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Adoptive transfer of Ag-specific T cells to prevent CMV disease after allogeneic stem-cell transplantation
Cytotherapy (2002) Vol. 4, No. 1, 3–10
Martin Dunitz
Taylor&Francis healthsciences
Concise review
Adoptive transfer of Ag-specific T cells to prevent CMV disease after allogeneic stem-cell transplantation F van Rhee1 and J Barrett2 1
Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2 Hematology Branch, NIH-NHLBI, Bethesda, MD, USA
Background
Discussion
Cytomegalovirus is a major cause of infectious morbidity and mortality after allogeneic stem-cell transplantation (allo-SCT).
The ability to raise CMV specific T cells on a clinical scale will have a major impact on the management of CMV post-allo-SCT, but
Farmacotherapy to prevent or treat CMV reaction and infection is only partially effective, and has considerable toxicity. Adoptive transfer of ex vivo generated CMV specific T cells is a new approach to the
will have to be compared to current pharmacological approaches. Further, the raising of CMV specific T cells may serve as a model, to generate other antigen specific T cells including other anti-viral
management of CMV post-allo-SCT.
and anti-tumor T cells.
Methods
Keywords
A comprehensive review of the published literature describing 1) the recovery of CMV immunity post-allo-SCT and 2) new strategies for the production of CMV specific T cells for adoptive immunotherapy.
Management of CMV post-allo-SCT, CMV specific T cells, adoptive immunotherapy.
Results
CMV specific T cells can be generated using a variety of systems comprising different antigen presenting cells and antigens.
CMV disease after allogeneic stem-cell transplantation CMV is a human herpes virus latent in 50–100% of the population as a circular plasmid in cells of the myeloid compartment [1,2].The virus is present in CD341 cells, DCs, myeloid cells, and monocytes [3,4]. In the first 3–4 months after an allogeneic stem-cell transplantation (alloSCT) about two-thirds of patients fail to develop a protective CD81 T-lymphocyte response against CMV. These immunocompromised individuals are at risk of CMV reactivation, which can cause fatal pneumonitis, gastroenteritis, retinitis, myelitis, myelosuppression and, occasionally, graft failure [5]. Recipient seropositivity, donor seronegativity, T-cell depleted transplants, unre-
lated donors and haplo-identical donors all increase the risk of CMV disease [6–8]. Until the development of effective antiviral drugs, mortality from CMV reactivation could reach 20%, and CMV is still one of the most common causes of infectious death post-allo-SCT. Ganciclovir and foscarnet are the agents most frequently used to prevent and treat CMV disease. Ganciclovir, in combination with i.v. Ig, has markedly improved the prognosis of established CMV pneumonitis [9,10]. However, antiviral agents are themselves not without risk. Pre-emptive or prophylactic administration of ganciclovir reduces the incidence of CMV disease in randomized studies, but at the cost of neutropenia and an increase in bacterial and fungal infection [11–16].
Correspondence to : Frits van Rhee, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 776, Little Rock, AR 72205, USA. © 2002 ISCT
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Continued treatment with antiviral drugs post-allo-SCT can cause drug resistance, and delay recovery of CMVspecific immunity [12,16–19]. As a consequence, late or persisting CMV disease (.100 days post-allo-SCT) has now become the most frequent manifestation of the virus in centers not using T-cell depletion. The financial impact of treating CMV is also considerable — a single episode of CMV disease can cost $23 000 [20].
Adoptive immunotherapy of CMV disease There is, therefore, a need for a better and cheaper approach to prevent CMV disease in allo-SCT patients. In healthy CMV seropositive individuals, CMV reactivation is prevented by a persisting frequency of 1 in 5000–20 000 circulating CMV-specific cytotoxic T cells (CTL), and a similar number of CMV-specific CD41 T cells [21]. This CTL response is lost immediately after allo-SCT, and restoration of the immune response can take months. Nevertheless, after allo-SCT CMV-specific CTLs can increase greatly — with up to 20% of circulating CD81 T cells being CMV-specific, as identified by peptide-loaded HLA tetramers [22]. Several studies indicate a direct correlation between the ability to mount an effective CMV-specific CTL response post-allo-SCT, fatal pneumonia and outcome [5,17,23]. These findings support the idea of preventing CMV reactivation after transplant, by the adoptive transfer of a protective dose of donor CMV-specific CTLs. In a ‘proof of principle’ study Walter and Riddell showed that CMV-specific CD81 T-cell clones, generated in vitro from the peripheral blood of donors, could indeed restore cellular immunity to CMV and prevent virus reactivation [24]. The approach used to generate T-cell clones was labor-intensive and impractical for widespread routine use. However, this pioneering trial showed that adoptive immunotherapy for CMV disease was feasible, effective and safe.
