Patients and collection of blood samples
Twenty-eight patients undergoing haplo-HSCT using RIC regimens at Shanghai Dao-pei Hospital between November 2012 and January 2015 were prospectively included in this study.
All patients underwent single haplo-HSCT from donors who were HLA-A*0201- or HLA-A*1101-positive. Nine patients were excluded because they relapsed (three cases) or were treated with donor lymphocyte infusion (six cases) within 100 days after transplantation; finally, 19 evaluable patients were enrolled in this study.
The donors for the 19 patients enrolled were positive for HLA-A*0201 (10 cases) or HLA-A*1101 (9 cases), the 28 healthy volunteers (all of them were long-term EBV carriers) carried HLA-A*0201 (9 cases) or HLA-A*1101 (19 cases), and the 49 negative controls were both HLA-A*0201- and HLA-A*1101-negative. Patients were divided into HLA-A*0201 and HLA-A*1101 groups according to the tissue type of their donors. The patients, their donors, healthy long-term EBV carriers, and negative controls all showed negative reactions in an EBV-IgM serum array and positive reactions in an EBV-IgG serum array. Allpatients were 100% donor types by bone punctures and chimerism examinations at + 30, + 60, and + 90 days after transplantation during their lifetimes.
Peripheral blood samples (5 mL) were collected from patients during routine laboratory blood draws. Blood samples from 28 healthy volunteers who were HLA-A*0201- or HLA-A*1101-positive were obtained to identify the baseline level of EBV-CTLs in healthy long-term EBV carriers. Blood samples from HLA-A*0201- and HLA-A*1101-negative volunteers were obtained and used as negative controls. Forty-eight blood samples were obtained from the 19 patients. In the HLA-A*0201 group, blood samples were obtained from five patients gave blood samples at + 30, + 60, and + 90 days; from four patients at + 30 and + 60 days; and from one patient at + 30 days after haplo-HSCT. One patient died at + 43 days because of invasive pulmonary aspergillosis, two patients died at + 74 and + 87 days after haplo-HSCT because of EBV-related diseases/PTLDs, and two patients were lost to follow-up at + 90 days (they did not die, and returned to follow-up at + 120 days). We found that the patient who died at + 43 days was a 100% donor type by bone puncture and short tandem repeat (STR) examination at + 30 days after transplantation. However, at + 33 days, this patient developed hyperthermia, pulmonary infection, and decreased blood cells and was found to have decreased chimerism to 23% of that of the donor recipient at + 37 days. Secondary graft failure was observed, which was thought to be related to the serious pulmonary fungal infection. In the HLA-A*1101 group, blood samples were obtained from six patients at + 30, + 60, and + 90 days and from three patients at + 30 and + 60 days; the other three patients died because of invasive pulmonary aspergillosis (one case), cytomegalovirus (CMV) pneumonia (one case), and EBV-related gastroenteritis (one case).
Assay to detect serum EBV-specific antibody status
The serum EBV-IgM and IgG statuses before HSCT in patients, their donors, the healthy long-term EBV carriers, and negative controls were detected using an anti-Epstein Barr virus (EBV-VCA) IgM human in vitro Enzyme-Linked Immunosorbent Assay (ELISA) kit and an EBV-VCA-IgG human in vitro ELISA kit (EK-Bioscience, Shanghai, China) according to the manufacturer’s instructions.
HLA-A, HLA-B, HLA-Cw, and HLA-DRB1 typing was performed using high-resolution DNA techniques according to the manufacturer’s instructions. The reagents (Special Monoclonal Tray-Asian HLA Class I and Micro SSP HLA Class I and II ABDR DNA Typing Tray; One Lambda, Canoga Park, CA) were commercially imported.
Bone marrow chimerism was detected every month for six months after haplo-HSCT. Chimerism was determined by one of two methods: DNA fingerprinting of STR, and chromosomal fluorescence in situ hybridization (FISH). Chimerism was evaluated by DNA fingerprinting of STR in recipient BM cells in sex-matched donor-recipient pairs; however, in sex-mismatched donor-recipient pairs, chimerism was analyzed by FISH.
