|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
TRANSPLANTATION
From Medizinische Klinik II, Institut für
Zellbiologie, and Medizinische Virologie,
Eberhard-Karls-Universität Tübingen, Germany.
Reconstitution of human cytomegalovirus (HCMV)-specific cytotoxic
T lymphocytes (CTLs), predominantly directed against pp65, provides
protective immunity for the development of HCMV disease after
allogeneic stem cell transplantation (SCT). To define pp65-derived CTL
epitopes that would allow sensitive detection of HCMV-specific immune
reconstitution, a computer-based epitope prediction was performed.
Peptide-specific CTL responses were assessed by interferon- Human cytomegalovirus (HCMV) infection continues to
be one of the most important infections in patients undergoing
allogeneic stem cell transplantation (SCT) and is associated with high
morbidity and mortality despite the introduction of new antiviral
treatment strategies.1-3 Whereas early-onset HCMV disease
(that occurring during the first 100 days after transplantation) can be
reduced significantly after introduction of pre-emptive or prophylactic antiviral therapy for HCMV infection, HCMV disease occurring after day
100 is now one of the leading causes of death after allogeneic SCT.3,4
Studies have demonstrated reconstitution of HCMV-specific immune
responses after allogeneic SCT to be protective against the development
of HCMV disease.5-8 In these studies, HCMV-specific T-cell
responses were assessed by standard cytotoxicity assays using
HCMV-infected autologous fibroblasts as target cells after repetitive
in vitro stimulations or by lymphoproliferation assays measuring
tritium-thymidine uptake after specific stimulation. These assays are
labor intensive and time intensive and not applicable routinely. Novel
flow cytometry-based techniques, as well as enzyme-linked immunospot
(ELISPOT) assays, allow rapid and sensitive quantification of
peptide-specific and protein-specific T cells.9-12
However, both techniques depend on defined epitopes for the detection
of HCMV-specific cytotoxic T lymphocytes (CTLs). Until now, only a
limited number of HCMV-peptide epitopes were defined by use of cloning
techniques13-15 and, more recently, by epitope prediction based on major histocompatibility complex (MHC) class I
motifs.16,17
In this study, HCMV epitopes were defined by the epitope-prediction
method provided by SYFPEITHI software18-20 and the ELISPOT technique. Reconstitution of HCMV-peptide-specific and
HCMV-protein-specific T-cell responses was associated with resolution
of HCMV infection. None of the patients with documented HCMV-specific
T-cell responses had onset of HCMV disease, indicating reconstitution
of protective immunity.
Patients
Patient characteristics are shown in Table
1. Conditioning therapy consisted of
fractionated total-body irradiation (TBI) (3 × 2 × 2 Gy on 3 successive days), cyclophosphamide (2 × 60 mg/kg body weight
[bw]), and etoposide (40 mg/kg bw). Alternatively, busulfan (16 mg/kg
bw given orally) and cyclophosphamide with cytosine arabinoside
(2 × 2 g/m2 body-surface area on 2 successive days) were
administered. Five patients who had relapse after autografting for
multiple myeloma received a reduced conditioning treatment consisting
of 2 Gy TBI, 2 × 20 mg/kg bw cyclophosphamide, and 5 × 30
mg/m2 fludarabine. One patient received a second graft
after immunosuppression therapy using total nodal irradiation and
thiotepa (2 × 5 mg/kg bw) after rejection of a first
T-cell-depleted allograft. Graft-versus-host disease (GVHD)
prophylaxis consisted of cyclosporin A administered according to serum
levels and antithymocyte globulin given for 3 days at a dose of 20 mg/kg bw 4 to 2 days before SCT in patients receiving a transplant from
a matched sibling donor or for 4 days (day 4 to day 1 before SCT) in
patients receiving a transplant from a matched unrelated donor. Five
patients received a CD34+-selected graft obtained with use
of the CliniMACS system (Miltenyi, Bergisch-Gladbach, Germany). All
patients received polymerase chain reaction (PCR)-based antiviral
therapy as reported previously.2 All patients gave
informed consent to donate blood for analysis of immune reconstitution
after allogeneic SCT.
