Absolute and human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T-cell counts were monitored in 38 solid organ (20 heart, 9 lung and 9 kidney) transplant recipients during the first year after transplantation by a novel assay based on T-cell stimulation with HCMV-infected autologous dendritic cells. According to the pattern of T-cell restoration occurring either within the first month after transplantation or later, patients were classified as either early (n = 21) or late responders (n = 17). HCMV-specific CD4+ and CD8+ T-cell counts were consistently lower in late compared to early responders from baseline through 6 months after transplantation. In addition, in late responders, while HCMV infection preceded immune restoration, HCMV-specific CD4+ restoration was significantly delayed with respect to CD8+ T-cell restoration. The number of HCMV-specific CD4+ and CD8+ T-cells detected prior to transplantation significantly correlated with time to T-cell immunity restoration, in that higher HCMV-specific T-cell counts predicted earlier immune restoration. Clinically, the great majority of early responders (18/21, 85.7%) underwent self-resolving HCMV infections (p = 0.004), whereas the great majority of late responders (13/17, 76.5%) were affected by HCMV infections requiring antiviral treatment (p = <0.0001). Simultaneous monitoring of HCMV infection and HCMV-specific T-cell immunity predicts T-cell-mediated control of HCMV infection.
Human cytomegalovirus (HCMV) is the major cause of viral infections in the post-transplantation period (1). HCMV infection may result in abortive infection or, in the absence of immune control or specific antiviral therapy, in disseminated infection, either symptomatic or asymptomatic. Symptomatic infection may eventually result in organ localization (gastrointestinal disease, pneumonia, hepatitis, retinitis). The severity of HCMV infection and the extent of organ involvement inversely correlate with the development (in primary infections of seronegative recipients) or the restoration (in reactivated infections of seropositive recipients) of an efficient CD4+ and CD8+ T-cell immune response (2,3). However, long-term prospective studies evaluating the kinetics of HCMV infection in relation to the presence or absence of a quantified HCMV-specific CD4+ and CD8+ T-cell response in solid organ transplant recipients are limited (4–8).
In the past, a major difficulty hampering the reliable quantification of functional HCMV-specific CD4+ T-helper and CD8+ cytotoxic T-lymphocytes was the complexity of the assays, which required the use of radioactive compounds (9–11). More recently, the HLA-peptide tetramer staining, ELISPOT and cytokine flow cytometry (CFC) technologies (12–15) have helped to resolve this issue offering a simplified and more rapid approach. However, the antigenic stimulation used in these assays generally consists of either synthetic peptides (16) or infected cell lysates. In this respect, while the individual response to single peptides is variable, the stimulus given by cell lysates is known to be much stronger for CD4+ as compared to CD8+ T cells (17). We recently developed a new methodological approach in order to provide a more comprehensive evaluation of the T-cell immune response in transplant recipients (18). This method is not limited by HLA-restriction, allows simultaneous expression of different viral proteins on the DC membrane, along with simultaneous quantification and functional evaluation of both HCMV-specific CD4+ and CD8+ T cells by CFC.
In the present report, we investigated the kinetics of HCMV infection and HCMV-specific CD4+ and CD8+ T-cell restoration during the first year after transplantation in a series of heart, lung and kidney transplant recipients. Results show that CD8+ precedes CD4+ T-cell restoration, yet long-term protection is conferred by both T-cell subpopulations, while the absence of T-cell (both CD4+ and CD8+) restoration is consistently associated with repeated episodes of reactivated infection.
Patients and Methods
From December 2003 to December 2004 a total of 38 patients were enrolled in the study following organ transplantation at the Department of Surgery of the University Hospital Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy. Inclusion criteria were: male and female subjects >18 years of age receiving a heart (n = 20), lung (n = 9) or kidney (n = 9) transplant and serological evidence of past HCMV infection in the organ recipient. Exclusion criteria were: recipients with HCMV negative serological status and subjects with a life expectancy less than 3 months. The study was approved by the local Ethics Committee and participants were asked to give written consent. Immunosuppressive drug regimens are reported in Table 1. Patients with allograft rejection episodes were treated with a daily bolus of intravenous methylprednisolone (1 g or 500 mg) for 3 days. In the four cases of steroid-resistant rejection, ATG was administered.
