非血缘脐血移植后CMV感染患者NK细胞表型和受体重建的特点研究
|
摘要
目的巨细胞病毒(Cytomegalovirus, CMV)感染是脐血移植后常见的一种并发症,但目前一些对CMV感染患者的免疫重建发现,CMV感染可能对造血干细胞移植(Hematopoietic stem cell transplantation)后的免疫重建起到一定的促进作用,因此CMV-DNA拷贝数在一定的范围内可以不进行治疗。但目前对非血缘脐血移植(Unrelated cord blood transplantation, UCBT)后发生CMV感染者的免疫重建研究较少,尤其是国内在此方面的研究更少。我们此项研究为了观察恶性血液病(Hematologic malignancies)患者进行UCBT后发生CMV感染者自然杀伤细胞(Natural killer cell, NK)的表型和受体重建特点,为日后进行CMV的免疫治疗提供依据和理论基础。
方法回顾性分析76例进行UCBT的恶性血液病患者,所有患者采用强化清髓性的预处理方案,预防移植物抗宿主病(Graft-versus-host disease, GVHD)的方案为环孢素(Cyclosporine)和吗替麦考酚酯(Mycophenolate mofetil, MMF)。因为研究需要,选择的病例均为脐血成功植入的患者。UCBT从植入时开始每周利用荧光定量PCR技术检测患者全血标本中的CMV-DNA拷贝数,根据有无发生CMV感染将患者分为两组。使用五色和四色流式细胞仪检测两组患者植入时、1月、2月、3月、6月、1年、2年、3年的NK细胞表型及受体谱的变化和重建情况,分析两组之间的差异,分析CMV感染患者的NK细胞表型及受体谱的重建特点。
结果
一、临床结果
1.CMV感染情况：CMV阳性组38例患者,CMV阴性组38例患者,CMV感染发生时间为32(18-125)天。与文献报道一致,CMV阳性患者在CMV-DNA拷贝数大于103给予治疗,首选的治疗药物为更昔洛韦及膦甲酸钠,经过抗病毒治疗后所有患者CMV-DNA均转阴,中位转阴时间为15(7-29)天,没有患者发生CMV病。
2.脐血移植物特征及造血恢复情况：因研究需要,选择的病例均为脐血成功植入的患者,其中CMV阳性组所含有核细胞数为3.9(2.0～15.4)×107/kg, CD34+细胞数2.7(1.2～28.4)×105/kg, CD56+细胞数1.5(0.38～10.4)×106/kg, CD3+细胞数2.6(0.77～11.8)×106/kg。CMV阴性组所含有核细胞数为4.3(2.1～16.2)×107/kg,CD34+细胞数2.9(1.3～29.3)×105/kg, CD56+细胞数1.6(0.35～11.0)×106/kg, CD3+细胞数2.5(0.8～12.0)×106/kg。CMV阳性组粒细胞植入中位时间为19(12～39)天,血小板植入的中位时间为32(14～79)天。CMV阴性组粒细胞植入中位时间为18(13～38)天,血小板植入的中位时间为35(15～83)天。两组在移植物特征及造血恢复时间方面均无差异,p均大于0.05。
3.植入前综合征(Pre-engraftment syndrome,PES)的发生情况：共发生PES53例(69.7%),其中CMV阳性组29例(76.3%),CMV阴性组24例(63.2%),两组无差异,p=0.21。PES发生的中位时间两组均为移植后7(5-15)天,均治疗后好转。
4.急性移植物抗宿主病(Graft-versus-host disease, GVHD)的发生情况：CMV阳性组共发生急性GVHD15例,其中Ⅰ度GVHD6例(15.8%)、Ⅱ度GVHD6例(15.8%)、Ⅲ度GVHD2例(5.3%)、Ⅳ度GVHD1例(2.6%)；CMV阴性组发生急性GVHD12例,其中Ⅰ度GVHD4例(10.5%)、Ⅱ度GVHD6例(15.8%)、Ⅲ度GVHD2例(5.3%)、Ⅳ度GVHD0例(0.0%)。两组在急性GVHD发生率方面无差异,p均大于0.05。
5.慢性GVHD的发生情况：CMV阳性组36例可评估的患者中发生4例慢性GVHD (11.1%),3例为局限型,1例死亡,CMV阴性组34例可评估的患者中发生3例慢性GVHD(8.8%),均为局限型,两组比较差异无显著性,p=1.0。
6.细菌及真菌感染情况：24例(31.6%)患者在移植后发生程度轻重不一的细菌及真菌感染,CMV阳性组为13例,CMV阴性组为11例,p=0.62。共发生细菌血流感染21例,包括大肠杆菌6例,肺炎克雷伯杆菌4例,缓征链球菌3例,粪肠球菌2例,白色葡萄球菌2例,草绿色链球菌1例,铜绿假单胞菌1例,小肠耶氏菌1例,丹毒丝菌1例。发生肺部侵袭性真菌感染3例。重症感染导致死亡的患者9例(11.8%),包括3例肺部侵袭性真菌感染和6例细菌感染患者,其中CMV阳性组5例,CMV阴性组4例,p=1.0。两组在细菌及真菌感染及死亡率方面均无统计学差异。
7.复发情况：随访至2014年3月1日,所有患者平均随访时间为518(59～2357)天,生存患者平均随访637(206～2357)天。共11例(14.5%)复发,均为高危患者,其中CMV阳性组4例(10.5%),CMV阴性组7例(18.4%),p=0.33,两组复发率比较无统计学差异。其中复发起源于骨髓的有6例,起源髓外的有5例。复发的患者中死亡的有6例,带病生存的患者有5例。
8.死亡及生存情况：截至2014年3月1日,共18例患者死亡,其中CMV阳性组10例,CMV阴性组8例,p=0.59,两组无统计学差异。死亡原因为细菌及真菌感染9例、复发6例、急性GVHD1例、慢性GVHD1例、其他原因1例。3年总生存率(Overall survival, OS)为74.1%,3年无事件生存率(Event-free survival, EFS)为66.8%。其中CMV阳性组和CMV阴性组的OS分别为68.3%和78.6%, p=0.56, CMV阳性组和CMV阴性组的EFS分别为61.0%和70.3%,p=0.82,均无统计学差异。
二、NK细胞免疫重建结果
1.淋巴细胞比例及绝对值变化：76例患者在UCBT后+60天淋巴细胞绝对值和比例均达到正常值水平,CMV阳性组和CMV阴性组在淋巴细胞比例和绝对值的比较无统计学意义,p均大于0.05。
2.NK细胞比例和绝对值重建情况：两组患者NK细胞占淋巴细胞比例均在造血植入时达到高峰,然后出现下降,但CMV阳性组较CMV阴性组下降缓慢,CMV阳性组比CMV阴性组NK细胞比例在移植后30天(32.3%±15.7VS20.3%±12.4,p=0.02)及60天(23.4%±12.8VS12.3%±6.5,p=0.04)明显升高,有统计学意义。CMV阳性组比CMV阴性组NK细胞绝对值在移植后30天(20.4×107/L±10.3VS10.2×l07/L±4.3, p=0.02)及60天(46.5×107/L±21.2VS23.4×107/L±11.05,p=0.02)明显升高,有统计学意义。
3. CD3-CD56brightNK细胞的重建:UCBT后+30天及+60天,CMV阴性组的CD3-CD56brightNK细胞比例形成高峰,后逐渐下降至正常,但CMV阳性组未形成明显高峰,故CMV阳性组的CD3-CD56brightNK细胞比例在+30天和+60天较CMV阴性组的CD3-CD56bright NK细胞比例明显减少,分别为(12.4%±5.7VS.2VS26.8%±9.5, p=0.02)。因UCBT后+30天和+60天CMV阳性组的NK细胞绝对值较高,CMV阳性组与CMV阴性组在CD3-CD56brightNK细胞绝对值方面无明显差异。CMV阳性组与CMV阴性组CD57+CD3-CD56bright、CD11b+CD3-CD56bright及CD27+CD3-CD56bright的表达比例及细胞绝对值均无明显差异。
4.CD3-CD56-CD16+细胞：CMV阳性患者与CMV阴性患者相比,CD3-CD56-CD16+细胞比例在+30天和+60天明显增加,分别为(17.6%±4.3VS8.1%±2.7,p=0.02)和(14.4%±2.9VS7.3%±2.2,p=0.03)。CMV阳性患者的CD3-CD56-CD16+细胞绝对值在+30天和+60天较CMV阴性患者的CD3-CD56-CD16+细胞绝对值明显增加,分别为(10.6×10-/L±3.3VS4.1×107/L±1.3, p=0.01)和(28.8×107/L8.2VS15.5×107/L±6.0,p=0.02),有统计学意义。
5.CD57+CD56-CD16+细胞：移植后+30天及+60天,CMV阳性组CD57在CD3-CD56-CD16+NK细胞上的表达比例较CMV阴性组升高,分别为(14.5%±4.2VS5.9%±1.7,p=0.02)和(20.9%±8.9VS6.8%±2.2,p=0.01), CMV阳性组CD3-CD56-CD16+CD57+细胞绝对值在移植后+30天(15.4×106/±3.3VS3.1×106/L±1.3,p=0.01)及+60天(60.2×106/L±11.2VS10.5×106/L±9.0,p=0.001)也明显高于CMV阴性组,CD11b及CD27表达无明显差异。
6.NKG2D受体的重建：与CMV阴性组相比,CMV阳性组的NK细胞表面的NKG2D受体重建加快。在移植后+30天和+60天,CMV阳性组的NKG2D比例明显高于CMV阴性组,分别为(84.6%±10.3VS56.8±8.2,p=0.02)和(92.1%±9.9VS62.3%±11.6,p=0.02).在移植后+30天和+60天,CMV阳性组的NKG2D受体绝对值也明显高于CMV阴性组,分别为(11.2×1O7/L±4.3VS5.7×107/L±2.3, p=0.04)和(42.8×107/L±11.2VS14.6×107/L±6.0,p=0.01).
