Inconsistent Responses of Cytomegalovirus‐Specific T Cells to pp65 and IE‐1 versus Infected Dendritic Cells in Organ Transplant Recipients
2007; Elsevier BV; Volume: 7; Issue: 8 Linguagem: Inglês
10.1111/j.1600-6143.2007.01890.x
ISSN1600-6143
AutoresDaniele Lilleri, Paola Zelini, Chiara Fornara, Giuditta Comolli, Giuseppe Gerna,
Tópico(s)Toxoplasma gondii Research Studies
ResumoAmerican Journal of TransplantationVolume 7, Issue 8 p. 1997-2005 Free Access Inconsistent Responses of Cytomegalovirus-Specific T Cells to pp65 and IE-1 versus Infected Dendritic Cells in Organ Transplant Recipients D. Lilleri, D. Lilleri Servizio di VirologiaSearch for more papers by this authorP. Zelini, P. Zelini Servizio di VirologiaSearch for more papers by this authorC. Fornara, C. Fornara Servizio di VirologiaSearch for more papers by this authorG. Comolli, G. Comolli Servizio di Virologia Laboratori Sperimentali di Ricerca-Area Biotecnologie, Fondazione IRCCS Policlinico San Matteo, Pavia, ItalySearch for more papers by this authorG. Gerna, Corresponding Author G. Gerna Servizio di Virologia * Corresponding author: Giuseppe Gerna, g.gerna@smatteo.pv.itSearch for more papers by this author D. Lilleri, D. Lilleri Servizio di VirologiaSearch for more papers by this authorP. Zelini, P. Zelini Servizio di VirologiaSearch for more papers by this authorC. Fornara, C. Fornara Servizio di VirologiaSearch for more papers by this authorG. Comolli, G. Comolli Servizio di Virologia Laboratori Sperimentali di Ricerca-Area Biotecnologie, Fondazione IRCCS Policlinico San Matteo, Pavia, ItalySearch for more papers by this authorG. Gerna, Corresponding Author G. Gerna Servizio di Virologia * Corresponding author: Giuseppe Gerna, g.gerna@smatteo.pv.itSearch for more papers by this author First published: 05 July 2007 https://doi.org/10.1111/j.1600-6143.2007.01890.xCitations: 41AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract CD4+ and CD8+ T cells specific for human cytomegalovirus (HCMV) and two immunodominant HCMV antigens (pp65 and IE-1) were monitored in 20 solid organ transplant recipients undergoing primary (n = 4) or reactivated (n = 16) HCMV infection during the first year after transplantation by using as a stimulator either HCMV-infected autologous dendritic cells (DCs) or pp65- or IE-1 peptide mixtures. Turnaround times for test performance were 7 days for infected DCs and 24 h for peptides. Using infected DCs, HCMV-specific T-cell restoration occurred in all patients for CD8+ and in 18/20 (90%) for CD4+ T-cell subpopulations, resulting in virus clearance from blood. Using peptide mixtures, T-cell responses were less frequently detected. In detail, 14 (70%) patients showed pp65-specific CD8+ T cells and 10 (50%) patients IE-1-specific CD8+ T cells, whereas pp65-specific CD4+ T cells were detected in 14 (70%) patients, and IE-1-specific CD4+ T cells in three (15%) patients only. Protection from HCMV infection was associated with the presence of a HCMV-specific T-cell response directed against multiple viral proteins, but not against pp65 or IE-1 only. In conclusion, the use of pp65 and IE-1 peptide mixtures for rapid monitoring of HCMV-specific T-cell responses in solid organ transplant recipients underestimates the actual T-cell immune response against HCMV. Introduction Human cytomegalovirus (HCMV) is the most important viral cause of morbidity and mortality in solid organ transplant recipients (SOTR) (1). HCMV infection may result in self-limiting infection or, in the absence of immune control or antiviral therapy, it can cause systemic and organ infections. The severity of HCMV infection and the extent of organ involvement inversely correlate with an efficient CD4+ and CD8+ T-cell immune response (2, 3). The majority of studies evaluating the kinetics of HCMV infection and HCMV-specific T-cell reconstitution in SOTR, while evaluating a CD4+ T-cell response against whole virus antigens (infected cell lysate) have focused on pp65-and IE-1 as targets for a CD8+ T-cell response (4-9). However, although these two antigens have been recognized as immunodominant for the CD8+ T-cell response to HCMV (10-12), other viral proteins are recognized by T cells (13, 14). In