Peptides The HCMV tegument protein pp65 has been identified as a major target antigen for CD8+ MHC class I-restricted, HCMV-specific CTLs.13,21 Therefore, potential HCMV pp65 peptides with a high probability of binding to HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*1101, HLA-B*0702, HLA-B*0801, and HLA-B*3501 were predicted by computer analysis as described previously.18 Briefly, the pp65 protein was screened against a matrix pattern that evaluates every amino acid within nonamer or decamer peptides fitting to the above-mentioned HLA class I motifs. Anchor residues are valued 10 and other residues 0 to 10, reflecting amino acid preferences for certain positions within the peptide. The theoretical maximum score for a candidate peptide is 36; scores for abundant natural ligands are typically between 32 and 34. Such motif predictions are available by using the database SYFPEITHI (http://www.syfpeithi.de).18The pp65-derived peptides were synthesized by using standard fluorenylmethyl chloroformate chemistry on an automated peptide synthesizer (432A; Applied Biosystems, Weiterstadt, Germany). Synthesis products were analyzed by reversed-phase high-performance liquid chromatography (HPLC; Varian Star; Zinsser, München, Germany) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (G2025A, Hewlett-Packard, Palo Alto, CA). Peptides with a purity of less than 80% were purified by preparative HPLC. For immune-reconstitution studies after allogeneic SCT, additional peptides derived from the pp65 protein (A*2402, AA 367-379, PTFTSQYRIQGKL; B*3502, AA 188-195, FPTKDVAL; and B*44XX, AA 512-521, EFFWDANDIY) and the pp150 protein (A*0301, AA 945-955, TTVYPPSSTAK) were synthesized.15 Cell lines The HLA-A2-expressing cell line T2 (174xCEM.T2 hybridoma, deficient in transporter associated with antigen processing [TAP] 1 and TAP2) was grown in RP10 medium (RPMI 1640 [Biochrome, Berlin, Germany] supplemented with 10% heat-inactivated fetal-calf serum [FCS] and 2% penicillin/streptomycin) and used to provide stimulator cells in the ELISPOT assay.Generation of monocyte-derived dendritic cells (DCs) Generation of DCs from peripheral blood mononuclear cells (PBMNCs) was performed as described previously.22-26 Briefly, PBMNCs were isolated by using Ficoll-Paque (Biochrome) density-gradient centrifugation of heparin-treated blood obtained from buffy-coat preparations of blood from healthy HCMV-seropositive blood donors registered at the local blood bank. All donors expressed the HLA class I alleles A*0101, A*0201, A*0301, A*1101, B*0702, B*0801, or B*3501.PBMNCs were plated (1 × 107 cells/3 mL per well) into
6-well plates (Becton Dickinson, Heidelberg, Germany) in RP10 medium. After 2 hours of incubation at 37°C, nonadherent cells were removed and the adherent blood monocytes (purity > 80%) were cultured in
RP10 medium supplemented with 100 ng/mL human recombinant
granulocyte-macrophage colony-stimulating factor (Leucomax; Novartis,
Basel, Switzerland), 1000 IU/mL interleukin (IL) 4 (R&D Systems,
Wiesbaden, Germany), and 10 ng/mL tumor necrosis factor Fresh medium and cytokines were added to the DC cultures every other day, and cell differentiation was monitored by using light microscopy. Analysis of surface markers as assessed by flow cytometry after 7 days of culture showed high levels of MHC class I and II expression, as well as CD80, CD83, CD86, CD40, and CD54 (data not shown), corresponding to characteristics of mature DCs. Short-term culture for expansion of peptide-specific CTLs in HCMV-seropositive individuals Autologous monocyte-derived mature DCs (5 × 105/mL) were pulsed with a mixture of 3 of the predicted synthetic peptides (10 µg/mL each), irradiated with 30 Gy, and incubated with 3 × 106 autologous PBMNCs in minimal essential medium (Biochrome) supplemented with 10%
heat-inactivated FCS and antibiotics. After 7 days of culture, cells
were restimulated weekly with irradiated autologous peptide-pulsed
PBMNCs, and human recombinant IL-2 (Lymphocult-T-LF; Biotest, Dreieich,
Germany) was added at a concentration of 10 U/mL every other day. After
2 restimulations, cells were analyzed for peptide specificity by
intracellular interferon- (IFN-) staining by using flow cytometry
after specific stimulation with individual peptides.
ELISPOT assay To test the predicted epitopes in a biologic assay, CD8+ T lymphocytes were isolated from HCMV-seropositive healthy donors and analyzed for reactivity to HCMV peptides derived from the pp65 matrix protein in an IFN--ELISPOT assay as described
previously.17 T2 cells were used as antigen-presenting
cells (APCs) for patients expressing HLA-A*0201, and autologous
monocyte-derived mature DCs were used for all other HLA restrictions.
Spot-forming cells were counted by using a KS-ELISPOT microscope
(Zeiss, Jena, Germany).11
Cell-surface and intracellular cytokine staining Intracellular cytokine staining for IFN- production was
performed as described previously.9 White blood cells were
obtained from healthy blood donors and patients after allogeneic SCT
selected on the basis of HCMV serostatus and HLA type. PBMNCs
(5 × 105) were stimulated overnight with peptide
solutions in the presence of brefeldin A (Sigma, Deisenhofen, Germany)
at a final concentration of 10 µg/mL each. To assess the precursor
frequencies of HCMV-specific CD4+ lymphocytes, stimulation
was alternatively performed by using HCMV antigen (1.25 µg/mL). The
next morning, cells were stained with monoclonal antibodies (mAbs)
directed against surface markers and fixed in Dulbecco
phosphate-buffered saline (DPBS) containing 2% formaldehyde (Sigma)
for 15 minutes at room temperature. After centrifugation at
400g for 5 minutes, cells were resuspended in DPBS
containing 0.5% saponin (Roth, Karlsruhe, Germany), 1% bovine serum
albumin (Roth) and anti-IFN- antibody (0.2 µg/well; Becton Dickinson). After intracellular IFN- staining, samples were analyzed on a fluorescence-activated cell-sorter scanner flow cytometer (FACSCalibur; Becton Dickinson) by using CellQuest software (Becton Dickinson).
