Cytomegalovirus (CMV) represents a major cause of morbidity after allogeneic stem cell transplantation (allo-SCT). Using interferon-gamma-enzyme-linked immunospot (ELISPOT) assay and HLA-peptide tetramers, we analysed 54 patients who received a reduced-intensity conditioning regimen, including fludarabine, busulphan and antithymocyte globulin (ATG), with the aim of defining essential elements of protective immunity to CMV. The cumulative incidence of CMV positive antigenaemia was 37% occurring at a median of 43 days (range, 7–104) after allo-SCT. In univariate analysis, conditioning regimen (ATG dose) and graft characteristics (graft source and CD3+ T-cell dose) significantly influenced CMV-specific immune recovery. A significant correlation (P=0.000002) was found between CMV-specific T cells detected by IFN-gamma ELISPOT assay and pp65-specific CD8+ T-cell frequency quantified by tetramers. CMV-specific CD8+ T cells presented a phenotype of effector cells (perforin and 2B4 positive). In multivariate analysis, bone marrow (BM) as a graft source was the only variable associated with an increased risk of CMV positive antigenaemia (P=0.0001) in line with the ELISPOT assay showing a higher frequency of functional CMV-specific effectors within peripheral blood stem cell grafts as compared to BM. Thus, early monitoring of CMV-specific immune recovery using sensitive new tools might prove useful for patient management after allo-SCT.
Cytomegalovirus (CMV) represents a major cause of morbidity in patients undergoing allogeneic stem cell transplantation (allo-SCT).1 Although antiviral prophylaxis has led to a significant reduction in early CMV disease,2 recovery of CMV-specific cytotoxic effectors is still necessary for the long-term control of CMV reactivation after allo-SCT. In an attempt to reduce procedure-related toxicity in patients not eligible for standard myeloablative allo-SCT, different reduced-intensity conditioning (RIC) regimens could show that durable donor cell engraftment can be achieved after RIC allo-SCT.3,4,5 All of these RIC protocols have been shown to be highly immunosuppressive, but because of the variability in the degree of myeloablation, the toxicity profile might vary from one protocol to another. RIC regimens including potentially T-cell depleting reagents, such as antithymocyte globulin (ATG) or Campath, have been shown to be associated with a high rate of early CMV positive antigenaemia.6,7
The introduction of new biologic tools such as the interferon-gamma-enzyme-linked immunospot (ELISPOT) assay or tetrameric HLA-peptide complexes has dramatically improved our understanding of virus-specific cytotoxic T-cell responses.8,9,10 Our current knowledge of CMV-specific immune recovery profile after allo-SCT is based primarily on analyses performed in the myeloablative allo-SCT setting using time- and labour-intensive standard cytotoxicity assays.11 Risk factors for CMV positive antigenaemia are still poorly defined after RIC, and limited information is available on factors influencing CMV-specific immune effectors recovery in this setting. This report describes the results of 54 patients who received an ATG-, fludarabine- and busulphan-based RIC for allo-SCT. The aim of this analysis was to better define essential elements of protective immunity to CMV, using the ELISPOT assay and HLA-peptide tetramers to study the relationship of functional T-cell responses to CMV-associated clinical events after RIC allo-SCT.
Patients and methods
Patients, donors and allo-SCT procedures
In all, 54 patients who received an RIC allo-SCT from HLA-identical donors for haematological and nonhaematological malignancies were included in this study. Written informed consent was obtained from each patient and donor. Clinical study design and end points, detailed allo-SCT procedures and supportive care measures are described elsewhere.12 The preparative regimen was adapted from that reported by Slavin et al.,3 with fludarabine 30 mg/m2 for 6 consecutive days, oral busulphan 4 mg/kg/day for 2 consecutive days and ATG (Thymoglobuline; Sangstat, Lyon, France) 2.5 mg/kg/day for 4, 3 or 1 day as indicated hereinafter (administered intravenously over 6–8 h between day –4 and –1). As part of the protocol, the ATG dose administered during conditioning was progressively decreased from initially 10 to 2.5 mg/kg. For comparison of ‘low’ vs ‘high’ ATG dose, the limit of 2.5 mg/kg was defined as the median ATG dose received by the patients. Therefore, patients receiving 10 or 7.5 mg/kg of ATG were considered as the ‘high’ ATG dose group, while patients receiving 2.5 mg/kg represented the ‘low’ ATG dose group.12 GVHD prophylaxis included cyclosporine A (CsA) only at a dose of 3 mg/kg/day by continuous intravenous infusion starting from day −2, and changed to twice daily oral dosing as soon as tolerated. CsA doses were adjusted to achieve blood levels between 150 and 250 ng/ml and to prevent renal dysfunction. CsA was tapered starting on day 90 if no GVHD appeared. A total of 13 patients in this series received a bone marrow (BM) graft collected under general anaesthesia, whereas the remaining 41 patients received peripheral blood stem cells (PBSC). For PBSC collection, donors were treated with granulocyte-colony-stimulating factor (G-CSF) at a dose of 10 μg/kg/day for 5 days. The day of BM or PBSC infusion was designated as day 0. The graft was analysed for content of haematopoietic progenitors and CD3+ lymphoid cells using standard flow cytometry procedures. Acute and chronic GVHD were graded according to standard criteria.13 Detailed analysis of GVHD features, evolution and treatments in this group of patients has already been reported.12