7.NKG2A受体的重建：CMV阳性组与CMV阴性组NKG2A受体比例均在植入时达到高峰,逐渐下降至稳定水平。与CMV阴性组相比,CMV阳性组的NK细胞表面的NKG2A比例下降更快。在移植后+30天,CMV阳性组的NKG2A比例已明显下降,而CMV阴性组的NKG2A比例在移植后+90天下降至稳定水平。故在移植后+30天和+60天,CMV阳性组的NKG2A比例明显低于CMV阴性组,分别为(44.7%±9.3VS82.1%±14.7,p=0.01)和(43.1%±13.9VS76.8%±15.3,p=0.03)。但是因为CMV阳性组在移植后NK细胞的绝对值重建较快,故CMV阳性组与CMV阴性组在NKG2A受体的绝对值方面无明显差异。
8.CD158a(KIR2DL1)受体的重建：CMV阴性组CD158a(KIR2DL1)受体比例重建较快,在移植后+30天达到了正常值水平,CMV阳性组在移植后+90天左右达到正常值水平。故在移植后+30天和+60天,CMV阳性组的CD158a受体比例明显低于CMV阴性组(8.3%±3.8VS17.2%±3.4,p=0.03；7.4%±3.6VS16.8%±4.5,p=0.03)。但是因为CMV阳性组在移植后NK细胞的绝对值重建较快,故CMV阳性组与CMV阴性组在CD158a受体的绝对值方面无明显差异。
9.CD158b受体的重建：CMV阳性组及CMV阴性组的CD158b受体比例在植入时均较高,但CMV阳性组在移植后+30天和+60天出现下降,而CMV阴性组在移植后+30天和+60天呈上升趋势。故移植后+30天和+60天,CMV阳性组和CMV阴性组的CD158b占NK细胞的比例分别为(17.3%±7.6VS40.7%±13.7)和(26.4%±8.9VS58.5%±16.5),p均为0.02,均存在显著性差异。因为CMV阳性患者的NK细胞绝对值较高,故CMV阳性组和CMV阴性组的CD158b绝对值无显著性差异。
10.NKP46受体的重建：CMV阳性组的NKP46受体在植入时、+30天及+60天所占比例均较高,CMV阴性组NKP46受体在植入时、+30天所占比例均较高,以后逐渐下降至稳定水平。移植后+30天和+60天,CMV阳性组比CMV阴性组的NKP46占NK细胞的比例明显升高,分别为(92.3%±10.4VS65.1%±10.6,p=0.02)和(86.4%±12.1VS58.3%±11.9,p=0.02),有统计学意义。移植后+30天和+60天,CMV阳性组比CMV阴性组的NKP46细胞的绝对值明显升高,分别为(18.8×107/L±5.3VS6.7×107/L±2.3,p=0.01)和(40.2×107/L±12.2VS13.6×107/L±4.0,p=0.01),均有统计学意义。说明CMV阳性组在移植后+30天和+60天NKP46受体的高峰更高。
结论CMV感染患者在CMV-DNA拷贝数大于103再进行抗病毒治疗,未增加患者的CMV病发生率及死亡率,生存情况与CMV阴性患者类似,国外有文献报道CMV感染可以减少HSCT后复发率,但可能因为病例数较少的原因,我们的研究中未发现两组在疾病复发率方面有统计学差异,以后可以扩大病例数继续研究。与CMV阴性组相比,CMV阳性组中NK细胞的比例及数量早期重建,代表NK细胞分泌功能的CD3-CD56bright细胞减少,代表NK细胞杀伤功能的CD3-CD56-CD16+细胞的增多,在CD3-CD56-CD16+CD57+在淋巴细胞上的表达升高。活化性受体包指NKG2D、NKP46重建加快、抑制性受体NKG2A、CD158a、CD158b重建减慢,说明CMV感染可以促进NK细胞的成熟,可能提高NK细胞的功能。
Objective CMV (Cytomegalovirus) is a verycommon complication after unrelated cord blood transplantation (UCBT), but currentlysome studies had showed CMV infection in patients can promote immune reconstitution after hematopoietic stem cell transplantation (HSCT). As CMV infection may play animportant role in immune reconstitution,patients can need no treatment if the number of CMV-DNA copies in a certain range.But the study of the immune reconstitution after UCBT with CMV-infectedindividualsis very less, especially the domestic research.Our study is to observe the natural killer cell (NK cell) phenotypic and receptor repertoire reconstitution of hematologic malignancies after unrelated cord blood transplantation in patients with cytomegalovirus (CMV) infection.
Methods A retrospective analysis of76patients who performed with UCBT and achieved successfully engraftment. All patients treated with intensive myeloablative conditioning regimen to promote engraftment, and the program to prevent graft-versus-host disease is cyclosporine combined with mycophenolate mofetil. Use fluorescence quantitative PCR in whole blood samples of patients to detect the copy number of CMV-DNA every week from engraftment after UCBT. The patients were divided into CMV-DNA positive group and CMV-DNA negative group according to whether develop CMV infection or not. Thirty-eight cases were in CMV-positive group and thirty-eight cases were CMV-negative group. Five-color and four-color flow cytometry was used to measure NK cells phenotypic and receptor repertoire reconstitution of all the patients in different intervals including the time of engraftment,+30days,+60days,+90days,+180days,1year,2years, and3 yearsafter UCBT and then analysis of the differences between the two groups.
Results
1Clinical outcome
(1) CMV infection:there were38patients in CMV-positive group and38patients in CMV-negative group.The median onset of CMV infection was32(18-125)dafter the transplantation. Consistent with the guidelines reported in the literature, Patients do not give the antiretroviral therapy unless the CMV-DNA copy number greater than103. Prophylaxis of CMV infection consisted ofacyclovir (5mg/kg twice daily) beginning on the next day after UCBT. The preemptive antiviral therapy wasinitiated with ganciclovir (5mg/kg, q12h) or foscarnet (60mg/kg, q12h). Sero-conversion was achieved in all the cases after treatment, witha median duration of15(7～29)d, no patients developed CMV disease.
(2) Umbilical cord blood graft characteristics and hematopoietic recovery:As the research needs, selected cases were all successfully engrafted with umbilical cord blood.Absolute neutrophil count contained in CMV-positive group was3.9(2.0～15.4)×107/kg, CD34+cells number was2.7(1.2～28.4)×105/kg, CD56+cell count was1.5(0.38～10.4)×106/kg, and CD3+cell count was2.6(0.77～11.8)×106/kg. Absolute neutrophil count contained in CMV-negative cells was4.3(2.1～16.2)×107/kg, CD34+cell count was2.9(1.3～29.3)×105/kg, CD56+was cell count1.6(0.35～11.0)×106/kg, CD3+cells number was2.5(0.8～12.0)×106/kg. The median time of granulocytes engraftment in CMV-positive group was19(12～39) d, and platelet engraftment was32(14～79) d. The median time of granulocytes engraftment in CMV-negative group was18(13～38) d, and platelet engraftment was35(15-83)d. There were no differences in graft characteristics and hematopoietic recovery time, p>0.05.
(3) Pre-implantation syndrome (PES):Fifty-three patients (69.7%) developed PES and the occurrence in CMV positive group was29cases (76.3%), and24cases (63.2%) inCMV-negative group, which had no differences between the two groups, p=0.21. The median time of PES occurrence was7(5～15) dafter transplantation.
(4) Acute graft-versus-host disease(GVHD):Fifteen of the38patients (39.5%) developed acute GVHD in CMV-positive group (grade I in6patients, grade II in6patients, grade Ⅲ in2patients and grade IV in1patients), and the cumulative incidence of grade Ⅲ to Ⅳ acute GVHD was7.9%(3of38). Twelve of the38patients (31.6%) developed acute GVHD in CMV-negative group (grade I in4patients, grade Ⅱ in6patients, grade Ⅲ in2patients and grade IV in0patients), and the cumulative incidence of grade Ⅲ to Ⅳ acute GVHD was5.3%(2of38).There was no difference in the incidence of acute GVHD between the two groups, p>0.05.
(5) Chronic GVHD:Eight of the36patients (11.1%) who could be evaluated developed chronic GVHDin CMV-positive group (3patients limited to the skin,1patient with extensive chronic GVHD and dead).Three of the34patients who could be evaluated developed chronic GVHD(8.8%) in CMV-negative group and all limited to the skin. There was no difference in the incidence of chronic GVHD between the two groups,p=1.0.
(6) Bacterial and fungal infections:There were24cases (31.6%) suffered bacterial and fungal infections after UCBT, including13cases(34.2%)in CMV-positive group and11cases(28.9%)in CMV-negative group, there was no significant difference between two groups,p=0.62.
(7) Relapse:The median follow-up after transplantation for the76patients was518(59-2357)d, and it was637(206-2357)dfor the patients who were alive.Eleven patients(14.5%)with high-risk disease relapsed, including4cases(10.5%)in CMV-positive group and7cases(18.4%)in CMV-negative group, there was no significant difference between two groups,p=0.33.
(8) Survival:As of March1,2014, a total of18patients died, including10cases in CMV positive patients and8cases in negative group, there was no difference between the two groups,p=0.59. The cause of death was9cases of bacterial and fungal infections,6cases of relapse,1case of acute GVHD,1case of chronic GVHD, and1case of other reasons. The3-year overall survival (OS) was74.1%and3-year event-free survival (EFS) was66.8%of all the patients. The OS were68.3%and78.6%in CMV-positive group and CMV-negative group respectively, p=0.56. The EFS were61.0%and70.3%in CMV-positive group and CMV-negative group respectively, p=0.82.
2NK cell reconstitution
(1)The percentage and absolute number of the lymphocyte:The percentage and absolute number oflymphocyteof all the76patients had reached the normal level from+60days after UCBT. CMV-positive group and CMV-negative group had no significant difference in the proportion and absolute number of lymphocytes after UCBT, p>0.05.
(2) The percentage and absolute number of NK cells:The peak of NK cell proportion and absolute number in CMV-positive group maintain longer compared with CMV-negative group. The NK cell proportion of CMV-positive group was significantly higher than CMV-negative group at+30days (32.3%±15.7VS20.3%±12.4, p=0.02)and+60days (23.4%±12.8VS12.3%±6.5,p=0.04)after UCBT, NK cells absolute value of CMV-positive group was also significantly higher than CMV-negative group at+30days (20.4×107/L±10.3VS10.2×107/L±4.3,p=0.02)and+60days (46.5×107/L±21.2VS23.4×107/L±11.0,p=0.02)after UCBT.
(3)CD3"CD56brightNK cells:The proportion of CD3-CD56bright NK cells in CMV-positive group was significant less compared to CMV-negative group at+30days(12.4%±5.7VS24.3%±8.7, p=0.02)and+60days(14.7%±3.2VS26.8%±9.5, p=0.02) after UCBT.Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences with the absolute numberof CD3-CD56bright NK cells inCMV positive group and CMV-negative group.The CD57, CD11b and CD27expression on CD56bright cells had no significant difference between CMV positive and CMV negative group.
(4) CD56-CD16+NK cell:The proportion of CD56-CD16+NK cell in CMV-positive group was significantly higher than CMV-negative groupat+30days (17.6%±4.3VS8.1%±2.7,p=0.02) and+60days (14.4%±2.9VS7.3%±2.2,p=0.03) after UCBT. The absolute number of NK cells in CMV-positive group was significantly higher than CMV-negative groupat+30days (10.6×107/L±3.3VS4.1×107/L±1.3,p=0.01) and+60days (28.8×107/L±8.2VS15.5×107/L±6.0,p=0.02) after UCBT.
