Despite prevention and viral control strategies, cytomegalovirus (CMV) infection in immunocompromised subjects in general is still a health problem (1, 2).
Viral strategies are mainly aimed at avoiding cytotoxic T lymphocytes and natural killer cells, which supports the theory that these two cell populations play a major role in virus clearance (3), as was previously suspected based on clinical studies (4).
The risk of CMV infection in kidney graft recipients is usually determined by donor and recipient pretransplant CMV serology and by immunosuppression regimes. This classifies patients at low, intermediate, and high risk of viral infection and helps establish a prevention strategy.
Measurement of humoral immune responses (CMV serology) as a tool to predict CMV disease is limited to pretransplant assessment. Current prediction algorithms that rely on pretransplant serostatus and posttransplant viral load testing are suboptimal for ascertaining the risk of CMV disease in certain patients. In recent years, it has become evident that CMV-specific T-cell immunity plays a critical role in controlling CMV infection in immunosuppressed patients (5). Therefore, the analysis of CMV-specific T-cell frequencies can reflect the patients’ ability to control the virus, predict patients at risk for developing viral replication or CMV disease after prophylactic treatment, and help clinicians define patient-specific prevention strategies (6).
Monitoring CMV-specific cellular immunity may involve assessing CD4 and CD8 T-cell responses because both subsets seem to play a fundamental role in the control of CMV infections (7–11). Protection from CMV following SOT has been shown to correlate with high frequencies of IE-1 but not pp65-specific CD8 T cells (12); however, these data contradict other studies that indicate that CD4 and CD8 pp65-specific T cells are also important for protecting against CMV disease (13–15).
A non-negligible percentage of seropositive kidney receptors considered at low or intermediate risk eventually develop CMV infection. A better understanding of susceptibility factors in these cases may help prevent severe infections.
The authors therefore performed a prospective analysis of CMV-specific immune cell responses in R+ patients with pre-emptive treatment and compared CD4 and CD8 T-cell responses in patients who developed CMV infection or CMV disease, or both, with those of patients who did not develop CMV infection or whose infection resolved spontaneously. T-cell responses to both pp65 and IE-1 CMV antigens were analyzed before transplant and during the first year posttransplant.
RESULTS
CMV Infection Outcomes
A total of 82 kidney transplants were performed in Hospital La Paz from April 2010 to November 2011. Eleven were not included in the study for various reasons. Fifty-six patients received universal prophylaxis, and 15 were treated with pre-emptive therapy to prevent CMV disease. The distribution of the patients related to both prevention strategies is shown in Figure S1 (see Figure S1, SDC,https://links.lww.com/TP/A910).
The 15 patients with no universal prophylaxis and at intermediate risk of CMV replication were analyzed. Six had CMV infection and one had CMV disease (viral syndrome) at 2.7±0.7 months on average (range 2–4 months) after transplant. These seven patients were treated with oral valganciclovir (dose adjusted to renal function), and immunosuppression therapy was reduced in five. Viral clearance, defined as two consecutive negative tests for pp65 antigenemia, was achieved on average after 40 days of valganciclovir treatment. No resistance to valganciclovir therapy was detected. One patient had recurrent infection.
The remaining eight patients did not have CMV disease or infection, and in five of these, no CMV replication was detected at any time. However, three showed positive antigenemia with values equal to or less than two positive cells per 2×105 leukocytes, which were not confirmed in subsequent tests. These patients were therefore considered free of CMV infection.
Table 1 shows the demographic characteristics of recipients and donors based on CMV infection in the first year posttransplant.
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TABLE 1: Recipient and donor characteristics
Those patients with CMV infection during the first year posttransplant showed decreased graft survival at 30 months (see Figure S2, SDC,https://links.lww.com/TP/A910) and increased incidence of comorbidities. Acute rejection episodes appeared in two patients. In one of them, CMV replication preceded the rejection episode, while in the second patient the rejection occurred before CMV reactivation.
CMV-Specific Cellular Immune Responses
Conflicting data have been reported concerning T-cell involvement in protection against CMV replication. It was therefore decided to analyze the specific response to IE-1 and pp65 CMV antigens in CD4 and CD8 T cells from seropositive kidney transplant recipients with pre-emptive treatment for CMV infection. Serial samples, including pretransplant samples, were analyzed at various time points.
Preliminary experiments in healthy control subjects and on pretransplant samples confirmed that individuals with CMV-negative serology did not show any specific response against IE-1 or pp65. As expected, most seropositive blood donors responded to both antigens. There was a subset of patients who, despite positive serology to CMV, showed no specific response to IE-1. However, positive responses to polyclonal stimulus, such as Staphylococcus enterotoxin B (SEB), were detected (see Figure S3, SDC,https://links.lww.com/TP/A910).