this respect, a recent study suggested that T cells directed towards pp65 and IE-1 cannot account for and do not correlate with the actual HCMV-specific T-cell pool (15). In addition, no correlation between presence of pp65- and IE-1-specific T cells and protection from late disease in liver transplant recipients developing primary HCMV infection was reported (16). Recently, a new methodological approach providing a more comprehensive evaluation of the HCMV-specific T-cell immune response (17) was adopted for the follow-up of HCMV-specific T cells in SOTR (18). This method is based on use of HCMV-infected dendritic cells (DCs) as the stimulus, allowing a more physiological activation of T cells, along with simultaneous quantification and functional evaluation of both HCMV-specific CD4+ and CD8+ T cells. In the present report, we investigated the kinetics of HCMV-specific as well as pp65- and IE-1-specific CD4+ and CD8+ T-cell restoration during the first year after transplantation in 16 HCMV-seropositive and four HCMV-seronegative heart, lung and kidney transplant recipients and in healthy controls. HCMV-specific T cells were determined by using HCMV-infected DC as a stimulus in conjunction with intracellular detection of interferon-γ (IFNγ) production, while mixtures of overlapping peptides spanning the entire pp65 or IE-1 proteins were used for determination of antigen-specific IFNγ-producing T cells. Patients and Methods Subjects Sequential blood samples were obtained from 20 patients receiving a heart (n = 9), lung (n = 8) or kidney (n = 3) transplantation at the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, between January and July 2004. Four HCMV-seronegative patients received an organ from a HCMV-seropositive donor (high-risk patients), while 16 HCMV-seropositive patients received a transplant from either a HCMV-seropositive or seronegative donor (low-risk patients). Following preemptive therapy administration, none of these patients developed HCMV disease (19). A total of 87 samples (3–7 per patient) were analyzed for a median follow-up of 359 (range 156–426) days after transplantation. Immunosuppression consisted of standard triple therapy including a calcineurin inhibitor (cyclosporine-A or tacrolimus), an anti-proliferative drug (azathioprine or mycophenolate mofetil) and steroids. As induction treatment, anti-thymocyte globulin (ATG) was given to ten patients, while three patients received anti-CD25 monoclonal antibodies (MAbs). Patients with allograft rejection episodes were treated with a daily bolus of intravenous methylprednisolone (1 g or 500 mg) for 3 days. In addition, ATG was administered to three patients with steroid-resistant rejection. As controls, 11 healthy subjects with serological evidence of remote HCMV infection and five HCMV-seronegative subjects were examined. HCMV infection diagnosis and treatment HCMV infection was defined as HCMV detection in blood or body tissues, whereas HCMV disease required documentation of HCMV infection along with symptoms and/or organ function abnormalities (20). We examined patients from a randomized study (19) aimed at evaluating a DNAemia (21) cut-off of 300 000 copies/mL whole blood for preemptive therapy of HCMV infections (22), in comparison with the antigenemia (23, 24) cut-off of 100 pp65-positive/2 × 105 leukocytes examined (25). Virological assays were performed weekly during the first 3 months post-transplant (twice a week during episodes of HCMV infection). Subsequently, tests were performed monthly unless otherwise indicated by clinical findings. In addition, viremia was routinely quantified to detect infectious virus by the shell vial technique (26). Intravenous ganciclovir (5mg/kg b.i.d.) was administered for preemptive therapy in both arms. Anti-HCMV prophylaxis was not given to any patient. When required, relapse episodes were similarly treated. The same preemptive approach was used in case of rejection episodes in either arm. The donor/recipient serostatus was determined by the enzyme-linked immunosorbent assay prior to transplantation using methods previously described (27). Stimulation of HCMV-specific or pp65- and IE-1-specific CD4+ and CD8+ T cells