Isotype control reagents were used to delineate positive and negative
populations. Unstimulated samples were analyzed to verify the effect of
the stimulation. Cell staining was performed by using fluorescein
isothiocyanate-conjugated (FITC), peridinin chlorophyll
protein-conjugated, or phycoerythrin (PE)-conjugated mouse mAbs
against the following surface markers: CD3, CD4, CD8, and human IFN- Cell staining with MHC-peptide tetrameric complexes MHC-peptide tetrameric complexes (kindly provided by R. Handgretinger, St Jude Children's Research Hospital, Memphis, TN) were used to assess the frequency of peptide-specific CD8+ T cells in leukapheresis products from 6 HCMV-seropositive family-member donors. PBMNCs (2-5 × 105) were stained in a 96-well plate with 20 µg/mL PE-labeled HLA-A2 or HLA-A1 tetramers (in 50 µL phosphate-buffered saline [PBS] with 10% FCS) and incubated for 15 minutes at 37°C. Cells were washed twice with 200 µL PBS and 10% FCS at 37°C. After tetramer staining, cells were incubated with anti-CD8 and anti-CD3 or anti-CD4 antibodies (FITC-CyChrome, PharMingen, San Diego, CA) in 200 µL PBS and 10% FCS for 15 minutes at 4°C. Cells were washed twice, resuspended in 200 µL PBS and 10% FCS, and analyzed in a FACSCalibur device.17Lymphoproliferation assay The proliferation assay was performed as described previously.8 HCMV antigen, phytohemagglutinin (Murex; Life Technology, Karlsruhe, Germany), and IL-2 (Biotest) were added at final concentrations of 1:400, 10 ng/mL, and 50 U/mL, respectively. A stimulation index of 3 was considered to indicate a positive lymphoproliferative response.Detection of HCMV infection by PCR and assessment of viral load in the blood All patients in the study were examined weekly by qualitative PCR assays using whole blood, beginning on day 0 and continuing until day 100 after SCT as described previously.2,4 Quantitative analysis of the HCMV DNA load was performed by using the standardized, commercially available COBAS Amplicor HCMV monitor test (Roche Diagnostics, Mannheim, Germany). Nucleic acid was extracted from 0.2 mL EDTA-anticoagulated plasma according to the protocol of the manufacturer. Specific primers amplified a 365-base-pair region of the HCMV DNA polymerase gene. The viral load was assessed by using an external standard and colorimetric amplicon detection. The lower detection limit of the assay was 400 genome equivalents (GE)/mL plasma, and linearity of the assay was achieved at between 400 and 2 × 105 GEs.
Epitope prediction Potential MHC class I-binding peptides from HCMV pp65 were predicted for the MHC class I restrictions HLA-A*0101, HLA-A*0201, HLA-A*0301, HLA-A*1101, HLA-B*0702, HLA-B*0801, and HLA-B*3501. Epitope prediction followed the rules reported previously and can be reproduced by following directions given at http://www.syfpeithi.de.18 Fifty peptides predicted to be the best binders were synthesized. HLA-A*0201-restricted peptides were tested for HLA-A2 binding by using the reconstitution assay (data not shown). However, none of the peptides were excluded from analysis, since the peptide-binding assay is not sensitive enough to detect binding of all natural ligands.Detection of HCMV-peptide-specific CTL responses in healthy HCMV-seropositive blood donors The ELISPOT assay was used to screen HCMV-seropositive healthy blood donors for the presence in blood of CD8+ T cells directed against the predicted HCMV pp65 peptide epitopes. Peptide-pulsed mature DCs were found to be potent stimulator cells, demonstrating a T-cell stimulatory capacity comparable to that of peptide-loaded T2 (data not shown). Thus, to achieve an optimal T-cell stimulation, peptide-loaded autologous mature DCs were used as stimulator cells for detection of non-HLA-A*0201-restricted CTLs.HLA-A*0201-positive donors had a dominant response to the NLVPMVATV