To test whether the lack of response was specific for IE-1 and whether cellular immunity was absent for other CMV antigens, the responses to IE-1 and pp65 CMV proteins in pretransplant blood samples from 15 R+ kidney transplant recipients at intermediate risk for CMV infection were compared. The patients were followed for 1 year, and CMV-specific responses were further analyzed at 1 month, 6 months, and 1 year posttransplant.
It was found that, in pretransplant samples, CD8 T-cell responses to IE-1 were significantly higher in patients who did not develop CMV infection by the 1-year follow-up posttransplant (Fig. 1A). In patients with positive responses to IE-1, CD8 T-cell responses predominated over CD4 T-cell responses. In line with this observation, no significant differences were found in the CD4 response to IE-1 in pretransplant samples (Fig. 1B), although a trend toward higher responses was found in patients who did not develop infection. There were also no significant differences in pretransplant CD4 and CD8 T-cell responses in patients who developed viral replication (Fig. 1C). In patients free of viral infection, a predominant response to IE-1 was detected pretransplant in the CD8 compartment (Fig. 1D).
FIGURE 1: T-cell response to IE-1 antigen in pretransplant samples. PBMCs isolated from patients before transplant were stimulated in vitro with peptide pools spanning IE-1 CMV antigen. The frequency of IFN-γ-producing CD8+ or CD4+ T cells was assessed by flow cytometry in selected CD3+ lymphocytes. A, box plots show the responses (median, 75th and 25th percentiles, and SEM) in CD8 T cells from patients who did or did not develop CMV infection posttransplant. B, responses in CD4 T cells. C, comparison of CD8 and CD4 response to IE-1 in patients with no CMV infection. D, comparison of CD8 and CD4 response to IE-1 in pretransplant samples from patients who developed CMV infection posttransplant. The Mann-Whitney U test was used to analyze data in panels A and B, and Wilcoxon t test was applied to data in panels C and D.
Conversely, no significant differences were found when the response to pp65 was analyzed in pretransplant samples in terms of further CMV infection. No predominant response was observed, either in the CD8 or CD4 subpopulation responses to pp65 (see Figure S4, SDC,https://links.lww.com/TP/A910).
CMV replication occurred in all cases during the first 4 months following kidney transplantation. A longitudinal analysis of CD8 and CD4 responses to IE-1 and pp65 showed that only the CD8 response to IE-1 was different among groups for the first 6-month follow-up (Fig. 2).
FIGURE 2: Evolution of IE-1 and pp65 T-cell responses in CMV-seropositive kidney transplant patients with pre-emptive treatment. PBMCs isolated from patients at different time points before or after transplant were stimulated in vitro with peptide pools spanning IE-1 or pp65 CMV antigens, and the frequency of IFN-γ producing CD8+ or CD4+ T cells was assessed by flow cytometry in selected CD3+ lymphocytes.
The data suggest that CD8 T-cell response to IE-1 is the relevant parameter for discriminating CMV infection risk among R+ patients with no prophylaxis. This hypothesis was confirmed by a receiver operating characteristics (ROC) analysis of data from pretransplant samples. In this analysis, only CD8 responses to IE-1 pretransplant were significant to predict CMV infection (AUC=0.029 P=0.010; 95% CI: 0.078–1.0) (Fig. 3). A ROC analysis was also used to identify cut-off levels of CD8 responses to IE-1 below which an increased risk of CMV infection would be indicated. Youden index was calculated and the maximum obtained corresponding to a cut-off of 0.055% of CD8+IFN-γ+ T cells in response to IE-1, with 100% specificity and 85.7% sensitivity (all patients whose CD8 T-cell response to IE-1 was ≤0.05% developed CMV infection). For values below 0.22%, 85.7% specificity and 50% sensitivity were calculated.
FIGURE 3: Receiver operating characteristic (ROC) analysis of IE-1 and pp65-specific T cells in pretransplant samples from patients who developed or did not develop CMV infection posttransplant. (AUC, area under the curve; CI, confidence interval).
DISCUSSION
Previous reports suggest that monitoring the immune response to viral antigens is important for maintaining (or withdrawing) prophylactic treatment in transplant patients at risk for CMV disease. Several publications have focused on CD4- and CD8-specific responses to CMV pp65 antigen (16–18). However, published data suggest that IE-1-specific lymphocytes would be more relevant for preventing CMV reactivation. Previous reports have suggested that monitoring pretransplant CD8 responses could predict future CMV replication in immunocompromised CMV-seropositive individuals (13). However, these studies included patients taking ganciclovir or valganciclovir prophylaxis, and some samples were taken after transplantation.