(5) CD57+CD56-CD16+cell:The CD57expression on CD3-CD56-CD16+NK cells of CMV-positive group was higher than CMV-negative group at+30days (14.5%±4.2VS5.9%±1.7, p=0.02) and+60days (20.9%±8.9VS6.8%±2.2,p=0.01) after UCBT. The absolute number of CD57+CD3-CD56-CD16+cell in CMV-positive group was significantly higher than CMV-negative groupat+30days (15.4×106/L±3.3VS3.1×106/L±1.3,p=0.01) and+60days (60.2×106/L±11.2VS10.5±106/L±9.0,p=0.001) after UCBT. The CD11b and CD27expression had no significantly difference between the two groups.
(6) NKG2D acceptor:The NKG2D reconstruction was more quickly in CMV-positive group. The proportion of NKG2Dacceptor in CMV-positive group was significant higher compared to CMV-negative group at+30days (84.6%±10.3VS56.8%±8.2, p=0.02) and+60days (92.1%±9.9VS62.3%±11.6, p=0.02) after UCBT. The absolute number of NKG2D in CMV-positive group was significantly more than CMV-negative group at+30days (11.2×107/L±4.3VS5.7×107/L±2.3,p=0.04) and+60days (42.8×107/L±11.2VS14.6×107/L±6.0,p=0.01) after UCBT.
(7)NKG2A acceptor:The proportion of NKG2A has significantly decreased around+30days in CMV-positive group while+90days in CMV-negative group after UCBT. Therefore, the proportion of NKG2A in CMV-positive group was significantly less than CMV-negative group at+30days (44.7%±9.3VS82.1%±14.7, p=0.01) and (43.1%±13.9VS76.8%±15.3, p=0.03) after UCBT.Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences with the absolute numberof NKG2AinCMV positive group and CMV-negative group.
(8) CD158a(KIR2DL1) acceptor:The CD158a (KIR2DL1) reconstruction was quicker in CMV-negative group than CMV-positive group. The proportion of CD158a (KIR2DL1) in CMV-positive group was significantly less than CMV-negative group at+30days (8.3%±3.8VS17.2%±3.43,p=0.03)and+60days (7.4%±3.6VS16.8%±3.5, p=0.03)after UCBT.Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences with the absolute numberof CD158a (KIR2DL1) inCMV positive group and CMV-negative group.
(9) CD158b(KIR2DL3) acceptor:The proportion of CD158b (KIR2DL3) cells in CMV-positive group was significantly higher than CMV-negative groupat+30days(17.3%±7.6VS40.7%±13.7,p=0.02) and+60days (26.4%±8.9VS58.5%±16.5, p=0.02) after UCBT. Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences of the absolute number of CD158b cells between the two groups.(10)NKP46acceptor:The proportion of NKP46cell in CMV-positive group were significantly higher than CMV-negative group at+30days (92.3%±10.4VS65.1%±10.6,p=0.02) and+60days (86.4%±12.1VS58.3%±11.9,p=0.02) after UCBT. The absolute number of NKP46cell in CMV-positive group was significantly higher than CMV-negative group at+30days(18.8×107/L±5.3VS6.7×107/L±2.3, p=0.01)and+60days (40.2×107/L±12.2VS13.6×107/L±4.0,p=0.01)after UCBT.
Conclusion
It did not increase the incidence of CMV disease and related death whenstartantiviral therapy whenCMV-DNA copy number is greater than103. Currently, some literature reported that CMV infection can reduce the relapse rate after HSCT, but our study found no significant difference between the two groups in relapse probably because of the small number of the cases.NK cells proportion and quantity reconstruction were earlier in CMV-positive group than CMV-negative group. The CD56bright cells which on behalf of secretory function decreased while CD56-CD16+cells which on behalf of cytotoxic function in increased. CD57expression on CD56-CD16+NK cells also increased. Activated receptor including NKG2D, NKP46reconstruction accelerated and inhibitory receptor like NKG2A, CD158a and CD158b reconstruction slowed which demonstrated that CMV infection can promote the maturation of NK cells.
方法回顾性分析76例进行UCBT的恶性血液病患者,所有患者采用强化清髓性的预处理方案,预防移植物抗宿主病(Graft-versus-host disease, GVHD)的方案为环孢素(Cyclosporine)和吗替麦考酚酯(Mycophenolate mofetil, MMF)。因为研究需要,选择的病例均为脐血成功植入的患者。UCBT从植入时开始每周利用荧光定量PCR技术检测患者全血标本中的CMV-DNA拷贝数,根据有无发生CMV感染将患者分为两组。使用五色和四色流式细胞仪检测两组患者植入时、1月、2月、3月、6月、1年、2年、3年的NK细胞表型及受体谱的变化和重建情况,分析两组之间的差异,分析CMV感染患者的NK细胞表型及受体谱的重建特点。
结果
一、临床结果
1.CMV感染情况：CMV阳性组38例患者,CMV阴性组38例患者,CMV感染发生时间为32(18-125)天。与文献报道一致,CMV阳性患者在CMV-DNA拷贝数大于103给予治疗,首选的治疗药物为更昔洛韦及膦甲酸钠,经过抗病毒治疗后所有患者CMV-DNA均转阴,中位转阴时间为15(7-29)天,没有患者发生CMV病。
2.脐血移植物特征及造血恢复情况：因研究需要,选择的病例均为脐血成功植入的患者,其中CMV阳性组所含有核细胞数为3.9(2.0～15.4)×107/kg, CD34+细胞数2.7(1.2～28.4)×105/kg, CD56+细胞数1.5(0.38～10.4)×106/kg, CD3+细胞数2.6(0.77～11.8)×106/kg。CMV阴性组所含有核细胞数为4.3(2.1～16.2)×107/kg,CD34+细胞数2.9(1.3～29.3)×105/kg, CD56+细胞数1.6(0.35～11.0)×106/kg, CD3+细胞数2.5(0.8～12.0)×106/kg。CMV阳性组粒细胞植入中位时间为19(12～39)天,血小板植入的中位时间为32(14～79)天。CMV阴性组粒细胞植入中位时间为18(13～38)天,血小板植入的中位时间为35(15～83)天。两组在移植物特征及造血恢复时间方面均无差异,p均大于0.05。
3.植入前综合征(Pre-engraftment syndrome,PES)的发生情况：共发生PES53例(69.7%),其中CMV阳性组29例(76.3%),CMV阴性组24例(63.2%),两组无差异,p=0.21。PES发生的中位时间两组均为移植后7(5-15)天,均治疗后好转。
4.急性移植物抗宿主病(Graft-versus-host disease, GVHD)的发生情况：CMV阳性组共发生急性GVHD15例,其中Ⅰ度GVHD6例(15.8%)、Ⅱ度GVHD6例(15.8%)、Ⅲ度GVHD2例(5.3%)、Ⅳ度GVHD1例(2.6%)；CMV阴性组发生急性GVHD12例,其中Ⅰ度GVHD4例(10.5%)、Ⅱ度GVHD6例(15.8%)、Ⅲ度GVHD2例(5.3%)、Ⅳ度GVHD0例(0.0%)。两组在急性GVHD发生率方面无差异,p均大于0.05。
5.慢性GVHD的发生情况：CMV阳性组36例可评估的患者中发生4例慢性GVHD (11.1%),3例为局限型,1例死亡,CMV阴性组34例可评估的患者中发生3例慢性GVHD(8.8%),均为局限型,两组比较差异无显著性,p=1.0。
6.细菌及真菌感染情况：24例(31.6%)患者在移植后发生程度轻重不一的细菌及真菌感染,CMV阳性组为13例,CMV阴性组为11例,p=0.62。共发生细菌血流感染21例,包括大肠杆菌6例,肺炎克雷伯杆菌4例,缓征链球菌3例,粪肠球菌2例,白色葡萄球菌2例,草绿色链球菌1例,铜绿假单胞菌1例,小肠耶氏菌1例,丹毒丝菌1例。发生肺部侵袭性真菌感染3例。重症感染导致死亡的患者9例(11.8%),包括3例肺部侵袭性真菌感染和6例细菌感染患者,其中CMV阳性组5例,CMV阴性组4例,p=1.0。两组在细菌及真菌感染及死亡率方面均无统计学差异。
7.复发情况：随访至2014年3月1日,所有患者平均随访时间为518(59～2357)天,生存患者平均随访637(206～2357)天。共11例(14.5%)复发,均为高危患者,其中CMV阳性组4例(10.5%),CMV阴性组7例(18.4%),p=0.33,两组复发率比较无统计学差异。其中复发起源于骨髓的有6例,起源髓外的有5例。复发的患者中死亡的有6例,带病生存的患者有5例。
8.死亡及生存情况：截至2014年3月1日,共18例患者死亡,其中CMV阳性组10例,CMV阴性组8例,p=0.59,两组无统计学差异。死亡原因为细菌及真菌感染9例、复发6例、急性GVHD1例、慢性GVHD1例、其他原因1例。3年总生存率(Overall survival, OS)为74.1%,3年无事件生存率(Event-free survival, EFS)为66.8%。其中CMV阳性组和CMV阴性组的OS分别为68.3%和78.6%, p=0.56, CMV阳性组和CMV阴性组的EFS分别为61.0%和70.3%,p=0.82,均无统计学差异。
二、NK细胞免疫重建结果
1.淋巴细胞比例及绝对值变化：76例患者在UCBT后+60天淋巴细胞绝对值和比例均达到正常值水平,CMV阳性组和CMV阴性组在淋巴细胞比例和绝对值的比较无统计学意义,p均大于0.05。
2.NK细胞比例和绝对值重建情况：两组患者NK细胞占淋巴细胞比例均在造血植入时达到高峰,然后出现下降,但CMV阳性组较CMV阴性组下降缓慢,CMV阳性组比CMV阴性组NK细胞比例在移植后30天(32.3%±15.7VS20.3%±12.4,p=0.02)及60天(23.4%±12.8VS12.3%±6.5,p=0.04)明显升高,有统计学意义。CMV阳性组比CMV阴性组NK细胞绝对值在移植后30天(20.4×107/L±10.3VS10.2×l07/L±4.3, p=0.02)及60天(46.5×107/L±21.2VS23.4×107/L±11.05,p=0.02)明显升高,有统计学意义。
3. CD3-CD56brightNK细胞的重建:UCBT后+30天及+60天,CMV阴性组的CD3-CD56brightNK细胞比例形成高峰,后逐渐下降至正常,但CMV阳性组未形成明显高峰,故CMV阳性组的CD3-CD56brightNK细胞比例在+30天和+60天较CMV阴性组的CD3-CD56bright NK细胞比例明显减少,分别为(12.4%±5.7VS.2VS26.8%±9.5, p=0.02)。因UCBT后+30天和+60天CMV阳性组的NK细胞绝对值较高,CMV阳性组与CMV阴性组在CD3-CD56brightNK细胞绝对值方面无明显差异。CMV阳性组与CMV阴性组CD57+CD3-CD56bright、CD11b+CD3-CD56bright及CD27+CD3-CD56bright的表达比例及细胞绝对值均无明显差异。
4.CD3-CD56-CD16+细胞：CMV阳性患者与CMV阴性患者相比,CD3-CD56-CD16+细胞比例在+30天和+60天明显增加,分别为(17.6%±4.3VS8.1%±2.7,p=0.02)和(14.4%±2.9VS7.3%±2.2,p=0.03)。CMV阳性患者的CD3-CD56-CD16+细胞绝对值在+30天和+60天较CMV阴性患者的CD3-CD56-CD16+细胞绝对值明显增加,分别为(10.6×10-/L±3.3VS4.1×107/L±1.3, p=0.01)和(28.8×107/L8.2VS15.5×107/L±6.0,p=0.02),有统计学意义。
5.CD57+CD56-CD16+细胞：移植后+30天及+60天,CMV阳性组CD57在CD3-CD56-CD16+NK细胞上的表达比例较CMV阴性组升高,分别为(14.5%±4.2VS5.9%±1.7,p=0.02)和(20.9%±8.9VS6.8%±2.2,p=0.01), CMV阳性组CD3-CD56-CD16+CD57+细胞绝对值在移植后+30天(15.4×106/±3.3VS3.1×106/L±1.3,p=0.01)及+60天(60.2×106/L±11.2VS10.5×106/L±9.0,p=0.001)也明显高于CMV阴性组,CD11b及CD27表达无明显差异。
6.NKG2D受体的重建：与CMV阴性组相比,CMV阳性组的NK细胞表面的NKG2D受体重建加快。在移植后+30天和+60天,CMV阳性组的NKG2D比例明显高于CMV阴性组,分别为(84.6%±10.3VS56.8±8.2,p=0.02)和(92.1%±9.9VS62.3%±11.6,p=0.02).在移植后+30天和+60天,CMV阳性组的NKG2D受体绝对值也明显高于CMV阴性组,分别为(11.2×1O7/L±4.3VS5.7×107/L±2.3, p=0.04)和(42.8×107/L±11.2VS14.6×107/L±6.0,p=0.01).