HCMV-specific CD4+ and CD8+ T cells were simultaneously stimulated by using autologous, monocyte-derived, HCMV-infected immature DCs (17). Following in vitro generation from peripheral blood mononuclear cells (PBMCs) (28), immature DCs were infected for 24 h with an endotheliotropic and leukotropic HCMV strain (VR1814) (29). HCMV-infected and mock-infected immature DCs were then co-cultured overnight with autologous PBMCs at a ratio of 1:20 in the presence of brefeldin-A to prevent cytokine release. To detect pp65- or IE-1-specific T cells, pools of 15 amino acid peptides (30) with an overlap of 11 amino acids spanning the entire HCMV proteins pp65 or IE-1 (Jerini AG, Berlin, Germany) were used as a stimulus. Peptides were used at a final concentration of 1μg/mL. One test volume of each peptide pool was resuspended in 100 μL RPMI 1640 and added to 1 × 106 PBMCs. Then, 0.5 μg of anti-CD28 and anti-CD49d MAbs were added. Cultures were then incubated at 37°C in a 5% CO2 atmosphere. After 1 h, 400 μL of RPMI 1640 + 10% FBS containing brefeldin-A at a final concentration of 10 μg/mL were added. Cells were incubated 16–18 h at 37°C in a 5% CO2 atmosphere in a slant position. As a control, cells were incubated with culture medium supplemented with anti-CD28 and anti-CD49d MAbs in the absence of peptide pools. Cytokine flow cytometry (CFC) analysis Following incubation with HCMV-infected DCs or peptide mixtures, PBMCs were tested for intracellular IFNγ production by CFC. PBMCs were washed and stained for 30 min on ice with FITC-conjugated MAb anti-CD8 (Beckman Coulter Immunotech Marseilles, France) and PC5-conjugated MAb anti-CD4 (Beckman Coulter Immunotech) in PBS+5% FBS, containing 5% human immunoglobulin and 0.01% sodium azide. In preliminary experiments, cells were stained in parallel with FITC-conjugated MAb anti-CD3 (Beckman Coulter Immunotech), PC5-conjugated MAb anti-CD4 (Beckman Coulter Immunotech) and APC-conjugated MAb anti-CD8 (Beckman Coulter Immunotech). Cells were then washed with PBS+5% FBS, fixed and permeabilized by using the FIX and PERM® kit (Caltag, Burlingham, CA), and stained for 45 min with PE-conjugated MAb anti-IFN-γ (Beckman Coulter Immunotech). Cells were resuspended in 1% paraformaldehyde and analyzed in a FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA) equipped with a 488 nm argon laser and a 635 nm red-diode laser, operating with the CellQuest software (Becton-Dickinson). As a routine, 1–2 × 105 viable lymphocytes were collected and at least 2.5 × 104 CD4+ and CD8bright T cells were analyzed as above. The frequency of CD4+ and CD8bright T cells producing IFNγ in response to HCMV stimuli were calculated by subtracting the value of the sample incubated with mock-infected DCs or culture medium (consistently < 0.05%) from the test value. Absolute CD3+CD4+ and CD3+CD8+ T-cell counts were determined by direct immunofluorescence using an EPICS-500XL flow cytometer (Beckman Coulter Inc, Fullertone, CA). The total number of HCMV- or antigen-specific CD4+ and CD8+ T cells was determined by multiplying the percentages of specific IFNγ-positive T cells by the relevant absolute CD4+ and CD8+ T-cell counts. Statistical analysis 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 Fisher's exact test. The correlation between two parameters was determined by calculating the correlation coefficient r. Curves of HCMV infection in blood in the post-transplantation period, as well as curves relevant to restoration of specific CD4+ and CD8+ T-cell immunity were determined by the Kaplan-Meier method, while differences between curves were determined by the log-rank test. Results HCMV- and viral protein (pp65 and IE-1)-specific CD4+ and CD8+ T-cell responses in healthy subjects As mentioned above, the frequency of pp65- and IE-1-specific T cells was determined in 11 healthy subjects with remote HCMV infection and in five HCMV-seronegative healthy subjects following stimulation with pp65 and IE-1 peptide mixtures. In preliminary experiments, it was observed that peptide stimulation induced a profound down regulation of CD3 expression on the surface of activated CD4+ T cells, thus leading to underestimation of the real frequency of antigen-specific