Strategies for producing T cells for adoptive immunotherapy To be applicable as a routine clinical procedure, as well as financially viable, the ideal protocol for generating CMVspecific T cells should be accomplishable under good manufacturing conditions in days, not weeks, without using live virus or Fetal Calf Serum (FCS). Furthermore, it would be necessary to generate CMV-specific CTLs in CMV-negative, as well as in CMV-positive SCT donors.
Finally, the CTLs should not exhibit allo-reactivity (Table 1). The components of the system to generate T cells for adoptive transfer are illustrated in Figure 1. The key variables that determine the characteristics of the cell product are: n The type of Ag-presenting cells (APCs) n The choice of Ag n The method of CMV-specific CTL induction and expansion to clinical scale n The selection step to eliminate alloreactive T cells and conserve CMV-specific cells.
Choice of APC Cells used as APC include fibroblasts, CD40 ligated B lymphocytes, EBV immortalized B cells, monocytes and DCs. CMV-infected fibroblasts lack HLA-Class II, and the co-stimulatory and accessory molecules required to make them professional APCs, even when treated with c -IFN. Fibroblasts cannot be used to stimulate primary immune responses from CMV seronegative donors, nor to raise CD41 cells. Since CMV-specific CD41 cells are essential to maintain the frequency of CMV-specific CD8, CTL strategies to generate sustained immune responses to CMV would require the use of APCs presenting CMV Ag both through MHC Class I and II, such as B cells, monocytes or DCs [5,17,25,26].
Choice of Ag The dominant CTL response to CMV in allo-SCT recipients is directed against structural viral proteins that are processed and presented by CMV-infected cells, without the necessity for endogenous viral replication or gene expression [17]. Recent studies, both in BMT recipients and in normal CMV seropositive normal individuals, have identified the viral matrix phosphoprotein pp65 as the major target for cytotoxic and helper T-cell responses [17,27–31] Furthermore, reactivity to pp65 protein appears to be largely independent of HLA, which suggests that the protein contains numerous immunogenic epitopes. Fine mapping of epitopes, with vaccinia viruses containing truncated pp65 sequences and pp65 recombinant proteins, has recently been carried out for HLA*0201, HLA*2402, HLA*0101, HLA*0301, HLA*1101, HLA B*0702, HLA B*3801/02, HLA*3801/2, HLA *B3502, HLA *A69801/2, HLA*B44xx, HLA-DR3 and DR11 [32–34]. Since pp65 matrix protein represents one of the most potent and
Adoptive transfer of Ag-specific T cells to prevent CMV disease
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Table 1. Ideal properties of a system to generate Ag-specific T cells for adoptive immunotherapy Desired effect Potent cytotoxicity for target cell — kills virus infected cell, tumor cell Helper function from CD4 cells — provides help for persistence of CTL Unwanted immune effects Non-alloreactive — does not cause GvHD No autoreactivity — does not cause autoimmune disease Practicalities Readily induced in vitro with simple Ags Induction technique not HLA restricted (protein or virus Ag not peptides) Short-term culture (e.g. 10–14 days) amplifies sufficient cells for therapeutic use Function after cryopreservation Detection technique for monitoring expansion and survival after transfer in vivo Simple selection system for Ag-specific cells (e.g. magnetic bead separation)
Antigen APC Peptide
T-cell induction
DC Protein
DNA construct
T-cell expansion IL-2 IL-12 IL-7
Monocyte macrophage
T-cell selection
LCL/B cell Viral lysate
IFN-g Artificial APC
Co-stimulation
TCR v-b
Virus
Tetramers Culture time
Figure 1. Components of a system to generate Ag-specific T cells.
broadly reactive Ags for the cellular immune response to CMV Ags critical for protective CMV immunity, it is, therefore, ideally suited for the generation of CMV-specific T-cell lines. In addition to pp65 peptides, other CMV Ag sources include commercial CMV-infected fibroblast lysates, crude or semi-purified, or recombinant pp65 protein. An
alternative method of generating CD41 and CD81 responses is the retroviral transfer of the pp65 gene into CD341 -derived DC. Retroviruses only integrate into dividing cells, the transduction is therefore performed with CD341 -selected cells stimulated to divide with stem-cell factor and GM-CSF [35]. More recently, Keever et al. used an adenovirus-pp65 vector for episomal
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expression in mature non-dividing, CD141 -derived DC [36]. Interestingly, the T-cell clones generated by this approach had similar HLA-restriction patterns, T-cell receptor usage and specificity. A primary concern of viral transfer is to maintain good manufacturing conditions in the stem-cell processing laboratory.