7.NKG2A受体的重建：CMV阳性组与CMV阴性组NKG2A受体比例均在植入时达到高峰,逐渐下降至稳定水平。与CMV阴性组相比,CMV阳性组的NK细胞表面的NKG2A比例下降更快。在移植后+30天,CMV阳性组的NKG2A比例已明显下降,而CMV阴性组的NKG2A比例在移植后+90天下降至稳定水平。故在移植后+30天和+60天,CMV阳性组的NKG2A比例明显低于CMV阴性组,分别为(44.7%±9.3VS82.1%±14.7,p=0.01)和(43.1%±13.9VS76.8%±15.3,p=0.03)。但是因为CMV阳性组在移植后NK细胞的绝对值重建较快,故CMV阳性组与CMV阴性组在NKG2A受体的绝对值方面无明显差异。
8.CD158a(KIR2DL1)受体的重建：CMV阴性组CD158a(KIR2DL1)受体比例重建较快,在移植后+30天达到了正常值水平,CMV阳性组在移植后+90天左右达到正常值水平。故在移植后+30天和+60天,CMV阳性组的CD158a受体比例明显低于CMV阴性组(8.3%±3.8VS17.2%±3.4,p=0.03；7.4%±3.6VS16.8%±4.5,p=0.03)。但是因为CMV阳性组在移植后NK细胞的绝对值重建较快,故CMV阳性组与CMV阴性组在CD158a受体的绝对值方面无明显差异。
9.CD158b受体的重建：CMV阳性组及CMV阴性组的CD158b受体比例在植入时均较高,但CMV阳性组在移植后+30天和+60天出现下降,而CMV阴性组在移植后+30天和+60天呈上升趋势。故移植后+30天和+60天,CMV阳性组和CMV阴性组的CD158b占NK细胞的比例分别为(17.3%±7.6VS40.7%±13.7)和(26.4%±8.9VS58.5%±16.5),p均为0.02,均存在显著性差异。因为CMV阳性患者的NK细胞绝对值较高,故CMV阳性组和CMV阴性组的CD158b绝对值无显著性差异。
10.NKP46受体的重建：CMV阳性组的NKP46受体在植入时、+30天及+60天所占比例均较高,CMV阴性组NKP46受体在植入时、+30天所占比例均较高,以后逐渐下降至稳定水平。移植后+30天和+60天,CMV阳性组比CMV阴性组的NKP46占NK细胞的比例明显升高,分别为(92.3%±10.4VS65.1%±10.6,p=0.02)和(86.4%±12.1VS58.3%±11.9,p=0.02),有统计学意义。移植后+30天和+60天,CMV阳性组比CMV阴性组的NKP46细胞的绝对值明显升高,分别为(18.8×107/L±5.3VS6.7×107/L±2.3,p=0.01)和(40.2×107/L±12.2VS13.6×107/L±4.0,p=0.01),均有统计学意义。说明CMV阳性组在移植后+30天和+60天NKP46受体的高峰更高。
结论CMV感染患者在CMV-DNA拷贝数大于103再进行抗病毒治疗,未增加患者的CMV病发生率及死亡率,生存情况与CMV阴性患者类似,国外有文献报道CMV感染可以减少HSCT后复发率,但可能因为病例数较少的原因,我们的研究中未发现两组在疾病复发率方面有统计学差异,以后可以扩大病例数继续研究。与CMV阴性组相比,CMV阳性组中NK细胞的比例及数量早期重建,代表NK细胞分泌功能的CD3-CD56bright细胞减少,代表NK细胞杀伤功能的CD3-CD56-CD16+细胞的增多,在CD3-CD56-CD16+CD57+在淋巴细胞上的表达升高。活化性受体包指NKG2D、NKP46重建加快、抑制性受体NKG2A、CD158a、CD158b重建减慢,说明CMV感染可以促进NK细胞的成熟,可能提高NK细胞的功能。
Objective CMV (Cytomegalovirus) is a verycommon complication after unrelated cord blood transplantation (UCBT), but currentlysome studies had showed CMV infection in patients can promote immune reconstitution after hematopoietic stem cell transplantation (HSCT). As CMV infection may play animportant role in immune reconstitution,patients can need no treatment if the number of CMV-DNA copies in a certain range.But the study of the immune reconstitution after UCBT with CMV-infectedindividualsis very less, especially the domestic research.Our study is to observe the natural killer cell (NK cell) phenotypic and receptor repertoire reconstitution of hematologic malignancies after unrelated cord blood transplantation in patients with cytomegalovirus (CMV) infection.
Methods A retrospective analysis of76patients who performed with UCBT and achieved successfully engraftment. All patients treated with intensive myeloablative conditioning regimen to promote engraftment, and the program to prevent graft-versus-host disease is cyclosporine combined with mycophenolate mofetil. Use fluorescence quantitative PCR in whole blood samples of patients to detect the copy number of CMV-DNA every week from engraftment after UCBT. The patients were divided into CMV-DNA positive group and CMV-DNA negative group according to whether develop CMV infection or not. Thirty-eight cases were in CMV-positive group and thirty-eight cases were CMV-negative group. Five-color and four-color flow cytometry was used to measure NK cells phenotypic and receptor repertoire reconstitution of all the patients in different intervals including the time of engraftment,+30days,+60days,+90days,+180days,1year,2years, and3 yearsafter UCBT and then analysis of the differences between the two groups.
Results
1Clinical outcome
(1) CMV infection:there were38patients in CMV-positive group and38patients in CMV-negative group.The median onset of CMV infection was32(18-125)dafter the transplantation. Consistent with the guidelines reported in the literature, Patients do not give the antiretroviral therapy unless the CMV-DNA copy number greater than103. Prophylaxis of CMV infection consisted ofacyclovir (5mg/kg twice daily) beginning on the next day after UCBT. The preemptive antiviral therapy wasinitiated with ganciclovir (5mg/kg, q12h) or foscarnet (60mg/kg, q12h). Sero-conversion was achieved in all the cases after treatment, witha median duration of15(7～29)d, no patients developed CMV disease.
(2) Umbilical cord blood graft characteristics and hematopoietic recovery:As the research needs, selected cases were all successfully engrafted with umbilical cord blood.Absolute neutrophil count contained in CMV-positive group was3.9(2.0～15.4)×107/kg, CD34+cells number was2.7(1.2～28.4)×105/kg, CD56+cell count was1.5(0.38～10.4)×106/kg, and CD3+cell count was2.6(0.77～11.8)×106/kg. Absolute neutrophil count contained in CMV-negative cells was4.3(2.1～16.2)×107/kg, CD34+cell count was2.9(1.3～29.3)×105/kg, CD56+was cell count1.6(0.35～11.0)×106/kg, CD3+cells number was2.5(0.8～12.0)×106/kg. The median time of granulocytes engraftment in CMV-positive group was19(12～39) d, and platelet engraftment was32(14～79) d. The median time of granulocytes engraftment in CMV-negative group was18(13～38) d, and platelet engraftment was35(15-83)d. There were no differences in graft characteristics and hematopoietic recovery time, p>0.05.
(3) Pre-implantation syndrome (PES):Fifty-three patients (69.7%) developed PES and the occurrence in CMV positive group was29cases (76.3%), and24cases (63.2%) inCMV-negative group, which had no differences between the two groups, p=0.21. The median time of PES occurrence was7(5～15) dafter transplantation.
(4) Acute graft-versus-host disease(GVHD):Fifteen of the38patients (39.5%) developed acute GVHD in CMV-positive group (grade I in6patients, grade II in6patients, grade Ⅲ in2patients and grade IV in1patients), and the cumulative incidence of grade Ⅲ to Ⅳ acute GVHD was7.9%(3of38). Twelve of the38patients (31.6%) developed acute GVHD in CMV-negative group (grade I in4patients, grade Ⅱ in6patients, grade Ⅲ in2patients and grade IV in0patients), and the cumulative incidence of grade Ⅲ to Ⅳ acute GVHD was5.3%(2of38).There was no difference in the incidence of acute GVHD between the two groups, p>0.05.
(5) Chronic GVHD:Eight of the36patients (11.1%) who could be evaluated developed chronic GVHDin CMV-positive group (3patients limited to the skin,1patient with extensive chronic GVHD and dead).Three of the34patients who could be evaluated developed chronic GVHD(8.8%) in CMV-negative group and all limited to the skin. There was no difference in the incidence of chronic GVHD between the two groups,p=1.0.