Conditioning regimens were as follows: intravenous administration of cytarabine (2.0 g/m2) once daily on days − 13 to − 12, fludarabine (30 mg/m2) once daily on days − 11 to − 7, busulfan (0.8 mg/kg, every 6 h) on days − 6 to − 4, and ATG (Fresenius, 5 mg/kg) once daily on days − 5 to − 2 (11 patients); or intravenous administration of cytarabine (2.0 g/m2) once daily on days − 14 to − 13, fludarabine (30 mg/m2) once daily on days − 12 to − 8, thiotepa (125 mg/m2) once daily on days − 8 to − 5, and ATG (Fresenius, 5 mg/kg) once daily on days − 5 to − 2 (eight patients).
For GVHD prophylaxis and management, each patient was administered cyclosporine A (CSA, 2.5 mg/kg twice daily, intravenously), a short course of methotrexate (15 mg/m2 once daily, intravenously, on day + 1 and 10 mg/m2 on days + 3 to + 5), and oral mycophenolate mofetil (7.5 mg/kg twice daily) from days + 1 to + 14 as prophylaxis for GVHD. CSA was withdrawn if the patients showed viral reactivation, including CMV or EBV, and did not have GVHD at that time. Methylprednisolone (0.25 mg/kg/day) was administered if patients developed aGVHD < grade II. Only two patients suffered from grade III aGVHD, which occurred before CSA was tapered.
Collection of hematopoietic stem cells was performed as follows: donor marrow and peripheral blood mobilized using granulocyte-colony stimulating factor (7.5 μg/kg/day, on day − 4 to day 02) were harvested on day 01 and 02, respectively.
Supportive care was similar for all patients. All blood products were irradiated and leukocyte-depleted. Antifungal prophylaxis was routinely administered with caspofungin during the conditioning process and voriconazole was administered after transplantation. Pneumocystis prophylaxis, typically trimethoprim-sulfamethoxazole, was administered until 6 months post-transplantion. Acyclovir, typically 400 mg orally twice per day, was administered until 24 months post-transplantation.
Monitoring of EBV reactivation in peripheral blood and definition of EBV-related diseases
The EBV-DNA copy number was determined by quantitative PCR (qPCR) using peripheral blood samples or biopsy specimens. Briefly, genomic DNA was isolated from 250 μL whole blood or digestive tissues with an Axyprep Mag tissue-blood genomic DNA kit (Axygen, Union City, CA,USA) according to the manufacturer’s instructions. qPCR was performed on an ABI 7300 thermal cycler using commercially available PCR kits for EBV (Daan Gene Technology, Guangzhou, China) and the results were shown as DNA copies/mL of whole blood, with a detection threshold of 500 copies/mL for EBV.
EBV-DNA copy numbers were monitored weekly during the first 30 days after haplo-HSCT and subsequently every other week until 3 months after transplantation or until EBV-DNA became undetectable in the peripheral blood.
EBV-DNAemia was defined as EBV-DNA loads of more than 500 copies/mL at two consecutive time points, without any signs or symptoms of EBV-related diseases, including probable and proven PTLDs. Probable PTLDs were defined as significant lymphadenopathy, hepatosplenomegaly, or other end-organ manifestations, accompanied by a high EBV-DNA blood load and the absence of tissue biopsy and other documented causes . Proven PTLDs were diagnosed as detection of EBV nucleic acids or EBV-encoded proteins in a tissue specimen, together with symptoms and/or signs from the affected organ .
