Cellular & Molecular Immunology
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Article
A Modified Peptide Stimulation Method for Efficient Amplification of Cytomegalovirus (CMV)-Specific CTLs Guangping Ruan1, Li Ma1, 3, Qian Wen1, Wei Luo1, Mingqian Zhou1 and Xiaoning Wang1, 2, 3 CMV-specific immunity is essential for control of human cytomegalovirus (HCMV) infection. Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the ex vivo production of cells for adoptive transfer is labor intensive and expensive. We report here a modified peptide stimulation method using CMV-specific epitope peptides to stimulate PBMCs for generation of CMV-specific CTLs. This method permits efficient amplification of CMV-specific CTLs and provides a large number of cells for FACS analysis from a single blood sample. Significantly, it achieves high frequencies of tetramer staining of CD8+ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. Thus, this approach expands and selects HLArestricted CMV-pp65-reactive T-cell lines of high specificity for potential adoptive immunotherapy. Cellular & Molecular Immunology. 2008;5(3):197-201.
Key Words: cytotoxic T lymphocyte, cytomegalovirus, tetramer assay
Introduction Human cytomegalovirus (HCMV) infections continue to be a significant clinical problem after stem cell transplants (SCTs) (1). Prophylactic or preemptive administration of ganciclovir (GCV) has reduced the incidence of early CMV disease significantly, but prolonged antiviral treatment may result in a delayed immune reconstitution, favoring the onset of CMV disease after day 100 (2-4). CD8+ cytotoxic T lymphocytes (CTLs) play an essential role in immunological protection from clinical cytomegalovirus (CMV) disease. Riddell et al. (5) have shown that protective immunity could be successfully transferred by the infusion of donor-derived CMV-specific CD8+ cytotoxic T-cell clones. However, the approaches used to generate these clones were complex, thus limiting their broad applications. Recently, Einsele et al. (6)
1 Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China; 2 School of Biosciences & Bioengineering, South China University of Technology, Guangzhou 510641, China; 3 Corresponding to: Dr. Li Ma, Tel: +86-20-6164-8322, Fax: +86-20-61648554, E-mail:
[email protected]; or Dr. Xiaoning Wang, Tel: +86-208711- 4254, Fax: +86-20-8754-7089, E-mail:
[email protected]
Received May 5, 2008. Accepted May 29, 2008. Copyright © 2008 by The Chinese Society of Immunology Volume 5
and Peggs et al. (7) have reported the use of donor-derived CMV-specific T cells generated by sensitization with CMV lysates loaded on either donor peripheral blood mononuclear cells (PBMCs) or monocyte-derived cytokine-activated dendritic cells, respectively. Nevertheless, the initial clinical results with each approach have been encouraging. However, concerns have been raised by regulatory agencies regarding the possibility that lysates of CMV-infected cells might contain live viral particles that could be transferred to the host. To address these concerns, we explored the use of CMV-pp65 peptide for sensitization and generation of CMVspecific T cells. In this report, we demonstrated that this method permited the rapid generation of CMV-specific T-cell lines that recognize immunodominant epitopes of CMV-pp65 presented by HLA class I alleles and can be detected by tetramers. We further demonstrated that the number of such cytotoxic T-cell lines was still high in PBMCs until day 30. The high percentage frequencies of CMV-specific CTLs provided the foundation for adoptive antigen specific CTL immunotherapy. In addition, it can be used as positive samples for the quality control of each tetramer batch produced.
Abbreviations: CTL, cytotoxic T lymphocyte; FACS, fluorescence-activated cell sorter; CMV, cytomegalovirus; PBMC, peripheral blood mononuclear cell; MHC, major histocompatibility complex; HLA, human leucocyte antigen, β2m, β2-microglobulin; IPTG, isopropyl-β-D-thiogalactopyranoside; Hc, heavy chain.
