Introduction

Despite the introduction of new antiviral treatment strategies, cytomegalovirus (CMV) remains one of the most frequent infections in stem cell transplant recipients.^{1, 2} CMV infection can cause life-threatening complications, especially in individuals with impaired T-cell-mediated immunity such as allogeneic stem cell transplantation (allo-SCT) recipients.³ All transplant recipients develop combined quantitative and functional immune system dysfunction that lasts for months after transplantation, and this contributes to an increased susceptibility to infections.^{3, 4} During this period about 60–70% of CMV-seropositive patients, and seronegative patients receiving stem cells from seropositive donors develop active CMV infection.^{5, 6} Risk factors as regards the development of CMV disease, include patient age, CMV status of the donors and the recipients, type of conditioning, development of graft-versus-host disease (GVHD) and number of incidences of CMV reactivation.^{6, 7, 8, 9}

CMV can today be controlled in most patients during the early follow-up period after SCT by close monitoring, using quantitative and qualitative polymerase chain reactions (PCRs) and pre-emptive therapy. On the other hand, these very sensitive monitoring techniques have a potentially negative effect in that antiviral therapy might be given unnecessarily to many patients.¹⁰ However, many patients do experience repeated reactivation and thereby need repeated courses of pre-emptive therapy, with an increased risk of side effects.

Previous studies on stem cell and organ transplant recipients and AIDS patients have shown the importance of cytotoxic T-lymphocytes (CTLs) in controlling CMV infection.^{11, 12}

Flow cytometry enables the direct visualization of cytokine production at the single cell level.¹³ Immunophenotyping of lymphocytes and intracellular cytokine staining allow estimation of specific T-cell immunity based on their phenotype and function. In this study, we employed this technique to monitor CMV-specific CD3+ and CD4+ T-cell responses after antigen stimulation. Our aim was to correlate transplantation factors with the recovery of cellular immunity in SCT patients at different time points after SCT. This might allow understanding of how CMV-specific T-lymphocyte reconstitution is influenced by those factors, and allow development of an algorithm for monitoring the specific immune status.

Materials and methods

Patients

Between January 2002 and December 2004, 52 patients who received allo-SCT for various haematological malignant and non-malignant disorders were included in this study. Four patients were excluded from the study because of insufficient numbers of samples. Thus, 48 patients were included in the analysis. The study was approved by the ethics committee at Karolinska University Hospital. The characteristics of the patients are shown in Table 1.

Table 1 - Patient data.

Full table

Conditioning regimens

The patients were conditioned as described previously¹⁴ either with myeloablative regimens (cyclophosphamide 60 mg/kg for two days combined with either 4 3 Gy total body irradiation (TBI) or busulphan 4 mg/kg 4 combined with anti-thymocyte globulin (ATG) (Thymoglobulin, Sangstat-Imtix, France) in patients with mismatched or unrelated donors) or three different reduced-intensity regimens. These regimens were fludarabine (30 mg/m²/day) for 6 days, combined with 4 mg/kg/day of busulfan for 2 days (total dose, 8 mg/kg) and ATG for 4 days. Patients with lymphoid malignancies were conditioned with fludarabine 30 mg/m²/day for 5 days combined with cyclophosphamide 30 mg/m²/day for 2 days, followed by TBI 3 Gy 2. In three patients, treosulphan 10–14 mg/m²/day was combined with fludarabine, given as above.

Intracellular INF- staining

Blood samples from the patients were obtained every second week after SCT during the first 3 months. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). The cells were washed twice in tissue culture medium RPMI (RPMI 1640-Hepes with Glutamax-I, Gibco-BRL, Grand Island, NY, USA) and resuspended at a concentration of 1 10⁶/ml in RPMI 1640, supplemented (10%) with heat-inactivated foetal calf serum. One million PBMCs were stimulated with CMV AD-169 antigen (1 g/ml; BioSite, Stockholm, Sweden) and incubated for 6 h at 37°C in 5% CO₂. Brefeldin A (10 g/ml, Sigma-Aldrich, St Louis, MO, USA) was added for the last 4 h of the incubation. As a negative control, we used R2 K-Ag HL cells (SMI, Stockholm, Sweden). As positive controls, cells stimulated with phorbol 12-myristate 13-acetate at 0.5 g/ml and ionomycin at 1 g/ml (both from SIGMA-Aldrich, Steinheim, Germany) were used. The cells were permeabilized and stained with fluorochrome-labelled anti-CD3, anti-CD4 and anti-interferon (INF)- antibodies (Becton Dickinson, San Jose, CA, USA) as described previously.¹⁵ The samples were analysed by flow cytometry using FACSCalibur (Becton Dickinson) equipment. During each analysis, 200 000 cells were collected. A level of 0.05% INF--producing T cells was interpreted as a positive result for this test.