Table 1. Characteristics of the 38 patients analyzed
Patient number (%)
CsA = cyclosporine-A; MMF = mycophenolate mophetil; FK = tacrolimus; ATG = antithymocyte globulin; α-CD25 MAb = anti-CD25 monoclonal antibody.
Median age at transplantation
55 (24–72) years
Steroid boli and ATG
HCMV infection diagnosis and treatment
HCMV infection was defined as HCMV isolation or detection in blood or body tissues, whereas HCMV disease required documentation of HCMV infection in blood or body tissues along with clinical symptoms and/or organ function abnormalities (19). Patients examined in this immunological study were included in a randomized controlled study aimed at evaluating a DNAemia cutoff for pre-emptive therapy of HCMV infections in comparison with the antigenemia cutoff in use in our department for several years. Thus, patients were randomly selected from each of the two arms using as a guide for pre-emptive therapy either antigenemia (20,21), with a cutoff of 100 pp65-positive leukocytes/2 × 105 leukocytes examined (22), or DNAemia (23) with a cutoff of 300 000 copies/mL whole blood (24). Antigenemia and DNAemia were performed once a week during the first 3 months post-transplant in the absence of HCMV infection, and twice a week during episodes of HCMV infection. In the following months, tests were performed once a month unless otherwise indicated by clinical findings. In addition, viremia was routinely quantified to detect infectious virus by the shell vial technique, as previously reported (25). Lower limits to methods employed were: 1 positive leukocyte/2 × 105 leukocytes examined for antigenemia and viremia, and 1000 DNA copies/mL blood for DNAemia. I.V. ganciclovir (GCV)(5 mg/kg/b.i.d) was administered for pre-emptive therapy in both arms. Anti-HCMV prophylaxis was not given to any patient. When required, relapse episodes were similarly treated. The same pre-emptive approach was used in the case of rejection episodes in either arm.
The donor/recipient serostatus was determined by the enzyme-linked immunosorbent assay prior to transplantation using methods developed in the laboratory (26).
Quantification of HCMV-specific CD4+ and CD8+ T cells by CFC
HCMV-specific CD4+ and CD8+ T-cells were simultaneously quantified by a novel method based on the use of autologous, monocyte-derived, HCMV-infected immature DC as previously reported (18). Following in vitro generation from peripheral blood mononuclear cells (PBMC) (27), immature DC were infected for 24 h with an endotheliotropic and leukotropic strain of HCMV (VR1814), as previously reported (28). HCMV-infected immature DC were then cocultured overnight with autologous PBMC at a ratio of 1:20 in the presence of brefeldin A to prevent cytokine release. Finally, PBMC were tested for the frequency of HCMV-specific CD4+ and CD8+ interferon-γ (IFN-γ)-producing T cells by the CFC assay (12,18). Absolute CD3+CD4+ and CD3+CD8+ T-cell counts were determined on heparinized peripheral blood samples by direct immunofluorescence flow cytometry (Beckman Coulter Inc., Fullertone, CA). The total number of HCMV-specific CD4+ and CD8+ T cells was determined by multiplying the percentages of HCMV-specific T cells positive for IFN-γ production by the relevant absolute CD4+ and CD8+ T-cell counts. Based on previously reported results, patients with HCMV-specific CD4+ and CD8+ T-cell counts greater than 0.4/μL blood were considered immune (18), while patients with T-cell counts lower than 0.2 cells/μL were found to be nonimmune, and patients with T-cell counts in the range of 0.2–0.4 cells/μL were considered to give equivocal results. Immunological restoration was routinely investigated at monthly intervals until day +180, then at days +270 and +360 after transplantation. All patients were monitored for at least 6 months and 37 of them completed the 1-year follow-up. The remaining patient died of respiratory distress due to rejection 9 months after transplant.