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Peptide Stimulation Method for Amplification of Specific CTLs
Materials and Methods
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Synthetic peptides for T-cell analysis and preparation of HLA-A2/peptide tetrameric complexes Since HLA-A2-restricted CTL epitopes of CMV antigens have been well characterized, synthetic peptides of this epitope was used in this study for CTL assays. Peptide NLVPMVATV was used to construct HLA-A2/peptide tetramers because this HLA-A2-restricted epitope could induce CTL activities. Another peptide RPPIFIRRL which was one of the HLA-B* 0702 affinity peptides was used as negative control. The HLA-A2/peptide tetrameric complexes were prepared by the method described previously (8). In brief, the bacterial strains expressing the HLA-A*0201 heavy chain (Hc) and light chain (β2-microglobulin, β2m) were kindly provided by Professor Xiao-Ning Xu from Oxford University (Oxford, UK). The strains were cultured overnight then inoculated into low-salt LB medium at a 1:10 ratio. The cell transformants were incubated at 37°C. At an optical density of OD600 = 0.5, the cells were induced by the addition of 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG). The cells were harvested after 5-h induction. The proteins expressed as inclusion bodies were isolated after cell disruption by sonication. Preliminary refolding of urea solubilized heavy chain and β2m was achieved by adding them to the refolding buffer, according to the urea-solubilized protein concentration, and the final protein concentration was adjusted to 2 mg/ml. Refolding was achieved by the dilution method in which the refolded β2m, refolded HLA-A2 heavy chain and viral peptides were mixed in the refolding buffer. Briefly, in 200 ml of refolding buffer, 3 mg of peptide (dissolved in 100 μl of DMSO) was added with vigorously stirring. After 20 min, 12 mg of β2m was added with vigorous mixing. After another 20 min, 12 mg of Hc was added and the mixture was stirred continuously for 48 h at 4°C. The refolded mixture was filtered through 0.8 μm filter paper, and then concentrated to a volume of less than 5 ml with an Amicon stir cell apparatus (membrane cutoff, 10 kDa). The buffer was then converted to biotinylation buffer using a disposable PD-10 column (Amersham Pharmacia Biotech). The concentrated mixture was biotinylated overnight at room temperature with BirA enzyme in the presence of biotin, ATP, pepstatin, and leupeptin. The refolded molecules were purified by fast protein liquid chromatography on a Superdex 75 column (Amersham Biosciences). The purity of each collected fraction was assessed by SDS-PAGE and immunoblotting. Fractions containing the biotinylated CMV peptide/HLA-A2 monomer complex were pooled and further concentrated through ultrafiltration with Millipore’s Amicon Ultra-15 centrifugal filter devices. Tetrameric molecules were formed by the addition of PE-labeled ExtrAvidin (Sigma) at a 4:1 molar ratio. The final tetrameric complexes were stored at 4°C in the dark until used for fluorescence-activated cell sorter (FACS) analysis. These tetramers were used for staining CMV-specific CD8+ T cells in the modified peptide stimulation method. Data were acquired with a FACSCalibur flow cytometer and analyzed with CellQuest software (both
6.72%
0.54%
19.90%
1.10%
Day 15
1.89%
18.56%
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CD8 FITC Figure 1. Expansion of CMV-specific CTLs by CMV-specific peptide stimulation in vitro. PBMCs (2 × 106) were stimulated with 50 μg/ml NLVPMVATV peptide, 50 μg/ml RPPIFIRRL peptide, or without peptide for 2 h, and then cultured for 11, 15 or 18 days as described in Materials and Methods. The percentage of CMV-specific CTLs (tetramer-PE and CD8-FITC double positive) was examined by flow cytometry.