Quantitative PCR for CMV DNA

All patients were monitored weekly for CMV by PCR. The real-time PCR used for quantification of CMV DNA from peripheral blood lymphocytes has been described previously.¹⁰ CMV disease was defined according to Ljungman et al.¹⁶

Pre-emptive antiviral therapy

Pre-emptive therapy with either intravenous ganciclovir or foscarnet was given as described by Reusser et al.¹⁷ A viral load of 100 genome copies/200 000 cells in one sample was used as the basis for initiating pre-emptive antiviral therapy. Treatment was given for 2 weeks at full dose (usually ganciclovir at 5 mg/kg 2) and if the CMV load was above either 10 (the first part of the study) or 100 genome copies, antiviral therapy was continued at a lower dose given as maintenance for another 2 weeks (ganciclovir 6 mg/kg 1; foscarnet 60 mg/kg 1) as published previously.¹⁷ Repeated courses were given as necessary, based on a new episode of CMV DNAemia. For the purpose of this study, a second reactivation was defined as occurring after the documentation of negative CMV PCR tests for at least 8 days in the absence of antiviral therapy.

Statistical analysis

All viral load results were transformed to log₁₀ values. The kinetics of viral load changes during the first course of antiviral therapy were calculated as the log₁₀ viral load at initiation of antiviral therapy minus the log₁₀ viral load of the last sample before antiviral therapy was discontinued, divided by the number of days of antiviral therapy.¹⁰ Fisher's exact test (two-tailed) was used to evaluate the effect of transplant parameters (source of stem cells, type of conditioning, donor type, CMV serological status) on CMV-specific T-cell function. Acute GVHD grade II–IV was included only if occurring before the time point of peak CMV viral load. Univariate analyses of the effects of transplant factors and CMV-specific immune responses on the peak viral load and viral load kinetics were performed by way of t-tests, and multivariate analyses were performed by means of linear regression with forward selection of variables.

Results

Overall, the CMV-specific immune responses were low with approximately half of the patients lacking a CMV-specific response at all studied time points during the first 12 weeks after transplantation (Figure 1).

Figure 1.

Percentage of CMV-specific CD4+ T cells at different time points after SCT.

Full figure and legend (11K)

Effects of pre-transplant factors on CMV-specific immunity after SCT

Patients who had undergone reduced-intensity conditioning (RIC) had nonsignificant tendencies to have more frequently both CD3+- (11/23; 48%; P=0.11) and CD4+-specific immunity (13/23; 56%) at 4 weeks after SCT than patients who had undergone myeloablative conditioning (MC) (CD3+ 4/20 (25%); CD4+ 7/20 (35%)). Similar tendencies were seen at 8 and 12 weeks after SCT (Figure 2).

Figure 2.

Percentage of CMV-specific CD4+ T cells in patients receiving RIC or MC at different time points after SCT.

Full figure and legend (16K)

Patients who had received transplants from seropositive donors tended more frequently to have detectable CMV-specific CD4+ cell function than patients who had received transplants from CMV seronegative donors at 8 weeks (11/22, 50% vs 3/8, 37%) and 12 weeks (15/27, 55% vs 4/11, 36%). As only six patients included in this study were CMV seronegative, the influence of recipient serological status could not be analysed. There was no effect on either CD3+ or CD4+ CMV-specific T-cell function by stem cell source, age, donor type or GVHD grade II–IV at any time point (data not shown).

Factors influencing the CMV peak viral load

The association between CMV antigen-induced INF- production and peak CMV viral load is shown in Table 2. Patients who had detectable INF- production from CD3+ T cells at 4 weeks after SCT had lower peak mean viral loads compared with patients with negative stimulation (P=0.03). There was a similar tendency as regards stimulation of CD4+ T cells (P=0.09). Analogous but nonsignificant tendencies towards lower peak viral loads in patients who had detectable CMV-specific T-cell immunity were observed at 8 and 12 weeks (Table 2). In multiple regression analysis, transplants from CMV seronegative donors were correlated to an increased log₁₀ peak viral load (r=0.68; P=0.03), whereas a detectable CD3+-specific T-cell response at 4 weeks after SCT was correlated to a lower viral load (r=-0.67; P=0.03) while the intensity of conditioning, donor type, or acute GVHD grade II-IV did not influence the peak CMV viral load.

Table 2 - INF- production by CD3+ and CD4+ cells and peak viral load.

Full table

Factors influencing the effect of antiviral therapy on the viral load

The only factor influencing the rate of decrease in viral load during antiviral therapy was grade II–IV acute GVHD (P=0.03), whereas the donor/recipient serological status, intensity of conditioning, type of donor or CMV-specific immune response did not.

