The patient studied in this report is a 44-yr-old male diagnosed with acute myeloid leukemia (M2 subtype) in December 2000. He responded to induction chemotherapy and achieved complete remission before undergoing allogeneic HSCT. In April 2001 he received a myeloablative preparative regimen consisting of 120 mg/kg cyclophosphamide and 1,400 cGy total body irradiation followed by infusion of G-CSF-mobilized blood stem cells from an HLA-identical female sibling. He remains in complete remission 2 yr after HSCT. His prophylactic immunosuppressive treatment included tacrolimus and methotrexate. 5 wk after transplantation, he developed acute GVHD of the skin (grade 2 GVHD). Acute GVHD responded to treatment with prednisone, daclizumab, and mycophenolate mofetil but did not resolve completely. He was subsequently treated for limited chronic GVHD with involvement of skin and oral mucosa with a combination of mycophenolate mofetil, tacrolimus, and prednisone. Chronic GVHD persists 2 yr after transplantation and the patient remains on immunosuppressive drugs.
Synthetic peptides were obtained from New England Peptide. Peptides were either unpurified (93 screening peptides), or >85% purity (DBX34-47, DBY34-47, DBX30-48, DBY30-48, DBX120-136, DBY118-134, DBX538-554, DBY536-552). Each peptide was reconstituted at 10 mg/ml DMSO and stored at -20 C.
Patient and allogeneic EBV-transformed B cell lines were generated by incubating PBMCs with supernatant from EBV-producing B95-8 marmoset cells. Once established, cell lines were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 4 mM glutamine, 1 mM sodium pyruvate, 10 mM Hepes, and antibiotics. T cell lines and clones were cultured in RPMI 1640 supplemented with 10% heat-inactivated human AB serum, glutamine, sodium pyruvate, Hepes, and antibiotics (referred to as T cell medium) in the presence of 100 U/ml recombinant IL-2. Medium was replenished twice weekly with fresh IL-2. Immature DCs were obtained as previously described. In brief, 40-60 x 106 PBMCs were incubated for 15 min in a 75-cm2 Costar culture flask (Corning Inc.). Nonadherent cells were removed by thoroughly washing the flask three times with medium. The remaining adherent cells were incubated for 1 wk in medium supplemented with 1,000 U/ml IL-4 (R&D Systems) and 1,000 U/ml GM-CSF (R&D Systems). Cells were then harvested, washed, and used as immature DCs. Maturation of DCs was achieved by incubating cells for 24 h with 25 ng/ml poly-IC (Amersham Biosciences) or 100 ng/ml LPS (Sigma-Aldrich).
A T cell line was generated by stimulating 6 x 106 PBMCs collected from the patient 16 mo after HSCT in the presence of peptide DBY34-50 at a final concentration of 25 mug/ml in the presence of 100 U/ml IL-2. 1 wk after stimulation, CD4+ T cells were immunopurified from the cell line using anti-CD4+ microbeads (Miltenyi Biotec) and subsequently cloned by limiting dilution on feeder cells consisting of irradiated patient EBV B cells (1.5 x 104 cells/well; 60 Gy) and allogeneic PBMCs (7 x 104 cells/well; 35 Gy) in the presence of 100 U/ml recombinant human IL-2. All of the T cell clones generated were assessed for specific reactivity with the DBY peptide. Clone 2F9 displayed strong reactivity and was further expanded under the same culture conditions with the addition of PHA (Invitrogen).
ELISPOT assays were performed as previously described. In brief, 1-2 x 105 PBMCs were incubated in duplicate cultures in the presence of 10 mug/ml different peptides in multiscreen-IP microplates (Millipore) previously coated with anti-IFN-gamma mAb (Mabtech). After an overnight incubation, plates were washed six times in PBS and incubated with a biotinylated anti-IFN-gamma mAb (Mabtech) at room temperature for 75 min. The plates were washed again and incubated with streptavidin alkaline phosphatase (Mabtech) for 45 min at room temperature. The plates were finally washed and spots were revealed using nitroblue tetrazolium and brom-chlor-indolyl phosphate color development substrates (Promega). Spots were counted in duplicate wells and results were presented as number of spot forming cells/106 PBMCs.
IFN-gamma secretion by T cell clone 2F9 was assessed by incubating 5 x 104 responder cells in 96-well, U-bottom microplates with 5 x 104 stimulator cells in 200 mul T cell medium containing 50 U/ml IL-2. Stimulator cells consisted of autologous or allogeneic EBV B cells pulsed with synthetic peptides (10 mug/ml or as otherwise stated) for 1 h at 37 C and washed twice to remove excess peptide. After 18 h of incubation, 50 mul/well supernatant was harvested and IFN-gamma was measured using an ELISA kit (Endogen) according to the manufacturer's instructions. In experiments with DCs, 10,000 immature DCs/well were incubated in 100 mul medium with or without LPS or poly-IC for 24 h at 37 C. 4 x 104 2F9 cells were then added to the culture in the presence of 50 U/ml IL-2, and secretion of IFN-gamma was measured after 18 h of incubation.
Target cells were labeled with 51Cr for 1 h at 37 C, washed three times, pulsed with the corresponding peptide for an additional hour at 37 C, washed three times again, and then plated at 2,000 cells/well in conical-bottom, 96-well microplates. Clonal 2F9 cells were added at varying effector/target ratios and 51Cr release was measured in the supernatant after 4 h of incubation at 37 C. In cold target inhibition experiments, HLA-DR*1501 female or male unlabeled matured DCs were incubated with 51Cr-labeled targets at various ratios for 1 h before adding the T cell clone.
Recombinant DBX and DBY protein production and purification has been described. In brief, full-length DBX and DBY cDNA were cloned using the Gateway cloning system (Invitrogen) into pDEST42 prokaryotic expression vector introducing V5 and 6 HIS Tag epitopes at the protein C terminus. After transformation in Escherichia coli, DBX and DBY expression was induced with isopropyl-beta-d-thiogalactopyranoside and proteins were purified from bacterial lysates by affinity chromatography on nickel columns (Invitrogen). For Western blotting, 5 mug of each protein was run by electrophoresis on a 4-12% polyacrylamide gel in denaturing conditions using the NuPAGE system (Invitrogen), transferred on a nitrocellulose membrane, and blocked for 1 h in blocking buffer (1X TBST, 5% nonfat dry milk). Membranes were incubated overnight with either horseradish peroxidase (HRP)-conjugated anti-V5 epitope mAb (Invitrogen) or patient serum diluted at 1:10 in blocking buffer, and then with HRP-conjugated goat anti-human IgG antisera (Jackson ImmunoResearch Laboratories). Proteins were subsequently detected using the "Western Lightning" chemiluminescence HRP reagents (PerkinElmer).
Antibodies to DBX and DBY recombinant protein, as well as DBX and DBY peptides, were measured by ELISA. In brief, high protein adherence Nunc-Immunoplates (Nunc) were coated overnight with either 5 mug/ml purified recombinant protein or 10 mug/ml synthetic peptides in carbonate-binding buffer, washed in TBST, and then blocked with 2% nonfat dry milk in TBST. Serum diluted in blocking buffer was then added to the plates and incubated either overnight at 4 C for the recombinant protein-coated plates, or for 3 h at 20 C for the peptide-coated plates. Plates were then washed in TBST, incubated for 1 h at 20 C with alkaline phosphatase-conjugated goat anti-human IgG antisera (Pierce Chemical Co.), diluted in blocking buffer, washed again, and developed using pNPP alkaline phosphatase substrate colorimetric reagents (Sigma-Aldrich). Optical density was read at 405 nm.