peptide, as reported previously by others.6,13,16 In
HLA-A*1101-positive donors, a strong release of IFN- No HCMV-peptide-specific CTLs were detectable by intracellular
IFN- In 6 of 7 HCMV-seropositive individuals expressing HLA-A*0201 and HLA-B*0702, a dominant HLA-B*0702-restricted CTL response to HCMV was observed (frequency of HLA-B*0702-restricted CTLs of 0.7%-3.3% versus frequency of HLA-A*0201-restricted CTLs of 0-0.1%). The dominance of the HLA-B*0702-restricted CTL response was also confirmed in 3 healthy individuals expressing HLA-B*0702 and HLA-A*0101 (frequency of HLA-B*0702-restricted CTLs of 0.75% to 3.3% versus frequency of HLA-A*0101-restricted CTLs of 0-0.1%). Thus, CTL precursor frequencies specific for a single peptide seem to vary according to the genetic background of individual patients. Frequency of HCMV-peptide-specific CD8+ T cells in leukapheresis products The frequency of HCMV-peptide-specific CD8+ T cells in leukapheresis products was assessed by using MHC-peptide tetrameric complexes in 6 HCMV-seropositive donors. In 5 donors expressing HLA-A*0201, a median of 2.24% (range, 0.19%-14.21%) of all CD8+ T cells showed positive staining with the A2 tetramer. In one donor expressing HLA-A*0101, 0.11% of all CD8+ T cells showed positive staining with the A1 tetramer. Because of the limited number of donors studied, it was not possible to make any correlation between the percentage of HCMV-specific CTLs in the donor and subsequent reconstitution after SCT.Reconstitution of CTLs specific for HCMV pp65 after allogeneic SCT In 19 patients at risk of HCMV infection, blood samples were drawn every month after allogeneic SCT to assess the recovery of HCMV-specific T-cell responses. The following T-cell assays were performed: peptide-specific CTL responses evaluated by ELISPOT or intracellular INF- staining with flow cytometry (n = 19), HCMV-protein-specific CD4+ T-cell responses assessed by
intracellular IFN- staining with flow cytometry (n = 18), and
lymphoproliferative responses after stimulation with HCMV protein
(n = 8).
HCMV-peptide-specific CD8+ T cells were observed in 14 of
19 patients at a median of 90 days after SCT (range, 35 to 234 days) and protein-specific CD4+ T cells in 11 of 18 patients at a
median of 90 days after SCT (range, 35 to > 180 days). Peak
counts of peptide-specific CD8+ T cells ranged from 0.14 to
60.6 cells/µL and peak counts of protein-specific CD4+ T
cells from 0.64 to 18.97 cells/µL, as assessed by intracellular IFN-
Patient 14 (Table 2), who received a transplant from a matched
unrelated donor after dose-reduced conditioning therapy, had a massive
expansion of HLA-A*0101-restricted, peptide-specific CTLs of up to
14.8% of all CD8+ T cells on day 63 and up to 1.4% of
HCMV-reactive CD4+ T cells on day 93 after SCT (Figure
1). This early HCMV-specific T-cell
reconstitution correlated with a short duration of viremia (3 weeks)
and a low peak viral load (1520 GE/mL). Patient 1 had a rapid
reconstitution of HCMV-specific CD8+ and CD4+ T
cells by day 35 after SCT and no detectable HCMV reactivation, even on
PCR assay. In the early posttransplantation period,
HLA-A*0201-restricted, CD8+ peptide-specific T cells
dominated, whereas on day 120 after SCT, similar numbers of
HLA-A*0201-restricted and HLA-B*0702-restricted, CD8+
peptide-specific T cells were documented (Figure
2A). Patient 9, who was given a
transplant of highly purified CD34+-selected peripheral
blood stem cells (2 × 105 CD3+ cells/kg bw)
from a 1-Ag-mismatched family member (donor, HLA-A*0301; recipient,
HLA-A*0201), showed early reconstitution of HLA-B*0702-restricted, peptide-specific T cells, whereas HLA-A*0201-restricted T cells were
detectable only at background levels (Figure 2B).