Differences between medians were determined by using the Mann-Whitney U-test for unpaired data, and the Wilcoxon test for paired data. Differences in proportions were tested using the Pearson's chi square test, while the Fisher's exact test was used to test differences in proportions when the total sample size was less than 30. All tests were 2-tailed. The correlation between two parameters was determined by calculating the correlation coefficient r. Curves of HCMV infection in blood in the posttransplantation period, as well as curves relevant to restoration of specific CD4+ and CD8+ T-cell immunity in late responders were determined by the Kaplan-Meier method, while differences between curves were determined by the log rank test. Receiver-operator curves (ROC) analysis was performed to identify levels of HCMV-specific CD4+ and CD8+ T-cells protective against HCMV infection reactivation at a time preceding the HCMV load peak, and to investigate pre-transplant levels predictive of antiviral treatment.
Early and delayed HCMV-specific CD4+ and CD8+ T-cell restoration
Apart from the type of transplantation, all transplant recipients were divided into two groups according to the time of HCMV-specific T-cell immunity restoration. Patients recovering both CD4+- and CD8+-specific immunity within 30 days after transplantation were named early responders, whereas patients recovering immunity in the following months were referred to as late (or delayed) responders. Of the 38 solid organ transplant recipients analyzed in this study, 21 (55.3%) were found to be early responders and 17 (44.7%) were late responders. At baseline, median absolute numbers of CD4+ and CD8+ T cells were not significantly different in the two groups of patients, whereas both T-cell subpopulations were significantly lower in number in late responders as compared to early responders throughout the entire year of follow-up after transplantation (Figures 1A and 2A).
HCMV-specific CD4+ T cells were lower in number before transplantation in late responders with respect to early responders and remained consistently lower for the entire study period (Figure 1B). Similarly, in late responders CD8+-specific T cells were significantly lower in number at baseline as well as at the following time points until 1 year after transplantation, when the number of HCMV-specific CD8+ T cells was comparable to that of early responders (Figure 2B).
In the early responder group, the absolute number of total and HCMV-specific CD4+ T cells did not change significantly throughout the study period with respect to baseline (median values at baseline 400, range 30–1500 cells/μL for total; and 3.0, range 0.21–26.68 cells/μL, for HCMV-specific CD4+ T cells). Similarly, total and HCMV-specific CD8+ T cells did not change in number with respect to baseline (median values at baseline 270, range 76–854; and 3.62, range 0.57–9.37 cells/μL, respectively) till day +180 for total, and day +360 for HCMV-specific CD8+ T cells, when they increased significantly. In the late responder group, the total CD4+ T-cell number was consistently lower than baseline values (median baseline value 526, range 27–888), as well as specific CD4+ T cells (median baseline value 0.80, range 0.05–1.50 cells/μL) until day +90, when matched with pretransplant levels. Total CD8+ T cells were similar in number to baseline values throughout the study, while HCMV-specific CD8+ T cells were significantly lower than baseline values (median baseline value 1.59, range 0.03–3.94) at day +30, and comparable to baseline from days +60 to +180, when they became higher. The level (high or low) of HCMV-specific CD4+ and CD8+ T cells detected at baseline substantially persisted throughout the study as shown by the significant correlation between baseline values and values of the two T-cell subpopulations until day +90 (p < 0.05, r= 0.39–0.70).
The analysis of the mean percent variation with respect to baseline in the two groups of early and late responders for absolute and HCMV-specific CD4+ and CD8+ T cells during the first year posttransplant showed that: (i) in early responders, absolute and specific CD4+ T cells had an overlapping trend, whereas specific CD8+ T cells increased more than absolute CD8+ T cells from day +30 (p = 0.05 at day +360); (ii) on the contrary, in late responders, initially (day +30) specific CD4+ showed a significant (p = 0.03) relative decrease with respect to total CD4+ T cells, whereas a relative increase in specific CD4+ and CD8+ T cells started from day +90 and reached a significant difference at day +360 (p = 0.04 for both CD4+ and CD8+ T cells) (Figure 3).
Kinetics of immune restoration
In the 17 late responders, HCMV-specific CD8+ recovered much earlier than CD4+ T cells. Following HCMV infection, the cumulative incidence of late responders progressively recovering CD4+- or CD8+-specific T-cell immunity is reported in Figure 4, showing the significantly earlier restoration of HCMV-specific CD8+ compared to CD4+ T cells (log rank test, p < 0.001). In detail, median time to HCMV detection in blood was 20 (6–117) days. In addition, median time to HCMV-specific CD8+ T-cell restoration was 81 (27–147) days, and to HCMV-specific CD4+ T-cell restoration was 179 (53–395) days (p < 0.001).
