The Study On Cytomegalovirus Specific Cytotoxic T-lymphocyte In Allogeneic Hematopoietic Stem Cell Transplant Recipients

ObjectiveCytomegalovirus (CMV) is the ubiquitous prototype member of theB-herpesvirinae. As with all members of the Herpesviridae, CMV is a very wildly human herpesvirus present in 40%-90% individuals, dependent on age and social class. The virus has the ability to persist in an immunocompetent host in a latent state after primary infection. Primary infection in an immunocompetent host is usually asymptomatic, but the virus has lifelong latency, so-called latent infection. Major factor for immune function destroyed in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients include pretreatment, graft-versus-host disease (GVHD) and immunodepressant, therefore ,CMV reactivation can cause CMV disease in 5~18% infected patients, such as interstitial pneumonitis; it has a mortality rate of 30% to 50%, despite treatment with ganciclovir, phosphonoformate and immunoglobulins. Prophylaxis with acyclovir or preemptive-treatment with ganciclovir can be only partially effective, although the majority of patients undergo prophylaxis or are treated effectively with these agents. Myelosuppression from ganciclovir may lead to graft failure. Continued treatment with antiviral drugs post-HSCT may induce drug resistance and delay reconstitution of CMV specific immunity in patients, resulting in late CMV disease increasing.The study on cellular immunity reconstitution of HSCT recipients was limited in the counts of T lymphocyte and its subgroup in the past. The detection of the frequency of T cell receptor recombination exicision circle (TREC) using quantitation polymerase chain reaction (Q-PCR) is a reliable test facility for evaluating thefunction of thymus in HSCT recipients, the function of T lymphocyte reconstitution is more importable than the counts of T lymphocyte reconstitution in cellular immunity reconstitution.. Proliferation essay of T lymphocyte and detection cytokine from T lymphocyte just evaluate non-specific cellular immunity.Evidence from studies in previously indicates that CMV-specific CD8+ cytotoxic T lymphocytes (CTL) have a predominant role in protection against CMV disease. It is important that evaluating and monitoring the activity of CMV specific CTL and the development of cellular immunologic response mediated by CMV specific CTL in HSCT recipients. For the object referred above, It is demand that detecting CMV specific CTL directly, including the counts, function and activity of CMV specific CTL. The counts of T lymphocyte subgroup and the proliferation assays for non-specific cellular immunity can not provide adequate information about CMV specific CTL.In this study, the introduction of HLA-peptide Pentamer (Pentamer) and enzyme-linked immunospot (ELISPOT) have allowed direct visualization of CMV specific CTL. The two new techniques are both highly specific and sensitive and has been applied to many areas of both human and murine immunology. The aim of this analysis is to better define essential elements of protective immunity to CMV, using HLA-peptide Pentamer and ELISPOT assay to study the counts, function and activity of CMV specific CTL in HSCT recipients, and to get the message of the reconstitution of CMV specific CTL. The expectation of this analysis is to provide evidence for selecting reasonable anti-CMV prophylactic treatment and anti-CMV therapy timely.Methods32 patients who received an allo-HSCT in 2003 to 2005 at the Nanfang Hospital and 9 donors(CMV-Ag positive , 2 donors) were eligible for study entry, median age is 29 (range, 11-52). Female/male ratio is 15/17. 