| Objective: Our study is to investigate the recovery of T cells,CD8 T cells subsets in 12 months after allogeneic hematopoietic stem cell transplantation(Allo-HSCT),to study the relationship with the clinical manifestations,such as the application of Antithymocyte Globulin(ATG),the occurrence of acute graft-versus-host disease(aGVHD)and the CMV infection.The purpose is to provide clinical evidence for the prevention and monitoring of aGVHD and CMV infection after transplantation.Methods:1 A retrospective analysis of the number of 44 patients underwent allo-HSCT from 2014.2 to 2016.12,in the Blood and Marrow Transplanation Center,Department of Hematology,The Second Hospital of Hebei Medical University.2 The clinical condition of patients: Age: the median age of patients was32.5 years old(13-54 years old).Gender: 26 cases were male,18 cases were femal.Diagnosis and staging: 33 cases were AL(23 of them were CR1,8 of them were more than CR2,2 case was NR),4 cases was SAA and 4 cases were MDS(Revised International prognostic scoring system(IPSS-R): all of them were high risk),2 cases were MDS-RAEB?,2 cases were MDS-RAEB?,1 case was atypical chronic myelogenous leukemia(aCML),1case was chronic myelogenous leukemia acceleration period,1 case was Lymphoma cell leukemia(LCL).The deadline of the observation:2016-12-31.The follow-up median time was 350 days(37-1248 days).HLA matched between donor and recipient:16 cases were HLA matched sibling donors,2cases were HLA matched unrelated donors,26 cases were HLA haploidentical related donors.Transplantation mode: haplo/MSD-HSCT use G-CSF primed bone marrow and peripheral stem cells(39/44 cases)or peripheral stemcells(3/44 cases),MUD-HSCT use G-CSF primed peripheral stem cells(2/44cases).Myeloablative conditioning regimen: people with severe aplastic anemia use CTX+ATG+BU±ATG,hematological malignancies use modified BU+CY or BU+CY+ATG/ATG-F.The average number of stem cells: MNC5.88±1.35*10^8/Kg(recipient weight),CD34+ 6.02±2.61*10^6/Kg(recipient weight).3 Control group: twenty-five healthy donors of Allo-HSCT.4 Detection time: recipients: at the time of 28 d,2,3,4.5,6,9,12 months after transplantation,control group: before G-CSF mobilization.5 Detection method: Collection of EDTA anticoagulation peripheral blood 4ml in transplant patients and healthy donors at every observation time point.FACS Cantoll ? flow cytometry was used to detect the changes of T lymphocyte subsets,assisted by Beijing Hester clinical inspection institute.Within 2 months after transplantation,collect EDTA anticoagulation peripheral blood 4ml two times a week,detection of CMV-DNA with PCR.6 Antibody combination: T lymphocyte: CD3,CD8+T lymphocyte:CD3/CD8,naive CD8+T lymphocyte: CD3/CD8/CD45 RA,memory CD8+T lymphocyte: CD3/CD8/CD45 RO.7 Diagnostic criteria: Diagnostic criteria for hematologic diseases are in line with the criteria for diagnosis and treatment of hematologic diseases.aGVHD diagnosis and classification criteria in line with the Seattle Fred Hutchinson Cancer Center classification.Within 100 days after transplantation is early stage of transplantation.CMV-DNA > 2×102 copies /ml for 2 times is CMV infection.CMV positive within 100 days after transplantation is defined as the early stage of CMV infection.8 Statistics analysis: Using two independent samples t-test and Mann-Whitney 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 Clinical information1.1 Implantation condition: Forty-four patients were successfully achieved myeloid reconstitution,of which 2 cases poor platelet engraftment.The median time for neutrophil engraftment was 11 days(range,9-19 days),for platelet recovery was 12 days(range,6-39 days).1.2 Infection and complications: Fifteen cases of pulmonary infection,the incidence is 34.1%;25 cases of infection is CMV and the incidence is 56.82%;one of them developed CMV viral encephalitis;5 of 44 cases have urinary tract infection(11.36%),9 cases of hemorrhagic cystitis,accounts for20.45%;suppurative otitis media 2 cases,the rate is 4.55%;1 case of severe maxillofacial region.26 cases of aGVHD(59.1%),among them,4 patients have III-IV a GVHD(9.1%).1.3 Relapse and death: 7 of 44 cases relapse,3 cases of hematologic relapse,recurrence of genetics account for 3 cases,the other one is extramed?llary relapse;7 cases died(15.91%),of which the primary disease recurrence 2 cases,because of ?-?a GVHD 3(6.82%),1 cases died of systemic inflammatory response syndrome(SIRS)and pulmonary infection,one of all patients died from hepatic ven?lar occlusive disease.1.4 Detection of T cell subsets: All 25 patients in control group were complete collection.At the time of 28 d,2,3,4.5,6,9,12 months after transplantation,the number of patients complete detection of T cell subsets were 40 cases,35 cases,31 cases,27 cases,23 cases,13 cases and 14 cases,separately.2 Recovery of T cells: Compared with the control group,T cells were reduced to the lowest at the 28 th day after transplantation(768.62±495.6/?l vs1747.6±552.17/?l,P<0.001),then gradually rised,3m was still significantly lower(P < 0.001),until 6 months after Allo-HSCT recovered to control level(1673.36±875.54/?l,P=0.348),12 th month the value was still at normal level(1902.31±888.84/?l vs 1747.6±552.17/?l,P=0.976).The results showed that T cell count was recovered at sixth month after transplantation.3 Recovery of CD8+T subgroup cells3.1 CD8+T cells: CD8 T cells recoverd