MHC class I-peptide pentameric complexes (pentamers) and reagents
Two EBV peptide epitopes were used: the HLA-A*0201-restricted epitope, GLCTLVAML, derived from the lytic cycle protein BMLF1 (amino acids 259–267), and the HLA-A*1101-restricted epitope SSCSSCPLSK, derived from the latent cycle protein, LMP2 (amino acids 340–349). The pentamers, HLA-A*0201/BMLF1-GLC and HLA-A*1101/LMP2-SSC, were purchased from ProImmune, Ltd. (Oxford, UK). Pentamers were labeled with phycoerythrin (PE). Monoclonal antibodies, including anti-CD3 (peridinin chlorophyll protein-[PerCP]), anti-CD8 (fluorescein isothiocyanate-[FITC]), and anti-CD19 (allophycocyanin-[APC]), were purchased from BD Biosciences (Franklin Lakes, NJ, USA). The experimental protocol and operation were recommended by the Pentamer Handbook from ProImmune, Ltd. (Oxford, UK). According to the protocol that cells obtained from a mismatched pentamer (irrelevant MHC allele and/or irrelevant peptide). can be used to control non-specific staining, and that exclusion of B cells is likely to reduce most of the non-specific background, we used both HLA-0201- and 1101-negative adult volunteers as negative controls and added anti-CD19 monoclonal antibody to remove B lymphocytes. Briefly, forward and side scatters were used to gate viable populations of cells, and CD3+CD19− cells were then gained to delete B lymphocytes. Next, CD3+CD19−CD8+ cells were gained as CD8+ T lymphocytes, and finally, pentamer-stained CD3+CD8+CD19− cells were gained as target cells for FACS analysis. The percentage of EBV-CTLs was calculated as follows: percentage EBV-CTLs = (percentage EBV-CTLs in patients or percentage EBV-CTLs in healthy long-term EBV carriers (healthy controls) – percentage EBV-CTLs in negative controls) × 100%.
Blood samples were collected from patients at + 30, + 60, and + 90 days after haplo-HSCT, whereas blood samples were collected only once from healthy volunteers as HLA-A*0201- or HLA-A*1101-positive and negative controls. We evaluated the performance of HLA-A*0201/BMLF1-GLC and HLA-A*1101/LMP2-SSC pentamers based on the percentages of EBV-specific CD8+ T cells by four-color flow cytometry. All experiments were performed in triplicate.
Pentamer staining assays
FACS lysing solution (BD Biosciences) was added to the blood samples to lyse the erythrocytes. Cells were washed twice and resuspended in phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS) and 0.1% sodium azide. The number of nucleated cells was counted under a microscope and adjusted such that each tube contained 1–2 × 106 nucleated cells. Titrated pentamers were then added to the samples. After incubation for 10 min at room temperature (25 °C) without light, titrated monoclonal antibodies (PerCP-, FITC-, or APC-conjugated CD3, CD8, or CD19, respectively) were added for surface staining. After incubation for 20 min at 4 °C without light, the cells were washed twice with PBS containing 1% FCS and 0.1% sodium azide and then stored in 1% paraformaldehyde at 4 °C. Samples were analyzed on a FACS Calibur instrument (BD Biosciences) within 6 h. At least 5000 live CD3+ lymphocytes were counted by flow cytometry. CellQuest (BD Biosciences) was used for analysis. The frequencies of pentamer-binding CD8+ T cells were calculated based on the percentages of CD3+CD8+CD19− T cells that bound to the pentamers. Blood samples from HLA-A*0201- and HLA-A*1101-negative patients were used as negative controls.
Statistical analysis was performed using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA). Quantitative data with normal distributions were expressed as the mean ± standard deviation. Continuous variables and quantitative data without normal distributions were expressed as the median and range. The percentages of pentamers between two groups were compared using independent sample t tests. Wilcoxon rank-sum tests were used to compare the frequencies of HLA-A*0201/BMLF1-GLC pentamers at + 60 days between patients with and without EBV-related diseases. Analysis of variance was used to compare the percentages of pentamer-reactive cells among multiple groups. All analyses were two-tailed and p < 0.05 was considered significant.