(6) Bacterial and fungal infections:There were24cases (31.6%) suffered bacterial and fungal infections after UCBT, including13cases(34.2%)in CMV-positive group and11cases(28.9%)in CMV-negative group, there was no significant difference between two groups,p=0.62.
(7) Relapse:The median follow-up after transplantation for the76patients was518(59-2357)d, and it was637(206-2357)dfor the patients who were alive.Eleven patients(14.5%)with high-risk disease relapsed, including4cases(10.5%)in CMV-positive group and7cases(18.4%)in CMV-negative group, there was no significant difference between two groups,p=0.33.
(8) Survival:As of March1,2014, a total of18patients died, including10cases in CMV positive patients and8cases in negative group, there was no difference between the two groups,p=0.59. The cause of death was9cases of bacterial and fungal infections,6cases of relapse,1case of acute GVHD,1case of chronic GVHD, and1case of other reasons. The3-year overall survival (OS) was74.1%and3-year event-free survival (EFS) was66.8%of all the patients. The OS were68.3%and78.6%in CMV-positive group and CMV-negative group respectively, p=0.56. The EFS were61.0%and70.3%in CMV-positive group and CMV-negative group respectively, p=0.82.
2NK cell reconstitution
(1)The percentage and absolute number of the lymphocyte:The percentage and absolute number oflymphocyteof all the76patients had reached the normal level from+60days after UCBT. CMV-positive group and CMV-negative group had no significant difference in the proportion and absolute number of lymphocytes after UCBT, p>0.05.
(2) The percentage and absolute number of NK cells:The peak of NK cell proportion and absolute number in CMV-positive group maintain longer compared with CMV-negative group. The NK cell proportion of CMV-positive group was significantly higher than CMV-negative group at+30days (32.3%±15.7VS20.3%±12.4, p=0.02)and+60days (23.4%±12.8VS12.3%±6.5,p=0.04)after UCBT, NK cells absolute value of CMV-positive group was also significantly higher than CMV-negative group at+30days (20.4×107/L±10.3VS10.2×107/L±4.3,p=0.02)and+60days (46.5×107/L±21.2VS23.4×107/L±11.0,p=0.02)after UCBT.
(3)CD3"CD56brightNK cells:The proportion of CD3-CD56bright NK cells in CMV-positive group was significant less compared to CMV-negative group at+30days(12.4%±5.7VS24.3%±8.7, p=0.02)and+60days(14.7%±3.2VS26.8%±9.5, p=0.02) after UCBT.Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences with the absolute numberof CD3-CD56bright NK cells inCMV positive group and CMV-negative group.The CD57, CD11b and CD27expression on CD56bright cells had no significant difference between CMV positive and CMV negative group.
(4) CD56-CD16+NK cell:The proportion of CD56-CD16+NK cell in CMV-positive group was significantly higher than CMV-negative groupat+30days (17.6%±4.3VS8.1%±2.7,p=0.02) and+60days (14.4%±2.9VS7.3%±2.2,p=0.03) after UCBT. The absolute number of NK cells in CMV-positive group was significantly higher than CMV-negative groupat+30days (10.6×107/L±3.3VS4.1×107/L±1.3,p=0.01) and+60days (28.8×107/L±8.2VS15.5×107/L±6.0,p=0.02) after UCBT.
(5) CD57+CD56-CD16+cell:The CD57expression on CD3-CD56-CD16+NK cells of CMV-positive group was higher than CMV-negative group at+30days (14.5%±4.2VS5.9%±1.7, p=0.02) and+60days (20.9%±8.9VS6.8%±2.2,p=0.01) after UCBT. The absolute number of CD57+CD3-CD56-CD16+cell in CMV-positive group was significantly higher than CMV-negative groupat+30days (15.4×106/L±3.3VS3.1×106/L±1.3,p=0.01) and+60days (60.2×106/L±11.2VS10.5±106/L±9.0,p=0.001) after UCBT. The CD11b and CD27expression had no significantly difference between the two groups.
(6) NKG2D acceptor:The NKG2D reconstruction was more quickly in CMV-positive group. The proportion of NKG2Dacceptor in CMV-positive group was significant higher compared to CMV-negative group at+30days (84.6%±10.3VS56.8%±8.2, p=0.02) and+60days (92.1%±9.9VS62.3%±11.6, p=0.02) after UCBT. The absolute number of NKG2D in CMV-positive group was significantly more than CMV-negative group at+30days (11.2×107/L±4.3VS5.7×107/L±2.3,p=0.04) and+60days (42.8×107/L±11.2VS14.6×107/L±6.0,p=0.01) after UCBT.
(7)NKG2A acceptor:The proportion of NKG2A has significantly decreased around+30days in CMV-positive group while+90days in CMV-negative group after UCBT. Therefore, the proportion of NKG2A in CMV-positive group was significantly less than CMV-negative group at+30days (44.7%±9.3VS82.1%±14.7, p=0.01) and (43.1%±13.9VS76.8%±15.3, p=0.03) after UCBT.Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences with the absolute numberof NKG2AinCMV positive group and CMV-negative group.
(8) CD158a(KIR2DL1) acceptor:The CD158a (KIR2DL1) reconstruction was quicker in CMV-negative group than CMV-positive group. The proportion of CD158a (KIR2DL1) in CMV-positive group was significantly less than CMV-negative group at+30days (8.3%±3.8VS17.2%±3.43,p=0.03)and+60days (7.4%±3.6VS16.8%±3.5, p=0.03)after UCBT.Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences with the absolute numberof CD158a (KIR2DL1) inCMV positive group and CMV-negative group.
(9) CD158b(KIR2DL3) acceptor:The proportion of CD158b (KIR2DL3) cells in CMV-positive group was significantly higher than CMV-negative groupat+30days(17.3%±7.6VS40.7%±13.7,p=0.02) and+60days (26.4%±8.9VS58.5%±16.5, p=0.02) after UCBT. Because the higher absolute number of NK cellsof CMV-positive group at+30days and+60days after UCBT, there were no significant differences of the absolute number of CD158b cells between the two groups.(10)NKP46acceptor:The proportion of NKP46cell in CMV-positive group were significantly higher than CMV-negative group at+30days (92.3%±10.4VS65.1%±10.6,p=0.02) and+60days (86.4%±12.1VS58.3%±11.9,p=0.02) after UCBT. The absolute number of NKP46cell in CMV-positive group was significantly higher than CMV-negative group at+30days(18.8×107/L±5.3VS6.7×107/L±2.3, p=0.01)and+60days (40.2×107/L±12.2VS13.6×107/L±4.0,p=0.01)after UCBT.
Conclusion
It did not increase the incidence of CMV disease and related death whenstartantiviral therapy whenCMV-DNA copy number is greater than103. Currently, some literature reported that CMV infection can reduce the relapse rate after HSCT, but our study found no significant difference between the two groups in relapse probably because of the small number of the cases.NK cells proportion and quantity reconstruction were earlier in CMV-positive group than CMV-negative group. The CD56bright cells which on behalf of secretory function decreased while CD56-CD16+cells which on behalf of cytotoxic function in increased. CD57expression on CD56-CD16+NK cells also increased. Activated receptor including NKG2D, NKP46reconstruction accelerated and inhibitory receptor like NKG2A, CD158a and CD158b reconstruction slowed which demonstrated that CMV infection can promote the maturation of NK cells.
引文
l.Mazharian Al,Mori J,Wang YJ,Heising S,Neel BQ Watson SP, Senis YA.Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shpl and Shp2 causes abnormal megakaryocyte development, platelet production, and function.Blood.2013 May 16;121(20):4205-20.
2.Amabile G1,Welner RS, Nombela-Arrieta C, D'Alise AM, Di Ruscio A,Ebralidze AK, Kraytsberg Y,Ye M,Kocher O,Neuberg DS,Khrapko K, Silberstein LE,Tenen DG. In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells. Blood.2013 Feb 21;121(8):1255-64.
3.Chervenick PA, Boggs DR. Patterns of proliferation and differentiation of hematopoietic stem cells after compartment depletion. Blood.1971 May;37(5):568-80.
4. Cheuk DK.Optimal stem cell source for allogeneic stem cell transplantation for hematological malignancies. World J Transplant.2013 Dec 24;3(4):99-112.
5. Ramakrishnan Al, Torok-Storb B, Pillai MM. Primary marrow-derived stromal cells:isolation and manipulation.Methods Mol Biol.2013; 1035:75-101.
6. Resnick IB1,Barkats C, Shapira MY, Stepensky P, Bloom AI, Shimoni A, Mankuta D, Varda-Bloom N, Rheingold L, Yeshurun M, Bielorai B, Toren A, Zuckerman T, Nagler A, Or R.Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC).Am J Blood Res.2013 Aug 19;3(3):225-38.
7.Anam K, Davis TA.Comparative analysis of gene transcripts for cell signaling receptors in bone marrow-derived hematopoieticstem/progenitor cell and mesenchymal stromal cell populations.Stem Cell Res Ther.2013 Sep 16;4(5):112.
8. Noel, D, et al. Muhipotent mesenchymal stromal ceils and im-mune toleranee[J]. Leuk Lymphoma,2007,48(7):P.1283-1289.
9. Lazarus, H M, et al. Cotransplantation of HLA-identical siblingculture-expanded mesenehymal stem cells and hematopoietic stemceils in hematologic malignancy patients[J]. Biol Blood Marrow. Transplant,2005,11(5): P.389-398.
10. Hashimoto S, Gm Y, Takeshita I, et al. [J]. Am J Respir Cut Can; Med, 2001,163:152-157.
11. Montanari M, Capelli D, Poloni A, et al.Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation:a comparison between controlled-rate freezing and uncontrolled-rate freezing at 80 degrees C. Transfusion.2003 Jan;43(1):42-49.
12. Statkute L, Verda L, Oyama Y, Mobilization, harvesting and selection of peripheral blood stem cells in patients with autoimmune diseases undergoing autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2007Mar;39(6):317-29.
13. Mahon FX, Deininger MW, et al. Selection and characterization of BCR-ABL positive cells lines with differential sensistivity to the tyrosine kinase inhibitor STI571:diverse mechanisms of resistance. Blood,2000;96:1070-1079.
14. Weisberg E, Griffin JD. Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines. Blood,2000; 95:3498-3505.
15. Byrne JL1, Carter GI, Ellis I, Haynes AP, Russell NH.Autologous GVHD following PBSCT, with evidence for a raft-versus-myeloma effect.Bone Marrow Transplant. 1997 Sep; 20(6):517-20.