Method of T-cell expansion, lines or clones? A more practical alternative is to generate ex vivo CMVspecific T-cell lines, instead of clones, from the donor. Virus-specific or protein-specific (as opposed to peptide-specific) T-cell lines have the advantage of containing both CD81 and CD41 T cells. EBV-transformed B lymphocytes (LCLs) have been used as stimulator cells for the raising of virus-specific T-cell lines. EBV-specific T-cell lines have already been raised successfully ex vitro and transfused to BMT patients with EBV-induced lymphoproliferative disorder post-allo-SCT [25]. Adoptive immunotherapy with EBV-specific CTLs has proved highly efficacious, and gene marking has demonstrated the long-term persistence of gene-marked CTLs that have the ability to respond to re-challenge with the virus [26]. LCLs express both HLA Class I and II molecules and a number of accessory molecules, and are highly efficient in stimulating immune responses that greatly facilitates the generation of EBV-specific T-cell lines. In one recent study, LCLs transduced with a retrovirus carrying the pp65 gene were used to modify autologous LCLs and present both CMV and EBV Ags, in order to elicit polyclonal, bispecific CTL responses [37]. However, this approach was not successful for the induction of responses from seronegative donors, which require DCs as professional APCs to initiate the response [38]. Similar approaches for the raising of bispecific T-cell lines have been developed, by infecting adenovirus into LCLs under the aegis of CD40 ligand, and mutated varieties of the human papilloma virus E7 protein, by lentivirus [38,39]. LCLs have a high proliferative rate and are ideally suited for modification with retrovirus.
fibroblast monolayers [40]. Only 5 of 10 cultures produced cultures that killed autologous CMV-infected fibroblasts or DCs at E:T ratios of 20:1. Some cultures were phenotyped, and consisted mostly of CD41 cells. The DCs were not matured with CD40L, which might have caused a relative lack of IL-12 [41,42]. However, preliminary results with these lines revealed that there is a broad T-cell repertoire, indicating that allo-reactive cells might not have been sufficiently deleted. Another disadvantage to this approach is that the lysates contain whole CMV, which can inhibit Ag processing and presentation [43–46]. The cultures contained only 107 cells at the end, which translates to a cell dose of only 1.4 3 105 T-cells/kg, which might not be enough, although the contribution in vivo to CMV specific cellular expansion is unknown. Definite advantages are that this method is not restricted to a certain HLA element; helper and cytotoxic T cells are provided, which should increase the longevity of the T cell; and that multiple Ags can be targeted [47]. More elegantly, DC can be pulsed with pp65 as an alternative to induce CMV-specific T-cell responses. However, pp65 protein is not commercially available, and synthesizing pp65 in the laboratory presents a significant problem due to the presence of a large hydrophobic epitope. In this issue of Cytotherapy, Santiago et al. describe how they overcome the problem of pp65 insolubility, by synthesizing a pp65–intermediate early-1 (IE-1) fusion protein, which is not only soluble but also contains additional antigenic sequences from the IE-1 protein.
Peptide Ag systems to induce CTL Difficulties with whole protein have led to current developments being made using peptides. CMV Ags, such as pp65, are processed into peptides by proteosomes, and subsequently transported to the endoplasmic reticulum for assembly of peptides with HLA Class I molecules, prior to expression at the cell surface. Many studies (including ours) have focused on the HLA-A*0201 restricted peptide pp65495–503, because of its identification as an immunodominant peptide and the high frequency of the HLA-A*0201 phenotype [37,48].
Non-peptide Ag systems to induce CTL CMV-specific CTLs have been generated by pulsing immature, donor monocyte-derived DCs with crude, inactivated, CMV Ag, consisting of lysates from human CMV-infected fibroblasts or human embryonic lung
Selecting Ag-specific T cells Unlike the affinity of an Ab for its Ag, the affinity of the T-cell receptor for a peptide presented by a single HLA molecule is very low. However, tetramers of HLA-Class
Adoptive transfer of Ag-specific T cells to prevent CMV disease
I molecules binding the relevant peptide have a much higher binding efficiency, and can be used to identify and select Ag-specific T cells [49]. Tetramers consist of four HLA Class I molecules, each folded around a restricted peptide ligand, enzymatically labeled with biotin, and bound together by an association with streptavidin. Fluorochrome labeling permits visualization of Ag-specific CTLs by flow cytometry [50]. Tetramer technology has been applied to characterize and isolate CTLs specific for influenza, HIV, simian immune deficiency virus, human T-cell lymphotropic virus and lymphocytic choriomeningitis virus [51–55]. Using tetramer technology, Ag-specific T cells labeled with HLA molecule/peptide complexes can be captured with immunomagnetic beads coated with antiPE Ab, or with immunomagnetic beads coated directly with HLA molecule/peptide complexes [56,57]. It is thus possible to directly isolate CMV-specific T-cells from leukapheresis products, with CMV peptide-HLA Class I tetramers [58]. Assuming a leukapheresis of 1–2 3 108 mononuclear cells/kg and 0.75–1.75% of CD81 cells from a seropositive donor are CMV-specific then, in theory, it should be possible to isolate around 1 3 105 CD81 cells/kg, CMV-specific T cells from a leukapheresis product (assuming some loss during processing). This amount of cells might be sufficient for adoptive immunotherapy purposes, but individual variation and the lack of circulating CMV-specific T cells in CMV seronegative donors would favor the use of an in vitro expansion step. Alternative methods of selection under evaluation are the use of an anti-IFN-c magnetic bead to select CTL stimulated by CMV to produce IFN-c . This method is elegant, however, it will require regulatory approval before clinical application. Since CMV-specific cells use the same Vb subtype in the context of a given HLA molecule, it would also be possible to sort expanded CMV-specific T cells to high purity using Abs to specific Vb families. However, we have found that Vb -selected T cells require time to recover function after selection. It may, in fact, be sufficient to exploit the attrition in culture of T cells not repeatedly stimulated by CMV-Ag, as a form of negative selection of non-CMV-specific T cells. In this issue of Cytotherapy, Einsele et al. describe the elimination of GvHD alloresponses, simply by a 3-week culture. In a similar manner, impure but non-alloreactive T cells expanded for CMV reactivity could be produced
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by first eliminating alloresponding T cells with immunotoxin, before expansion with CMV Ag [60].