Among factors potentially affecting HCMV-specific T-cell restoration, the following were considered: absolute number of HCMV-specific CD4+ and CD8+ T cells/μL at baseline, type of transplant, immunosuppressive drug regimen evaluated according to the different mechanism of drug action or class, presence or absence of induction treatment and presence or absence of rejection episodes. Within the limits of the relatively small number of patients examined, the only factor that was significantly associated with early restoration of T-cell-mediated specific response was the number of HCMV-specific CD4+ and CD8+ T cells detected at baseline. Median baseline HCMV-specific CD4+ T-cell number/μl was 3.00 (0.21–26.68) in early responders versus 0.80 (0.05–1.50) in late responders (p < 0.01), while median number/μl of CD8+ T cells was 3.62 (0.57–93.76) in early responders versus 1.59 (0.03–3.94) in late responders (p = 0.02). In particular, two late responders (receiving in the past immunosuppressive therapy) were negative for both HCMV-specific CD4+ and CD8+ T cells before transplantation, while five additional late responders showed an equivocal response for either HCMV-specific CD4+ or CD8+ T cells (Figures 1B and 2B).
Total CD4+ T-cell counts were significantly lower (p < 0.001) in patients receiving ATG induction than in patients receiving no ATG induction until day +90, while HCMV-specific CD4+ T cells were not significantly different in the two groups. On the other hand, both total and HCMV-specific CD8+ T cells were significantly lower (p < 0.05) in patients receiving ATG at day +30. However, of the 21 patients receiving ATG induction, 10 (47.6%) were early, and 11 (52.4%) were late responders, with no significant difference.
HCMV-specific T-cell response and control of viral infection
During the entire follow-up period, no case of HCMV disease was observed. However, HCMV infection was diagnosed in all 38 patients examined (Table 2) by detection of positive antigenemia (in 19/21 early and 17/17 late responders), or DNAemia (in 21/21 early and 16/17 late responders). In addition, viremia was positive in 8/21 early, and 16/17 late responders. However, only three (14.3%) early responders had to be treated with antiviral drugs, whereas as many as 13 (76.5%) late responders were treated (p < 0.001) after reaching the cutoff for pre-emptive therapy, and four of them had relapsing infections. Median antigenemia, DNAemia and viremia levels were 185 (20–310) pp65-positive leukocytes, 2419 (27–9120) DNA copies and 10 (1–180) infectious leukocytes in patients treated versus 6 (0–60), 152 (0–2297) and 0 (0–15) in protected patients (p < 0.001 for all viral markers).
Table 2. Relationship between HCMV T-cell response and control of viral infection
Type of HCMV-specific T-cell response
No. of patients with HCMV infection (%)
However, in the early responder group, 3/21 patients (14.3%), all lung transplant recipients, received anti-rejection treatment the second month after transplantation, with loss of HCMV-specific CD4+ T cells in two of them and development of HCMV infection requiring antiviral treatment. In these patients, pharmacological control of HCMV infection was followed by recovery of specific CD4+ T cells with no subsequent relapse. On the other hand, four (23.5%) late responders did not reach the viral load cutoff required for antiviral treatment, and four (23.5%) faced repeated episodes of HCMV infection due either to very late restoration of T-cell immunity or to anti-rejection treatment. Furthermore, six late responders showed a detectable HCMV-specific CD8+ T-cell response as early as day +30. However, five of them reached the viral load cutoff prior to HCMV-specific CD4+ T-cell restoration and, thus, had to be treated.
ROC analysis of HCMV-specific CD4+ and CD8+ T cells was performed prior to the peak of HCMV infection (i.e. 32, 24–142, days after transplantation) in view of identifying patients protected from HCMV infection requiring antiviral treatment (i.e. not reaching antigenemia or DNAemia cutoff for pre-emptive therapy). This analysis showed that both HCMV-specific CD4+ and CD8+ quantitative assays are accurate enough to identify protected patients. In detail, the area under the curve (AUC) was 0.80 for CD4+ and 0.75 for CD8+ when examining all 38 patients. However, these values rose to 0.82 and 0.85, respectively, when the 11 patients undergoing steroid therapy to treat rejection, in concomitance with HCMV infection, were excluded from the analysis. As shown in Table 3, a cutoff of 0.4 T cells/μL for both HCMV-specific CD4+ and CD8+ T cells displayed the maximal diagnostic (predictive) value. In addition, ROC analysis showed that pre-transplant levels of HCMV-specific CD4+ and CD8+ T cells were a poorer indicator when to start antiviral treatment than the relevant levels examined prior to the peak of infection (AUC values: 0.69 and 0.52, respectively).
