In order to evaluate whether we could predict reactivation of CMV by monitoring the number of CMV-specific cytotoxic T-lymphocytes (CTL), tetramer analysis was performed in 37 patients who underwent hematopoietic stem cell transplantation (HSCT). The results disclosed that the mean number of CMV-specific CTL at day 30 did not differ among patients who developed CMV antigenemia (22/μl) and those who did not (12/μl). Serial tetramer analysis showed that 21% of the patients had >10/μl CMV-specific CTL at the first detection of CMV antigenemia and 67% of the patients had more than 10/μl CMV-specific CTL at the onset of CMV disease. Intracellular staining upon stimulation by CMV lysates and peptide in patients with CMV colitis revealed that both IFN-γ producing CD4+ and CD8+ lymphocytes were suppressed at the onset of CMV colitis (1.6 and 8/μl), which increased with recovery of the disease (19 and 47/μl). These data suggest that it is difficult to predict CMV reactivation solely by the number of CMV-specific CTL. We suggest that additional functional analysis by intracellular cytokine assay may be useful for immunomonitoring against CMV.
Reactivation of CMV is one of the major complications in patients undergoing hematopoietic stem cell transplantation (HSCT) and is significantly related to morbidity and mortality despite the recent development of potent antiviral medications.1, 2 The decision to administer antiviral therapy is currently based on the clinical risk and the detection of viremia by various methods including PCR for CMV-derived DNA or CMV antigenemia assay. However, treatment with antiviral drugs such as ganciclovir and foscarnet increases the risk for secondary graft failure and other infectious complications due to myelotoxicity. To optimize the therapy with minimum drug exposure, it is important to monitor the recovery of CMV-specific immunity accurately. For this purpose, tetramer-based monitoring of CMV-specific cytotoxic T-cells (CTL) has been widely performed in patients with an HLA-A02 or HLA-B07 serotype.3, 4, 5, 6, 7, 8, 9, 10, 11 Some of the results have demonstrated that the reconstitution of CMV-specific CTL as evaluated by quantitative tetramer to levels >10–20/μl is adequate for protection against CMV infection.5, 6, 7 However, some patients with CMV-specific CTL above this level still experience CMV reactivation.9 It has also been reported that the cellular response to CMV in immunosuppressed patients reflects functional impairment,10 and CMV reactivation following HSCT has been shown to be associated with the presence of dysfunctional CMV-specific T-cells.11 Therefore, by itself, the quantification of CMV-specific CTL seems to be insufficient and a simultaneous qualitative analysis of CMV-specific lymphocytes is needed. Furthermore, it is essential that we should develop a universal monitoring method, which is not limited to HLA to cover larger populations, since an epitope that is potent enough for immunomonitoring is not obtained in some HLA types such as HLA-A24.12 In this study, simultaneous functional analysis of CMV-specific lymphocytes by intracellular cytokine assay upon stimulation with CMV lysate and antigen peptide were performed with tetramer-based CTL quantification in patients who underwent HSCT to identify an optimal monitoring system.
Materials and methods
CMV seropositive patients with an HLA-A*0201 or HLA-A*0206 genotype who had undergone allogeneic non-T-cell depleted-HSCT between February 2002 and May 2005 were included in this study. Patients were eligible with the availability for 160 days of follow-up. The study was approved by the Ethics Committee and a written informed consent was given by all patients. Peripheral blood samples were obtained at days 30±7 and 60±7 after transplantation. When patients agreed to additional sampling, additional samples were obtained every 2–3 weeks. The median age of studied patients was 52 (21–68). The genotype for HLA-A02 in 37 eligible patients was HLA-A*0201 in 20 patients, HLA-A*0206 in 16 patients and both the HLA-A*0201 and HLA-A*0206 genotypes in one patient. Nine patients received BMT from an unrelated donor, two received BMT from a related donor and the remaining 26 received peripheral blood HSCT from a related donor. With regard to the conditioning regimen, 11 patients received a conventional regimen that included 120 mg/kg CY plus 16 mg/kg BU or 120 mg/kg CY plus 12 Gy of TBI, whereas 26 received a reduced-intensity regimen with 0.66 mg/kg cladribine (2-chlorodeoxyadenosine) plus 8 mg/kg BU or 180 mg/m2 fludarabine plus 8 mg/kg BU. For patients who received a graft from an unrelated donor or DNA-mismatched donor, 4 Gy of TBI or 5 mg/kg of rabbit antithymocyte globulin (ATG) were added to reduced-intensity conditioning.