The number of HCMV-specific CD8+ T cells in the peripheral blood of patients expressing the HLA alleles A*0101, A*0201, or B*0702 was correlated with the viral load in the blood as assessed by quantitative PCR assay in 45 blood samples. In 11 of 13 blood samples with more than 5 peptide-specific CD8+ T cells/µL, no HCMV DNA was detectable, whereas HCMV DNA was detectable in 23 of 32 blood samples with a lower frequency of HCMV-specific CTLs (data not shown). Reconstitution of HCMV-peptide-specific CD8+ T cells in 14 patients was found to be associated with rapid clearance of HCMV infection (r2 = 0.89, P < .0001 on linear
regression analysis with GraphPad Prism, version 2.0 [GraphPad
Software, San Diego, CA]; Figure 3A). A
similar correlation was documented for reconstitution of HCMV-specific
CD4+ T cells and clearance of viral DNA from the blood in
11 patients (r2 = 0.61, P = .0045 on linear
regression analysis; Figure 3B). Thus, the duration of viremia was
shorter in 12 patients who had HCMV-specific T-cell responses on day
100, with only 1 of 12 patients having viremia for more than 4 weeks
compared with 3 of 7 patients lacking HCMV-specific T-cell responses on
day 100 (P = .07 on
HCMV-specific T-cell response in patients more than 150 days after allogeneic SCT Eight patients were assessed for HCMV-specific T-cell responses late after allogeneic SCT. HCMV-peptide-specific CD8+ T cells were detectable in 6 of the 8, and HCMV-protein-specific CD4+ T cells were observed in 3 of 6 patients analyzed. Late-onset HCMV disease developed in 3 of the 8 patients, 2 of whom presented with prolonged viremia (8 weeks) and a high peak viral load (> 5 × 104 GE/mL blood) before day 100 (patients 21 and 23 on Table 3). In the third patient, HCMV-specific T cells were completely eliminated by high-dose corticosteroids and OKT3 (patient 15 on Table 3; Figure 4). HCMV disease was fatal in 2 patients lacking HCMV-specific T-cell responses (patients 15 and 23 on Table 3; Figure 4). The only patient who survived HCMV pneumonia showed recovery of an HCMV-specific CTL response.
Despite major advances in the diagnosis and treatment of HCMV infection, HCMV disease remains a major cause of transplant-related morbidity and mortality after allogeneic SCT. Protective immunity toward HCMV infection is maintained by HCMV-specific CTLs. The viral structural matrix protein pp65 has been identified as a major target antigen for HCMV-specific, MHC class I-restricted CTLs derived from the blood of healthy individuals.14,21 With use of HLA class I-restricted T-cell clones, the HLA-A*0201-restricted peptide NLVPMVATV was defined,13,14 and this epitope was later also characterized by epitope prediction based on MHC class I motifs.16,17 However, only a few HCMV CTL epitopes have been characterized
previously. In an attempt to define pp65-derived CTL epitopes that
allow rapid and sensitive detection of HCMV-specific immune reconstitution after transplantation, a computer-based epitope prediction was performed. By screening HCMV-seropositive healthy individuals for IFN- Because of the high frequency of CTLs specific for HCMV pp65
NLVPMVATV among peripheral blood lymphocytes in HCMV-seropositive and
HLA-A*0201-expressing individuals, this peptide has been described as
an immunodominant pp65 peptide antigen.14,16
Interestingly, we found a dominance of the HLA-B*0702-restricted CTL
response compared with the HLA-A*0101-restricted and the
HLA-A*0201-restricted CTL response in HCMV-seropositive healthy
individuals. In some HCMV-seropositive healthy individuals expressing
these HLA restrictions, even a complete absence of
HLA-A*0201-restricted CTLs was observed. Thus, CTL precursor
frequencies specific for a single peptide seem to vary according to the
genetic background of individual patients. Obviously,
HLA-B*0702-restricted, HCMV-specific CTL responses seem to dominate
the HLA In addition, other HCMV proteins, such as the IE1 protein and pp150, have also been described as potential targets of HCMV-specific CTLs.10,15,27 The tegument protein pp65 was defined as the major target of HCMV-specific CTLs in studies using fibroblasts infected with the laboratory strain AD169.13,15,21 However, this might have reflected a nonphysiologic overexpression of this protein in the specific culture system rather than a clear equal immunodominance in nature. Further characterization of HCMV-peptide epitopes for other HCMV proteins, such as pp150 and IE1, with the approach described here will help to further improve our understanding of protective HCMV immunity. The introduction of novel techniques such as the ELISPOT assay
and intracellular cytokine staining after specific stimulation has
improved epitope mapping as well as the detection of peptide-specific CTLs present at a low frequency in peripheral
blood.9,12,28 In this study, we used intracellular IFN- Ganciclovir prophylaxis is considered an effective strategy for preventing HCMV disease early after transplantation. However, ganciclovir has important toxic side effects, most importantly myelosuppressive and immunosuppressive effects,32 and it has been found to delay the development of HCMV-specific CTLs, thereby leading to late-stage interstitial pneumonia after cessation of drug treatment.29 A large prospective trial could not show a survival benefit in patients receiving ganciclovir prophylaxis, in spite of an impressive reduction in HCMV infection and disease most likely due to a delayed HCMV-specific immune reconstitution.33 Our analysis of HCMV-specific immune reconstitution in a cohort of patients receiving pre-emptive antiviral treatment based on sensitive detection methods shows that short courses of antiviral treatment for documented active HCMV infection allow rapid reconstitution of HCMV-peptide-specific CTLs in most patients, at least in those given a transplant from an HCMV-seropositive donor and even after transplantation of highly purified CD34+ stem cells or stem cells from unrelated donors. Our data indicate that with the introduction of novel methods (such as intracellular cytokine staining or assessment of class I and class II tetrameric HLA-peptide complexes for well-defined peptides) that allow rapid and sensitive assessment of CTL precursor frequencies and a broader characterization of HCMV-peptide epitopes for many HLA restrictions, HCMV-specific immune reconstitution can be quantified easily. The potential use of these techniques for detection of HCMV-specific CTLs depends on the presence of frequent HLA alleles in the general population. If such frequency exists, identifying a limited number of CTL epitopes might allow assessment of HCMV-specific CTL precursor frequencies in most patients.15 In a recent analysis,34 definition of only 5 CTL epitopes from HCMV protein pp65 was considered sufficient to cover more than 97% of the Dutch population. In addition, loading of these peptides on monocyte-derived mature DCs allows in vitro generation of HCMV-specific T cells from a proportion of HCMV-seropositive and HCMV-seronegative donors.17,35 In this study, we demonstrated that epitope prediction by MHC peptide
motifs and an IFN-
We thank M. Pirinen and F. Frank for excellent technical assistance.