CMV-specific T-cell immunity and CMV disease

Only three of the 48 patients developed CMV disease; two had CMV gastrointestinal disease and one had CMV retinitis. None of these patients had detectable CMV-specific cytokine production in the test closest to the time of development of CMV disease (data not shown).

Discussion

Whereas CMV infection results in no pathology in an immunocompetent host, it reactivates and might bring about life-threatening complications in immunocompromised patients.³ In contrast to patients treated with high-dose chemotherapy and autologous SCT, patients who undergo allo-SCT are at a much higher risk of CMV infection, even after immune reconstitution, owing to the delayed recovery of T-cell and B-cell functions.⁵ Several studies of CMV-specific T-cell responses have shown that restoration of CTLs depends on the recovery of CD4+ cells and that recovery of CD4+ CMV-specific lymphoproliferative responses is obligatory for endogenous reconstitution of CTLs.^{18, 19}

Even if recipients have normal total lymphocyte counts within 2 months after SCT, they have abnormal CD4/CD8 T-cell ratios, reflecting their decreased CD4 and increased CD8 T-cell counts.²⁰

It has been increasingly recognized during the last decade that the timing of CMV disease has changed so that patients develop it later after SCT. Boeckh et al.²¹ identified several risk factors for late CMV disease such as antigenaemia developing before day 100 after SCT, presence of chronic GVHD, lymphocytopenia, low number of CD4+ cells and absence of a CMV-specific T-cell response.²¹

In the recently published study by Lilleri et al.,²² it was suggested that, in a mainly paediatric population, recipient seropositivity is the major factor influencing CMV-specific immune reconstitution. Too few recipients were CMV seronegative in our study to allow assessment of the impact of recipient seropositivity. Donor seropositivity might be a factor, but the number of patients included in our study was too low to allow any conclusion regarding this factor. Development of reliable methods to evaluate the immune status against CMV might therefore be clinically important for the management of CMV reactivation in SCT patients.

Several possible techniques to study immunity to CMV exist, including detection of CMV-specific tetramers and functional assays such as intracellular cytokine staining and ELISPOT. It is possible that, depending on the choice of assay, different results might be obtained. Ozdemir et al.²³ combined tetramer staining and assessment of cytokine production to demonstrate that patients who developed CMV antigenaemia had a lower proportion of tumour necrosis factor--producing tetramer staining CMV-specific T cells than those who did not. In our study, we aimed to investigate anti-CMV immunity by measuring the population of INF--producing CD3+ and CD4+ T cells, stimulated in vitro with CMV antigen AD-169 and investigated by means of flow cytometric assay for intracellular cytokine staining.

The number of studied patients was unfortunately too low to show clear effects of transplant factors on the presence of CMV-specific immunity after SCT. However, some suggestive effects were seen. Patients who underwent allo-SCT with RIC tended to have a higher incidence of specific CD3+ and CD4+ T cells early after SCT, suggesting either better retainment of pre-transplant immunity or transfer of specific donor T cells. This would be in accordance with the previously published observation that the use of non-MC is associated with stronger CMV-specific immunity early after SCT.²⁴ According to previously published reports, reconstitution of CMV immunity is significantly earlier in patients who receive RIC without ATG than in patients receiving RIC with ATG or conventional conditioning.^{25, 26} In our study, this was difficult to analyse, as only 19% of the patients receiving RIC SCT from human leucocyte antigen-identical siblings did not receive ATG. A similar tendency was observed when comparing patients who received transplants from CMV seropositive vs seronegative donors. CMV-specific CD8+ T cells have been demonstrated to be derived from the donor.²⁷ Gratama et al.²⁸ also showed that the number of CMV-specific CTLs transferred from seropositive donors was inversely correlated to the number of recurrent CMV infections after SCT. Our results suggest that CMV-specific CD4+ T cells can also be transferred from the donor, influencing the recovery of CMV-specific immunity after transplantation.

In a previous study, we showed that having acute GVHD, grade II–IV and having a CMV-negative donor influenced the CMV viral load.¹⁰ In the present study, we again documented the effect of donor status on peak viral load and in addition found that the detection of INF- production by T cells after CMV stimulation at 4 weeks after SCT was associated with a lower peak viral load. This supports the notion of a direct controlling effect on CMV replication kinetics by CMV-specific T cells.

Our study shows that INF- secretion after CMV stimulation is a potentially useful marker of functional CMV immunity in SCT patients. This might be useful for further improvement of clinical management of CMV infection after allo-SCT.

References

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Acknowledgements

We thank Hernán Concha Quezada for skilful technical support with flow cytometry. This study was supported by the Swedish Cancer Fund and research funds of the Karolinska Institutet. Gayane Avetisyan was the recipient of a scholarship from the Swedish Institute.