CD4+ T cells, when IFNγ+ cells were selected for from CD3+/CD4+ lymphocytes instead of CD4+ lymphocytes. This phenomenon did not occur to the same degree with CD8+ T cells, that is, no significant difference was found in the IFNγ+ cell frequency when determined within either CD3+/CD8+ or CD8bright lymphocyte subpopulations (data not shown). Thus, in subsequent experiments, cells were not stained for CD3 expression and only CD4+ and CD8bright lymphocytes (to exclude NK cells) were analyzed. On the basis of results obtained from the stimulation of PBMCs from seronegative and seropositive healthy subjects with the two peptide mixtures, samples with IFNγ-positive CD4+ and CD8+ T cells < 0.2/μL blood were considered non-responsive, samples with IFNγ-positive CD4+ and CD8+ T cells >0.4/μl blood were considered responsive, while values between 0.2 and 0.4 IFNγ-positive CD4+ and CD8+ T cells/μl blood were considered equivocal. Results obtained following the stimulation of PBMCs with either HCMV-infected DCs, or pp65 and IE-1 are reported in Figure 1. All but one, of the 11 HCMV-seropositive controls (91%) showed a positive CD4+ T-cell response to pp65, whereas six (55%) responded to IE-1 (one of these gave an equivocal response). As for CD8+ T cells, 9/11 subjects (82%) showed a positive response to pp65 (one equivocal response) and 7/11(64%) to IE-1 (two equivocal responses). All HCMV-seropositive subjects tested showed a positive HCMV-specific CD4+ and CD8+ T-cell count, as determined by infected DC stimulation, including those with negative CD8+ T-cell response to pp65 (n = 2) and IE-1 (n = 4), and subjects with negative CD4+ T-cell response to both pp65 (n = 1) and IE-1 (n = 5). Figure 1Open in figure viewerPowerPoint Absolute levels of CD4+ (A) and CD8+ (B) T cells specific for HCMV-infected DCs, pp65 and IE-1 in HCMV seropositive and seronegative healthy subjects. The mean+/-SD inter-assay coefficient of variation was 32.48+/-16.84% for CD4+ T cells and 25.35+/-6.27% for CD8+ T cells. HCMV and antigen-specific CD4+ and CD8+ T-cell responses in SOTR At the end of follow-up, 18/20 (90%) SOTR showed a positive CD4+ T-cell response to infected DCs. CD4+ T cells specific for pp65 were detected in 14 (70%) patients, whereas response to IE-1 was found in three (15%) SOTR only. Two patients were lost to follow-up before development of CD4+ T-cell response to infected DCs (156 and 247 days post-transplantation, respectively), but were able to control infection by restoration of HCMV-specific CD8+ T cells. As expected, these two patients did not show CD4+ T cells specific for either pp65 or IE-1. All 20 patients developed a CD8+ T-cell response to infected DCs, whereas 14 (70%) and 10 (50%) showed a CD8+ T-cell response to pp65 and IE-1, respectively (Figure 2A). Figure 2Open in figure viewerPowerPoint Response to different stimuli in solid organ transplant recipients (SOTR). (A) Number of patients showing CD4+ and CD8+ T-cell response to the different HCMV stimuli (DCs, pp65 and IE-1) at the end of follow-up. (B) Among patients with CD4+ (n = 18) and CD8+ (n = 20) T cells responding to infected DCs at the end of follow-up, the relative frequencies of response to pp65, IE-1, both or neither protein are reported. (C–D) Cumulative detection of HCMV-, pp65- and IE-1-specific CD4+ (C) and CD8+ (D) T cells in 16 HCMV-seropositive SOTR suffering from HCMV reactivation. (E–F) Cumulative detection of specific CD4+ (E) and CD8+ (F) T cells in four HCMV-seronegative SOTR undergoing primary HCMV infection. The relative frequencies of response to either pp65, IE-1, both proteins or neither one among patients with CD4+ and CD8+ T cells responding to infected DCs at the end of the follow-up are reported in Figure 2B. Among the 18 patients showing a positive CD4+ T-cell response to infected DCs, 11 (61%) responded also to pp65, three (17%) patients responded to both pp65 and IE-1 (in two cases the response to IE-1 appeared later with respect to pp65) and four (22%) did not show CD4+ T cells specific for either viral protein. As for the CD8+ T-cell response (20/20 patients responded to infected DCs), six (30%) showed a positive response to pp65, two (10%) to IE-1, 