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Keywords:

  • tetramer;
  • cytomegalovirus;
  • magnetic selection;
  • CD8 T cell;
  • marrow transplant

Abstract

  1. Top of page
  2. Abstract
  3. Methods and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Cytomegalovirus (CMV) reactivation and disease remains an important clinical problem for patients after allogeneic stem cell transplantation. Impaired cellular immune control of viral replication is responsible for viral reactivation, and transfer of CMV-specific T cells from transplant donors can be effective in providing protection. Recent reports have indicated that the frequency of CMV-specific CD8+ T cells in the peripheral blood of healthy donors is surprisingly high. Here we demonstrate that by using a combination of human leucocyte antigen (HLA) Class I-peptide tetramers and magnetic selection it is possible to select CMV-specific T cells from CMV antibody-positive individuals to high purity. Reliable purification of CMV-specific T cells up to 99·8% of CD8+ cells was possible within hours, even when starting with a precursor frequency of < 0·1% of peripheral blood CD8+ T cells. CMV-specific T cells remained functional after the selection process. This novel form of antigen-specific T-cell selection should facilitate the selection of T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. In addition, this technique could potentially be applied to many antigens including against other infective agents and tumour-specific antigens.

Despite the introduction of antiviral agents, cytomegalovirus (CMV) status and reactivation remains an important problem for patients with severe immunosuppression after allogeneic bone marrow or peripheral progenitor cell transplantation (Maltazou et al, 1999; Broers et al, 2000; Osarogiagbon et al, 2000; Craddock et al, 2001). The reconstitution of CD8+ T cells is essential to prevent CMV disease in both animal and human studies (Couriel et al, 1996; Podlech et al, 1998). These findings have led to the adoptive transfer of donor-derived CD8+ T cells into transplant patients with encouraging results (Walter et al, 1995). One report suggests CD8 T -cells may be useful in established CMV disease (Witt et al, 1998). Unfortunately this approach has not been adopted widely as CMV-specific T-cell culture remains a challenging laboratory procedure.

Human leucocyte antigen (HLA)-peptide ‘tetramers’ are fluorescent reagents that allow the direct visualization of antigen-specific T cells (Altman & Moss, 1996). They consist of individual peptide epitopes refolded with HLA class I protein and bind to the CD8+T cells specific for that particular epitope (see Fig 1). They allow the direct quantification of antigen-specific lymphocytes and have been applied widely in human and murine immunology. Recently, tetramers have been used to demonstrate that a frequency of CMV-specific CD8+ T cells in the peripheral blood of healthy immunocompetent donors is high. A mean of around 1% of CD8+ T cells bind to tetramers containing immunodominant peptide epitopes derived from CMV (Gillespie et al, 2000).

image

Figure 1.  T-cell-tetramer-magnetic bead complex.

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We have investigated the possibility of harvesting CMV-specific T cells from healthy CMV antibody-positive individuals for possible use in immunotherapy by direct selection using a combination of Phycoerythrin (PE) conjugated HLA-Class I tetramers and anti-PE magnetic cell sorting. Such an approach should allow the development of antigen-specific donor leucocyte infusion without laboratory culture. As the selected T-cell populations are specific for a single epitope, it is predicted that this approach would produce a highly specific effector response without side-effects such as graft-versus-host disease.

Methods and methods

  1. Top of page
  2. Abstract
  3. Methods and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Subjects Ten HLA-A*0201 CMV antibody-positive donors, three HLA-A*0201 CMV antibody-negative donors and seven HLA-B*0702 CMV-positive donors were used in the study. All donors were healthy laboratory workers.

Peripheral blood mononuclear cell (PBMC) preparation Heparinized blood (20 ml) was taken from healthy CMV antibody-positive laboratory donors. PBMC were isolated using Ficoll–Hypaque density gradient centrifugation and cultured in Roswell Park Memorial Institute (RPMI) medium with 10% fetal calf serum and 100 U/ml penicillin and 100 µg/ml streptomycin.

Major histocompatability complex (MHC) class I tetramer production Soluble MHC-peptide tetramers were produced using standard approaches (Altman & Moss, 1996). Briefly, recombinant HLA-A*0201 and HLA-B*0702 heavy-chain and β2-microglobulin protein were produced by prokaryotic expression in Escherichia coli. Monomeric HLA–peptide complexes were folded in vitro in the presence of appropriate peptide and biotinylated using BirA enzyme before purification using gel filtration and ion exchange chromatography. The peptide epitopes were the HLA-A*0201 restricted NLVPMVATV epitope (amino acids 495–503 of the lower matrix protein, pp65) and the HLA-B*0702 epitope, TPRVTGGGAM (amino acids 417–426 of the lower matrix protein pp65). These peptide epitopes have been shown to be immunodominant in the CD8 T-cell response to CMV (Wills et al, 1996). HLA–peptide tetramers were made by adding biotinylated protein to streptavidin-PE at a ratio of 4:1.