from Becton Dickinson). Reagents The formulation of the complete medium used in the modified peptide stimulation method (see below) was RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 25 mM HEPES, 2 mM L-glutamine, 1% sodium pyruvate, 0.05 mM 2-mercaptoethanol, and antibiotics (all from GIBCO). Modified peptide stimulation method to amplify CMV-specific CTLs The experimental procedures of the modified peptide stimulation culture method are as follows. PBMCs were isolated with Ficoll gradient centrifugation from sequencespecific PCR-defined HLA-A*0201-positive donor peripheral blood. The cells were washed twice with RPMI 1640 medium. Briefly, 0, 5, 10, 20, 40 μl NLVPMVATV peptide (1 mg/ml) was added to 100, 95, 90, 80, 60 μl PBMCs (2 × 106) and incubated at 37°C for 2 h, and the final concentrations of peptide were 0, 50, 100, 200, 400 μg/ml, respectively. At the same time, parallel studies were performed with the RPPIFIRRL peptide or with no peptide as matched controls. Then 2 ml of RPMI 1640 containing 10% FCS was added to each sample and the samples were cultured in 24-well plates at 37°C in a humidified incubator with 5% CO2. The concentration of IL-2 is gradually increased, which is different from other approaches previously published. On day 1, IL-2 was added to a final concentration of 25 units/ml. On
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Cellular & Molecular Immunology
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donor 3
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Figure 2. The percentage of CMV specific-CTL in three HLA-A*0201 subjects on day 19. PBMCs from 3 HLA-A*0201 donors were stimulated for 2 h with 50 μg/ml NLVPMVATV peptide, 50 μg/ml RPPIFIRRL peptide, 50 μg/ml HLA-A*0201NLVPMVATV monomer or without peptide, respectively, and cultured for 19 days as described in Materials and Methods. The percentages of CMV-specific CTLs were determined by tetramers.
Figure 3. The percentage of CMV specific-CTL in donor 3 at different days. PBMCs from HLA-A*0201 donor 3 were stimulated for 2 h with 50 μg/ml NLVPMVATV peptide, 50 μg/ml RPPIFIRRL peptide, 50 μg/ml HLA-A*0201-NLVPMVATV monomer or without peptide, respectively, and cultured for different days as described in Materials and Methods. The percentages of CMVspecific CTLs were determined by tetramers.
day 3, IL-2 was added to a final concentration of 50 units/ml. On day 5, IL-2 was supplemented to a final concentration of 100 units/ml. On day 8, 800 μl of the old medium was aspirated from each well and replaced with 1 ml of RPMI 1640 plus 10% FCS containing 100 units/ml of IL-2 and cultivation was continued. On days 11, 15 and 18, an aliquot of cells was aspirated to detect CMV-specific CTLs by FACS analysis. After the samples had been aspirated, the contents of the wells were replenished with fresh complete medium containing 100 units/ml IL-2. The longest culture time is 30 days. The experiments were repeated at least three times with similar results.
stimulation by 50 μg/ml NLVPMVATV peptide for 2 h and then cultured for 11 days. After stimulation by 100, 200, 400 μg/ml NLVPMVATV peptides for 2 h then cultured for 11 days, the percentages of CMV-specific CTLs were 2.47%, 1.24% and 1.05%, respectively. The percentage of CMVspecific CTLs was 0.53% without peptide stimulation. When stimulated by different concentrations of unrelated control peptides, the percentages of CMV-specific CTLs were 0.28%, 0.49% and 0.46%, respectively (data not shown). So a conclusion can be drawn that the percentage of CMV-specific CTLs was increased greatly after stimulation by 50 μg/ml CMV-specific peptide. The extent of increase was much larger than those without peptide or with unrelated control peptide (Figure 1). The percentage of CMV-specific CTLs reached 19.90% after stimulation by 50 μg/ml NLVPMVATV peptides for 2 h and then cultured for 15 days. After stimulation by 100, 200, 400 μg/ml NLVPMVATV peptides for 2 h, and then cultured for 15 days, the percentages of CMV-specific CTLs were 5.02%, 2.94% and 3.17%, respectively. When without peptide stimulation, the percentage of CMV-specific CTLs was 0.54%. If unrelated control peptide were added, the percentage of CMV-specific CTLs was 1.10%. So these results suggested that the percentage of CMV-specific CTLs was increased higher after stimulation by 50 μg/ml CMV-specific peptide than those stimulated by peptides at other concentrations. The extent of increase was much larger than that without peptide stimulation or with unrelated control peptide. The flow cytometric analysis indicated that the percentage of CMV-specific CTLs reached 18.56% after stimulation by 50 μg/ml NLVPMVATV peptides for 2 h and then cultured for 18 days. After stimulation by 100, 200, 400 μg/ml NLVPMVATV peptides for 2 h, and then cultured for 18 days, the percentages of CMV-specific CTLs were 5.54%, 3.57% and 4.38%, respectively. While without peptides added, the percentage of CMV-specific CTLs was 1.89%.