Table 3. ROC curve analysis of HCMV-specific CD4+ and CD8+ T-cell levels defined as protection from HCMV infection requiring antiviral treatment (i.e. from infection reaching antigenemia or DNAemia cutoff for preemptive therapy) in the 27 patients not receiving antirejection therapy
T-cell cutoff level
Sens = sensitivity; Spec = specificity; PPV = positive predictive value; NPV = negative predictive value; AUC = area under the curve.
Simultaneous monitoring of HCMV infection and HCMV-specific immune response
Representative examples of simultaneous monitoring of HCMV infection and HCMV-specific immune response in two heart and two lung transplant recipients are reported in Figure 5A–D. Very early restoration of T-cell immunity controlled HCMV infection from the beginning of the posttransplant period of a heart transplant recipient (Figure 5A). In another heart transplant recipient behaving as a late responder, a slight delay in the restoration of T-cell immunity was associated with a single HCMV infection peak, that was treated with GCV (Figure 5B). Steroid-treated acute rejection in one lung transplant recipient was associated with the loss of HCMV-specific CD4+ T-cell responses and the first HCMV infection peak which was treated with GCV. In the same patient, multiple subsequent self-resolving peaks concomitant with steroid- or ATG-treated rejection episodes were observed, which were eventually controlled by the restoration of complete T-cell immunity (Figure 5C). Finally, in another lung transplant recipient, a very long delay in the restoration of HCMV-specific and, in particular, CD4+ T-cell immunity was associated with recurrent episodes of HCMV infection, requiring multiple courses of antiviral treatment to control HCMV infection (Figure 5D).
In solid organ transplant recipients, HCMV infection typically occurs beyond the first month after transplantation. The severity of HCMV infection is greater, as a rule, in patients with primary infection (6,29,30), but may evolve toward HCMV disease also in patients with reactivated infection. This is generally due to the lack of specific immunity in D+R− patients, and to the loss of specific immunity in D+R+ patients, as shown in the present study. Thus, it is critical to establish whether HCMV-specific T-cell immunity is present 30 days after transplantation. This time point was selected with the intent to predict protection against HCMV infection prior to the development of the great majority of HCMV infections in transplanted patients. Results of our study indicate that patients lacking specific cellular immunity within the first month after transplantation often develop HCMV infections requiring antiviral treatment. Conversely, patients with restored specific cellular immunity within the first month after transplantation, generally develop abortive HCMV infection.
In this study, we took advantage of a newly developed methodology, which uses autologous dendritic cells infected with a wild strain of HCMV 24 h prior to PBMC stimulation (18). By using this method, peptides derived from late (structural) viral proteins present in virions and dense bodies are processed by the endocytic pathway. In addition, after viral and cellular membrane fusion, peptides are processed through the endoplasmic reticulum-Golgi pathway, and are transported to the cell membrane for presentation to PBMC. As a result, both MHC class II-peptide complexes mostly activating CD4+ T cells, and MHC class I-peptide complexes mostly activating CD8+ T cells are formed. Finally, neosynthesized immediate-early and early viral proteins are processed and complexed with MHC class I molecules. Therefore, multiple epitopes for each protein may be presented to PBMC, thus providing a more comprehensive evaluation of T-cell immune restoration.