In this study, CD4 and CD8 T-cell responses to the main CMV T-cell targets pp65 and IE-1 in kidney transplant patients at intermediate risk for CMV infection were analyzed, before and at various times after organ transplantation, using the production of IFN-γ in each cell subset, as assessed by intracellular flow cytometry, as a readout.
Despite positive serology in all individuals analyzed pretransplant, approximately 50% had practically negative cellular response to early CMV antigen IE-1. It was found that CD8 responses to IE-1 predominate over CD4 responses, which is similar to that reported in previous publications (19). Accordingly, the main differences between the two patient groups (with or without CMV infection) were found within the CD8 subpopulation, with absence of IE-1-responsive CD8 T cells pretransplant in R+ patients who developed CMV infection upon immunosuppression after kidney transplantation. When the CD8 T-cell response to pp65 was analyzed, there were no significant differences between the two groups.
Similar data was reported by Bunde et al. (12) who analyzed the response to IE-1 and pp65 in 23 heart and 4 lung CMV-seropositive transplant recipients. Although they reached similar conclusions, some of the initial determinations were performed during the first days posttransplantation (range 0–4) when patients were already undergoing induction treatment.
Various methods have been used to evaluate CMV-specific cellular immune responses, such as intracellular flow cytometry to evaluate IFN-γ production, ELISA, ELIspot, and proliferation assays (6). A recent published study showed that Quantiferon assessment of T-cell responses to several CD8 predominant epitopes could predict CMV disease risk in pretransplant patients (20). However, the analysis included patients at intermediate to high risk of CMV with a low frequency of R+ patients with CMV replication. Moreover, Quantiferon contains a pool of peptides presented by the most frequent HLA-I alleles, and results for this assay may be negative for patients with uncommon HLA types.
This study included 15 CMV-seropositive kidney transplant recipients with no prophylactic treatment. The first sample was obtained pretransplant and before any induction treatment in all cases, which strengthens the conclusion that, in addition to serostatus, IE-1 immunity pretransplant may be useful for risk assessment in SOT recipients.
Patients who developed CMV infection had not developed IE1-specific CD8 T-cell responses after 6 months. This may be a result of the idiosyncratic characteristics of the patients, but it is also possible that valganciclovir (administered to treat the infection) may hamper the development of a proper immune response by inhibiting viral replication. This lack of response was not likely a result of immunosuppression, as average responses to pp65 were similar to those of patients who did not develop CMV infection.
IE-1 is an immediate early antigen expressed at the onset of CMV replication. Conversely, pp65 is expressed in late infectious viral particles. It has been reported that during latency, stochastic episodes of desilencing of the viral genome lead to the expression of IE-1 transcripts, which in mice is accompanied by an activation of effector CD8 T cells specific to a peptide derived from the IE-1 protein. These findings were combined in the silencing/desilencing and immune-sensing hypothesis of virus latency and reactivation. This hypothesis proposes that IE-1 gene expression leads to cell surface presentation of IE-1 and that its recognition by CD8 T cells terminates virus reactivation. This hypothesis has been proven in a murine model of CMV infection (21) and is the basis of a phenomenon called “memory inflation”. A hallmark of CD8 T-cell response to CMV is the observation that with increasing time during latency CD8 T cells specific to certain viral epitopes encoded by “antigenicity-determining transcripts expressed in latency (ADTELs)” increase in numbers (22). It is thus tempting to speculate that patients with longer exposure to these ADTELs-derived proteins, which would include IE-1 as a major representative, would have a higher frequency of protective IE-1-specific CD8 T cells. An alternative explanation for the scarcity of such lymphocytes in seropositive patients is the availability of HLA-I alleles able to present such antigens to T cells (19, 23).
In conclusion, our results confirm previous data and suggest that CD8 T-cell immunity to IE-1 pretransplant (but not to pp65) protects against CMV replication in immunocompromised patients with no antiviral prophylaxis and at intermediate risk of CMV infection. These results may have implications for the design of clinical strategies for preventing CMV infection, as this assay could be useful for guiding CMV prevention strategies in transplant recipients. However, our study is limited by its single-center nature and the small sample size. Additional studies are needed including more patients and different centers to confirm this hypothesis. Although we have used an antigenemia assay for monitoring CMV infection, these studies could be performed in patients monitored by PCR assessment of DNA copy numbers to estimate CMV viral load because of good correlation between these techniques (24).
Our study’s strengths include its prospective nature and sample homogeneity. The use of peptide libraries enables the assessment of responses in every patient, regardless of HLA genotype.