rapidly,and reached the control level at 28 th day after transplantation(549.44±356.47/?l vs 675.60±253.66/?l,P=0.14),3m was still at contral group level.At sixth month,ninth month,twelfth month,CD8+T cells was significantly higher than that of the control group(6m 1171.9±526.84/?l?9m 1011.67±406.69/?l?12m 1214.62±558.4/?l),P values were 0.000,0.004,0.001,respectively.3.2 Naive CD8+T cell: The naive CD8 T cells count recovered slowly after transplantation,at 28 th day(120.3±71.98/?l vs 300.4±125.65/?l,P<0.001),3m had not yet recovered(200.71±99.38/?l vs 300.4±125.65/?l,P=0.004),returned to the normal level at sixth month(287.95±183.61/?l vs300.4±125.65/?l,P=0.787).12 m maintained at normal levels,P>0.05.The res?lts explained that the naive CD8 T cells were stable reconstruction after 6months of transplantation.3.3 Memory CD8+T cell: 28 days after Allo-HSCT,there was no difference in memory CD8+T cells from the control group(288.4±174.45/?l vs258.8±110.73/?l,P=0.828),the count was significantly higher after 2months(487.33±251.86/?l vs 258.8±110.73/?l,P < 0.001),then the count remained stable.The results prompted that the memory CD8 T cells were not significantly reduced after transplantation,and can rapidly exceed the normal level,which may be related to the stimulation of allogeneic antigen.4 Recovery of CD8+T subgroup cells associated with CMV virus infection after early Allo-HSCT.Twenty-five cases of CMV infection in 100 days after transplantation,the median infection time was 35 days(range,10-90 days).CMV infection group CD8+T cells count were higher than the uninfected group at 28 d.Contrarily,were all lower than the uninfected group at second month,third month and 4.5 month,but there were all no statistical differences.The trend of the T cell,naive CD8+T cell and memory CD8+T cell is same as CD8+T cell.Only two months after Allo-HSCT,there was significant difference between the naive CD8 T cell infected group and uninfected group,and the P value is 0.034.It was suggested that the naive CD8 T cell reconstitution delayed may be associated with CMV infection.5 Recovery of CD8+T subgroup cells associated with the occurrence of aGVHD.There were 26 cases with different degrees of aGVHD afterAllo-HSCT.The median time was 27 days(range,12-90 days).22 cases suffered from mild aGVHD,4 cases of severe aGVHD.T cells,CD8 T cells and memory CD8 T cells counts in the aGVHD group were all higher than those in the non-aGVHD group at 28 th day and the second month.The third month,the 4.5th month and the sixth month were lower than non-aGHVD group.And the memory CD8+T cells in 2th month were significantly higher than that in non-aGVHD group(P=0.042).Except for the sixth month,the naive CD8+T cell were all higher in the aGVHD group than in the non-aGVHD group.However,there was no significant difference between the two groups.The results indicated that memory CD8 T cells were significantly increased when aGVHD occurred.Compared with control group,CD8 T cells in 28 d non-aGVHD group was significantly lower than that in control group(P=0.016).At the time of the third month,the number of naive CD8+ T cell in the aGVHD group was no difference between the control group,P values was0.061,but non-aGVHD group was still significantly lower than it(P=0.003).It suggested that the occurrence of aGVHD could accelerate the speed of naive CD8 T cell reconstitution.6 The application of ATG and the CD8+T cell subsets.6.1 Thity-five of 44 cases patients applied ATG in conditioning regimen.Monitoring the recovery of cell subsets at 28 d,2m,3m.There was no difference in T cell and CD8 T cell subsets between ATG group and non-ATG group in the first 3 months after transplantation.The naive CD8 T cells in non-ATG group were restored to the control level in second month(251.25±136.58/?l vs 300.4±125.65/?l,P=0.296),while the ATG group was still lower than that of the control group to 3m(P < 0.05).Compared with the naive CD8 T cells recovered to normal level in 44 patients at +6m,It can be concluded that ATG can inhibit the naive CD8 T cells in the early stage of transplantation.6.2 In the 18 cases treatment of HLA matched group,there were 9 cases applied ATG in conditioning regimen.There was no significant difference between the two groups at the detection time point of the first 6 months whenmentioned the T cells and the naive CD8 T cell count.The values of the CD8+T cells and the memory CD8+T cells in non-ATG group were still higher than that of the ATG group until 4.5 month after Allo-HSCT.Only the memory CD8+T cells in the third month and the 4.5th month after transplantation were statistically different,P value was 0.03,0.006,separately.The rest of the time due to a small number of cases,we did not do statistical analysis.Conclusion:1 The CD8+T cell and memory CD8+T cell recovered rapidly and reached to the normal level at first month after allo-HSCT.T cells and naive CD8+T cells recovered to the level of the control group at sixth month.In the early stage of transplantation,T cells were reconstructed mainly by CD8+T cells,and CD8+T cells were mainly reconstructed by memory T cells.2 The occurrence of aGVHD could significantly increase the memory CD8+T cells and accelerate the speed of naive CD8 T cells reconstitution.3 ATG can inhibit the reconstruction of the naive CD8+T cells in the early stage of transplantation. |
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