16. Bolanos-Meade J1, Wu J, Logan BR, Levine JE, Ho VT, Alousi AM, Weisdorf DJ, Luznik L; Blood and Marrow Transplant Clinical Trials Network. Lymphocyte phenotype during therapy for acute graft-versus-host disease:a brief report from BMT-CTN 0302. Biol Blood Marrow Transplant.2013 Mar;19(3):481-5.
17. Gluckman E, Broxmeyer HE, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med 1989; 321:1174-1178.
18. Section on Hematology/Oncology and Section on Allergy/Immunology. Cord Blood Banking for Potential Future Transplantation. Pediatrics,2007; 119:165-170.
19. Yamaguchi M1,Ebina S, Kashiwakura I.Involvement of placental/umbilical cord blood acid-base status and gas values on the radiosensitivity of humanfetal/neonatal hematopoietic stem/progenitor cells. J Radiat Res.2013 Mar 1;54(2):277-84.
20. Geyer MB1, Jacobson JS, Freedman J, George D, Moore V, van de Ven C, Satwani P, Bhatia M, Garvin JH, Bradley MB, Harrison L, Morris E, Della-Latta P,Schwartz J, Baxter-Lowe LA, Cairo MS.A comparison of immune reconstitution and graft-versus-host disease following myeloablative conditioning versus reduced toxicity conditioning and umbilicalcord bloodtransplantation in paediatric recipients.Br J Haematol.2011 Oct;155(2):218-34.
21. Liu J1,He J, Chen S, Qin F, Wang F,Xu G,Zhu F, Lv H, Yan L. Cord Blood Banking and Transplantation in China:A Ten Years Experience of a Single Public Bank.Transfus Med Hemother.2012 Feb;39(1):23-27.
22. Mouzaki A1, Panagoulias I, Raptis G, Farri-Kostopoulou E.Cord blood leptin levels of healthy neonates are associated with IFN-y production by cord blood T-cells. PLoS One.2012;7(7):e40830.
23. Risdon G, Gaddy J, Stehman FB,Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation. Cell Immunol. 1994; 154(1):14-24.
24. Parmar S1, Liu X2,Tung SS2, Robinson SN2, Rodriguez G2, Cooper LJ3, Yang H4, Shah N2, Yang H2, Konopleva M4, Molldrem JJ2, Garcia-Manero G4,Najjar A5,Yvon E2,McNiece I2,Rezvani K2,Savoldo B6, Bollard CM7, Shpall EJ2. Cytotherapy. Third-party umbilical cord blood-derived regulatory T cells prevent xenogenic graft-versus-host disease.2014 Jan;16(1):90-100.
25. Parody R1, Martino R, Rovira M, Vazquez L, Vazquez MJ, de la Camara R, Blazquez C, Fernandez-Aviles F, Carreras E, Salavert M, Jarque I, Martin C, Martinez F, Lopez J, Torres A, Sierra J, Sanz GF. Severe infections after unrelated donor allogeneic hematopoietic stem cell transplantation in adults:comparison of cord blood transplantation with peripheral blood and bone marrow transplantation. Biol Blood Marrow Transplant.2006 Jul;12(7):734-48.
26. Pera A1, Campos Cl, Corona Al, Sanchez-Correa B2, Tarazona R2, Larbi A3, Solana R1.CMV Latent Infection Improves CD8+ T Response to SEB Due to Expansion of Polyfunctional CD57+ Cells in Young Individuals. PLoS One.2014 Feb 12;9(2):e88538.
27. Boeckh M, Leisenring W, Riddell SR, Bowden RA, Huang ML, Myerson D, Stevens-Ayers T, Flowers ME, Cunningham T, Corey L. Latecytomegalovirus disease and mortality in recipientsof allogeneic hematopoietic stem cell transplants:importance of viral load and T-cell immunity.Blood,2003, 101(2):407-414.
28. Mikulska M, Raiola AM, Bruzzi P, Varaldo R, Annunziata S, Lamparelli T, Frassoni F, Tedone E, Galano B, Bacigalupo A, Viscoli C. CMV infection after transplant from cord blood compared to other alternative donors:the importance of donor-negative CMV serostatus.Biol Blood Marrow Transplant.2012; 18(1):92-99.
29. Gerdemann U,Vera JF, Rooney CM,Leen AM. Generation of multivirus-specific T cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant. J Vis Exp.2011 May 27; (51).2736-2741.
30.Rocha V,Labopin M,Sanz G,Arcese W,Schwerdtfeger R,Bosi A,Jacobsen N,Ruutu T,de Lima M,Finke J,Frassoni F,Gluckman E; Acute LeukemiaWorking Party of EuropeanBlooandMarrowTransplant Group;Eurocord-Netcord Registry.Transplantsof umbilical-cord blood or bone marrow from unrelateddonors in adults with acute leukemia.N Engl J Med 2004; 351(22):2276-2285.
31. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR, Kaufman DS. Human embryonic stem cellsdifferentiate into a homogeneous population of natural killer cellswith potent in vivo antitumor activity. Blood,2009,113 (24):6094-6101.
32. Tong J, Sun Z, Liu H, Geng L, Zheng C, Tang B, Song K, Yao W, Liu X.Risk factors of CMV infection in patients after umbilical cord blood transplantation:a multicenter study in China. Chin J Cancer Res.2013; 25(6):695-703.
33.Michael Boeckh, Per Ljungman. How I treat cytomegalovirus in hematopoietic cell transplant recipients. Blood.2009; 113:5711-5719.
34. Teunissen EA, de Raad M, Mastrobattista E. Production and biomedical applications of virus-like particles derived from polyomaviruses.J Control Release. 2013; 172(1):305-321.
35.Blyth E, Clancy L, Simms R,Ma CK, Burgess J, Deo S, Byth K,Dubosq MC, Shaw PJ, Micklethwaite KP, Gottlieb D J.Donor-derivedCMV-specificTcellsreduce therequirement forCMV-directedpharmacotherapyafter allogeneicstemcelltransplantation.Blood. 2013; 121(18):3745-58.
36.Tang Y1, Li X, Wang M, Zou Q, Zhao S, Sun B, Xu L, Jiang Y.Increased numbers of NK cells, NKT-like cells, and NK inhibitory receptors in peripheral blood of patients with chronic obstructive pulmonary disease.Clin Dev Immunol. 2013;2013:721782.
37. Yeap SKI, Omar AR, Ho WY, Beh BK, Ali AM, Alitheen NB.Rhaphidophora korthalsii modulates peripheral blood natural killer cell proliferation, cytokine secretion and cytotoxicity. BMC Complement Altern Med.2013 Jun 24;13:145.
38.Cooper MA1, Fehniger TA, Caligiuri MA.The biology of human natural killer-cell subsets. Trends Immunol.2001;22(11):633-40.
39. De Colvenaer V1, Taveirne S, Hamann J, et al. Continuous CD27 triggering in vivo strongly reduces NK cell numbers. Eur J Immunol.2010; 40(4):1107-17.
40. Fu B, Tian Z, Wei H. Subsets of human natural killer cells and their regulatory effects. Immunology.2014 Apr;141(4):483-9.
41. Farag SS, Fehniger TA, Ruggeri L, et al. Natural killer cell receptors:new biology and insights into the graft versus leukemia effect. Blood,2002, 100(6):1935-1947.
42. Goodridge JP1, Burian A, Lee N, Geraghty DE.HLA-F and MHC class I open conformers are ligands for NK cell Ig-like receptors. J Immunol.2013 Oct 1;191(7):3553-62.
43. Ljunggren HG, Karre K.In search of the 'missing self':MHC molecules and NK cell recognition. Immunol Today,1990,11(7):237-244.
44. Nagarajan N2, Lind KC2, Kanaseki T3. Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum.Shastri N1, Curr Opin Immunol.2014 Feb;26C:123-127.
45. Borrego F, Kabat J, Kim DK, et al. Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol Immunol,2002,38(9):637-660.
46. Ruggeri L, Capanni M, Mancusi A, et al. Alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation. Blood Cells Mot Dis,2004, 33(3):216-221.
47. Wilson MJ, Torkar M, Haude A, et al.Plasticity in the organization and sequences of human KIR/ILT gene families[J]Proc Natl Acad Sci USA, 2000,97(9):4778-4783.
48. Palmer JM1, Rajasekaran K, Thakar MS, Malarkannan S.Clinical relevance of natural killer cells following hematopoietic stem cell transplantation. J Cancer. 2013;4(1):25-35.
49. Merino AM1, Sabbaj S, Easlick J, Goepfert P, Kaslow RA, Tang J.Dimorphic HLA-B signal peptides differentially influence HLA-E- and natural killer cell-mediated cytolysis of HIV-1-infected target cells. Clin Exp Immunol.2013 Dec;174(3):414-23.
50.Roda-Navarro P1, Reyburn HT.The traffic of the NKG2D/Dap10 receptor complex during natural killer (NK) cell activation.J Biol Chem.2009 Jun 12;284(24):16463-72.
51. Gonzalez S1, Groh V, Spies T. Immunobiology of human NKG2D and its ligands. Curr Top Microbiol Immunol.2006;298:121-38.
52.Ullrich E1, Koch J2, Cerwenka A3, Steinle A4. New prospects on the NKG2D/NKG2DL system for oncology.Oncoimmunology.2013 Oct 1;2(10):e26097.
53. Costello RT1, Boehrer A, Sanchez C, Mercier D, Baier C, Le Treut T, Sebahoun G Differential expression of natural killer cell activating receptors in blood versus bone marrow in patients with monoclonal gammopathy..Immunology.2013 Jul;139(3):338-41.
54. Fauriat C, Just-Landi S, Mallet F,et al. Deficient expression of NCR in NK cells from acute myeloid leukemia:Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction.Blood.2007; 109(l):323-30.
55.Huang XJ, Wang Y, Liu DH, Xu LP, Chen H, Chen YH, et al.Modified donor lymphocyte infusion (DLI) for the prophylaxis of leukemia relapse after hematopoietic stem cell transplantation in patients with advanced leukemia--feasibility and safety study.J Clin Immunol 2008; 28:390-397.
56.Park M1, Lee SH, Lee YH, et al. Pre-engraftment syndrome after unrelated cord blood transplantation:a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant.2013; 19(4):640-6.
57.Sakiyama M1, Kami M, Hori A,et al. Regimen-related toxicity following reduced-intensity stem-cell transplantation (RIST):comparison between Seattlecriteria and National Cancer Center Common Toxicity Criteria (NCI-CTC) version 2.0.Bone Marrow Transplant.2004; 34(9):787-94.
58. Ljungman P, Perez-Bercoff L, Jonsson J, et al.Risk factors for the development of cytomegalovirusdisease after allogeneic stem cell transplantation.Haematologica,2006,91(1):78-83.
59. Eid AJ, Bakri SJ, Kijpittayarit S, et al.Clinical features and outcomes of cytomegalovirusretinitis after transplantation. Transpl InfectDis.2008, 10(1):13-18.
