| Objective: To research the restoration of natural killer cells after allogeneic hematopoietic stem cell transplantation. At the same time, to analysis the correlation of KIR genotypes of NK cell to the clinical manifestations,such as the incidence of acute graft versus host disease and the overall survival, etc.Methods:1 A number of 38 patients underwent allo-HSCT from 2014-2 to 2015-12, in the Blood and Marrow Transplantation Center, Department of Hematology, The Second Hospital of Hebei Medical University. 2 The clinical condition of patients. 2.1 The recovery of NK cells: Age: the median age of patients was 31.5 years old(3-56 years old).Gender:17 cases were male, 21 cases were femal. Diagnosis and staging: 22 cases were AL( 16 of them were CR1, 5 of them were ? CR2,1 of them were NR), 6 cases were MDS(4 cases were MDS-RAEB?,2 cases were MDS-RAEB?, Revised International prognostic scoring system(IPSS-R): all of them were high risk,all of them were NR),2 cases were Lymphoma cell leukemia(LCL),7 cases were SAA,1 case was a CML.The deadline for research(2015-12-31).HLA match for donor/recipient:2 cases were MUD HLA10/10,17 cases were MSD HLA 10/10,19 cases were haplo-HSCT. Transplantation mode: 2 cases underwent MUD, 17 cases underwent MSD, 19 cases underwent haplo-HSCT. Unrelated donors used peripheral stem cells, haplo-HSCT/MSD used bone marrow and peripheral stem cells(31/36)/peripheral stem cells(5/36). Control group: 25 cases healthy donors. The average number of stem cells:MNC 5.84 ±136*108/Kg, CD34+ 7.03±3.62*106/Kg(Recipient weight).2.2 The study of KIR: Age: the median age of patients was 32.5 years old(3-52 years old). Gender: 14 cases were male, 14 cases were femal. Diagnosis and staging: 17 cases were AL, 4 cases were MDS, 1 cases were Lymphoma cell leukemia(LCL), 5 cases were SAA, 1 case was a CML. The deadline for research(2015-12-31). HLA match for donor/recipient: 2 cases were MUD HLA10/10, 17 cases were MSD HLA 10/10,19 cases were haplo-HSCT. Transplantation mode: 2 cases underwent MUD, 10 cases underwent MSD, 16 cases underwent haplo-HSCT. Unrelated donors used peripheral stem cells, haplo-HSCT/MSD used bone marrow and peripheral stem cells(21/26)/peripheral stem cells(5/26). the average number of stem cells: MNC 6.17±1.31*108/Kg, CD34+ 7.64±3.28*106/Kg(Recipient weight). 2.3 Diagnosis disease: Disease diagnosis standard reference “blood disease diagnosis and curative effect of standard”[1]. Diagnosis and classification standard of a GVHD conform to Seattle's Fred Hutchinson cancer center classification method[2]. The definition of early virus infection after transplantation: within 100 days after allo-HSCT, testing patients' peripheral blood copy number of virus(concluding EBV-DNA?CMV-DNA?JCV-DNA?BKV-DNA, etc.) when the copy number was greater than 1.0×10^3, we thought there was virus infection. 3 Sample collection: before transplantation and at the time of +1, 2, 3, 4.5, 6, 9, 12 months after transplantation,collected patients' EDTA anticoagulated peripheral blood 4ml.The control group collected peripheral blood before stem cell mobilization. 4 FACS Cantoll ? flow cytometry was used to analyse immune cell subsets' changes. Antibody combination: NK cells(CD16+CD56), B cells(CD19), Tlymphocytes(CD3, CD3+CD4, CD3+CD8). 5 The detection of KIR genotypes for donor/recipients used sequence specific primer polymerase chain reaction(PCR- SSP). The “shanghaidishuobeiken medical examination center” assisted us to detect it. 16 types of KIR genotypes were contained.6 Statistics: Applying two independent sample t-test to compare two group measurement date. Count date comparison between groups used chi-square test. When P<0.05, we thought that difference was statistically significant.Results:1 Recovery of NK cells : CD16+CD56+NK cells count recovered rapidly after allo-HSCT, at 1 month after transplantation recovered to the same level as that in control group(448.29±343.36/ul vs 329.32±174.58/ul, P=0.119). 