A practical approach to the generation of CMV-specific T cells — a feasibility exercise We have established a system for generating sufficient CMV-specific CTLs for adoptive immunotherapy of HLA-A*0201 allo-SCT recipients using 200 mL of donor blood. Monocyte-derived DCs were cultured in media supplemented with autologous plasma (AP-DCs), IL-4, and GM-CSF. The use of FCS is still allowed by the FDA, although it is immunogenic and might be infectious. Several groups have demonstrated that AP-DCs and FCS-DCs from normal donors have similar morphology, immunophenotype, and functional activity. We have determined the optimal time for pulsing DCs cultured in autologous plasma with whole proteins, including the immunogenic CMV-derived pp65 protein. Furthermore, we have made CMV-specific cytotoxic T lymphocytes, using the immunodominant pp65 peptide 495–503. We confirmed the binding of this peptide to HLA-A*0201 and determined the appropriate peptide dose for generating CMV-specific cytotoxic T-cells. Donor monocytes are used to generate DCs in medium with autologous plasma (AP), IL-4, GM-CSF and CD40 ligand. The DCs are pulsed with the immunodominant HLA-A*0201 restricted CMV peptide pp65495–503, and incubated with donor T cells. These cultures were re-stimulated twice with peptide-pulsed lymphoblastoid cell lines (LCL) or CD40-ligated B cells, and purified with PElabeled pp65495–503/HLA-A* 0201 tetramers by flow sorting, or with anti-PE paramagnetic beads. The pure tetramer-positive population was then rapidly expanded to obtain sufficient cells for clinical immunotherapy. The expanded CTLs were . 80% pure, of memory phenotype, with a Tc1 cytokine profile. They efficiently killed CMV-infected fibroblasts, and peptidepulsed targets expressed the integrin VLA-4, suggesting that the CTLs could cross endothelial barriers [61]. This technique is reproducible and could be used for generating CMV-specific CTLs to prevent CMV disease after allogeneic blood and BM transplantation. The HLA tetramer also allows for the monitoring of the fate of the adoptively transferred CTLs, by staining peripheral blood samples with the CMV peptide/HLA tetramers for CMV-specific T cells.
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Towards simpler strategies for adoptive Tcell transfer Besides the immediate goal of protecting patients from CMV disease, the development of adoptive T-cell transfer has two important, but more distant goals. Firstly, the goal of devising simple economical ways to stimulate, expand and select Ag-specific T cells for the clinic could be applied to many viruses that cause post-transplant problems — CMV, VZV, HSZ adenovirus and viruses responsible for hemorrhagic cystitis. Secondly, the approach serves as a model for developing practical techniques for generating tumor-specific T cells for the clinic. Here, the problem remains to identify useful tumorspecific Ags but, in principle, the lessons learned from generating CMV-specific T cells should be widely applicable.