Submitted March 22, 2001; accepted January 10, 2002.
Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 510, project B3) and from the Federal Ministry of Education and Research (01KS9602) and the Interdisciplinary Center of Clinical Research Tübingen, project C2.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hermann Einsele, Medizinische Klinik II,
Otfried-Müller-Stra
1. Ljungman P, Engelhard D, Link H, et al. Treatment of interstitial pneumonitis due to cytomegalovirus with ganciclovir and intravenous immune globulin: experience of European Bone Marrow Transplant Group. Clin Infect Dis. 1992;14:831-835[Medline] [Order article via Infotrieve].
2.
Einsele H, Ehninger G, Hebart H, et al.
PCR monitoring after BMT to reduce the incidence of CMV disease and the duration and side effects of antiviral therapy.
Blood.
1995;86:2815-2820
3.
Boeckh M, Bowden RA, Gooley T, Myerson D, Corey L.
Successful modification of a pp65 antigenemia-based early treatment strategy for prevention of cytomegalovirus disease in allogeneic marrow transplant recipients.
Blood.
1999;93:1781-1782 4. Einsele H, Hebart H, Kauffmann-Schneider C, et al. Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection. Bone Marrow Transplant. 2000;25:757-763[CrossRef][Medline] [Order article via Infotrieve].
5.
Ljungman P, Lonnqvist B, Wahren B, Ringden O, Gahrton G.
Lymphocyte responses after cytomegalovirus infection in bone marrow transplant recipients
6.
Reusser P, Riddell SR, Meyers JD, Greenberg PD.
Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease.
Blood.
1991;78:1373-1380
7.
Walter EA, Greenberg PD, Gilbert MJ, et al.
Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.
N Engl J Med.
1995;333:1038-1044 8. Krause H, Hebart H, Jahn G, Müller CA, Einsele H. Screening for CMV-specific T cell proliferation to identify patients at risk of developing late onset CMV disease. Bone Marrow Transplant. 1997;19:1111-1116[CrossRef][Medline] [Order article via Infotrieve]. 9. Kern F, Surel IP, Brock C, et al. T-cell epitope mapping by flow cytometry. Nat Med. 1998;4:975-978[CrossRef][Medline] [Order article via Infotrieve]. 10. Kern F, Faulhaber N, Frommel C, et al. Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol. 2000;30:1676-1682[CrossRef][Medline] [Order article via Infotrieve].
11.
Herr W, Schneider J, Lohse AW, Meyer-zum Büschenfelde KH, Wölfel T.
Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor 12. Waldrop SL, Pitcher CJ, Peterson DM, Maino VC, Picker LJ. Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J Clin Invest. 1997;99:1739-1750[Medline] [Order article via Infotrieve]. 13. Wills MR, Carmichael AJ, Mynard K, et al. The human cytotoxic (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol. 1996;70:7569-7579[Abstract].
14.
Diamond DJ, York J, Sun JY, Wright CL, Forman SJ.
Development of a candidate HLA-A*0201 restricted peptide-based vaccine against human cytomegalovirus infection.
Blood.
1997;90:1751-1767 15. Longmate J, York J, La Rosa C, et al. Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes. Immunogenetics. 2001;52:165-173[CrossRef][Medline] [Order article via Infotrieve].
16.
Solache A, Morgan CL, Dodi AI, et al.
Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.
J Immunol.