Magnetic activated cell sorting (MACS Magnetic cell sorting has been described previously (Radbruch et al, 1994). Briefly, PBMC were pelleted after washing twice in phosphate-buffered saline (PBS) (containing 0·5% bovine serum albumin plus 2 mmol/l EDTA). An aliquot of cells were used for controls and baseline flow cytometry. The remainder of the cells were suspended in 80 µl of PBS with 2·5 µl of fluorescein isothiocyanate (FITC)-conjugated anti-CD8 monoclonal antibody (Becton Dickinson, Oxford, UK) and 2·5 µl of either HLA-A*0201 or HLA-B*0702 CMV tetramer. Incubation was at room temperature for 30 min. The cells were washed twice in PBS, resuspended in 80 µl of PBS and 20 µl/107 cells of super-paramagnetic microbeads conjugated with monoclonal mouse anti-PE antibodies (Miltenyi Biotec, Birmingham, UK).

Tetramer bound cells were positively selected using MS+ columns and a MiniMACS magnetic separator. The selection process was repeated using the positive cells from the first selection.

Flow cytometry Flow Cytometry was performed on a Coulter EPICS XL (High Wycombe, UK) with Coulter System II v2·13 software. To identify rare populations a target of 5 × 106 events were counted. Post-acquisition listmode data files were analysed using WinMDI v2·8 software.

Enzyme-linked immunospot (ELIspot) assay for interferon-γ production Donor 6 and 13 were selected for functional studies to represent HLA-A*0201 and HLA-B*0702 alleles respectively. Unselected PBMC, negatively selected and positively selected cells were tested for interferon-γ production. The near complete purity of CMV-specific T cells found after two selections prevented the ELIspot from detecting a response as no antigen presenting cells remain in the population. For this reason, cells were selected cells once only prior to ELIspot assay.

The ELIspot assay has been previously described (Czerkinski et al, 1988). Briefly a millipore MAIP N45 plate was coated with 50 µl of monoclonal anti-interferon and left at room temperature for 3 h. Excess antibody was washed off thoroughly and 0·2 × 106 cells were added to each well. The appropriate CMV peptide (10 µl of 20 µg/ml) was added. Phytohaemagglutinin (PHA) was used as a positive control. The plates were incubated at 37°C and 5% CO2 overnight. On the second day, non-adherent cells were washed off. The remaining cells were stained with a biotinylated monoclonal anti-interferon and then with a streptavidin-bound alkaline phosphatase. The plates were developed with the Biorad Alkaline Phosphatase conjugate substrate kit. The wells were read manually by counting the spots in each well under a ×10 microscope. Each spot is produced by a single interferon-γ secreting T cell. All tests were in duplicate and the mean value taken.

Results

  1. Top of page
  2. Abstract
  3. Methods and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Staining of CD8+ T cells with CMV-specific tetramers

CMV-specific T cells were detected in eight out of 10 HLA-A*0201 antibody-positive donors (Table I) and six of the seven HLA-B*0702 antibody-positive donors (Table II). The frequency of CTL ranged from 0·03 to 3% of the CD8+ population on initial staining.

Table I.   Tetramer-positive cells in A2 donors.
 Percentage of CD8Percentage of all gated cells
Pre selectionPost 1st selectionPost 2nd selectionPre selectionPost 2nd selection
HLA-A*0201 CMV + donor
 10·22760·0517
 20·326970·0445
 30·66·9860·253
 40·23·6530·048·5
 50·664990·0294
 61·56199·80·3795
 70·030·1130·011·8
 82·532970·581
Mean0·831·977·60·2149
Median0·62991·40·1349
HLA-A*0201 CMV negative donor
 900000
 1000000
 1100000
Table II.   Tetramer-positive cells in B7 donors.
 Percentage of CD8+Percentage of all gated cells
HLA-B*0702 CMV + donorPre selection1st selection2nd selectionPre selection2nd selection
1346980·8683
20·516960·0865
70·073·7930·0225
12130980·0768
13346990·1485
14112950·1865
Mean1·426970·2365
Median123970·1666

The average number of CD8+ T cells binding the HLA-B*0702 tetramer was greater than those binding the HLA-A*0201 tetramer (mean 1·4% versus 0·6%), which is in line with previous results (Gillespie et al, 2000).

Magnetic selection of tetramer-binding CD8+T cells

After staining of PBMC with tetramer, magnetic beads coated with an anti-PE monoclonal antibody were added to the cells (Miltenyi ®). After incubation the cells were applied to a selection column and underwent either one or two rounds of positive selection on a magnetic column (Tables I and II). FACS analysis of the positively selected fraction showed a significant increase in the percentage of tetramer-binding cells.

Two representative flow cytometry plots are shown in Figs 2 and 3. Figure 2(A and B) demonstrates a rare population of 0·07% of CD8 T cells being enriched to 93% from donor 7. Figure 3(A and B) shows a more typical starting frequency of 1·5% being enriched to 99·8%.

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Figure 2.  Donor 7, B7 CMV-specific T-cell selection.