References 1
2
3
4
5
6
7
8
9
Gerberderling JL. Incidence and prevalence of human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and cytomegalovirus among health care personnel at risk for blood exposure: final report from a longitudinal study. J Infect Dis 1994;170:1410–17. Bevan IS, Daw RA, Day PJ et al. Polymerase chain reaction for detection of human cytomegalovirus infection in a blood donor population. Br J Haematol 1991;78:94–9. Bolovan-Fritts C, Mocarski ES, Wiedeman JA. Peripheral blood CD141 cells from healthy subjects carry a circular conformation of latent cytomegalovirus genome. Blood 1999;93:394–8. Hahn G, Jores R, Mocarski ES. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci USA 1998;95:3937–42. Reusser P, Riddell SR, Meyers JD et al. Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease. Blood 1991;78:1373–80. Meyers JD, Flournoy N, Thomas ED. Risk factors after human bone marrow transplantation. J Infect Dis 1986; 153:478–88. Enright H, Haake R, Weisdorf D et al. Cytomegalovirus pneumonia after bone marrow transplantation: risk factors and response to therapy. Transplantation 1993;55:1339–46. Reed EC, Bowden RA, Dandliker PS et al. Treatment of cytomegalovirus pneumonia with ganciclovir and intravenous immunoglobulin in patients with bone marrow transplant. Ann Intern Med 1988;109:783–8. Reusser P, Attenhofer R, Hebart H et al. Cytomegalovirusspecific T-cell immunity in recipients of autologous peripheral blood stem cell or bone marrow transplants. Blood 1997;89:3873–9.
10 Goodrich JM, Mori M, Gleaves CA et al. Early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation. N Engl J Med 1991;325:1601–7. 11 Schmidt GM, Horak DA, Niland JC et al. A randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants [The City of Hope–Stanford–Syntex CMV Study Group]. N Engl J Med 1991;324:1005–11. 12 Winston DJ, Ho WG, Bartoni RN et al. Ganciclovir prophylaxis of cytomegalovirus infection and disease in allogeneic bone marrow transplant recipient. Results of a placebo-controlled, double blind trial. Ann Intern Med 1993;118:179–84. 13 Boeckh M, Gooley TA, Myerson D et al. Cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus ganciclovir at engraftment: a randomized doubleblind study. Blood 1996;88:4063–71. 14 Goodrich JM, Bowden RA, Fisher L et al. Ganciclovir prophylaxis to prevent cytomegalovirus infection after allogeneic marrow transplant. Ann Intern Med 1993;118:173–8. 15 Salzberger B, Bowden RA, Hackman RC et al. Neutropenia in allogeneic marrow transplant recipients receiving ganciclovir for prevention of cytomegalovirus disease: risk factors and outcome. Blood 1997;90:2502–8. 16 Knox KK, Drobyski WR, Carrigan DR. Cytomegalovirus isolate resistant to gancoclovir and foscarnet from a marrow transplant patient. Lancet 1991;337:1292–3. 17 Li C-R, Greenberg PD, Gilbert MJ et al. Recovery of HLArestricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood 1994;83:1971–9. 18 Slavin MA, Bindra RR, Gleaves CA et al. Ganciclovir sensitivity of cytomegalovirus at diagnosis and during treatment of cytomegalovirus pneumonia in marrow transplant recipients. Antimicrob Agents Chemother 1993;37:1360–3. 19 Reusser P, Cordonnier C, Einsele H et al. for the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation (EBMT). European Survey of herpes virus resistance to antiviral drugs in bone marrow transplant recipients. Bone Marrow Transplant 1996;17:813–19. 20 Griffiths RI, Bass EB, Powe NR et al. Factors influencing third party payer cost for allogeneic BMT. Bone Marrow Transplant 1993;12:43–8. 21 Borysiewicz LK, Graham S, Hickling S et al. Human cytomegalovirus-specific T-cytotoxic T cells: their precursor frequency and stage specificity. Eur J Immunol 1988;18:269–75. 22 Cwynarski K, Ainsworth J, Cobbold M et al. Direct visualization of cytomegalovirus-specific-T-cell reconstitution after allogeneic stem cell transplantation. Blood 2001; 97:1232–40.