8 (40%) to both pp65 and IE-1 (in two patients the development of the response to IE-1 was delayed, whereas in another patient pp65-specific CD8+ T cells appeared later), while four patients (20%) responded to infected DCs alone. In Figures 2C and D, the cumulative detection of CD4+ and CD8+ T cells specific for the different stimuli in the 16 HCMV-seropositive SOTR (reactivated infections) are reported. Again, a significantly higher proportion of patients showed a positive CD4+ and CD8+ T-cell response to infected DCs during follow-up as compared to both pp65 and IE-1 peptide pools (p < 0.01). In addition, a significantly higher number of patients showed a CD4+ T-cell response to pp65 as compared to IE-1 (p < 0.01), whereas no significant difference in the proportion of patients with CD8+ T cells specific for pp65 or IE-1 was found. The cumulative detection of CD4+ and CD8+ T cells specific for the different stimuli in the four HCMV-seronegative SOTR receiving transplantation from a HCMV-seropositive donor (and developing primary HCMV infection) is shown in Figures 2E and 2F. The number of patients examined is too small to draw any conclusion about the kinetics of T-cell specific development for the different stimuli after primary infection. The correlation between the number of CD4+ or CD8+ T cells responding to HCMV-infected DCs and to pp65 or IE-1 peptides is reported in Figure 3. Although the correlation was significant for both T-cell subpopulations and both peptide pools analyzed, r values were rather low. Figure 3Open in figure viewerPowerPoint Correlation of HCMV-specific and pp65- (A) or IE-1- (B) specific CD4+, and HCMV-specific and pp65- (C) or IE-1- (D) specific CD8+ T-cell response in 87 blood samples from 20 solid organ transplant recipients. Early HCMV-specific T-cell response and control of viral infection All 20 patients examined developed an active HCMV infection in blood at a median time of 24 days, range 6–117 days, after transplantation. Virus was cleared spontaneously without the need for antiviral intervention in nine patients, whereas in the other 11 patients (four of them undergoing primary HCMV infection) DNAemia or antigenemia reached the cut-off for preemptive therapy. Antiviral treatment was given, leading to virus clearance from blood in all 11 patients. Following preemptive therapy, in no patient was HCMV overt disease diagnosed. In a previous prospective study, it was found that early (within the first month) detection of HCMV-specific T cells, in the absence of subsequent anti-rejection treatments, correlates strongly with spontaneous HCMV clearance from blood (18). Thus, we correlated the early (30 days after transplantation) presence of HCMV-, pp65- and IE-1-specific T cells with spontaneous control of HCMV infection (immune protection) or need for preemptive therapy (risk for developing HCMV disease). As reported in Table 1, 8/9 patients with self-resolving infection showed early presence of HCMV-specific CD4+ and CD8+ T cells. The last patient showed a delayed appearance of HCMV-specific CD8+ and CD4+ T cells at day +117 and +189, respectively, following a delayed HCMV infection (day +117). Conversely, only five and three patients had CD8+ T cells responding to pp65 and IE-1, respectively, at day +30, whereas one patient only had pp65-specific and none showed IE-1-specific CD4+ T cells. Table 1. Relationship between early (day +30) HCMV-, pp65- and IE-1-specific T-cell response and control of viral infection Type of HCMV infection Number of patients with early (day +30) T-cell response to: DCs pp65 IE-1 Self-resolving CD4+ 8/91 1/9 0/9 CD8+ 8/91 5/91 3/9 Treated CD4+ 3/112 0/112 0/112 CD8+ 3/112 1/11 2/11 1One patient had delayed HCMV infection and simultaneous development of HCMV-specific immunity. 