CMV antigenaemia diagnosis and pre-emptive CMV therapy
All blood products were filtered and irradiated, but not CMV screened. In the first 100 days post allo-SCT, patients were assessed twice per week for CMV infection by antigenaemia assay (CINAkit, Argene Biosoft, France). This method uses a monoclonal antibody pool that recognises the lower matrix structural phosphoprotein pp65. With this semiquantitative technique, results were reported as the number of antigen positive cells in a positive specimen.6 A patient was considered positive when having at least two infected cells out of 2 × 105 leucocytes, in order to initiate pre-emptive ganciclovir therapy. All patients with a positive CMV antigenaemia received front-line preemptive ganciclovir therapy (5 mg/kg intravenously twice daily) for 14 days. None of the patients received maintenance therapy after pre-emptive therapy. CMV disease was defined as described previously.2
IFN-gamma ELISPOT assay
IFN-gamma ELISPOT assay was performed in PVDF-bottomed-well microplates (Millipore MAIPS45, Millipore, Bedford, MA, USA). Plates were coated overnight with antibody to human IFN-gamma (clone B-B1, Diaclone, Besançon, France) and washed. PBMC were thawed and then added at 105/well and incubated in triplicate with either CMV lysates (1/10 final dilution), or control lysates (BioWhittaker, Verviers, Belgium) or 10 μg/ml HCMVpp65/HLA-A2 peptide (sequence NLVPMVATV). After incubation for 20 h at 37°C, cells were removed and spots were revealed with a second biotinylated antibody to human IFN-gamma (clone B-G1, Diaclone) followed by streptavidine-alkaline phosphatase (Bio-Rad, Marnes la Coquette, France) and BCIP/NBT substrate (Sigma). Spots were counted using the KS Elispot reader (Zeiss, Germany). The results obtained from healthy CMV seronegative donors were used to determine the threshold for the absence of CMV-specific effectors in the ELISPOT assay. PBMCs from CMV seropositive healthy donors (Etablissement Français du Sang, Marseille, France) were isolated on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and used as healthy controls.
Flow cytometry analysis and tetramer staining
Standard flow cytometry procedures were described elsewhere.14,15 For tetramer binding assay, T cells were labelled for 30 min at room temperature with PE-conjugated iTAG™ pp65CMV/HLA-A*0201 tetramer (Beckman-Coulter) or APC-conjugated pp65CMV/HLA-A*0201 tetramer (a kind gift from P Coulie, LICR, Brussels, Belgium), washed and stained with the indicated surface markers (Beckman-Coulter) for 30 min at 4°C. FITC-, PE-, PC5- or APC-conjugated monoclonal antibodies against CD3, CD8, CD45RA, CD27, CD28 and 2B4 were all purchased from Beckman-Coulter. The results obtained from healthy HLA-A*0201 CMV seronegative donors were used to determine the threshold for the absence of CMV-specific effectors in the tetramer staining assay. Intracellular staining of perforin was performed using the CytoStain kit and FITC-conjugated antiperforin (Pharmingen).16 Samples were analysed on a FACSCalibur using CellQuest software (BD Biosciences, Le Pont de Claix, France).
Detailed statistical methods were described elsewhere.13 All data were computed using SPSS for Windows (SPSS, Inc., Chicago, IL, USA) and SEM software (SILEX, Mirefleurs, France). The Mann–Whitney test was used for comparison of continuous variables. Categorical variables were compared using the χ2 test. The probability of developing a CMV positive antigenaemia was determined by calculating the cumulative incidence.17 The association of time to CMV positive antigenaemia with relevant variables was evaluated in a multivariate analysis, with the use of Cox proportional hazards regression model.18
Patient characteristics and CMV positive antigenaemia risk factors are shown in Table 1. In this series, the cumulative incidence of CMV positive antigenaemia was 37% (95% confidence interval (CI), 24–50%), occurring at a median of 43 days (range, 7–104) after allo-SCT. During the follow-up period, none of the eight CMV low-risk cases (both donor and recipient CMV seronegative, Table 1) developed a positive CMV antigenaemia compared to the remaining 46 intermediate or high-risk patients, further confirming the potent protective effect of a negative CMV donor–recipient serostatus against the development of CMV antigenaemia.19 In CMV seropositive donor–recipients pairs, stem cell source, CD3+ cells in the graft, and the ATG dose received during conditioning were the only variables significantly associated with the risk of CMV positive antigenaemia in univariate analysis (Table 1).