Statistical analysis Statistical analysis (two-way analysis of variance (ANOVA)) was carried out using SPSS for Windows Version 10.0 software. Multiple comparisons were used Tukey HSD method and SNK method.
Results The modified peptide stimulation procedure increases tetramer staining frequencies Peptide-specific CD8+ T cells could be visualized by the staining of tetrameric complexes in fresh PBMCs and after peptide stimulation using the culture procedure of the modified peptide stimulation method. The staining frequencies were significantly increased after in vitro stimulation with CMV-specific peptide. Conceivably, the tetramer staining for the epitope-specific CD8+ T cells after in vitro peptide stimulation was more easily detected than those of fresh PMBCs. Detection of CMV-specific CTLs after peptide stimulation for eleven, fifteen, eighteen days The percentage of CMV-specific CTLs reached 6.72% after Volume 5
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When adding unrelated control peptide, the percentage of CMV-specific CTLs was 1.02%. These results indicated that the percentage of CMV-specific CTLs was increased greatly after stimulation by 50 μg/ml CMV-specific peptide. The extent of increase was much higher than those without peptide or with unrelated control peptide (Figure 1). Further verification of modified peptide stimulation procedure Another three HLA-A*0201 subjects were selected. Their PBMCs were divided into four tubes, stimulated for 2 h by 50 μg/ml NLVPMVATV peptide, 50 μg/ml RPPIFIRRL peptide, 50 μg/ml HLA-A*0201-NLVPMVATV monomer or without peptide, respectively. Culture procedure is the same as before. On day 19, the frequency of CMV-specific CTLs was determined by tetramers (Figure 2). Two-way analysis of variance results yielded F = 13.019 (p = 0.005). The result indicated that the frequencies of CMV-specific CTLs for the four stimulation methods were statistically significantly different. The use of CMV-specific pp65 peptide to stimulate PBMCs resulted in the highest percentage among the four stimulation methods. The results of multiple comparison indicated after 50 μg/ml NLVPMVATV peptide stimulation, the percentage of CMV-specific CTLs has significant difference when compared to other three groups, p < 0.05. The statistical results indicated our peptide stimulation procedure was effective. We investigated one sample for 30 days, and the results suggested that CMV-specific CTLs maintain high percentage with the 30 day mark (Figure 3).