2Three patients lost specific immunity in the subsequent month following steroid treatment for acute rejection. Among the 11 patients with HCMV infection reaching the cut-off value for preemptive therapy, only three showed early appearance of HCMV-specific CD4+ and CD8+ T cells. However, these three patients lost HCMV-specific CD4+ T cells in the subsequent month following anti-rejection treatment, reacquiring HCMV-specific CD4+ T cells between 3 and 6 months after transplant. None of these three patients had pp65- or IE-1-specific CD4+ T cells at day +30, whereas one showed IE-1-specific CD8+ T cells and another both IE-1- and pp65-specific T cells (Table 1). Kinetics of HCMV infection and HCMV-specific immune response The virological and immunological follow-up of four patients with recurrent (A–C) or primary (D) infection is reported in Figure 4. In patient A, HCMV-specific CD4+ and CD8+ T cells were already detected 1 month after transplantation, and HCMV infection resolved spontaneously in the absence of antiviral intervention. Both CD4+ and CD8+ T cells specific for pp65 (but not for IE-1) were detected during the entire follow-up. Figure 4Open in figure viewerPowerPoint Virological and immunological follow-up of four solid organ transplant recipients. Ganciclovir (GCV) treatment courses are indicated by open bars and arrows indicate steroid or anti-thymocyte globulin (ATG) treatment for rejection. HTR = heart transplant recipient; LTR = lung transplant recipient. Patient A had a self-resolving HCMV infection due to prompt appearance of HCMV-specific CD4+ and CD8+ T cells. Both CD4+ and CD8+ T cells specific for pp65 (but not for IE-1) were detected during the entire follow-up. Patient B developed HCMV infection requiring treatment in the absence of HCMV specific CD4+ and CD8+ T cells. Then HCMV-specific CD8+ (first) and CD4+ (later) T cells appeared, allowing long-term disappearance of HCMV infection. Only a borderline CD4+ T-cell response to pp65 was detected. Patient C lost the previously detectable HCMV-specific CD4+ and CD8+ T cells after treatment for acute rejection and developed HCMV infection requiring preemptive therapy. After restoration of HCMV-specific T cells, CD8+ T cells were preserved notwithstanding subsequent courses of anti-rejection treatment, thus permitting resolution of an episode of recurrent infection. Neither pp65- nor IE-1-specific T cells were detected during follow-up. Patient D underwent primary HCMV infection, which rapidly cleared after GCV treatment, with subsequent development of HCMV- (and IE-1-) specific CD8+ T cells and HCMV-specific CD4+ T cells; pp65-specific CD8+ T cells were not detected, while both pp65- and IE-1-specific CD4+ T cells appeared 1 year after transplantation. Patient B was lacking HCMV-specific CD4+ and CD8+ T cells 30 days after transplantation and developed HCMV infection with high viral load requiring treatment. Then, HCMV-specific CD8+ (after the second month) and CD4+ (after 1 year) T cells appeared, with no subsequent relapse of HCMV infection. As for the antigen-specific T cells, only a borderline CD4+ T-cell response to pp65 was detected 1 year after transplantation. Patient C lost the previously detectable HCMV-specific CD4+ and CD8+ T cells after steroid treatment for acute rejection and developed HCMV infection reaching the cut-off for preemptive therapy. After restoration of HCMV-specific T cells, subsequent courses of anti-rejection treatment (including ATG) led to disappearance of specific CD4+ T cells, while specific CD8+ T cells were still present. A concomitant single episode of recurrent infection was spontaneously cleared. Neither pp65- nor IE-1-specific T cells were detected during follow-up. Finally, patient D was a HCMV-seronegative heart recipient from a HCMV-seropositive donor and developed HCMV primary infection in the first month after transplantation. HCMV was rapidly cleared from blood by ganciclovir treatment, followed by prompt development of HCMV-specific CD8+ T cells and slightly delayed appearance of specific CD4+ T cells. IE-1-specific CD8+ T cells were readily detectable, while pp65-specific CD8+ T cells did not appear during the entire follow-up. Both pp65- and IE-1-specific CD4+ T cells were detected later (1 year after transplantation) as compared to HCMV-specific CD4+ T cells. Administration of steroid boli as anti-rejection treatment 90 days after transplantation led to reappearance of HCMV in blood but, in the presence of an effective HCMV-specific immune response, infection recurrence was spontaneously cleared. Discussion The lack of a reconstituted