| 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.Results1Clinical 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.ConclusionIt 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. |
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