2 Recovery of NK cells and a GVHD: At 1 to 3 months after transplantation, CD16+CD56+ NK cells in patients with a GVHD were significantly lower than that in those without a GVHD(1m 222.60±148.45/ul vs 581.06 ± 358.64/ul; 2m 199.70 ± 139.48/ul vs 474.76 ± 267.28/ul; 3m 143.88±80.59/ul vs 333.50±180.32/ul). P =0.010, 0.002, 0.003, respectively. Difference was statistically significant. 3 Recovery of NK cells and the relapse: Among 38 patients, 6 cases of them relapse(18.4%). 4 cases of them monitored NK cells count to 3 months. NK cells in relapse group were lower than the NK cells in patients without relapse(1m 205.00±182.18/ul vs 555.90±337.03/ul; 2m 244.00±89.61/ul vs 402.18±281.19/ul; 3m 165.00±61.37/ul vs 281.11±181.86/ul). P =0.040, 0.039, 0.043, respectively. Difference was statistically significant. But at the following month, we did not analysis it because there was too little data. 4 Recovery of NK cells and virus infection: 12 patients in 38 patients with virus infection, NK cells count in patients with virus infection group was obviously lower than the group of patients without virus infection(1m 269.42±257.35/ul vs 644.73±309.92/ul; 2m 189.83±130.74/ul vs 519.33±251.10/ul; 3m 143.40±83.42/ul vs 365.50±170.84/ul).P=0.020, 0.000, 0.001, respectively. There was statistically significant. 5 KIR genotype for donor/recipient and a GVHD. In the 28 patients,who had received allo-HSCT,(1) 64.3%(18/28) of donor/recipient KIR were completely identical,(2) 17.9%(5/28) were the recipients KIR genotype containing the donors,(3) 14.3%(4/28) were the donors KIR containing the recipients,(4) 3.5%(1/28)were completely different.The graft versus host (GVH) direction KIR-matched group was 82.1%(23/28), while the KIR-mismatched group shared only 17.9%(5/28). In patients from KIR-matched group, the incidence of a GVHD was 60.9%(14/23), and in patients from KIR-mismatched group, the incidence of a GVHD was 0%(P=0.020). There was statistically significant. The OS rate of the two groups were 69.6%(16/23) and 80%(4/5), respectively(P=0.552). There was no statistically significant. 6 KIR genotype and a GVHD. Among 28 patients, the group donor A/A and group donor B/X the incidence of a GVHD was 65%(13/20)vs12.5%(1/8) and the OS rate was(65%(13/20) vs87.5%(7/8))(P=0.016, P=0.240). The former had statistically significant, but the OS had not. 7 28 patients who had received allo-HSCT, the rate of a GVHD of the group donor KIR-2DS1+ was 14.28%(1/7), the group donor KIR-2DS1-a GVHD incidence was 61.9%(13/21)(P=0.038). The difference was statistically significant. The OS rate of the two groups were 85.7 %(6/7) and 66.7%(14/21), respectively(P=0.327). There was no statistically significant difference.Conclusion: 1 CD16+CD56+ NK cells recovered to normal at 1 month after allo-HSCT. 2 Patients who had acute GVHD, NK cells recovered more slowly than those who had non-a GVHD. 3 NK cells recovered much later in patients with virus infection which we detected than those patients without virus infection. 4 The GVH direction KIR-matched group had lower incidence of a GVHD than the KIR-mismatched group. 5 The incidence of a GVHD of the group donor B/X was lower than the group donor A/A. 6 The group donor KIR-2DS1+ had lower incidence of a GVHD than group donor KIR-2DS1-. |
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