Adoptive transfer of Ag-specific T cells to prevent CMV disease
23 Quinnan GV, Kirmani NG, Rook AH et al. Cytotoxic T cells in cytomegalovirus infection. N Engl J Med 1982;307:6–13. 24 Walter EA, Greenberg PD, Gilbert MJ et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow transplantation. N Engl J Med 1995;333:1038–44. 25 Rooney C, Colton-Smith C, Loftin S et al. Use of gene modified virus specific T lymphocytes to control Epstein–Barr virus related lymphoproliferation. Lancet 1995;345:9–13. 26 Heslop HE, Ng YC, Li C et al. Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat Med 1996;2:551–5. 27 McLauglin-Taylor E, Pnade H, Forman SJ et al. Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD81 virus-specific cytotoxic T lymphocytes. J Med Virol 1994;43:103–10. 28 Beninga J, Kropff B, Mach M et al. Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response. J Virol 1995;76:153–60. 29 van Zanten J, Harmsen MC, van der Giessen M et al. Proliferative T cell responses to four human cytomegalovirus (HCMV)-specific proteins in healthy subjects and solid organ transplant recipients. J Infect Dis 1995;172:872–9. 30 Boppana SB, Britt WJ. Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology 1996;222:293–6. 31 Wills MR, Carmichael AJ, Mynard K et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65–specific CTL. J Immunol 1996;70:7569–79. 32 Weekes MP, Wills MR, Mynard K, Carmichael AJ. The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J Virol 1999;73:2099–108. 33 Diamond DJ, York J, Sun JY et al. Development of a candidate HLA-A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997;90:1751–67. 34 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;163:5512–18. 35 Reeves ME, Royal RE, Lam JS et al. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res 1996;56:5672–7. 36 Keever-Taylor CA, Margolis D, Sandford G et al. Generation of cytomegalovirus-specific cytolytic T cell lines and clones using pp65 transfected dendritic cells. Blood 1999:94[abstract 667,52a]. 37 Qi S, Pollok KE, Burton RL et al. Simultaneous ex vivo expansion of cytomegalovirus and Epstein–Barr virusspecific cytotoxic T lymphocytes using B-lymphoblastoid
38
39
40
41
42
43
44
45
46
47
48
49
50
51
9
cell lines expressing cytomegalovirus pp65. Blood 1999; 94:3242. Regn S, Raffegerst S, Chen X et al. Ex vivo generation of cytotoxic T lymphocytes specific for one or two distinct viruses for the prophylaxis of patients receiving an allogeneic bone marrow transplant. Bone Marrow Transplant 2001;27:53–64. Spencer HT, Galloway A, Pirisi-Creek L et al. Generating cytotoxic T lymphocytes against cervical cancer with lentivirus-transduced antigen-presenting cells. Third Annual Meeting of the American Society of Gene Therapy. Mol Ther 2000;1:S262 [Abstract 718]. Peggs K, Verfuerth S, Mackinnon S. Induction of cytomegalovirus (CMV)-specific T-cell responses using dendritic cells pulsed with CMV antigen: a novel culture system free of live CMV virions. Blood 2001;97:994–1000. Cella BM, Scheidegger D, Palmer-Lehmann K et al. Ligation of CD40 on dendritic cells triggers production of high level of interleukin-12 and enhances T cell stimulatory capacity: T:T help via APC activation. J Exp Med 1996;184:747–52. Bhardwaj N, Seder RA, Reddy A et al. IL-12 in conjunction with dendritic cells enhances antiviral CD81 CTL responses in vitro. J Clin Invest 1996;98:715–22. Ahn K, Angulo A, Ghazal P et al. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci USA 1996;93:10990–5. Koszinowski UH, Reddehase MJ, Del Val M. Principles of cytomegalovirus antigen presentation in vitro and in vivo. Semin Immunol 1992;4:71–9. Wiertz EJHJ, Jones TR, Sun L et al. The human cytomegalovirus US11 gene product dislocates MHC Class 1 heavy chains from the endoplasmic reticulum to the cytosol. Cell 1996;84:769–79. Hengel H, Flohr T, Hammerling GJ et al. Human cytomegalovirus inhibits peptide translocation into the endoplasmic reticulum for MHC class assembly. J Gen Virol 1996;77:2287–96. Kern F, Surel IP, Faulhaber N et al. Target structures of the CD1 -T-cell response to human cytomegalovirus: the 72kilodalton major immediate-early protein revisited. J Virol 1999;73:8179–84. Ellis JM, Henson V, Slack R et al. Frequencies of HLA-A2 alleles in five US population groups. Hum Immunol 2000;61:334–40. Corr M, Slanetz AE, Boyd LF et al. T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity. Science 1994;265:946–9. Altman JD, Moss PAH, Goulder PJR et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996; 274:94–6. Dunbar PR, Ogg GS, Chen et al. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood. Curr Biol 1998;8:413–16.
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52 Ogg GS, Jin X, Bonhoeffer S et al. Quantitation of HIV1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998;279:2103–6. 53 Kuroday MJ, Schmitz JE, Barouch DH et al. Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected Rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J Exp Med 1998;187:1373–81. 54 Bieganowska K, Hollsberg P, Buckle GJ et al. Direct analysis of viral-specific CD81 T cell with soluble HLA-A2/ Tax11-19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy. J Immunol 1999;162:1765–71. 55 Gallimore A, Glithero A, Godkin A et al. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J Exp Med 1998;187:1383–93. 56 Clave E, Jin SWA, Bradstock K et al. Use of a CMV pp65specific HLA tetramer to select and expand CMV-specific CD81 CTL. Blood 1999;94:155a.