Diagnostic tests for CMV infection and CMV disease
CMV seropositivity was assessed by the detection of IgG antibodies to CMV late antigen. All patients and 31 donors (84%) were seropositive for CMV. CMV antigenemia was monitored weekly after engraftment to day 60, and at longer intervals thereafter, by using the immunocytochemical detection of pp65 antigen in leukocytes. Test results were considered to be positive when more than one cell per 50 000 leukocytes was positively stained. CMV disease was diagnosed clinically, with confirmation by biopsy of the involved organ. Pre-emptive antiviral therapy was given with an antigenemia of more than 10 positive cells per 50 000 leukocytes, which we defined as high antigenemia. The initial therapy was ganciclovir 5 mg/kg once per day, which was adjusted according to the follow-up CMV antigenemia value.
Peptide and CMV antigen
A >80% pure HLA-A02-binding peptide NLVPMVATV (AA 495–503, referred to as NLV peptide) from the CMV pp65 phosphoprotein was obtained using high-performance liquid chromatography (Qiagen, Tokyo, Japan).
Tetramer staining was performed as recently described.13 Briefly, 5 μl CD8-FITC, CD4-PC5, CD19-PC5, CD13-PC5 and 2 μl PE-conjugated tetrameric HLA-A*0201 NLV peptide complex (CMV-tetramer), purchased from Beckman Coulter Inc. (Fullerton, CA, USA), were added to 100 μl heparinized blood and incubated for 30 min. After RBC were lysed and washed twice, the cells were fixed and acquired on a flow cytometer (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ, USA). More than 20 000 cells in the lymphocyte gate were acquired and analyzed using Cellquest software. The CD4−, CD19−, CD13− and CD8+ CMV-tetramer-positive fraction of the lymphocyte gate was defined as CMV-specific CTL.
Intracellular cytokine assay
Intracellular cytokine staining was performed as recently described14 with the following modifications. Peripheral whole blood (1 ml) was stimulated for 6 h at 37 °C with 10 μg/ml NLV peptide or 1 μg/ml CMV lysate (Advanced Biotechnologies, Colombia, MD, USA), in the presence of costimulatory monoclonal antibodies, CD28 and CD49d (Becton Dickinson, 1 μg/ml each). Breferdin A (Sigma, St Louis, MO, USA; 10 μg/ml) was added for the last 4 h of incubation. Positive and negative controls were obtained by stimulating the cells with 10 μg/ml staphylococcal enterotoxin B or phosphate-buffered saline. Samples were lysed, permeabilized and stained with 2.5 μl CD69-FITC, 20 μl IFN-γ-PE, 0.6 μl CD3-APC and 10 μl CD8− or CD4− PerCP. More than 10 000 cells in the lymphocyte gate were acquired and analyzed using an FACS Calibur. The cells were gated on the CD3+ fraction of the lymphocyte gate and the proportion of IFN-γ and CD8 or CD4 was analyzed. CD69 was used as a marker for activated T-cells.
The difference between groups was compared with the Wilcoxon–Mann–Whitney U-test and the probabilities of P<0.05 were defined as statistically significant.
CMV antigenemia was observed in 27 patients (73%) between day 23 and day 56 (median, day 34) after transplantation; 13 (35%) of them had a peak antigenemia level of >10/50 000 leukocytes (high antigenemia) which required ganciclovir therapy and four (11%) subsequently developed CMV disease. The median number of leukocytes and lymphocytes were 3500 (1300–17200)/μl and 576 (228–3333)/μl at day 30 and 3900 (1400–9700)/μl and 1018 (192–6790)/μl at day 60, respectively. The median percentages of CD4+ and CD8+/lymphocytes were 35% (7–64%) and 38% (20–83%) at day 30 and 25% (6–37%) and 52% (27–83%) at day 60, respectively.
The tetramer analysis showed that the mean and median number of CMV-specific CTL at day 30 was, respectively, 11 and 1.9/μl for patients without CMV antigenemia, 23 and 7.8/μl for those with antigenemia, 33 and 15/μl for those with peak antigenemia <10/50 000, 12 and 3.7/μl for those with high antigenemia, and 21 and 2.4/μl for those who developed CMV disease. There was no significant correlation between the number of CMV-specific CTL and the incidence or severity of CMV antigenemia (P>0.05) (Figure 1).
To further evaluate the accurate number of CMV-specific CTL at the onset of CMV antigenemia, serial analysis of CMV-specific CTL was performed weekly in 14 patients (Figures 2 and 3). Patient's characteristics are shown in Table 1. CMV antigenemia was observed in 12 patients, and five of them (UPN1-5) developed high antigenemia, including three (UPN1-3) with CMV colitis. The mean and median number of CMV-specific CTL at the first detection of CMV antigenemia was 21/μl and 4.7 (0–100)/μl in the 12 patients, and three (UPN2, 13, 14) showed >10/μl. For those who did not require antiviral therapy (UPN6-14), the number of CMV-specific CTL was widely ranged. While UPN6-8 showed <10/μl throughout the observation time, the maximum CTL count was >200/μl for UPN12-14. The number of CMV-specific CTL for UPN1 and UPN2 who developed CMV colitis showed >10/μl, which was 14 and 80/μl when diarrhea occurred, and 88 and 63/μl, respectively at the time of colon biopsy which proved CMV colitis.