The ELISPOT assay was used to screen patients for the presence of CMV-specific T cells in blood at different times post allo-SCT as compared to healthy CMV-seropositive donors. In the group of CMV seropositive donor–recipients pairs, a high number of IFN-gamma producing cells specific for CMV comparable to healthy donors could be detected as early as day 30 after allo-SCT. Despite a slight but not statistically significant decrease by day 60, possibly due to immunosuppressive treatments for grade 2–4 acute GVHD (median time to onset of acute GVHD, 30 (range, 17–74) days), a sustained response was achieved within the first year post allo-SCT (Figure 1a). Interestingly, patients experiencing CMV positive antigenaemia displayed a trend towards a decreased number of spot-forming T cells, suggesting a protective effect of CMV-specific effectors against infection (Figure 1b). Moreover, patients receiving a BM graft or experiencing grade 2–4 acute GVHD also had a trend towards a decreased number of spot-forming T cells in the first month after allo-SCT (Figure 1c and d), supporting the fact that graft characteristics and conditioning regimen significantly influence CMV-specific immune recovery. Figure 2 illustrates the specific course of three patients from this series. The patient shown in Figure 2a diagnosed with AML did not develop any sign of GVHD and was off immunosuppressive therapy early after allo-SCT. This patient had an early and sustained recovery of CMV-specific effectors without any positive CMV antigenaemia. In contrast, the patient shown in Figure 2b, another AML patient, did not recover any CMV-specific effectors by day 28 post allo-SCT. He developed severe grade 4 acute GVHD at day 34, requiring highly immunosuppressive therapy (high-dose prednisone >2 mg/kg and ATG). This patient experienced 2 consecutive positive CMV antigenaemias at days 56 and 104. The first CMV specific effectors from this patient were only observed simultaneously to immunosuppressive therapy decrease (less than 0.5 mg/kg dose of prednisone) by day 155 after allo-SCT. The multiple myeloma patient shown in Figure 2c had a chronic evolution of CMV positive antigenaemia with CMV-specific effectors evolution being closely dependent on GVHD-related immunosuppressive therapies.
The frequency of CMV-peptide-specific CD8+ T cells was also assessed by MHC-peptide tetramers in 16 HLA-A2 patients from this series. As shown in Figure 3, a significant correlation (P=0.000002) could be observed between the frequency of IFN-gamma producing cells as assessed by ELISPOT and pp65-specific CD8+ T-cell frequency quantified by tetramer staining, suggesting that a significant proportion of CMV-specific CD8+ effectors recovering after ATG-based RIC allo-SCT is functional. Table 2 shows absolute numbers of pp65-specific CD8+ T cells detected from 12 HLA-A2 patients included in this analysis, and time of detection of CMV positive antigenaemia and acute GVHD onset. These data suggest that in addition to their functional status, a minimal circulating level of pp65-specific CD8+ T cells might be necessary for an efficient protection against CMV positive antigenaemia (Table 2).
Finally, on the phenotypic level, pp65-specific CD8+ T had an effector phenotype that was mainly CCR7−, CD45RA− (at least for half of the cells), CD27− and CD28− as previously described , and the expression of cytotoxicity markers 2B4 and intracellular perforin (Figure 4).20,21,22,23
In multivariate analysis performed in the 46 CMV seropositive donor–recipient pairs, the use of BM as stem cell source was the only variable significantly associated with an increased risk of CMV positive antigenaemia (P=0.0001; RR, 5.9; 95% CI, 2.4–14.7). In view of this result, we assessed the frequency of CMV-specific effectors contained within 17 grafts (seven BM and 10 PBSC) available from this cohort. Although not statistically significant because of the limited number of samples analysed, our results show that PBSC grafts are likely to contain a higher number of functional CMV-specific effectors compared to BM (Figure 5), supporting a putative protective effect of PBSC transplantation against CMV positive antigenaemia, at least in the early period following allo-SCT.