32 patients were: acute myelogenous leukemia(9 patients), acute lymphoblastic leukemia(4 patients), chronic myelogenous leukemia(17 patients) and non-Hodgkin's lymphoma(2 patients). CMV-IgG serologic status: donor (-) / recipient (-) had 30 patients and donor (+) / recipient (-) had 2 patients.Blood samples from recipients and donors were analyzed for CMV antigen by using immunohistochemistry. All patients were tested every other weekly for CMV antigen after haematogenesis reconstitution, and were tested monthly for CMV antigen after 6 months. On continuous twice detections of CMV -Ag (+) (antigenemia assessment results, >3-5 CMV antigen cells/50 000 peripheral blood mononuclear cells [PBMC]), ganciclovir (GCV) or phosphonoformate (PFA) therapy was begun until CMV -Ag (-).Blood samples were collected from HSCT recipients who were HLA-A2 positive pre-HSCT and at 3, 6, 9, 12 months post-HSCT. If possible, blood was also obtained from the donor pre-HSCT. Detection of CMV-specific CTL (HLA-A2-pp65495-503 specific CTL) using HLA-A2-pp65495-503 pentamer complexes was performed with a fluorescence activated cell-sorter flow cytometer.T lymphocyte subgroup reconstitution was analysised at the same time.IFN-gamma ELISPOT assay was performed in 96 bottomed-well microplates. Plates were coated overnight with antibody to human IFN-gamma, and then washed plates. Heparin treated blood was collected from recipients post-HSCT, PBMC were isolated by using Ficoll-Isopaque density gradient centrifugation. PBMC were thawed and then added at 4xlO5/well and incubated in triplicate with CMV pp65(10ug/ml) , After incubation for 20 h at 37 °C, cells were removed and spots were revealed with a second biotinylated antibody to human IFN-gamma followed by streptavidine alkaline phosphatase and BCIP/NBT substrate . Spots were counted using the Elispot reader. All data were computed using SPSS 10.0 for windows.Results1. There were complete data in 29/32 HSCT recipients. 24/29 patients(82.8%) suffered CMV antigenemia post-HSCT once or more times. The median time of onset CMV antigenemia is at 78 day post-HSCT(range, 31-476 day). No patients developed CMV disease.2. When expressed in absolute counts, the median value was 0.014xl09/L(range, 0.0004 ~0.1370xl09/L) of HLA-A2-pp65-specific CTL were detectable in the peripheral blood of 9 healthy donors tested. When expressed as a fraction of CD8+ T cells, the median value was 0.92 % (range,0.01—8.13%) of CD8+ T cells.When expressed in absolute counts, the median value was 0.023xl09/L(range, 0.001 ~ 2.488xlO9/L) of HLA-A2-pp65-specific CTL were detectable in the peripheral blood of 21 patients tested. When expressed as a fraction of CD8+ T cells, the median value was 6.275% (range,0.91~32.27% ) of CD8+ T cells.3. Monitoring reconstitution of HLA-A2-pp65-specific CTL post- HSCT: CpThe counts of HLA-A2-pp65-specific CTL were continuously increased in 12month post-HSCT. The absolute counts of HLA-A2-pp65-specific CTL at 3 month post-HSCT are lower than at 6 month (P=0.000) ,9 month (P=0.022) , 12month (P=0.013) obviously.?The absolute counts of HLA-A2-pp65-specific CTL at 3 month post-HSCT are lower than pre-HSCT (P=0.015).(3)The absolute counts and the percentages (of CD8+ T cells) of HLA-A2-pp65-specific CTL at 12 month post-HSCT were more than pre-HSCT (P=0.008, P=0.026).?The reconstitution of HLA-A2-pp65-specific CTL post-HSCT of 3 patients whose testing more than 3 times were showed increasing or decreasing individually, because they had affected by some reasons.4. Analysis of CMV specific CTL by ELISPOT?the median value of spots was 46(range, 13-65) in 22 patients. The numbers of spots within 6 months post-HSCT is lower than after 6 months obviously (P= 0.004).?the frequency of HLA-A2-pp65-specific CTL by pentamer staining and IFN-y producing T cells stimulated by the pp65 peptide in the ELISPOT assay are correlated (P=0.038) in the whole group (n=10) of HLA-A2 patientsConclusions1. In this study, the absolute counts and the percentages (of CD8+ T cells) of HLA-A2-pp65-specific CTL in health donors by pentamer were more than the reported other studies by tetramer. CMV-Ag(+) of donor maybe is a correlated factor for the reconstitution of HLA-A2-pp65-specific CTL post-HSCT in HSCT recipients.2. The counts of HLA-A2-pp65-specific CTL were continuously increased post-HSCT, and it grew more quick from 3 month to 6 month than after 6 month. Using pentamer and Elispot, It is clearly that the reconstitution of the function ofCMV specific CTL were notable after 6 month post-HSCT , and CMV specific CTL were lowest within 3 month post-HSCT.3. The reconstitution of HLA-A2-pp65-specific CTL post-HSCT of different patients were showed increasing or decreasing individually.