It has been demonstrated that in patients coexpressing HLA-A02 and HLA-B07, CMV-specific cellular immune responses restricted by HLA-B07 dominate those restricted by HLA-A02, possibly because CD8+ T cells specific for dominant epitopes are able to suppress immune responses to less favored epitopes.3 The allele frequency of HLA-B07 is low (5.2%) among Japanese15 and only one patient coexpressed HLA-B07 in this study. We did not exclude this patient (UPN14) from the analysis because the number of HLA-A02-restricted CMV-specific CTL in this patient was 9.5/μl on day 30 and the maximum value reached 243/μl on day 128 suggesting that the coexpression of HLA-B07 seems not to have affected the immunoresponse of HLA-A2 in this patient.
Intracellular cytokine assay
Upon stimulation with CMV lysate, intracellular IFN-γ staining among five patients (UPN1–5) who developed high antigenemia and required antiviral therapy showed that the mean number of IFN-γ-producing cells was 3.6 (0–6.7)/μl at day 60, which subsequently increased to 72 (15–250)/μl at day 160. As for three patients with CMV colitis (UPN1–3), only one patient (UPN2) had detectable level of IFN-γ-producing cells (4.8/μl) at the time of disease onset and were undetectable for the other two patients, which remained negative until day 90 for UPN1. The mean number of IFN-γ+ cells subsequently increased to 19 (5–38)/μl after recovery from CMV disease (Figures 2, 4a and d). Among the patients who did not require antiviral therapy, the IFN-γ-producing cells were all >10/μl at day 60.
When stimulated with CMV peptide, IFN-γ-producing cells numbered 8 (0–16)/μl at the time of disease onset with a subsequent increase to 47 (15–95)/μl after recovery from CMV disease (Figures 4b and e).
Regarding the phenotype of IFN-γ-producing cells, median of 81% (76–100) were CD4+ and <20% were CD8+ upon stimulation by CMV lysate. The staining of IFN-γ was brighter in CD4+ than in CD8+ cells and CD69 was positive for both CD4+ and CD8+ fraction. IFN-γ-producing cells were CD69 low positive and median of 42% (25–68) were CD8+, while the rest were CD8−/CD4– phenotype upon CMV peptide stimulation.
Our results showed that it is difficult to predict CMV infection by the number of CMV-specific CTL alone as this did not correlate with the incidence and severity of CMV infection. While UPN1 and UPN2 developed CMV colitis after the recovery of sufficient number of CTL, UPN6, UPN7 and UPN8 did not require antiviral therapy despite low CMV-specific CTL. These results showed that CMV disease could occur after HSCT even in patients with >10/μl CMV-specific CTL as evaluated by tetramer assay, which has been considered to be sufficient to protect against CMV infection.5, 6, 7
CMV-specific CTL emerged immediately following the detection of antigenemia in most patients, suggesting that CMV infection can be a trigger for the recovery of CMV-specific immunity. However, UPN9 had recovery of CMV-specific CTL at day 60 even though his CMV antigenemia and CMV DNA as evaluated by PCR were negative throughout the course.