Further research is warranted to study whether the development of CMV infection after prophylaxis in patients at high risk correlates with deficiencies in CD8 T-cell responses to IE-1 and to analyze whether this assay would be appropriate for monitoring immune cell response to maintain or withdraw antiviral treatment in such patients.
MATERIALS AND METHODS
Patients
A longitudinal prospective analysis was performed. The study included patients undergoing kidney transplantation at University Hospital La Paz from April 2010 to November 2011 who were at intermediate risk of CMV replication and were undergoing pre-emptive therapy with valganciclovir as a prevention strategy for CMV infection. The patients’ CMV antigen responses were assessed. The study was approved by the Institutional Review Board, and all patients gave their written informed consent.
Immunosuppressive treatment consisted of triple therapy with steroids, tacrolimus, and mofetil mycophenolate for all patients. The induction treatment is shown in Table 1. Acute rejection episodes were classified according to the Banff working group classification (25), and patients received proper immunosuppressive treatment according to the rejection type.
Monitoring of CMV Replication
Virological follow-up and diagnosis of CMV replication was conducted by evaluating pp65 antigenemia weekly during the first month, biweekly during the second and third months, and once a month from the fourth month. Positivity was established when at least five positive cells per 2×105 leukocytes were detected, and pre-emptive treatment with oral valganciclovir was started with a pp65 antigenemia evaluation once a week. Valganciclovir was discontinued after two successive negative tests.
Patients with CMV replication were classified as asymptomatic (infection) or symptomatic (disease) according to the definitions published in the international consensus document for infectious diseases (26).
Monitoring of CMV-Specific T-Cell Responses
Peptides
Pools of overlapping 15-mer ACTS_human, HCMV-IE-1, and pp65 peptides (ProMix) were purchased from ProImmune (Oxford, UK).
Antibodies
CD28, CD49d, CD3-PerCP, CD4-FITC, CD8-APC, and IFN-γ-PE antibodies were purchased from Miltenyi Biotec.
PBMC Preparation and Stimulation for Ex Vivo Analysis of CMV-pp65 and IE-1-Responsive T Cells
Blood samples were collected in EDTA, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Hypaque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) density gradient centrifugation. In vitro stimulation was performed as previously described (27). Briefly, CMV pp65, IE-1, or control ProMix peptides (1 μg/mL) were added to 2 million cryopreserved PBMCs in 0.5 mL RPMI-1640 medium (Lonza) containing 10% heat-inactivated FCS (CM). Costimulatory antibodies to CD28 and CD49d (Miltenyi) were added to the cultures to 1 μg/mL each at the same time as peptides. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere in loosely covered Falcon 2025 tubes (BD Biosciences). After 2 hours, 0.5 mL CM containing brefeldin A (BFA; Sigma-Aldrich) was added to the cultures for a final concentration of 2.5 μg/mL BFA. The cells were incubated overnight before intracellular flow cytometry analysis of IFN-γ production. Parallel cultures were established: a negative control in the presence of a peptide library representing human actin (ACTS-human) and a positive stimulation control with Staphylococcus enterotoxin B (Sigma-Aldrich).
Flow Cytometry Analysis
For intracellular flow cytometry analysis of IFN-γ production, cells were stimulated overnight as described above, washed and labeled for 20 minutes with CD3-PerCP, CD4-FITC, and CD8-APC antibodies before permeabilization (FACS permeabilization solution 2; BD), followed by labeling with IFN-γ-PE for 30 minutes. Half of the sample was stained with PE-IgG1 control isotype. Flow analysis was performed in a FACScalibur flow cytometer (Becton Dickinson) and analyzed with CellQuest Pro Software (BD). At least 100,000 events were collected for each condition.
Statistical Analysis
Statistical analysis was performed with SPSS and GraphPad programs. For the description of quantitative variables, the results are expressed as mean±SD values or as median and ranges where appropriate. Patient data were compared using the chi-square test for categorical variables and the Mann-Whitney U test and Wilcoxon test for continuous variables, when appropriate. The incidence of kidney graft survival according to CMV replication was calculated using Kaplan-Meier curves and the log rank. The cut-off levels of IFN-γ-producing T cells associated with the best specificity and sensitivity were analyzed by means of ROC curve analysis with Youden test. P values less than 0.05 were considered statistically significant.
ACKNOWLEDGMENTS
The authors thank Rosario Madero from the Statistics Department of IdiPAZ for her invaluable help with ROC curve analysis of the data.
The authors would like to thank Juliette Siegfried and her team at ServingMed.com for their editing of the manuscript.
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