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image

Figure 3.  Donor 6, A2 CMV-specific T-cell selection.

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Table I shows the data from tetramer selections on HLA-A*0201 donors. Table II shows the data from B7 donors. The median purity of CMV-specific T cells after two column selection in the HLA-A*0201 donors was 91% (range 13–99·8%). In donor 7 the population enriched to 13% enabled the demonstration of an immune response below the limits detectable using standard tetramer staining and flow cytometry. We are confident that this was a genuine population of CMV-specific T cells as no CMV-specific T cells were detected in the three HLA-A*0201 CMV antibody-negative donors. The HLA-B*0702 response was consistently stronger (mean 1·4% of all CD8+) than that of HLA-A*0201 (mean 0·6% of all CD8+), and the final purity achieved from the HLA-B*0702 donors was therefore generally better (mean 96·5%) than from HLA-A*0201 donors (mean 77·6%). The final purity achieved was proportional to the starting population, which indicated the reproducibility of the selection process (Fig 4). It is clear that in order to achieve a very pure population two selection columns are required.

image

Figure 4.  Selection of Tetramer Positive T cells.

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Functional study of tetramer-binding cells

Cells were tested for effector function in an ELIspot assay both before selection and following elution from the magnetic column. The positively selected cells gave a strong positive response confirming interferon-γ production post selection (Fig 5).

image

Figure 5.  ELIspot for Interferon-γ using unselected, negatively and positively selected cells.

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T cells stained with tetramer followed by subsequent addition of anti-PE microbeads may well be subject to considerable cross-linking of membrane receptors. It was considered possible that this might lead to cell activation and this was assessed by performing ELIspots on donor 8 and donor 14. PBMC were incubated with either peptide, specific tetramer or control tetramer of a different class I allele or specific tetramer plus MACS beads. PBMC from the HLA-A*0201-positive donor 8 were stimulated by pp65495−503 peptide, HLA-A*0201:495–503 tetramer and HLA-A*0201:495–503 tetramer plus beads but not by the HLA-B*0702 tetramer. In contrast, PBMC from donor 14 were stimulated by pp65417−426 peptide, HLA-B*0702:417–426 tetramer and HLA-B*0702:417–426 tetramer plus magnetic beads, but not by HLA-A*0201 tetramer (Fig 6).

image

Figure 6.  ELIspot for Interferon-γ using different stimuli.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

CD8 T cell reconstitution is essential to prevent CMV disease after bone marrow and progenitor cell transplantation (Couriel et al, 1996; Podlech et al, 1998). Adoptive transfer studies of anti-CMV CD8 T cells in human transplant recipients has not been generally adopted because of the difficulty in generation of these cells in virus-stimulated culture.

We have demonstrated that functional antigen-specific CD8+ cells can be harvested and purified from the peripheral blood of normal donors using HLA–peptide tetramers combined with magnetic selection. The median purity achieved was > 91% of CD8+ cells for A2 donors and 97% for B7 donors, the two antigenic epitopes used in this study. The final purity was dependent on the frequency of antigen-specific cytotoxic T-lymphocytes (CTL) in the starting population and in order to achieve high purity a second selection step was generally required. However, satisfactory purification of CMV-specific T cells was achieved in all donors in whom tetramer staining cells were demonstrable in PBMC. Positively selected T cells remained functional after selection as demonstrated by their ability to secrete interferon-γ in an ELIspot assay. In addition, we noted that HLA–peptide tetramer alone was also capable of stimulating T cells in an antigen-specific manner. Although multimeric ligands have been shown to be capable of stimulating T-cell signalling (Boniface et al, 1998), we believe this to be the first demonstration of a functional T-cell response to addition of tetramer. However, T-cell activation by free peptide cannot be completely ruled out as peptide may be released from unstable HLA–peptide monomers in the tetramer preparation.

Currently, we are developing this CD8 antigen-specific selection method using the CliniMACS system to harvest sufficient CTL to be used therapeutically. This novel form of CTL selection should prove ideal for isolating antigen-specific T cells for cellular immunotherapy to treat or prevent CMV disease after transplantation. After transfusion into the patient in vivo expansion of T cells is likely to occur although the possible need for CD4+ T cell help cannot be definitely excluded. The purity achieved in the selection system is clearly sufficient to exert a specific anti-CMV response without limited other effects such as graft-versus-host disease. Ultimately, it may be necessary to transfuse multiple T-cell populations against different viral epitopes to reduce mutation of the virus and immune escape. The marrow donor, recipient or possibly even third party donor matched at a single Class I allele, could be used as a source of anti-CMV cells. Potentially, this technique could be applied to many antigens including against other infective agents and tumour specific antigens.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Dr R. Keenan is supported by a Birmingham Heartlands Hospital Haematology Research Fund, The Peel Medical Research Fellowship and The Royal College of Physicians Saltwell-Will Medical Research Fellowship.

References

  1. Top of page
  2. Abstract
  3. Methods and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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