60. Zheng C, Tang B, Yao W, et al.Comparison of unrelated cord blood transplantation and HLA-matched sibling hematopoietic stem cell transplantation for patients with chronic myeloid leukemia in advanced stage.Biol Blood Marrow Transplant.2013; 19(12):1708-12.
61. Charrier E1, Cordeiro P, Brito RM, et al. Reconstitution of maturating and regulatory lymphocyte subsets after cord blood and BMT in children.Bone Marrow Transplant.2013; 48(3):376-82.
62.Locatelli F1, Kabbara N, Ruggeri A, et al. of patients with hemoglobinopathies given either cord blood or bone marrow transplantation from an HLA-identical sibling. Blood.2013 Aug 8; 122(6):1072-8.
63. Crisinel PA, Duval M, Crisinel DT, et al. Risk of cytomegalovirus infection and disease after umbilical cord blood transplantation in children. Can J Infect Dis Med Microbiol.2013 Spring; 24(1):ell-5.
64. Juan Tong, Zimin Sun, Huilan Liu, et al. The risk factors of CMV infection in patients after umbilical cord blood transplantation:a multicenter study in China. ChinJCancer Res.2013; 25(6):695-703.
65. Boeckh M, Gooley TA, Myerson D, et al. Cytomegalovirus pp65antigenemia-guided early treatment with ganciclovirversus ganciclovir at engraftment after allogeneicmarrow transplantation:a randomizeddouble-blind study. Blood,1996,88(10):4063-4071.
66. Emery VC, Sabin CA, Cope AV, et al. Application of viral-loadkinetics to identify patients who develop cytomegalovirusdisease after transplantation. Lancet,2000,355(9220):2032-2036.
67. Zaia JA, Sun JY, Gallez-Hawkins GM, et al.The effect of single and combined activating killer immunoglobulin-like receptor genotypes on cytomegalovirus infection and immunity after hematopoietic cell transplantation.Biol Blood Marrow Transplant.2009; 15(3):315-25.
68. Inokuchi T, Kato J, Hiraoka S, et al.Long-term follow-up of ulcerative colitis patients treated on the basis of their cytomegalovirus antigen status. World J Gastroenterol.2014;20(2):509-17.
69. Benkhoucha M, Molnarfi N, Schneiter G, et al.The neurotrophic hepatocyte growth factor attenuates CD8+ cytotoxic T-lymphocyte activity.J Neuroinflammation.2013;10:154.
70. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR, Kaufman DS. Human embryonic stem cellsdifferentiate into a homogeneous population of natural killer cellswith potent in vivo antitumor activity. Blood,2009,113 (24):6094-6101.
71. Campos C, Pera A2, Sanchez-Correa B3, et al. Effect of age andCMVonNKcellsubpopulations. Exp Gerontol.2014 Jan 17.
72. Della Chiesa M1,Falco M,Podesta M,et al. Phenotypicandfunctionalheterogeneityofhuman NKcellsdevelopingafterumbilical cord blood transplantation:a role for human cytomegalovirus? Blood.2012; 119(2):399-410.
73. Guma M, Budt M, Saez A, et al.Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts.Blood.2006; 107(9):3624-31.
74. Della Chiesa M1,Falco M, Bertaina A, et al.Human Cytomegalovirus Infection Promotes Rapid Maturation of NK Cells Expressing Activating Killer Ig-like Receptor in Patients Transplanted with NKG2C-/- Umbilical Cord Blood. J Immunol.2014 Feb 15;192(4):1471-9.
75. Della Chiesa M, Muccio L, Moretta A.et al. CMV induces rapid NK cell maturation in HSCT recipients.Immunol Lett.2013; 155(1-2):11-3.
2.Amabile G1,Welner RS, Nombela-Arrieta C, D'Alise AM, Di Ruscio A,Ebralidze AK, Kraytsberg Y,Ye M,Kocher O,Neuberg DS,Khrapko K, Silberstein LE,Tenen DG. In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells. Blood.2013 Feb 21;121(8):1255-64.
3.Chervenick PA, Boggs DR. Patterns of proliferation and differentiation of hematopoietic stem cells after compartment depletion. Blood.1971 May;37(5):568-80.
4. Cheuk DK.Optimal stem cell source for allogeneic stem cell transplantation for hematological malignancies. World J Transplant.2013 Dec 24;3(4):99-112.
5. Ramakrishnan Al, Torok-Storb B, Pillai MM. Primary marrow-derived stromal cells:isolation and manipulation.Methods Mol Biol.2013; 1035:75-101.
6. Resnick IB1,Barkats C, Shapira MY, Stepensky P, Bloom AI, Shimoni A, Mankuta D, Varda-Bloom N, Rheingold L, Yeshurun M, Bielorai B, Toren A, Zuckerman T, Nagler A, Or R.Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC).Am J Blood Res.2013 Aug 19;3(3):225-38.
7.Anam K, Davis TA.Comparative analysis of gene transcripts for cell signaling receptors in bone marrow-derived hematopoieticstem/progenitor cell and mesenchymal stromal cell populations.Stem Cell Res Ther.2013 Sep 16;4(5):112.
8. Noel, D, et al. Muhipotent mesenchymal stromal ceils and im-mune toleranee[J]. Leuk Lymphoma,2007,48(7):P.1283-1289.
9. Lazarus, H M, et al. Cotransplantation of HLA-identical siblingculture-expanded mesenehymal stem cells and hematopoietic stemceils in hematologic malignancy patients[J]. Biol Blood Marrow. Transplant,2005,11(5): P.389-398.
10. Hashimoto S, Gm Y, Takeshita I, et al. [J]. Am J Respir Cut Can; Med, 2001,163:152-157.
11. Montanari M, Capelli D, Poloni A, et al.Long-term hematologic reconstitution after autologous peripheral blood progenitor cell transplantation:a comparison between controlled-rate freezing and uncontrolled-rate freezing at 80 degrees C. Transfusion.2003 Jan;43(1):42-49.
12. Statkute L, Verda L, Oyama Y, Mobilization, harvesting and selection of peripheral blood stem cells in patients with autoimmune diseases undergoing autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2007Mar;39(6):317-29.
13. Mahon FX, Deininger MW, et al. Selection and characterization of BCR-ABL positive cells lines with differential sensistivity to the tyrosine kinase inhibitor STI571:diverse mechanisms of resistance. Blood,2000;96:1070-1079.
14. Weisberg E, Griffin JD. Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines. Blood,2000; 95:3498-3505.
15. Byrne JL1, Carter GI, Ellis I, Haynes AP, Russell NH.Autologous GVHD following PBSCT, with evidence for a raft-versus-myeloma effect.Bone Marrow Transplant. 1997 Sep; 20(6):517-20.
16. Bolanos-Meade J1, Wu J, Logan BR, Levine JE, Ho VT, Alousi AM, Weisdorf DJ, Luznik L; Blood and Marrow Transplant Clinical Trials Network. Lymphocyte phenotype during therapy for acute graft-versus-host disease:a brief report from BMT-CTN 0302. Biol Blood Marrow Transplant.2013 Mar;19(3):481-5.
17. Gluckman E, Broxmeyer HE, Auerbach AD, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling. N Engl J Med 1989; 321:1174-1178.
18. Section on Hematology/Oncology and Section on Allergy/Immunology. Cord Blood Banking for Potential Future Transplantation. Pediatrics,2007; 119:165-170.
19. Yamaguchi M1,Ebina S, Kashiwakura I.Involvement of placental/umbilical cord blood acid-base status and gas values on the radiosensitivity of humanfetal/neonatal hematopoietic stem/progenitor cells. J Radiat Res.2013 Mar 1;54(2):277-84.
20. Geyer MB1, Jacobson JS, Freedman J, George D, Moore V, van de Ven C, Satwani P, Bhatia M, Garvin JH, Bradley MB, Harrison L, Morris E, Della-Latta P,Schwartz J, Baxter-Lowe LA, Cairo MS.A comparison of immune reconstitution and graft-versus-host disease following myeloablative conditioning versus reduced toxicity conditioning and umbilicalcord bloodtransplantation in paediatric recipients.Br J Haematol.2011 Oct;155(2):218-34.
21. Liu J1,He J, Chen S, Qin F, Wang F,Xu G,Zhu F, Lv H, Yan L. Cord Blood Banking and Transplantation in China:A Ten Years Experience of a Single Public Bank.Transfus Med Hemother.2012 Feb;39(1):23-27.
22. Mouzaki A1, Panagoulias I, Raptis G, Farri-Kostopoulou E.Cord blood leptin levels of healthy neonates are associated with IFN-y production by cord blood T-cells. PLoS One.2012;7(7):e40830.
23. Risdon G, Gaddy J, Stehman FB,Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation. Cell Immunol. 1994; 154(1):14-24.
24. Parmar S1, Liu X2,Tung SS2, Robinson SN2, Rodriguez G2, Cooper LJ3, Yang H4, Shah N2, Yang H2, Konopleva M4, Molldrem JJ2, Garcia-Manero G4,Najjar A5,Yvon E2,McNiece I2,Rezvani K2,Savoldo B6, Bollard CM7, Shpall EJ2. Cytotherapy. Third-party umbilical cord blood-derived regulatory T cells prevent xenogenic graft-versus-host disease.2014 Jan;16(1):90-100.
25. Parody R1, Martino R, Rovira M, Vazquez L, Vazquez MJ, de la Camara R, Blazquez C, Fernandez-Aviles F, Carreras E, Salavert M, Jarque I, Martin C, Martinez F, Lopez J, Torres A, Sierra J, Sanz GF. Severe infections after unrelated donor allogeneic hematopoietic stem cell transplantation in adults:comparison of cord blood transplantation with peripheral blood and bone marrow transplantation. Biol Blood Marrow Transplant.2006 Jul;12(7):734-48.
26. Pera A1, Campos Cl, Corona Al, Sanchez-Correa B2, Tarazona R2, Larbi A3, Solana R1.CMV Latent Infection Improves CD8+ T Response to SEB Due to Expansion of Polyfunctional CD57+ Cells in Young Individuals. PLoS One.2014 Feb 12;9(2):e88538.
27. Boeckh M, Leisenring W, Riddell SR, Bowden RA, Huang ML, Myerson D, Stevens-Ayers T, Flowers ME, Cunningham T, Corey L. Latecytomegalovirus disease and mortality in recipientsof allogeneic hematopoietic stem cell transplants:importance of viral load and T-cell immunity.Blood,2003, 101(2):407-414.