1999;163:5512-5518 17. Kleihauer A, Grigoleit U, Hebart H, et al. Ex vivo generation of human cytomegalovirus-specific cytotoxic T cells by peptide-pulsed dendritic cells. Br J Haematol. 2001;113:231-239[CrossRef][Medline] [Order article via Infotrieve]. 18. Rammensee HG, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999;50:213-219[CrossRef][Medline] [Order article via Infotrieve]. 19. Rammensee HG, Falk K, Rötzschke O. Peptides naturally presented by MHC class I molecules. Annu Rev Immunol. 1993;11:213-244[CrossRef][Medline] [Order article via Infotrieve]. 20. Rammensee HG, Bachmann J, Stevanovic S. MHC Ligands and Peptide Motifs. Austin, TX: Landes Bioscience; 1997. 21. Mc Laughlin-Taylor E, Pande H, Forman SJ, et al. Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus specific cytotoxic T lymphocytes. J Med Virol. 1994;43:103-110[Medline] [Order article via Infotrieve].
22.
Porgador A, Gilboa E.
Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are potent inducers of cytotoxic T lymphocytes.
J Exp Med.
1995;182:255-260
23.
Brossart P, Stuhler G, Flad T, et al.
Her-2/neu derived peptides are tumor-associated antigens expressed by human renal cell and colon carcinoma lines and are recognized by in vitro induced specific cytotoxic T lymphocytes.
Cancer Res.
1998;58:732-736
24.
Brossart P, Heinrich KS, Stuhler G, et al.
Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies.
Blood.
1999;93:4309-4317
25.
Sallusto F, Lanzavecchia A.
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor
26.
Zhou LJ, Tedder TF.
CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.
Proc Natl Acad Sci U S A.
1996;93:2588-2592 27. Gyulai Z, Endresz V, Burian K, et al. Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs. J Infect Dis. 2000;181:1537-1546[CrossRef][Medline] [Order article via Infotrieve].
28.
Appay V, Nixon DF, Donahoe SM, et al.
HIV-specific CD8+ T cells produce antiviral cytokines but are impaired in cytolytic function.
J Exp Med.
2000;192:63-75
29.
Li CR, Greenberg PD, Gilbert MJ, Goodrich JM, Riddell SR.
Recovery of HLA-restricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis.
Blood.
1994;83:1971-1979
30.
Cwynarski K, Ainsworth J, Cobbold M, et al.
Direct visualization of cytomegalovirus-specific T-cell reconstitution after allogeneic stem cell transplantation.
Blood.
2001;97:1232-1240
31.
Gratama JW, van Esser JW, Lamers CH, et al.
Tetramer-based quantification of cytomegalovirus (CMV)-specific CD8+ T lymphocytes in T-cell-depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection.
Blood.
2001;98:1358-1364 32. Bowden RA, Digel J, Reed EC, Meyers JD. Immunosuppressive effects of ganciclovir on in vitro lymphocyte responses. J Infect Dis. 1987;156:899-903[Medline] [Order article via Infotrieve].
33.
Goodrich JM, Bowden RA, Fisher L, Keller C, Schoch G, Meyers JD.
Ganciclovir prophylaxis to prevent cytomegalovirus disease after allogeneic marrow transplant.
Ann Intern Med.
1993;118:173-178 34. Schipper RF, van Els CA, D'Amaro J, Oudshoorn M. Minimal phenotype panels. A method for achieving maximum population coverage with a minimum of HLA antigens. Hum Immunol. 1996;51:95-98[CrossRef][Medline] [Order article via Infotrieve].
35.
Szmania S, Galloway A, Bruorton M, et al.
Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA tetramers.
Blood.
2001;98:505-512
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  A. Schub, I. G. Schuster, W. Hammerschmidt, and A. Moosmann CMV-Specific TCR-Transgenic T Cells for Immunotherapy J. Immunol., November 15, 2009; 183(10): 6819 - 6830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Crough and R. Khanna Immunobiology of Human Cytomegalovirus: from Bench to Bedside Clin. Microbiol. Rev., January 1, 2009; 22(1): 76 - 98. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sacre, S. Nguyen, C. Deback, G. Carcelain, J.-P. Vernant, V. Leblond, B. Autran, and N. Dhedin Expansion of Human Cytomegalovirus (HCMV) Immediate-Early 1-Specific CD8+ T Cells and Control of HCMV Replication after Allogeneic Stem Cell Transplantation J. Virol., October 15, 2008; 82(20): 10143 - 10152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Brennan and S. R. Burrows A mechanism for the HLA-A*01-associated risk for EBV+ Hodgkin lymphoma and infectious mononucleosis Blood, September 15, 2008; 112(6): 2589 - 2590. [Full Text] [PDF]
|
|
|
|
 |
|
 C. Solano, I. Benet, M. A. Clari, J. Nieto, R. de la Camara, J. Lopez, J. C. Hernandez-Boluda, M. J. Remigia, I. Jarque, M. L. Calabuig, et al. Enumeration of cytomegalovirus-specific interferon{gamma} CD8+ and CD4+ T cells early after allogeneic stem cell transplantation may identify patients at risk of active cytomegalovirus infection Haematologica, September 1, 2008; 93(9): 1434 - 1436. [Full Text] [PDF]
