and prostaglandin/E2 (1 µg/ml, Sigma Chemical, St. Louis, MO, USA).

PHA-transformed blast cells were generated by stimulation of CD4+ selected T cells with PHA (1 µg/ml), and maintained in culture by repeated addition of IL-2 (10 ng/ml/wk).

DC or PHA blasts were either infected with Ad2/ESO or v.v. ESO at 1000 infection units/cell at 37°C in 250 µl serum-free medium (X-VIVO-15, BioWhittaker, Walkersville, MD, USA), or pulsed with peptides at 10 µg/ml, with recombinant proteins at 20 µg/ml, or with tumor cell lysates prepared by three freeze-thaw cycles of 1 x 10⁶ tumor cells, and cultured for 24 h. APCs were then washed twice and used as targets in ELISPOT assays at 3 x 10⁴ cells/well.

ELISPOT assay

Flat-bottomed, 96-well nitrocellulose-lined plates (Millipore MultiScreen, Millipore, Bedford, MA, USA) were coated with IFN-gamma mAb (2 µg/ml, 1-D1K, MABTECH, Stockholm, Sweden) and incubated overnight at 4°C. After washing with PBS, plates were blocked with 10% human AB serum for 1 h at 37°C. Typically, 2.5 x 10⁴ CD4+ or CD8+ presensitized effector T cells (see above) were incubated with 5 x 10⁴ target cells, which were pulsed with NY-ESO-1 peptide, recombinant protein, NY-ESO-1+ tumor cells lysates, or transduced with the viral vector constructs Ad2/ESO or v.v. ESO. Effector and target cells were incubated for 20 h in RPMI medium 1640 supplemented with 10% human serum. After incubation, the plates were thoroughly washed with 0.05% Tween 20 in PBS to remove cells, and biotinylated secondary IFN-gamma mAb (0.2 µg/ml, 7-B6-1-biotin, MABTECH) was added to each well. After incubation for 2 h at 37°C, the plates were washed and developed with streptavidin-alkaline phosphatase (1 µg/ml, MABTECH) for 1 h at room temperature. After washing, substrate (5-bromo-4-chloro-3-indolyl phosphate nitroblue tetrazolium, Sigma) was added and the plates incubated for 5-10 min. After washing, the dark-violet spots were counted under the microscope (14, 16).

Cytotoxicity assay

Cytotoxicity against NY-ESO-1 peptide-pulsed NW1539 EBV-B cells and tumor cell lines before and after treatment with IFN-gamma was determined in standard chromium release assays as described (32).

Immunophenotyping of CD4+ T cell clones

The expression of cell surface markers was determined by FACS® analysis using FITC-conjugated anti-CD4, anti-CD8, anti-CD25, anti-CD69, and anti-CTLA4 antibodies (Beckman Coulter, Marseille, France). FITC-conjugated antimouse IgG1 was used as a negative control. 1-5 x 10⁵ CD4+ T cells were labeled with the appropriate antibody in PBS for 20 min (4°C), washed, and analyzed using a FACS® (Epics XL, Beckman Coulter).