On the other hand, intracellular analysis revealed that IFN-γ production in both CD4+ and CD8+ T lymphocytes was depressed in patients with high antigenemia or CMV disease and this had subsequently recovered at disease resolution. Functional analysis methods for CMV-specific immune response by flow cytometry have been established,16 and it was reported that patients who developed CMV disease after SCT had no detectable IFN-γ production by CD3+/4+ T-cells upon CMV AD-169 antigen stimulation.17 It has also been demonstrated that levels of IFN-γ-producing CD4+ cells less than one cell/μl and CD8+ less than three cells/μl upon stimulation by CMV-infected autologous dendritic cells are not protective against recurrent infection.18 As assessed by IFN-γ ELISPOT assay, the threshold level for protection against CMV reactivation was estimated as over one cell/μl peripheral blood upon CMV pp65 peptide stimulation.19 The number of IFN-γ-producing cells upon CMV lysate stimulation were above ten cells/μl among patients whose antigenemia was <10/50 000 cells in our study, which may be sufficient for protection against CMV reactivation. It is difficult to determine the exact threshold level for protection against CMV since IFN-γ production differs among various stimulating agents. Also the magnitude of response is higher in the cytokine flow cytometry assay while the cytokine flow cytometry assay was less likely than the ELISPOT assay to detect low-level responses.20
Several studies on HIV-infected patients have shown the availability of analyzing the phenotype and other cytokine production of virus-specific T-cells such as IL-2, TNF-α.21, 22, 23 It has been demonstrated that virus-specific T-cells, which produce both IFN-γ and IL-2 are important in virus-specific immunity, and that IFN-γ/IL-2 secreting CD8+ T-cells were CD45RA−/CCR7− phenotype and correlated with that of proliferating T-cells, whereas single IFN-γ-secreting cells were either CD45RA−/CCR7− or CD45RA+/CCR7−.22 Another study has shown that immunorestored patients had increased levels of circulating CMV-specific CD8+ T-cells with ‘early’ (CD27+/CD28+/CD45RA+, CD27+/CD28+/CD45RA−) and ‘intermediate’ (CD27−/CD28+/CD45RA−) phenotype.23 Only IFN-γ production was assessed in our study, however higher-order flow cytometry might have added more discriminatory value. Foster et al.24 demonstrated that CMV-specific CD4+ T-helper cells show the same reconstitution kinetics as CD8+ CTL. Thus, functional analysis of lymphocytes upon lysate stimulation that can be used to assess both CD4+ and CD8+ cells is a useful tool for monitoring T cell immunity against CMV in patients after HSCT. This method is more widely applicable than peptide stimulation or tetramer assay, since it is not restricted to HLA or a single epitope. However, peptide stimulation and tetramer assay may still be a major procedure in the analysis of CD8+ T-cells, since tetramers are widely applied to adoptive immunotherapy of CMV25 and the dominant population of IFN-γ-producing cells upon lysate stimulation was CD4+. Previous study has demonstrated that flow cytometry following stimulation of PBMC with pp65 and immediate early (IE)-1 peptide pools consisted of 15-aa peptides was highly sensitive and specific in predicting the presence of recognized epitope in the respective proteins.26 Furthermore, it has been shown that IE-1-specific responses were more important in protective immunity than pp65-specific responses in heart and lung transplant recipients.27 The stimulation with comprehensive peptide pools might have better assessed both functional CD4+ and CD8+ T-cell responses. Further study is needed to identify whether IE-1 is more important than pp65 in allogeneic HSCT patients, and the significance of IE-1 in Japanese population with low allele frequency of HLA-A1 (1.8%), -B7 (5.2%) or -B8 (<1%),15 which is known to present IE-1 epitopes.
It is likely that the patients who did not have CMV reactivation despite low CMV-specific CTL had sufficient T-cell immune-recovery against CMV since the number of intracellular IFN-γ positive cells upon CMV lysate stimulation was as high as that in patients who had recovered from CMV reactivation. As for CD8+ T cells in these patients, CTL against other CMV-epitopes besides NLV might have helped to protect against CMV. It is reported that the recovery of CMV specific T-cells is earlier in patients who received reduced-intensity conditioning compared to conventional regimen and this was delayed by the use of ATG.19, 28 Additionally, the graft source and CD3+ T-cell dose significantly influence the recovery of CMV-specific immunity.28 The difference of immune recovery according to the conditioning regimen and graft source was not demonstrated in this study, probably due to heterogeneous patients and small sample size. Functional depression of the lymphocytes due to corticosteroid for GVHD seems to be the major cause of CMV infection as documented in all patients with high antigenemia. Moreover, 75% of the patients with CMV disease were receiving more than 1 mg/kg/day of methylprednisolone (mPSL), while among those who did not require antiviral therapy, only 13% had received 1 mg/kg/day or more mPSL. The influence of corticosteroid on the number of CMV-specific CTL is controversial. Some studies have reported that a significant reduction of CMV-specific CTL occurred with corticosteroid therapy.6, 7, 8 Others have shown that the frequency and the absolute number of CMV-specific CD8+ T cells were similar in patients receiving corticosteroids and those who didn't, while the CMV-specific CD8+ T cells showed decreased cytokine production.10, 11 Our result was consistent with the latter observation that while the number of CMV-specific CTL does not decrease significantly with corticosteroid therapy, IFN-γ production of CMV-specific CTL is severely suppressed. Therefore, concomitant assessment of T-cell function is essential in patients after HSCT, especially in those who are receiving corticosteroid therapy.
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Morita-Hoshi, Y., Heike, Y., Kawakami, M. et al. Functional analysis of cytomegalovirus-specific T lymphocytes compared to tetramer assay in patients undergoing hematopoietic stem cell transplantation. Bone Marrow Transplant 41, 515–521 (2008) doi:10.1038/sj.bmt.1705932
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