57 Mavroudis DM, Barrett J. Separating GvHD and GvL reactions. In: Barrett AJ and Jiang Y-Z, editors. Graft versus leukemia. New York: Marcel Dekker Publishers, 2000:435–46. 58 Luxembourg AT, Borrow P, Teytom L et al. Biomagnetic isolation of antigen-specific CD81 T cells usable in immunotherapy. Nature Biotechnol 1998;16:281–5. 59 Brosterhus H, Brings S, Leyendeckers H, Manz RA. Enrichment and detection of live antigen-specific CD4(1 ) and CD8(1 ) T cells based on cytokine secretion. Eur J Immunol 1999;29:4053–9. 60 Asemissen AM, Nagorsen D, Keilholz U. Flow cytometric determination of intracellular or secreted IFNc for the quantification of antigen reactive T cells. J Immunol Methods 2001;251:101–8. 61 Keenan R, Ainsworth J, Assenmacher M et al. Magnetic selection of cytomegalovirus-specific CTLs for use in antigen-specific donor leukocyte infusion. Blood 1999;94:320a. 62 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;98:505–12.
Martin Dunitz
Taylor&Francis healthsciences
Concise review
Adoptive transfer of Ag-specific T cells to prevent CMV disease after allogeneic stem-cell transplantation F van Rhee1 and J Barrett2 1
Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR, USA 2 Hematology Branch, NIH-NHLBI, Bethesda, MD, USA
Background
Discussion
Cytomegalovirus is a major cause of infectious morbidity and mortality after allogeneic stem-cell transplantation (allo-SCT).
The ability to raise CMV specific T cells on a clinical scale will have a major impact on the management of CMV post-allo-SCT, but
Farmacotherapy to prevent or treat CMV reaction and infection is only partially effective, and has considerable toxicity. Adoptive transfer of ex vivo generated CMV specific T cells is a new approach to the
will have to be compared to current pharmacological approaches. Further, the raising of CMV specific T cells may serve as a model, to generate other antigen specific T cells including other anti-viral
management of CMV post-allo-SCT.
and anti-tumor T cells.
Methods
Keywords
A comprehensive review of the published literature describing 1) the recovery of CMV immunity post-allo-SCT and 2) new strategies for the production of CMV specific T cells for adoptive immunotherapy.
Management of CMV post-allo-SCT, CMV specific T cells, adoptive immunotherapy.
Results
CMV specific T cells can be generated using a variety of systems comprising different antigen presenting cells and antigens.
CMV disease after allogeneic stem-cell transplantation CMV is a human herpes virus latent in 50–100% of the population as a circular plasmid in cells of the myeloid compartment [1,2].The virus is present in CD341 cells, DCs, myeloid cells, and monocytes [3,4]. In the first 3–4 months after an allogeneic stem-cell transplantation (alloSCT) about two-thirds of patients fail to develop a protective CD81 T-lymphocyte response against CMV. These immunocompromised individuals are at risk of CMV reactivation, which can cause fatal pneumonitis, gastroenteritis, retinitis, myelitis, myelosuppression and, occasionally, graft failure [5]. Recipient seropositivity, donor seronegativity, T-cell depleted transplants, unre-
lated donors and haplo-identical donors all increase the risk of CMV disease [6–8]. Until the development of effective antiviral drugs, mortality from CMV reactivation could reach 20%, and CMV is still one of the most common causes of infectious death post-allo-SCT. Ganciclovir and foscarnet are the agents most frequently used to prevent and treat CMV disease. Ganciclovir, in combination with i.v. Ig, has markedly improved the prognosis of established CMV pneumonitis [9,10]. However, antiviral agents are themselves not without risk. Pre-emptive or prophylactic administration of ganciclovir reduces the incidence of CMV disease in randomized studies, but at the cost of neutropenia and an increase in bacterial and fungal infection [11–16].
Correspondence to : Frits van Rhee, Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 776, Little Rock, AR 72205, USA. © 2002 ISCT
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Continued treatment with antiviral drugs post-allo-SCT can cause drug resistance, and delay recovery of CMVspecific immunity [12,16–19]. As a consequence, late or persisting CMV disease (.100 days post-allo-SCT) has now become the most frequent manifestation of the virus in centers not using T-cell depletion. The financial impact of treating CMV is also considerable — a single episode of CMV disease can cost $23 000 [20].
Adoptive immunotherapy of CMV disease There is, therefore, a need for a better and cheaper approach to prevent CMV disease in allo-SCT patients. In healthy CMV seropositive individuals, CMV reactivation is prevented by a persisting frequency of 1 in 5000–20 000 circulating CMV-specific cytotoxic T cells (CTL), and a similar number of CMV-specific CD41 T cells [21]. This CTL response is lost immediately after allo-SCT, and restoration of the immune response can take months. Nevertheless, after allo-SCT CMV-specific CTLs can increase greatly — with up to 20% of circulating CD81 T cells being CMV-specific, as identified by peptide-loaded HLA tetramers [22]. Several studies indicate a direct correlation between the ability to mount an effective CMV-specific CTL response post-allo-SCT, fatal pneumonia and outcome [5,17,23]. These findings support the idea of preventing CMV reactivation after transplant, by the adoptive transfer of a protective dose of donor CMV-specific CTLs. In a ‘proof of principle’ study Walter and Riddell showed that CMV-specific CD81 T-cell clones, generated in vitro from the peripheral blood of donors, could indeed restore cellular immunity to CMV and prevent virus reactivation [24]. The approach used to generate T-cell clones was labor-intensive and impractical for widespread routine use. However, this pioneering trial showed that adoptive immunotherapy for CMV disease was feasible, effective and safe.