28. Mikulska M, Raiola AM, Bruzzi P, Varaldo R, Annunziata S, Lamparelli T, Frassoni F, Tedone E, Galano B, Bacigalupo A, Viscoli C. CMV infection after transplant from cord blood compared to other alternative donors:the importance of donor-negative CMV serostatus.Biol Blood Marrow Transplant.2012; 18(1):92-99.
29. Gerdemann U,Vera JF, Rooney CM,Leen AM. Generation of multivirus-specific T cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant. J Vis Exp.2011 May 27; (51).2736-2741.
30.Rocha V,Labopin M,Sanz G,Arcese W,Schwerdtfeger R,Bosi A,Jacobsen N,Ruutu T,de Lima M,Finke J,Frassoni F,Gluckman E; Acute LeukemiaWorking Party of EuropeanBlooandMarrowTransplant Group;Eurocord-Netcord Registry.Transplantsof umbilical-cord blood or bone marrow from unrelateddonors in adults with acute leukemia.N Engl J Med 2004; 351(22):2276-2285.
31. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR, Kaufman DS. Human embryonic stem cellsdifferentiate into a homogeneous population of natural killer cellswith potent in vivo antitumor activity. Blood,2009,113 (24):6094-6101.
32. Tong J, Sun Z, Liu H, Geng L, Zheng C, Tang B, Song K, Yao W, Liu X.Risk factors of CMV infection in patients after umbilical cord blood transplantation:a multicenter study in China. Chin J Cancer Res.2013; 25(6):695-703.
33.Michael Boeckh, Per Ljungman. How I treat cytomegalovirus in hematopoietic cell transplant recipients. Blood.2009; 113:5711-5719.
34. Teunissen EA, de Raad M, Mastrobattista E. Production and biomedical applications of virus-like particles derived from polyomaviruses.J Control Release. 2013; 172(1):305-321.
35.Blyth E, Clancy L, Simms R,Ma CK, Burgess J, Deo S, Byth K,Dubosq MC, Shaw PJ, Micklethwaite KP, Gottlieb D J.Donor-derivedCMV-specificTcellsreduce therequirement forCMV-directedpharmacotherapyafter allogeneicstemcelltransplantation.Blood. 2013; 121(18):3745-58.
36.Tang Y1, Li X, Wang M, Zou Q, Zhao S, Sun B, Xu L, Jiang Y.Increased numbers of NK cells, NKT-like cells, and NK inhibitory receptors in peripheral blood of patients with chronic obstructive pulmonary disease.Clin Dev Immunol. 2013;2013:721782.
37. Yeap SKI, Omar AR, Ho WY, Beh BK, Ali AM, Alitheen NB.Rhaphidophora korthalsii modulates peripheral blood natural killer cell proliferation, cytokine secretion and cytotoxicity. BMC Complement Altern Med.2013 Jun 24;13:145.
38.Cooper MA1, Fehniger TA, Caligiuri MA.The biology of human natural killer-cell subsets. Trends Immunol.2001;22(11):633-40.
39. De Colvenaer V1, Taveirne S, Hamann J, et al. Continuous CD27 triggering in vivo strongly reduces NK cell numbers. Eur J Immunol.2010; 40(4):1107-17.
40. Fu B, Tian Z, Wei H. Subsets of human natural killer cells and their regulatory effects. Immunology.2014 Apr;141(4):483-9.
41. Farag SS, Fehniger TA, Ruggeri L, et al. Natural killer cell receptors:new biology and insights into the graft versus leukemia effect. Blood,2002, 100(6):1935-1947.
42. Goodridge JP1, Burian A, Lee N, Geraghty DE.HLA-F and MHC class I open conformers are ligands for NK cell Ig-like receptors. J Immunol.2013 Oct 1;191(7):3553-62.
43. Ljunggren HG, Karre K.In search of the 'missing self':MHC molecules and NK cell recognition. Immunol Today,1990,11(7):237-244.
44. Nagarajan N2, Lind KC2, Kanaseki T3. Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum.Shastri N1, Curr Opin Immunol.2014 Feb;26C:123-127.
45. Borrego F, Kabat J, Kim DK, et al. Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol Immunol,2002,38(9):637-660.
46. Ruggeri L, Capanni M, Mancusi A, et al. Alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation. Blood Cells Mot Dis,2004, 33(3):216-221.
47. Wilson MJ, Torkar M, Haude A, et al.Plasticity in the organization and sequences of human KIR/ILT gene families[J]Proc Natl Acad Sci USA, 2000,97(9):4778-4783.
48. Palmer JM1, Rajasekaran K, Thakar MS, Malarkannan S.Clinical relevance of natural killer cells following hematopoietic stem cell transplantation. J Cancer. 2013;4(1):25-35.
49. Merino AM1, Sabbaj S, Easlick J, Goepfert P, Kaslow RA, Tang J.Dimorphic HLA-B signal peptides differentially influence HLA-E- and natural killer cell-mediated cytolysis of HIV-1-infected target cells. Clin Exp Immunol.2013 Dec;174(3):414-23.
50.Roda-Navarro P1, Reyburn HT.The traffic of the NKG2D/Dap10 receptor complex during natural killer (NK) cell activation.J Biol Chem.2009 Jun 12;284(24):16463-72.
51. Gonzalez S1, Groh V, Spies T. Immunobiology of human NKG2D and its ligands. Curr Top Microbiol Immunol.2006;298:121-38.
52.Ullrich E1, Koch J2, Cerwenka A3, Steinle A4. New prospects on the NKG2D/NKG2DL system for oncology.Oncoimmunology.2013 Oct 1;2(10):e26097.
53. Costello RT1, Boehrer A, Sanchez C, Mercier D, Baier C, Le Treut T, Sebahoun G Differential expression of natural killer cell activating receptors in blood versus bone marrow in patients with monoclonal gammopathy..Immunology.2013 Jul;139(3):338-41.
54. Fauriat C, Just-Landi S, Mallet F,et al. Deficient expression of NCR in NK cells from acute myeloid leukemia:Evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction.Blood.2007; 109(l):323-30.
55.Huang XJ, Wang Y, Liu DH, Xu LP, Chen H, Chen YH, et al.Modified donor lymphocyte infusion (DLI) for the prophylaxis of leukemia relapse after hematopoietic stem cell transplantation in patients with advanced leukemia--feasibility and safety study.J Clin Immunol 2008; 28:390-397.
56.Park M1, Lee SH, Lee YH, et al. Pre-engraftment syndrome after unrelated cord blood transplantation:a predictor of engraftment and acute graft-versus-host disease. Biol Blood Marrow Transplant.2013; 19(4):640-6.
57.Sakiyama M1, Kami M, Hori A,et al. Regimen-related toxicity following reduced-intensity stem-cell transplantation (RIST):comparison between Seattlecriteria and National Cancer Center Common Toxicity Criteria (NCI-CTC) version 2.0.Bone Marrow Transplant.2004; 34(9):787-94.
58. Ljungman P, Perez-Bercoff L, Jonsson J, et al.Risk factors for the development of cytomegalovirusdisease after allogeneic stem cell transplantation.Haematologica,2006,91(1):78-83.
59. Eid AJ, Bakri SJ, Kijpittayarit S, et al.Clinical features and outcomes of cytomegalovirusretinitis after transplantation. Transpl InfectDis.2008, 10(1):13-18.
60. Zheng C, Tang B, Yao W, et al.Comparison of unrelated cord blood transplantation and HLA-matched sibling hematopoietic stem cell transplantation for patients with chronic myeloid leukemia in advanced stage.Biol Blood Marrow Transplant.2013; 19(12):1708-12.
61. Charrier E1, Cordeiro P, Brito RM, et al. Reconstitution of maturating and regulatory lymphocyte subsets after cord blood and BMT in children.Bone Marrow Transplant.2013; 48(3):376-82.
62.Locatelli F1, Kabbara N, Ruggeri A, et al. of patients with hemoglobinopathies given either cord blood or bone marrow transplantation from an HLA-identical sibling. Blood.2013 Aug 8; 122(6):1072-8.
63. Crisinel PA, Duval M, Crisinel DT, et al. Risk of cytomegalovirus infection and disease after umbilical cord blood transplantation in children. Can J Infect Dis Med Microbiol.2013 Spring; 24(1):ell-5.
64. Juan Tong, Zimin Sun, Huilan Liu, et al. The risk factors of CMV infection in patients after umbilical cord blood transplantation:a multicenter study in China. ChinJCancer Res.2013; 25(6):695-703.
65. Boeckh M, Gooley TA, Myerson D, et al. Cytomegalovirus pp65antigenemia-guided early treatment with ganciclovirversus ganciclovir at engraftment after allogeneicmarrow transplantation:a randomizeddouble-blind study. Blood,1996,88(10):4063-4071.
66. Emery VC, Sabin CA, Cope AV, et al. Application of viral-loadkinetics to identify patients who develop cytomegalovirusdisease after transplantation. Lancet,2000,355(9220):2032-2036.
67. Zaia JA, Sun JY, Gallez-Hawkins GM, et al.The effect of single and combined activating killer immunoglobulin-like receptor genotypes on cytomegalovirus infection and immunity after hematopoietic cell transplantation.Biol Blood Marrow Transplant.2009; 15(3):315-25.
68. Inokuchi T, Kato J, Hiraoka S, et al.Long-term follow-up of ulcerative colitis patients treated on the basis of their cytomegalovirus antigen status. World J Gastroenterol.2014;20(2):509-17.
69. Benkhoucha M, Molnarfi N, Schneiter G, et al.The neurotrophic hepatocyte growth factor attenuates CD8+ cytotoxic T-lymphocyte activity.J Neuroinflammation.2013;10:154.
70. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR, Kaufman DS. Human embryonic stem cellsdifferentiate into a homogeneous population of natural killer cellswith potent in vivo antitumor activity. Blood,2009,113 (24):6094-6101.
71. Campos C, Pera A2, Sanchez-Correa B3, et al. Effect of age andCMVonNKcellsubpopulations. Exp Gerontol.2014 Jan 17.
72. Della Chiesa M1,Falco M,Podesta M,et al. Phenotypicandfunctionalheterogeneityofhuman NKcellsdevelopingafterumbilical cord blood transplantation:a role for human cytomegalovirus? Blood.2012; 119(2):399-410.
73. Guma M, Budt M, Saez A, et al.Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts.Blood.2006; 107(9):3624-31.
74. Della Chiesa M1,Falco M, Bertaina A, et al.Human Cytomegalovirus Infection Promotes Rapid Maturation of NK Cells Expressing Activating Killer Ig-like Receptor in Patients Transplanted with NKG2C-/- Umbilical Cord Blood. J Immunol.2014 Feb 15;192(4):1471-9.
75. Della Chiesa M, Muccio L, Moretta A.et al. CMV induces rapid NK cell maturation in HSCT recipients.Immunol Lett.2013; 155(1-2):11-3.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |