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Journal of Virology, October 2008, p. 10143-10152, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.00688-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Expansion of Human Cytomegalovirus (HCMV) Immediate-Early 1-Specific CD8⁺ T Cells and Control of HCMV Replication after Allogeneic Stem Cell Transplantation

Karim Sacre,¹ Stéphanie Nguyen,^1,2 Claire Deback,³ Guislaine Carcelain,¹ Jean-Paul Vernant,² Véronique Leblond,² Brigitte Autran,^1* and Nathalie Dhedin^1,2

Laboratoire d'Immunologie Cellulaire et Tissulaire, INSERM U543, AP-HP, Université Pierre et Marie Curie-Paris6, Paris, France,¹ Service d'Hématologie Clinique, Hôpital Pitié-Salpêtrière, AP-HP, Université Pierre et Marie Curie-Paris6, Paris, France,² Laboratoire de Virologie, EA 2387, Université Pierre et Marie Curie-Paris6, Paris, France³

Received 27 March 2008/ Accepted 28 July 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recovery of human cytomegalovirus (HCMV)-specific T immunity is critical for protection against HCMV disease in the early phase after allogeneic stem cell transplantation (SCT). Using an enzyme-linked immunospot assay with overlapping 15-mer peptides spanning pp65 and immediate-early 1 HCMV proteins, we investigated which HCMV-specific CD8⁺ gamma interferon-positive (IFN-⁺) T-cell responses against pp65 and IE-1 were associated with control of HCMV replication in 48 recipients of unmanipulated HLA-matched allografts at 3 months (M3) and 6 months (M6) after SCT and in 23 donors. At M3 after SCT, the magnitude of the pp65-specific IFN--producing CD8⁺ T-cell response was greater in recipients than in donors, regardless of HCMV status. In contrast, expansion of IE-1-specific CD8⁺ T cells at M3 was associated with protection against HCMV, and no patient with this expansion had HCMV replication at M3. At M6, the number of HCMV-specific CD8⁺ T cells against both pp65 and IE-1 had expanded in all recipients, regardless of their previous levels of HCMV replication. The recipients' HCMV-specific CD8⁺ T cells already detectable in related donors were predominantly targeting pp65. In contrast, in 40% of the cases, the HCMV-specific CD8⁺ T cells in recipients involved new CD8⁺ T-cell specificities undetectable in their related donors and preferentially targeting IE-1. Taken together, these results showed that the delay in reconstituting IE-1-specific CD8⁺ T cells is correlated with the lack of protection against HCMV in the first 3 months after SCT. They also show that IE-1 is a major antigenic determinant of the early restoration of protective immunity to HCMV after SCT.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Because cellular immunity is severely impaired after allogeneic stem cell transplantation (SCT), uncontrolled human cytomegalovirus (HCMV) replication is frequent in the posttransplantation period and causes significant morbidity and mortality (17, 23). Thus far, the best known marker for protection against HCMV disease in the first months after SCT is the size of the HCMV-specific CD8⁺ T-cell population (4, 21, 30, 32). Although recent data show that HCMV-specific T cells in healthy HCMV-positive individuals may be directed against a wide variety of HCMV proteins (8, 37), the key antigen targeted by HCMV-specific CD8⁺ T cells in immunosuppressed patients remains controversial.

Both the structural pp65 and the nonstructural immediate-early 1 (IE-1) HCMV proteins are now regarded as dominant T-cell targets (28, 31). IE-1 was considered irrelevant to immune protection (11, 12, 21) until recently, however. Accordingly, most research on post-SCT protective immunity against HCMV focused on pp65 and showed a positive correlation between immune reconstitution and an increased number of pp65-specific T cells (3, 5, 14, 16). On the other hand, a recent study of heart and lung transplant recipients showed that, in the early period after transplantation, a high frequency of IE-1-specific, but not of pp65-specific, T cells was correlated with protection against HCMV disease (2). Our group analyzed the parameters of the CD8⁺ T-cell responses to HCMV associated with restoration of protection after recovery from HCMV retinitis in human immunodeficiency virus (HIV)-infected patients and found that a key role was played by the breadth and repertoire of the IE-1-specific CD8⁺ T cells among the anti-HCMV CD8 cells (35). These observations call into question the specificity of CD8⁺ T cells associated with restored protection against HCMV after SCT.

Restoration of T-cell immunity after SCT is a slow process (7, 25). After an unmanipulated T-cell-replete allograft, recipient T cells may be derived from either mature transferred donor T cells (24, 26) or graft donor precursor cells differentiated in the recipient's thymus (7, 10, 18, 34). During the first 6 months after SCT, protective immunity against HCMV is known to come from mature donor T cells coinfused with the graft (3, 10). In this case, immune protection may be mediated by transplanted donor memory T cells or by transplanted donor T cells that expand only in the recipient environment (7, 10, 13, 25, 26). No studies have yet determined the relative contributions of memory and newly expanded transferred CD8⁺ T cells to the restoration of protective immunity against HCMV in SCT recipients and the balance between specificity for pp65 and IE-1 in these cells.

In this study, we evaluated pp65- and IE-1-specific CD8⁺ T-cell responses in 48 HCMV-positive (HCMV⁺) unmanipulated allograft recipients and 23 HCMV⁺ healthy SCT donors. We found a correlation between the lack of IE-1-specific T cells and an absence of control of HCMV replication at 3 months (M3) after SCT. In addition, at 6 months (M6) after SCT, IE-1-specific CD8⁺ T-cell responses expanded in recipients with controlled HCMV replication. That is, the expansion of IE-1-specific CD8⁺ T cells associated with protection appeared to be delayed. Moreover, around 40% of the HCMV-specific CD8⁺ T cells in recipients with controlled HCMV replication at M6 after SCT were new cells that expanded only in the recipient environment and predominantly targeted the IE-1 protein. Our findings challenge the conventional wisdom according to which pp65 is the key protein for control of HCMV replication after SCT. They suggest instead that IE-1 is a major determinant of the restoration of protective immunity against HCMV.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study populations. Samples of peripheral blood mononuclear cells (PBMCs) were obtained from 71 individuals, including 23 healthy SCT donors and 48 recipients of unmanipulated T-cell-replete HLA-matched allografts. Samples were prospectively collected at either M3 or M6 after SCT, and samples from five recipients were taken at both M3 and M6. The recipients were divided into four groups according to their HCMV clinical status and the timing of blood sampling, as described below and in Table 1. Overall, 69% (n = 33) of allografts were from HLA-identical sibling donors, and the conditioning regimen was myeloablative in 75% (n = 36) of cases. All 48 recipients received graft-versus-host disease (GVHD) prophylaxis, which included cyclosporine and methotrexate in 79% (n = 38) of the cases. Acute and chronic GVHD were treated with steroids. Written informed consent was obtained from each patient.

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TABLE 1. Characteristics of study subjects

HCMV status. The HCMV serological status was defined by the presence of HCMV-specific immunoglobulin G antibodies. Overall, before SCT, 41 SCT recipients were HCMV positive and 7 were HCMV negative but received allografts from HCMV-positive donors. Of the 41 HCMV-positive donors, 23 were tested. HCMV replication was monitored weekly in all recipients, from day 0 to day 100, and twice monthly from day 100 to day 180, by HCMV pp65 antigenemia and/or whole-blood HCMV PCR assays. The HCMV pp65 antigenemia assays used the Cinakit (Argene Biosoft, Varilhes, France) according to the manufacturer's recommendations. This test indirectly detects immunofluorescence of the pp65 HCMV internal matrix phosphoprotein in peripheral blood leukocytes. HCMV pp65-positive cells were counted on duplicate stained slides and reported as the number of positive cells per 200,000 leukocytes. Real-time PCR was performed on the ABI Prism 7000 instrument (Applied Biosystems, Courtaboeuf, France), with TaqMan technology applied as previously described (29). The procedure had a sensitivity of 25 copies of HCMV DNA per ml of initial whole blood. Immunocytology measurements (pp65 antigenemia) and HCMV PCR on whole blood are concordant methods for monitoring HCMV replication in immunosuppressed patients (6). On detection of HCMV reactivation (>1,000 copies per milliliter of whole blood on two successive HCMV PCRs 1 week apart or more than two pp65-positive cells per slide), standard (preemptive) treatment with ganciclovir was administered at a dose of 5 mg/kg of body weight intravenously twice a day for 14 days. No recipients received prophylactic anti-HCMV therapy.

PBMC preparation and storage. PBMCs were isolated by standard Ficoll-Hypaque density gradient centrifugation (Pharmacia) on fresh blood samples and immediately cryopreserved in fetal calf serum (Gibco BRL, Life Technology) containing 10% dimethyl sulfoxide (Merck) in liquid nitrogen. The cryopreserved cells were stored in liquid nitrogen until they were used. Trypan blue exclusion after the cells were thawed showed a mean viability of 85%.

Chimerism analysis. Testing for the presence of allogeneic hematopoietic cells in whole-blood samples from allograft recipients took place in the molecular biology laboratory of the Henri-Mondor Hospital's central hematology laboratory, directed by Dominique Bories. Real-time quantitative PCR assays and Taqman probe technology were used, along with primers specific for selected informative human DNA sequence polymorphism, as described previously (1). This real-time PCR technique has been shown to produce rapid, accurate, and robust quantification of mixed chimerism, with a sensitivity of at least 0.1%. Independent real-time PCR was performed for specific donor and recipient alleles. The sensitivity is close to 0.01%, depending on the marker, and was at least 0.1% for all markers.

Peptides. The synthetic peptides were 15 amino acids in length, overlapped by 10 amino acids, and spanned the pp65 and IE-1 HCMV proteins. They were obtained from the INSERM "Action-Thématique-Concertée" on antiviral immunity. The 110 pp65 peptides were clustered in 11 pools of 10 contiguous peptides each (pp65 pools 1 to 11), and the 96 IE-1 peptides were clustered in 8 pools of 10 contiguous peptides each (IE-1 pools 1 to 8) and in 2 pools of 8 contiguous peptides each (IE-1 pools 9 and 10). Each pool covered a sequence of 59 amino acids, except for IE-1 pools 9 and 10, which covered sequences of 49 and 50 amino acids, respectively.

ELISPOT assays. The enzyme-linked immunospot (ELISPOT) assay was described previously (36). In brief, 96-well plates (Multiscreen Immobilon-P filtration plates; Millipore) were coated with human monoclonal anti-gamma interferon (IFN-) immunoglobulin G1 (Mabtech). After washings and blockage with 10% fetal calf serum, PBMCs were added at a concentration of 10⁵ per well in duplicate or triplicate wells for each experimental condition. Pools of overlapping 15-mer peptides spanning HCMV pp65 and HCMV IE-1 (100 µl at a final concentration of 2 µg/ml) or RPMI 1640⁺ (RPMI 1640 supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, and antibiotics; negative control) or phytohemagglutinin (Gibco BRL) (control antigen) were added to the appropriate wells. The plates were incubated for 18 h at 37°C in a 5% CO₂ atmosphere and then washed. An anti-IFN- biotinylated detection antibody (Mabtech) was added to the wells. After washings, streptavidin-alkaline phosphatase (Amersham Biosciences) and then substrate were added, and the plates were incubated at room temperature. Spot-forming cells (SFCs) were counted with an automated ELISPOT reader (Carl Zeiss MicroImaging). The results were expressed as the mean number of SFCs/10⁶ PBMCs after subtraction of the mean negative control values. All peptide pools that induced an IFN- response of >50 SFCs/10⁶ PBMCs above background (for baseline testing) or triple the background (for postculture testing) were considered putative CD8⁺ T-cell epitopes.

Ex vivo expansion of HCMV-specific memory T cells. PBMCs were cultured for 6 days in the presence of pp65 and IE-1 peptide pools recognized by the T cells of the genotypically HLA-identical sibling recipients. The culture medium consisted of RPMI 1640 supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, antibiotics (RPMI 1640⁺), and 10% human AB serum. The cells were cultured at 1 x 10⁶ PBMCs/well in 1,000 µl medium in 24-well plates at 37°C and 5% CO₂. Peptide pools (final concentration, 2 µg/ml) were added on day zero. Cultures in the presence of medium and irrelevant peptides from pp65 or IE1 served as negative controls. On day 2, interleukin 2 (IL-2) was added at 2 IU/ml. On day 4, the cells were cultured in RPMI 1640⁺ supplemented with 10% human AB serum without peptides or IL-2. On day 6, the cells were restimulated for 18 h in ELISPOT assays as described above, with the pp65 and IE-1 peptide pools used for expansion.

By comparing the results of ELISPOT assays in recipients ex vivo and in related donors ex vivo and after culture, we defined three distinct HCMV-specific T-cell subpopulations in recipients. First, recipient HCMV-specific T cells that recognized peptide pools to which the related-donor T cells had previously responded ex vivo were defined as "expanded T cells." In addition, recipient HCMV-specific T cells that recognized peptide pools to which related-donor T cells had responded only after culture were defined as "quiescent T cells." Finally, recipients' HCMV-specific T cells that recognized peptide pools to which related-donor T cells did not respond either ex vivo or after culture were defined as "newly expanded T cells".

Cell surface and intracellular staining by flow cytometry. The flow cytometry assay was previously described (27). One million PBMCs were incubated for 6 h in the presence of either peptide pools detected as epitopes in the ELISPOT assay at a final concentration of 2 µg/ml or control antigen (phytohemagglutinin; Gibco-BRL) or RPMI 1640⁺ (negative control). Brefeldin A (Sigma-Aldrich) was added to the culture for the final 5 hours. After washings, PBMCs were incubated for 15 min at room temperature with the fluorescence-labeled conjugated monoclonal antibodies anti-CD3-allophycocyanin, anti-CD8-peridinin chlorophyll protein, anti-CD27-fluorescein isothiocyanate, and anti-CD28-phycoerythrin (Becton Dickinson). After washings, the cells were fixed with buffered formaldehyde acetone solution, permeabilized with 0.1% saponin-50 mM D-glucose, incubated, stained with anti-IFN--allophycocyanin (BD Biosciences), washed, and fixed in phosphate-buffered saline-0.5% bovine serum albumin. Flow cytometric analysis was performed immediately with a FACScalibur (Becton Dickinson). The frequency of CD8⁺ IFN-⁺ cells within the lymphocyte gate was assessed with Cellquest software (Becton Dickinson). Data files contained a mean of 75,000 events positive for CD8 fluorescence per lymphocyte gate. The results were considered positive if >0.1% of lymphocytes were IFN- positive after the negative control values were subtracted.

Statistical analysis. The nonparametric Mann-Whitney test and Fisher's exact test were used to compare continuous and dichotomous variables, respectively, between groups. P values of <0.05 were considered to be statistically significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
HCMV status in the study subjects. At M3 and M6 after SCT, we analyzed HCMV-specific CD8⁺ T cells in 48 allogeneic SCT recipients subdivided into four groups according to their HCMV status. An episode of active HCMV (AHCMV⁺) replication without invasive disease was detected at M3 post-SCT in 23 recipients, all immediately treated for 2 weeks by anti-HCMV therapy. Among them, the HCMV-specific T cells of 11 were analyzed the day before anti-HCMV therapy (AHCMV⁺), while for 12 they could be analyzed only 3 months later, when HCMV was undetectable (prior-AHCMV⁺ [pAHCMV⁺]). No HCMV replication was detectable after SCT in the remaining 25 patients, analyzed at M3 (AHCMV^–; n = 12) or M6 (pAHCMV^–; n = 13) (Table 1). Five recipients were analyzed at both M3 and M6, including three who were AHCMV⁺/pAHCMV⁺ and two who were AHCMV^–/pAHCMV^–. In addition, nine pairs of HLA-identical donor/recipient siblings were evaluated at M6, including three who were pAHCMV⁺ (Table 2). Except for CD4 cell counts, which were significantly lower in AHCMV⁺ subjects than in the other groups, no significant differences were observed between study groups, including a myeloablative conditioning regimen, GVHD treatment prophylaxis, and steroid use (Table 1).

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TABLE 2. Characterictics of HLA-identical sibling donor/recipient pairsa

A lack of IE-1-specific CD8⁺ T cells is associated with uncontrolled HCMV replication. We performed ELISPOT analysis of the IFN--producing T cells directed against 15-mer peptides encompassing the HCMV-pp65 and IE-1 proteins in samples from HCMV⁺ donors and recipients. A flow cytometry analysis of intracellular cytokine production performed in eight of these individuals showed that IFN- was produced mainly by CD8⁺ T cells in response to the dominant pp65 and IE-1 peptides, with a mean of 82.4% ± 14.9% IFN-⁺ CD8⁺ T cells (Table 3). At M3 post-SCT, the magnitude of the pp65-specific CD8⁺ T-cell response was significantly greater in both AHCMV⁺ and AHCMV^– recipients than in healthy donors (mean, 1,404 ± 1,187 SFCs/10⁶ PBMCs in AHCMV⁺ recipients [P = 0.016], 1,414 ± 842 in AHCMV^– recipients [P = 0.003], and 524 ± 689 in donors) (Fig. 1A). The numbers of pp65-specific CD8⁺ T cells at M3 did not differ significantly between recipients with HCMV replication (AHCMV⁺) and those without it (AHCMV^–) (P = 0.689). At M6, the numbers of pp65-specific CD8⁺ T cells remained higher in pAHCMV+ and pAHCMV^– recipients than in donors (mean, 1,8242 ± 2,362 [P = 0.003] and 1,501 ± 1,103 [P = 0.025] SFCs/10⁶ PBMCs, respectively), with no difference between the two SCT groups (P = 0.807) (Fig. 1A). Overall, the pp65 responses remained stable over time and did not differ for either group between M3 and M6 (AHCMV⁺ versus pAHCMV⁺, P = 0.975; AHCMV^– versus pAHCMV^–, P = 0.686).

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TABLE 3. Differentiation of distinct subsets of HCMV-specific CD8+ T cells

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FIG. 1. Distinct magnitudes of pp65- and IE-1-specific CD8+ T-cell responses in recipients at M3 and M6 after SCT and in healthy donors. Each dot represents the number of HCMV pp65-specific (A) and IE-1-specific (B) IFN- spots (per 106 PBMCs) detected in the ELISPOT assay per subject in each study group. At M3 after SCT, there were active HCMV replication (AHCMV+; n = 11) and spontaneous control of HCMV replication (AHCMV–; n = 12). At M6 after SCT, all recipients controlled HCMV replication after an episode of uncontrolled HCMV replication (pAHCMV+; n = 12) or no episode (pAHCMV–; n = 13). , donors; , recipients at M3; , recipients at M6. Horizontal lines represent the mean magnitude of response per subject in each study group.

In contrast, when measured at M3, the IE-1-specific CD8⁺ T-cell population was found to be expanded only in AHCMV^– (1,510 ± 1,430 SFCs/10⁶ PBMCs; P = 0.001), but not in AHCMV⁺ (305 ± 494; P = 0.48), recipients compared to donors (228 ± 638). Accordingly, the number of IE-1-specific CD8⁺ T cells was higher in AHCMV^– than in AHCMV⁺ recipients (P = 0.017) and was associated with control of HCMV replication (Fig. 1B). On the other hand, at M6, the IE-1-specific T-cell population had expanded and was greater for both groups of SCT recipients than in donors (pAHCMV⁺, 1,446 ± 1,576 SFCs/10⁶ PBMCs, P = 0.0003; pAHCMV^–, 1,614 ± 2,069 SFCs/10⁶ PBMCs, P = 0.004), while no difference was observed between the two SCT groups (P = 0.849) (Fig. 1B). The number of IE-1-specific CD8⁺ T cells was significantly lower in AHCMV⁺ recipients at M3 than in pAHCMV⁺ recipients at M6 (P = 0.007). In three recipients who had an episode of HCMV replication at M3 and were tested at both M3 and M6, the IE-specific cell population expanded only at M6 (Fig. 2A). In contrast, the IE-1 response expanded early, was visible at M3, and persisted at M6 for the two recipients with spontaneously controlled HCMV after SCT who were tested at M3 and M6 (Fig. 2B).

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FIG. 2. Delayed expansion of IE-1-specific CD8+ T cells. Each bar represents the number of HCMV pp65-specific and IE-1-specific IFN- spots (per 106 PBMCs) detected in the ELISPOT assay for five recipients examined at M3 and M6 after SCT. (A) Three recipients (AHCMV+/pAHCMV+) had experienced an episode of active HCMV replication at M3 and controlled the virus at M6. (B) Two recipients (AHCMV–/pAHCMV–) spontaneously controlled HCMV replication after SCT.

These results show an early and durable expansion of the pp65-specific CD8⁺ T-cell population in SCT recipients compared with healthy donors regardless of HCMV status and thus suggest that the magnitude of the response against pp65 is not related to protection against HCMV. In contrast, the lack of IE-1-specific CD8⁺ T-cell expansion is associated with the lack of control of HCMV replication in the early period after SCT, but expansion of that cell population occurred several weeks after treatment of the acute HCMV episode.

Restoration of protection against HCMV 6 months after SCT involves expansion of new-HCMV-specific CD8⁺ T cells. We then investigated the mechanism of expansion of HCMV-specific CD8⁺ T cells in SCT recipients. The overall (anti-pp65 plus IE-1) magnitude of the HCMV-specific CD8⁺ T-cell response was higher in recipients 6 months after SCT (3,274 ± 2,696 SFCs/10⁶ PBMCs) than in the healthy donors (753 ± 1,008 SFCs/10⁶ PBMCs) (P < 0.0001) (Fig. 3A). To further investigate the origin of the HCMV-specific T-cell expansion at M6, we focused our study on the nine HCMV-positive HLA-identical sibling recipient/donor pairs. In all cases the chimerism analysis determined that the recipients' PBMCs were of donor origin (Table 2). The magnitude of the pp65- plus IE-1-specific CD8⁺ T-cell response was six times greater in recipients (2,955 ± 2,029 SFCs/10⁶ PBMCs) than in their related donors (495 ± 352 SFCs/10⁶ PBMCs) (P < 0.0001) (Fig. 3B). This greater response resulted partly from broader HCMV-specific T-cell responses, recognizing a mean of 6 ± 2.7 HCMV peptide pools (10 peptides per pool) per recipient compared with 2.1 ± 1.1 peptide pools per related donor (P = 0.0003) (data not shown).

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FIG. 3. Expansion of new HCMV-specific CD8+ T cells at M6 after SCT. (A) Numbers of all HCMV (pp65 plus IE-1)-specific IFN- cells detected in the ELISPOT assay per subject (tested in all recipients at M6 after SCT and in healthy donors). , donors; , recipients at M6. Horizontal lines represent the mean magnitude of response per subject in each study group. (B) HCMV (pp65 plus IE-1)-specific IFN- spots (per 106 PBMCs) in nine donors and related recipients. In pair 6, no HCMV-specific response was detectable in the donor. Recipients 3*, 4*, and 9* each had an episode of uncontrolled HCMV replication at M3 after SCT. All nine recipients controlled HCMV replication without any HCMV-specific treatment for 6 months after SCT. *, P < 0.05.

We then investigated the origin of the CD8⁺ T cells that recognized these additional peptide pools in recipients by testing whether the peptide pools recognized by recipients' T cells were recognized in the ELISPOT assay by their related donors' HCMV-specific CD8⁺ T cells after a peptide-specific in vitro expansion. First, all HCMV peptide pools recognized ex vivo by both donors and related-recipient cells gave rise to expansion of the donor CD8⁺ T-cell population after in vitro culture. These cells, which recognized HCMV peptides common to both donors and recipients, accounted for 56% (1,655 ± 1,291 SFCs/10⁶ PBMCs) of all recipients' HCMV-specific CD8⁺ T cells and were designated "expanded" HCMV-specific CD8⁺ T cells. Second, some peptide pools that were recognized ex vivo by recipient cells were not recognized ex vivo by their related donors' cells but were recognized after an in vitro culture. The corresponding cells, designated "quiescent" HCMV-specific CD8⁺ T cells, accounted for only 5% (161 ± 192 SFC/10⁶ PBMCs) of all recipients' HCMV-specific CD8⁺ T cells. Finally, some peptide pools were recognized ex vivo by recipient CD8⁺ T cells but could not be recognized by the related-donor CD8⁺ T cells, either ex vivo or after culture. The corresponding cells, designated "newly expanded" HCMV-specific CD8⁺ T cells, accounted for 39% (1,138 ± 1,135 SFC/10⁶ PBMCs) of all recipient HCMV-specific CD8⁺ T cells (Fig. 4A).

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FIG. 4. Recipients' newly expanded HCMV-specific CD8+ T cells preferentially target IE-1. Shown are the numbers of HCMV-specific IFN--producing cells detected in the ELISPOT assay per recipient from each of the nine pairs (A) that were directed against pp65 (B) and that were directed against IE-1 (C). The bars represent responses that depend on "newly expanded," "quiescent," and "expanded" CD8+ T cells. Recipients R3*, R4*, and R9* had an episode of uncontrolled HCMV replication at M3 after SCT. *, P 0.05.

When analyzing these HCMV-specific CD8⁺ T cells by intracellular cytokine detection, we observed that in recipients both expanded and newly expanded HCMV-specific CD8⁺ T-cell subsets displayed a predominant CD27^±/CD28^– phenotype (Table 3).

Newly recipient-expanded HCMV-specific CD8⁺ T cells preferentially target IE-1. We then compared the relative contributions of the expanded, quiescent, and newly expanded HCMV-specific CD8⁺ T cells to the anti-pp65 and IE-1 responses. In recipients, 74% of the pp65-specific CD8⁺ T cells (mean, 2,151 ± 1,216 SFCs/10⁶ PBMCs) were expanded (1,581 ± 1,271), 4% were quiescent (90 ± 113), and 22% (481 ± 584) were newly expanded (Fig. 4B). In contrast, in the IE-1-specific CD8⁺ T-cell population (mean, 1,035 ± 1,213 SFCs/10⁶ PBMCs), only 10% were expanded (mean, 109 ± 231) and 9% were quiescent (mean, 92 ± 204), while 81% were newly expanded (mean, 834 ± 1,074) (Fig. 4C). Therefore, the anti-pp65 responses were composed of significantly more expanded than newly expanded HCMV-specific CD8⁺ T cells (P = 0.052). In contrast, more newly expanded than expanded HCMV-specific CD8⁺ T cells (P = 0.049) recognized IE-1 in recipients.

Next, we determined the composition of the dominant HCMV-specific response (i.e., the peptide pool-specific CD8⁺ T cells for which the magnitude of response was highest) for each recipient (Fig. 5). In recipients 2, 3, 6, 7, and 9, two of whom (R3 and R9) were pAHCMV⁺, the dominant response was directed against pp65 and was mediated, in all except recipient 6, by expanded T cells. In contrast, in recipients 1, 4, 5, and 8 (one, R4, was pAHCMV⁺), the dominant responses targeted IE-1 and were always mediated by newly expanded T cells. Finally, we observed that in three pairs (pairs 1, 5, and 8) donors had immunodominant pp65-specific CD8⁺ T cells while the immunodominance of their related recipients switched to IE-1-specific CD8⁺ T cells due to expansion of newly expanded IE-1-specific CD8⁺ T cells. No HCMV replication was detectable after SCT in these three recipients.

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FIG. 5. Relative contributions of expanded-donor, quiescent-donor, and new-recipient CD8+ T cells to the HCMV-specific CD8+ T cells in recipients. In each of the nine pairs, the bars represent the number of specific IFN- spots/106 PBMCs detected in the ELISPOT assay against each distinct pp65 and IE-1 peptide pool in donors (black bars; bottom part) and related recipients (top part). The bars represent responses depending on "newly expanded," "quiescent," and "expanded" CD8+ T-cells subsets. Recipients R3*, R4*, and R9* each had an episode of uncontrolled HCMV replication at M3 after SCT.

Therefore, IE-1 triggers immunodominance of newly expanded HCMV-specific CD8⁺ T cells, while expanded CD8⁺ T cells preferentially recognize pp65.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study of HCMV-specific T-cell reconstitution in recipients of unmanipulated allogeneic SCT shows that it is the CD8⁺ T-cell response directed against IE-1, and not against pp65, that correlates with early protection against HCMV after SCT. Moreover, the restoration of durable control of HCMV after SCT is associated with transfer of donors' CD8⁺ T cells that primarily target IE-1 and expand in the recipients' environment.

This study compared restoration of HCMV-specific CD8⁺ T cells in recipients spontaneously controlling HCMV within the first 6 months post-SCT and in those experiencing an early HCMV reactivation at M3 who were rapidly and efficiently treated with anti-HCMV therapy. Although a limitation of our study is its cross-sectional design, which compares HCMV-specific CD8⁺ T cells at M3 and M6, the characteristics of the study groups did not differ, except for a lower CD4⁺ T-cell count in AHCMV⁺ recipients. This difference is consistent with previous reports showing that restoration of HCMV-specific cytotoxic T lymphocytes appears to depend on the level of CD4⁺ T-cell recovery in the early period after both allogeneic SCT and kidney transplantation (9, 15, 21) or after recovery from HCMV retinitis in HIV-infected patients treated with antiretroviral therapy (35). Moreover, the profiles observed in the five recipients examined at both M3 and M6 were similar at M3 to those of the patients observed only at M3 and at M6 to those observed only at M6.

Most of the HCMV-specific T-cell responses detected in our study were CD8⁺ T cells. The flow cytometry analysis focused only on the dominant peptide pool recognized by patients' cells. It has been reported, however, that some CD4⁺ T cells also respond to pp65 and IE-1 peptide stimulation (2, 37), and we cannot rule out the possibility that our approach underestimates the contribution of CD4⁺ T cells to defense against HCMV.

We found, as reported by others (14, 21, 32), that the magnitude of the pp65-specific CD8⁺ T-cell response was significantly higher in recipients than in donors. However, this expansion of pp65-specific CD8⁺ T cells is not associated with control of HCMV replication, while the early expansion of recipients' IE-1-specific CD8⁺ T cells at M3 was.

These findings are consistent with the report of an association between IE-1-specific CD8⁺ T cells and protection against HCMV pneumonitis in recipients of lung or heart transplants (2). Expansion of IE-1-specific CD8⁺ T cells was also delayed in recipients whose HCMV replication was ultimately controlled after a transient preemptive anti-HCMV intervention. At M6, all recipients with controlled HCMV, regardless of previous HCMV replication, had higher levels of HCMV-specific CD8⁺ T cells than did donors. This may reflect some subclinical HCMV reactivation, as suggested previously (15). Indeed, breakthrough subclinical HCMV reactivation in SCT recipients may expose the immune system to a higher HCMV antigenic pressure than in healthy individuals.

A study recently emphasized the inconsistent responses of HCMV-specific T cells to pp65 and IE-1 compared with infected autologous dendritic cells in some patients after solid-organ transplantation (22). The number of T cells responding to HCMV-specific dendritic cells was nonetheless positively correlated with the number of T cells detected by overlapping using a pp65 and IE-1 strategy. This point was of major concern, because it can be very complex to conduct a comprehensive and exhaustive analysis of the HCMV-specific T-cell response in clinical settings: a more pragmatic analysis focusing on the most immunogenic peptide and achieving an acceptable correlation with protection would be particularly valuable.

A recent study of orthotopic liver transplant (OLT) recipients at high risk of HCMV reactivation (donor HCMV⁺/recipient HCMV^–) suggests, however, that the presence or absence of T-cell responses is not predictive of HCMV disease or plasma viremia (20). These unexpected results were noticeably different from those reported for HCMV-seropositive SCT recipients, in whom the protective role for the HCMV-specific T-cell response was first described. However, the restoration of protective immunity after unmanipulated SCT depends not only on the expansion of preexisting memory T cells in recipients, as observed after OLT, but rather mostly on the expansion of naive transferred donor T cells. Restoration of protective immunity after SCT is therefore quite different than restoration after OLT. On the other hand, the ability to secrete IFN- may not be a sufficient indicator of a protective T-cell response, and additional assays of effector function (e.g., IL-2, tumor necrosis factor alpha, and degranulation) or homing abilities may be necessary to better define protective T-cell immunity to HCMV (19).

We showed that the T-cell responses that expanded against both pp65 and IE-1 in recipients at M6 after SCT originated from different populations of donor T cells. While all the HCMV-specific CD8⁺ T cells that expanded in donors were also detectable in recipients at M6 and were primarily directed against pp65, the reverse was not true: 40% of the recipients' total HCMV-specific CD8⁺ T cells expanded in the recipient's environment only and were primarily directed against IE-1. Chimerism studies confirmed that all recipients' peripheral blood lymphocytes derived from the donor cells. Since the rebound of the thymus usually occurs after 6 months (7, 25), we hypothesize that the new HCMV-specific CD8⁺ T cells already expanded in recipients at M6 were derived from naive transferred donor T cells. We suggest that these cells are primed and expanded in the recipient's environment because of the higher HCMV antigenic stimulation occurring in these severely immunosuppressed patients. However, we did not observe any difference in the differentiation profiles of these newly recipient-expanded HCMV-specific CD8⁺ T cells and their donor-expanded counterparts. Finally, we observed that 90% of the HCMV-specific CD8⁺ T cells measured at M6 in one HCMV-positive recipient of an HCMV-negative HLA-identical sibling donor were directed against IE-1 and associated with protection at M6 (data not shown).

This preferential induction of IE-1-specific CD8⁺ T cells in the recipients in the context of occult or detectable HCMV replications and the association we report between IE-1-specific T cells and HCMV control may reflect the kinetics of these proteins' production during the life cycle of the virus. IE proteins are the first to be expressed on HCMV reactivation (28) and continue to be produced in cells that harbor latent HCMV infection (28, 33). As a consequence, cells that express IE products only may not be recognized by pp65-specific T cells, while IE-1-specific T cells can confer protection.

Taken together, our findings suggest that the HCMV antigenic pressure occurring in these severely immunosuppressed patients may favor IE-1 presentation and polarize the newly recruited CD8⁺ T cells toward IE-1 recognition. These results are in accordance with others that show that continuous recruitment of naive T cells contributes to the heterogeneity of antiviral CD8⁺ T cells during persistent infection (38). The differences observed between AHCMV⁺ and AHCMV^– recipients in terms of their CD4 cell counts also suggest that the ability to recruit naive T cells directed against IE-1 may depend on CD4 help. Similarly, we had previously shown that restoration of HCMV-specific CD8⁺ T cells after recovery from HCMV retinitis in HIV-coinfected patients depended upon the restoration of the CD4 count allowed by antiretroviral therapy (35).

In conclusion, our findings emphasize the key role played by CD8⁺ T cells directed against the IE-1 protein in the control of HCMV replication early after SCT. They also demonstrate that new IE-1-specific CD8⁺ T cells expanding only in recipients exposed to HCMV replication contribute to the restoration of immune protection against HCMV. The IE-1 immunodominance of newly recipient-expanded CD8⁺ T cells after unmanipulated SCT provides support for the hypothesis that antigen-driven processes govern the restoration of immune protection against HCMV by allowing continuous recruitment of naive T cells directed against HCMV IE-1.

 
The study was supported by grants from the Programme Hospitalier de Recherche Clinique (PHRC), the Action Thématique Concertée (ATC) INSERM on antiviral immunity, and the Fondation Médicale pour la Recherche (FRM).

We thank D. Olive (Centre Paoli Calmettes, Marseille, France), who coordinates the INSERM-ATC, and his colleagues, who prepared all peptide samples; T. Benissad for assistance in organizing patient samples; and J. A. Cahn for help with manuscript preparation.

We have no conflicting financial interests.

 
* Corresponding author. Mailing address: Laboratoire d'Immunologie Cellulaire et Tissulaire, INSERM U543, Hôpital Pitié-Salpêtrière, 83 Bld de l'Hôpital, 75651 Paris Cedex 13, France. Phone: 33-1-42-17-74-81. Fax: 33-1-42-17-74-90. E-mail: brigitte.autran{at}psl.aphp-paris.fr

Published ahead of print on 6 August 2008.

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Journal of Virology, October 2008, p. 10143-10152, Vol. 82, No. 20
0022-538X/08/$08.00+0     doi:10.1128/JVI.00688-08
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