In this study, we analysed CMV-specific T-cell recovery following ATG-based RIC. Different factors were shown to influence this recovery. The risk of CMV antigenaemia was associated with an absence of CMV-specific effectors. In this setting of ATG-based RIC, our data support a model where functional CMV-specific T cells can recover relatively early after allo-SCT. The latter is at odds with another study suggesting recovery of dysfunctional CMV-specific T cells after allo-SCT,9 but correlates with other studies showing recovery of functional CMV-specific effectors with a protective effect against CMV reactivation.24,25 These discrepancies are likely to be explained by different patients' features or allo-SCT settings, such as preparative regimens or post allo-SCT immunosuppression. The combination of pp65-specific CD8+ T cells by tetramer quantification, with a functional ELISPOT assay (detection of both CD4+ and CD8+ CMV-specific effectors when CMV lysate is used), allowed us to depict the pattern of CMV immune recovery in a large RIC allo-SCT series. In our study, the use of ATG as part of the preparative regimen could provide a certain level of in vivo T-cell depletion, modulating the kinetics of immune reactions and reconstitution after allo-SCT.12 Although this study was not originally powered to detect the exact impact of ATG dose on CMV immune recovery and no individual variations in ATG pharmacokinetics were monitored, in univariate analysis a high ATG dose correlated with a significantly higher incidence of CMV positive antigenaemia. This is in line with previous studies from the standard myeloablative T-cell depletion setting, showing that combined in vivo/ex vivo T-cell depletion would influence the incidence of early active CMV infection and disease in the depleted patients.26,27 Although in this study the majority of patients receiving BM also received higher doses of ATG during conditioning, in multivariate analysis, the stem cell source (BM vs PBSC) was the only predictive factor for the development of positive CMV antigenaemia. In addition to the well-known quantitative differences already shown between BM and PBSC grafts as for T-cell composition, these data suggest that protection against CMV positive antigenaemia might be conferred by specific T lymphocytes transferred with the graft, at least in the early period after allo-SCT. Although the number of patients included in this study was relatively low for depicting other predictive factors, this result further supports the major influence of stem cell source in transplant-related events. Despite a relatively high incidence of CMV positive antigenaemia, none of the patients included in our study developed clinical signs of CMV disease. This is different from a recent large case–control study from the Seattle group suggesting that late-onset CMV disease still occurs after RIC allo-SCT.28 However, such differences can be explained by the differing and variable immunosuppressive potential of the major RIC regimens investigated at different centres. The Seattle RIC regimen, including fludarabine and low dose total body irradiation, used a combination of CsA and mycophenolate mofetil (MMF) for GVHD prophylaxis,28 while our ATG-based regimen used CsA alone. It has already been shown that the combination of MMF and CsA had potent synergistic immunosuppressive effects on T cells.29,30 Thus, it is possible that CsA/MMF combination would alter CMV-specific immune recovery favouring delayed onset of CMV disease, which is not necessarily the case of CsA alone, which still allows the emergence of functional CMV-specific effectors following allo-SCT. The latter further enhances the need for stringent biological monitoring for the assessment of the potential advantages of the different RIC regimens in comparison with standard regimens.31 Moreover, and although we showed previously that ATG-based RIC allo-SCT is usually associated with early full donor chimerism,32 our study did not address the potential impact of donor–recipient chimerism on CMV specific effectors recovery, where residual recipient CMV-specific effectors could contribute to CMV-specific immunity. This issue warrants further investigation. Overall, although further large-scale prospective studies are still needed, we envision that for individual patients, rapid assessment of CMV-specific immune recovery early after allo-SCT might prevent unnecessary routine ganciclovir prophylaxis impairing CMV-specific T-cell recovery in some patients,33 but can help to better identify those patients at high risk of CMV reactivation who may benefit from pre-emptive therapy or adoptive transfer of CMV-specific T cells or to eliminate the need for prolonged monitoring of CMV antigenaemia.34
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This study was supported by the ‘Action Thématique Concertée (ATC) biothérapie’ (INSERM; coordinator: D Olive) and the ARECA project of the Association pour la Recherche sur le Cancer (ARC; coordinator: D Blaise) and by grants from the ‘Association Sang pour Cent la Vie’ (Paris, France) and from the ‘Ligue Departementale contre le Cancer du Gard’ (Nimes, France) (to MM). MM was also supported by the ‘Association Mediterraneenne pour le Developpement de la Transplantation’ (Marseille, France). We thank the ‘Association pour la Recherche sur le Cancer (ARC)’, the ‘Ligue Nationale contre le Cancer’ and the GEFLUC for their generous and continued support for our ongoing work. We thank the nursing staff for providing excellent care for our patients and S Jut-Landi and N Baratier for excellent technical assistance. We also thank the following physicians at the Institut Paoli-Calmettes for their dedicated patient care: A Gonçalves, F Viret, AC Braud, N Vey, GL Damaj, V Ivanov, AM Stoppa, RT Costello, JM Schiano de Collela, A Charbonnier, R Bouabdallah, D Coso, C Chabannon, G Novakovitch and P Ladaique.
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Mohty, M., Mohty, A., Blaise, D. et al. Cytomegalovirus-specific immune recovery following allogeneic HLA-identical sibling transplantation with reduced-intensity preparative regimen. Bone Marrow Transplant 33, 839–846 (2004). https://doi.org/10.1038/sj.bmt.1704442
- Allogeneic stem cell transplantation
- reduced-intensity preparative regimen
- immune recovery
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