|
|
|
|
|
|
D. Lilleri, C. Fornara, A. Chiesa, D. Caldera, E. P. Alessandrino, and G. Gerna Human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in adult allogeneic hematopoietic stem cell transplant recipients and immune control of viral infection Haematologica, February 1, 2008; 93(2): 248 - 256. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Asemissen, U. Keilholz, S. Tenzer, M. Muller, S. Walter, S. Stevanovic, H. Schild, A. Letsch, E. Thiel, H.-G. Rammensee, et al. Identification of a Highly Immunogenic HLA-A*01-Binding T Cell Epitope of WT1 Clin. Cancer Res., December 15, 2006; 12(24): 7476 - 7482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Lilleri, G. Gerna, C. Fornara, L. Lozza, R. Maccario, and F. Locatelli Prospective simultaneous quantification of human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in young recipients of allogeneic hematopoietic stem cell transplants Blood, August 15, 2006; 108(4): 1406 - 1412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Elkord, P. E. Williams, H. Kynaston, and A. W. Rowbottom Differential CTLs specific for prostate-specific antigen in healthy donors and patients with prostate cancer Int. Immunol., October 1, 2005; 17(10): 1315 - 1325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Bunde, A. Kirchner, B. Hoffmeister, D. Habedank, R. Hetzer, G. Cherepnev, S. Proesch, P. Reinke, H.-D. Volk, H. Lehmkuhl, et al. Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells J. Exp. Med., April 4, 2005; 201(7): 1031 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Seggewiss, K. Lore, E. Greiner, M. K. Magnusson, D. A. Price, D. C. Douek, C. E. Dunbar, and A. Wiestner Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner Blood, March 15, 2005; 105(6): 2473 - 2479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Li Pira, L. Bottone, F. Ivaldi, R. Pelizzoli, F. Del Galdo, L. Lozzi, L. Bracci, A. Loregian, G. Palu, R. De Palma, et al. Identification of new Th peptides from the cytomegalovirus protein pp65 to design a peptide library for generation of CD4 T cell lines for cellular immunoreconstitution Int. Immunol., May 1, 2004; 16(5): 635 - 642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kondo, Y. Akatsuka, K. Kuzushima, K. Tsujimura, S. Asakura, K. Tajima, Y. Kagami, Y. Kodera, M. Tanimoto, Y. Morishima, et al. Identification of novel CTL epitopes of CMV-pp65 presented by a variety of HLA alleles Blood, January 15, 2004; 103(2): 630 - 638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Roback, M. S. Hossain, L. Lezhava, J. W. Gorechlad, S. A. Alexander, D. L. Jaye, S. Mittelstaedt, S. Talib, J. E. Hearst, C. D. Hillyer, et al. Allogeneic T Cells Treated with Amotosalen Prevent Lethal Cytomegalovirus Disease without Producing Graft-versus-Host Disease Following Bone Marrow Transplantation J. Immunol., December 1, 2003; 171(11): 6023 - 6031. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Meijer, G. J. Boland, and L. F. Verdonck Prevention of Cytomegalovirus Disease in Recipients of Allogeneic Stem Cell Transplants Clin. Microbiol. Rev., October 1, 2003; 16(4): 647 - 657. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chakrabarti, D. W. Milligan, D. Pillay, S. Mackinnon, K. Holder, N. Kaur, D. McDonald, C. D. Fegan, H. Waldmann, G. Hale, et al. Reconstitution of the Epstein-Barr virus-specific cytotoxic T-lymphocyte response following T-cell-depleted myeloablative and nonmyeloablative allogeneic stem cell transplantation Blood, August 1, 2003; 102(3): 839 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Nakai, S. Mineishi, M. Kami, Y. Kanda, R. Tanosaki, Y. Takaue, C. Junghanss, R. Storb, and M. Boeckh Chimerism induction and delayed onset of cytomegalovirus (CMV) infection after allogeneic reduced-intensity stem cell transplantation (RIST) Blood, September 18, 2002; 100(7): 2674 - 2675. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-enzyme-linked
immunospot assay and flow cytometry in healthy individuals and
in patients after allogeneic stem cell transplantation
Top
Abstract
Introduction
2 testing). In 4 of the
14 patients with a documented HCMV peptide-specific CD8+
T-cell response, recurrence of HCMV infection was documented later,
after intensification of immunosuppression for GVHD. In 2 of these 4 patients, HCMV disease developed. Notably, both patients in whom
late-onset HCMV disease developed had an HCMV-peptide-specific CD8+ T-cell response but no protein-specific
CD4+ T-cell response 80 to 100 days after SCT (Table 
A*0101-restricted and HLA-A*0201-restricted CTL
response in most HCMV-seropositive healthy individuals.
e 10, D-72076 Tübingen, Germany;
e-mail: 
a one-year follow-up.
Transplantation.
1985;40:515-520