A major finding of our study was that both absolute and HCMV-specific CD4+ and CD8+ T-cell counts in late responders were significantly and consistently lower compared to early responders. In addition, in late responders the restoration of HCMV-specific CD4+ was significantly delayed with respect to the CD8+ T-cell response. These results appeared to be related to the level of HCMV-specific CD4+ and CD8+ T cells detected at baseline, i.e. absolute numbers of HCMV-specific CD4+ and CD8+ T cells greater than 3/μL at baseline predicted a high probability of preserving the pretransplant HCMV-specific immune response at day +30, whereas lower numbers of HCMV-specific T cells at baseline predicted with high probability the loss of specific pre-transplant T-cell immunity at day +30, with recovery of specific cellular immunity occurring only in the following months. In this respect, we found that two late responders had no HCMV-specific T cells before transplantation, and five of them had a borderline response, whereas all HCMV-seropositive healthy people showed HCMV-specific T cells >0.4/μL (18). From this data, it can be argued that the underlying disease or the relevant immunosuppressive therapy (often steroid-based) may have impaired in the past HCMV-specific T-cell immunity. Given the results of this study, it would seem reasonable that these patients receive antiviral prophylaxis from the time of transplantation to prevent (early) HCMV disease.
In previous studies, it was shown that both HCMV-specific CD4+ and CD8+ T-cell responses were elicited in solid organ transplanted patients. However, immunological testing was often restricted to single or few time points, and designed to quantify either CD8+ or CD4+ T cells (31–33). Notwithstanding these limitations, the early appearance of HCMV-specific CD8+ T cells has been repeatedly reported (5,17,33), as well as the long-term protection conferred by HCMV-specific CD4+ T cells (4,17,33). In primary HCMV infections, a dominance of HCMV-specific CD8+ T cells was reported in contrast to a relative predominance of virus-specific CD4+ T cells in persistent or latent infection (17). In addition, it was shown in asymptomatic renal transplant recipients with primary infection that the HCMV-specific CD4+ precedes the HCMV-specific CD8+ T-cell response, whereas in symptomatic patients the HCMV-specific CD4+ T-cell response was delayed, in the presence of a CD8+ T-cell response that was not protective (4). These findings appear to be in keeping with the results of the present study. In fact, all patients (excluding patients with rejection) with HCMV-specific CD4+ T cells at day +30 were protected, while all patients with a delayed CD4+ T-cell response were at risk for HCMV infection requiring antiviral treatment, even in the presence of specific CD8+ T cells. Thus, HCMV-specific CD4+ T cells appear to play a major role in long-term protection from HCMV infection.
Qualitative phenotypic and functional defects of CD4+ and CD8+ T cells following anti-rejection and steroid treatment remain to be fully defined (34). However, apart from these conditions, cutoffs emerging from this study for both CD4+ and CD8+ T cells appear to be protective. Within the limits of the small sample size of this study, the AUC values of the ROC analysis provide statistical support to these conclusions, while clinical follow-up data appear to validate immunological laboratory testing predictions. From the clinical standpoint, it appears reasonable to conclude that simultaneous immunological and virologic follow-up of individual patients may improve management of HCMV infections in transplanted patients (5,8), thereby avoiding treatment of patients with apparently efficient T-cell immunity. Given the ongoing debate between prophylaxis and preemptive therapy for reducing costs of HCMV infection in transplant recipients, the identification of a cohort of patients that are less likely to develop HCMV reactivation may suggest (in the absence of rejection) discontinuation of HCMV monitoring. On the other hand, the identification of another cohort of patients that are more likely to develop HCMV reactivation may prompt the use of either anti-viral prophylaxis or preemptive therapy. In this study, it was found that an early drop post-transplant of HCMV-specific CD4+ and CD8+ T cells in late responders is often associated with a drop in total CD4+ and CD8+ T cells. This finding can be utilized by centers unable to measure HCMV-specific T cells to identify patients at high risk of worse outcome of HCMV infection.
Results of our study show that short-term T-cell immune restoration is protective against HCMV infection, whereas delayed specific T-cell restoration is the basis for recurrent HCMV infections often requiring antiviral treatment. Patients not developing T-cell immunity within 6 months after transplantation are eligible for adoptive T-cell immunotherapy.
The authors wish to thank all the technical staff of the Servizio di Virologia, Barbara Castiglioni and Benedetta Nocita for samples and data collection, Daniela Sartori for preparation of the manuscript, and Laurene Kelly for revision of the English.
This work was partially supported by grants from Ministero della Salute, Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, 89269/A, 80541 and 80425; and by grants from the Fondazione Cariplo 93005.