Patients and donors
The study was approved by the Ethics Committee at Beijing Friendship Hospital approved by the ethical committee of the Beijing Friendship Hospital. Written, Informed consent was obtained from each patient and/or their family or guardian before the treatment began.
A retrospective case-control study of refractory EBV-HLH patients with GPBSCs infusion from HLA-mismatched donors after chemotherapy (as GPBSCs group) and sole chemotherapy (as control group) was performed. The patients of control groups were randomly selected from refractory EBV-HLH patients who underwent salvage therapy during the same period. Patients who enrolled in this study fulfilled the following criteria: (1) patients satisfied HLH-2004 criteria at time of initial diagnosis (the NK cell activity was tested by flow cytometry, ZL 201610013454.1) ; (2) high values for EBV-DNA copies in the peripheral blood (>5.0*102 copies/ml) (tested with PCR assay); (3) primary HLH was excluded by HLH-related defective gene proteins PRF, GrB, XIAP, SAP, Munc13-4, Munc18-2, STX11, Rab27a, ITK, CD27, AP3B1 and whole genome sequencing if necessary, and lymphoma was excluded by repeated pathological biopsy of the focal area if there is an abnormal increase in FDG activity tested by positron emission tomography-computed tomography (PET-CT), and bone marrow biopsy; (4) treated with HLH-94 regimen (etoposide 150 mg/m2 twice weekly for 2 weeks and then weekly) and dexamethasone (initially 10 mg/m2 for 2 weeks followed by 5 mg/m2 for 2 weeks, 2.5 mg/m2 for 2 weeks, 1.25 mg/m2 for one week, and one week of tapering)  no less than 2 weeks before enrollment and did not achieve at least PR; (5) Patients are unable to perform allo-HSCT at that time point due to lack of donors, financial, patient and/or families’ refusal, and hesitating or other reasons such as economic limitation.
EBV infection was confirmed by identifying significantly increased EBV-DNA copies in the peripheral blood. In the absence of accepted diagnostic criteria, refractory HLH was defined according to previous research findings  and our clinical experience as failure to achieve at least PR according to an evaluation 2 weeks after receiving HLH-94 induction therapy.
Before the GPBSCs infusion, the HLA matching of donors and recipients was tested. The HLA matching of the GPBSCs infusion group was HLA-Mismatched with relative donors. All the donors were haplo-identical and at least 5/10.
Two kinds of treatment methods for patients: (1) GPBSCs group: GPBSCs infusion from HLA-mismatched donors after chemotherapy, the infusion of the cells was performed at 36 hours after chemotherapy regimen on day 0; (2) control group: only with chemotherapy. Chemotherapy regimen includes the salvage therapy: DEP regimen (liposomal doxorubicin 25 mg/m2 day 1; etoposide 100 mg/m2 was administered once on the first day of every week; methylprednisolone 15 mg/kg days 1 to 3, 2 mg/kg days 4 to 6, 1 mg/kg days 7 to 10, 0.75 mg/kg days 11 to 14, 0.5 mg/kg days 15 to 21, and 0.4 mg/kg days 22 to 28) and L-DEP regimen (PEG-aspargase 2000 U/m2 on day 5; liposomal doxorubicin (doxorubicin hydrochloride liposome injection) 25 mg/m2/day, day 1; etoposide 100 mg/m2/day on the first day of every week; and methylprednisolone 15 mg/kg/day for days 1 to 3, 0.75 mg/kg/day for days 4 to 7, and 0.25 mg/kg/day for days 8 to 10).
Mobilization and apheresis of donor peripheral mononuclear cells
Apheresis of HLA-mismatched donor peripheral mononuclear cells was carried out after the donor was subcutaneously injected with 5ug/kg G-CSF twice a day for 5 days. Donor cells were divided into aliquots and were cryopreserved in liquid nitrogen, but freshly collected cells were used in the first course.
Detection of Donor Chimerism and Donor Microchimerism
All patients in the GPBSCs infusion group were assessed for donor chimerism after infusion. Eight patients were evaluated for donor microchimerism at least 1 week and 2 weeks after infusion.
Evaluation criteria and observed indicators
Efficacy was evaluated 2 and 4 weeks after initiating therapy, according to the criteria proposed by Marsh et al. Complete response (CR) was defined as the normalization of all quantifiable symptoms and laboratory markers of HLH, including levels of soluble CD25, ferritin, and triglyceride; hemoglobin levels; neutrophil and platelet counts; and alanine aminotransferase (ALT) levels. Partial response(PR)was defined as improvement in two or more of the following quantifiable symptoms and laboratory markers by 2 weeks: 1.5-fold decrease in soluble CD25 response; ferritin and triglyceride decreases of at least 25%; an increase of at least 100% to >0.5*109/L in patients with an initial neutrophil count of <0.5 *109/L; an increase by at least 100 % to >2.0*109/L in patients with an initial neutrophil count of 0.5 to 2.0 × 109/L; and a decrease of at least 50% in patients with initial ALT levels >400 U/L. Additionally, the subject's body temperature had to have reverted to normal ranges for either CR or PR to be diagnosed. Failure to achieve PR was defined as no response.
The observational indicators included symptoms and laboratory findings which were indicted in evaluation criteria. EBV-DNA copies in peripheral blood were also observed.
Survival times were calculated from the date of diagnosis of refractory EBV-HLH. All patients were followed up until death or 1 March 2018, whichever occurred first.
SPSS 22.0 (IBM, USA) statistical software was adopted, data that fit a normal distribution are presented as average± standard deviation, and those that did not are presented as median and range. T-test (two-sided) was used for data that fit a normal distribution and homogeneity of variance, and Wilcoxon rank sum test was used for others. Kaplan–Meier survival curves were used to analyze the patients’ survival and the log-rank test to evaluate survival time. P<0.05 was considered to denote a significant difference. Sample size calculation using PASS 15 Power Analysis and Sample Size Software (2017, NCSS, LLC. Kaysville, Utah, USA) with the β=0.1 and α=0.05 (two independent proportions).