Discussion CMV is a human double-stranded DNA 230 kb β-herpesvirus. After primary infection, CMV is not eliminated from the host but remains in a latent state within the precursors of granulocyte, macrophage and dendritic cell. CD8+ cytotoxic T cells play a key role in immunological protection from clinical CMV disease, as delayed reconstitution of CMV-specific CD8+ T cells has been correlated with CMV disease in allogeneic bone marrow transplant recipients. Therefore, the detection of CMV-specific CD8+ T cells has important clinical significance. Before the advent of tetramer assay, the most commonly used quantitative system available to count virus-specific CTLs was limiting dilution analysis (LDA) (9). The standard 51 Cr release CTL assay is, at best, semi-quantitative and measures the property of a population of cells, which may be polyclonal and multi-specific. The LDA assay was subject to variability which probably reflected the frequency of specific cells, their in vitro expansion potential, and the ability to lyse appropriate targets. Compared with the results obtained by the enzyme-linked immunospot (ELIspot) assay of interferon-γ (IFN-γ) for direct ex vivo functional T-cell analysis, the frequencies of circulating virus-specific CD8+ T cells were significantly underestimated by LDA. However, the ELIspot assay might not reflect the true number of virus-specific T cells because such quantification of cells depended on cytokine production. By contrast, the introduction of MHC Volume 5
class I-peptide tetramers that bind to the TCRs of Ag-specific T cells now permits enumeration of specific CD8+ T cells regardless of their effector function and provides additional information on their phenotype if combined with staining for cell surface markers and intracellular cytokines. Therefore, the tetramer assay should be regarded as complementary to other functional assays of T cell activity, such as LDA and the IFN-γ ELIspot assays. In this paper, we have shown the immunological results on 4 HLA-A*0201 donors by using tetramers. Since HLAA2 is the most polymorphic and most common HLA phenotype found in various ethnic populations. About 30% Chinese people is HLA-A2 subtype, and HLA-A*0201 allele frequency is 48.9% of HLA-A2 subtype. So we studied on HLA-A*0201 donors, and our studies were representative. The relatively low number of the epitope-specific CD8+ T cells in the peripheral blood of these donors was increased by the modified peptide stimulation culture method, making the cellular staining with MHC class I peptide-tetrameric complexes more facile. Our study showed that after peptide and IL-2 stimulation the modified peptide stimulation method significantly expanded the pool of viral antigen-specific T cells to an amount sufficient for phenotypic quantification by tetramer-peptide complex staining as well as with various functional assays. In the last 5 to 10 years, results from several phase 1 and small phase 2 trials have provided evidence that adoptive transfer of virus-specific T cells can prevent and effectively treat chemotherapy refractory monoclonal EBV lymphomas and prevent the development of clinically significant CMV infections (5, 6, 10, 11). However, the techniques used to sensitize virus-specific T cells in vitro need culture of the T cells with virus-infected autologous cells, such as CMVvirus-infected skin fibroblasts (10), EBV-transformed B cells (12), or DCs transduced with retroviral or adenoviral vectors to express specific viral proteins or immunogenic peptides (13). Such approaches have proved to be both time- and labor-intensive and difficult to implement or sustain. Alternative sensitization strategies using DCs pulsed with lysates of CMV-infected cells have shown promise, but such lysates are difficult to standardize and have raised regulatory concerns that they might contain and transmit live virus. The current standard approach to generate antigen-specific CTLs involves their isolation and culture in vitro using significant quantities of autologous dendritic cells. These and other related approaches invariably involve expensive, labor intensive, and cumbersome ex vivo production of large numbers of CTLs for potential adoptive transfer, a process susceptible to contamination at every step. The lack of an optimal method to produce antigenspecific CTLs has hindered the further development of adoptive immunotherapy. To address several of these limitations, we have explored the use of CMV-specific epitope peptides for the generation of CMV-specific CTLs. These peptides are synthetic, sterile, and can be manufactured under good manufacturing practice (GMP) conditions. In this study, we prepared the tetramers with the CMV pp65 peptide epitope, which can then be used to detect CMV-specific
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CTLs in the peripheral blood from HLA-A*0201 people. We designed a method that would efficiently generate CMVspecific CTLs, one that needs only simple equipment and reagent, and the concentration of IL-2 is gradually increased which is different from the other approaches previously published (7, 12, 13). IL-2 is necessary for T cell growth. But if the concentration of IL-2 is high, non-specific amplification of T cells will occur. On the other hand, if the concentration of IL-2 is low, T cell will died. So in our method, the concentration of IL-2 is gradually increased. We used the CMV-specific pp65 epitope peptide in combination with IL-2 to induce CMV-specific CTLs of PBL from 1% to 20%, accounting for more than 40% of CD8+ T cells. The cultivation and amplification of CMV-specific CTLs in vitro by modified peptide stimulation method will provide the foundation for adoptive immunotherapy applications.
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Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (30771952, 30771971), the Program for New Century Excellent Talents in University (NCET-07-0410) and the Natural Science Foundation of Guangdong Province (07117783).
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