specific T-cell-mediated immune response in seropositive patients (or the impaired development of a primary immune response in seronegative subjects) is the trigger for HCMV infection and disease in transplant recipients. Thus, the determination of HCMV-specific CD4+ and CD8+ T-cell responses is a major tool to assess the risk of developing HCMV disease or the ability to control infection in the absence of antiviral treatment in individual patients. By taking advantage of a newly developed methodology, using autologous DC infected with a wild-type HCMV strain as a stimulus for ex vivo detection of HCMV-specific T cells (17), a prospective study allowing monitoring of HCMV-specific immunity in SOTR was recently conducted (18). The study showed that patients lacking specific cellular immunity within the first month after transplantation often develop HCMV infection requiring antiviral treatment, whereas patients with early restoration of specific cellular immunity generally develop abortive HCMV infection. Limitations to our method are the biological variability of a cell-based assay, the relatively long time-lapse required for performing the entire procedure (1 week) and the potential difficulty in generating DCs from severely leukopenic patients. With this in mind, we decided to compare the direct stimulation of PBMCs with overlapping peptide mixtures of HCMV proteins pp65 and IE-1 with the stimulation provided by infected DCs. Turnaround times were 24 h for the peptide assay vs. 7 days for the DC assay. It was found that the great majority of healthy subjects with remote HCMV infection had CD4+ (91% of controls) and CD8+ (82% of controls) T cells reactive with pp65-derived peptides, whereas 55% and 64% of controls showed a CD4+ and CD8+ T-cell response against IE-1, respectively. However, all subjects that did not show a T-cell response against either one of the two proteins did respond to HCMV-infected DCs. This confirms that in some subjects the HCMV-specific T-cell repertoire is directed towards other viral proteins than pp65 and IE-1, and some subjects do not generate a detectable memory T-cell pool recognizing these two immunodominant HCMV antigens (14, 15, 17). It can be speculated that DC infection with HCMV-infected cell culture supernatant (29) may enable DC to load on MHC class I and II molecules peptides derived from neosynthesized viral proteins (such as IE-1) as well as from late proteins present in dense bodies (such as pp65, pp71, pp150, pp28, gB and gH), entering DCs with the same mechanism as virions (31). All the above-mentioned viral proteins were shown at our laboratory to be exposed on the DC membrane 24 h after infection (unpublished results). However, it is still uncertain and should be further investigated which antigens are actually presented to T cells by DCs 24 h after infection. We observed the same picture by the follow-up analysis of HCMV-specific immune response in SOTR. Although a significant correlation was found between results of the CFC assays when performed using infected DCs as a stimulus or either one of the two antigens, the r coefficients were rather low. Again, it was found that, at the end of follow-up, about 20% of patients analyzed did not show CD4+ and CD8+ T cells specific for either pp65 or IE-1, even in the presence of a clear-cut T-cell response to infected DCs. This indicates that the analysis of the T-cell response directed towards pp65 and IE-1 underestimate the actual protective immunity against HCMV in these patients. As mentioned above, in a previous study of HCMV-specific immune response in SOTR, it was found that early (within the first month) detection of HCMV-specific T cells, in the absence of subsequent anti-rejection treatments, correlates strongly with protection, that is, with spontaneous HCMV clearance from blood (18). In the present cohort, we confirm that nearly all patients with self-resolving infection (8/9) had HCMV-specific CD4+ and CD8+ T cells 1 month after transplantation, whereas only a minor proportion of patients had pp65- or IE-1-specific CD8+ T cells and only one showed pp65-specific CD4+ T cells at this early
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