Strategies for producing T cells for adoptive immunotherapy To be applicable as a routine clinical procedure, as well as financially viable, the ideal protocol for generating CMVspecific T cells should be accomplishable under good manufacturing conditions in days, not weeks, without using live virus or Fetal Calf Serum (FCS). Furthermore, it would be necessary to generate CMV-specific CTLs in CMV-negative, as well as in CMV-positive SCT donors.
Finally, the CTLs should not exhibit allo-reactivity (Table 1). The components of the system to generate T cells for adoptive transfer are illustrated in Figure 1. The key variables that determine the characteristics of the cell product are: n The type of Ag-presenting cells (APCs) n The choice of Ag n The method of CMV-specific CTL induction and expansion to clinical scale n The selection step to eliminate alloreactive T cells and conserve CMV-specific cells.
Choice of APC Cells used as APC include fibroblasts, CD40 ligated B lymphocytes, EBV immortalized B cells, monocytes and DCs. CMV-infected fibroblasts lack HLA-Class II, and the co-stimulatory and accessory molecules required to make them professional APCs, even when treated with c -IFN. Fibroblasts cannot be used to stimulate primary immune responses from CMV seronegative donors, nor to raise CD41 cells. Since CMV-specific CD41 cells are essential to maintain the frequency of CMV-specific CD8, CTL strategies to generate sustained immune responses to CMV would require the use of APCs presenting CMV Ag both through MHC Class I and II, such as B cells, monocytes or DCs [5,17,25,26].
Choice of Ag The dominant CTL response to CMV in allo-SCT recipients is directed against structural viral proteins that are processed and presented by CMV-infected cells, without the necessity for endogenous viral replication or gene expression [17]. Recent studies, both in BMT recipients and in normal CMV seropositive normal individuals, have identified the viral matrix phosphoprotein pp65 as the major target for cytotoxic and helper T-cell responses [17,27–31] Furthermore, reactivity to pp65 protein appears to be largely independent of HLA, which suggests that the protein contains numerous immunogenic epitopes. Fine mapping of epitopes, with vaccinia viruses containing truncated pp65 sequences and pp65 recombinant proteins, has recently been carried out for HLA*0201, HLA*2402, HLA*0101, HLA*0301, HLA*1101, HLA B*0702, HLA B*3801/02, HLA*3801/2, HLA *B3502, HLA *A69801/2, HLA*B44xx, HLA-DR3 and DR11 [32–34]. Since pp65 matrix protein represents one of the most potent and
Adoptive transfer of Ag-specific T cells to prevent CMV disease
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Table 1. Ideal properties of a system to generate Ag-specific T cells for adoptive immunotherapy Desired effect Potent cytotoxicity for target cell — kills virus infected cell, tumor cell Helper function from CD4 cells — provides help for persistence of CTL Unwanted immune effects Non-alloreactive — does not cause GvHD No autoreactivity — does not cause autoimmune disease Practicalities Readily induced in vitro with simple Ags Induction technique not HLA restricted (protein or virus Ag not peptides) Short-term culture (e.g. 10–14 days) amplifies sufficient cells for therapeutic use Function after cryopreservation Detection technique for monitoring expansion and survival after transfer in vivo Simple selection system for Ag-specific cells (e.g. magnetic bead separation)
Antigen APC Peptide
T-cell induction
DC Protein
DNA construct
T-cell expansion IL-2 IL-12 IL-7
Monocyte macrophage
T-cell selection
LCL/B cell Viral lysate
IFN-g Artificial APC
Co-stimulation
TCR v-b
Virus
Tetramers Culture time
Figure 1. Components of a system to generate Ag-specific T cells.
broadly reactive Ags for the cellular immune response to CMV Ags critical for protective CMV immunity, it is, therefore, ideally suited for the generation of CMV-specific T-cell lines. In addition to pp65 peptides, other CMV Ag sources include commercial CMV-infected fibroblast lysates, crude or semi-purified, or recombinant pp65 protein. An
alternative method of generating CD41 and CD81 responses is the retroviral transfer of the pp65 gene into CD341 -derived DC. Retroviruses only integrate into dividing cells, the transduction is therefore performed with CD341 -selected cells stimulated to divide with stem-cell factor and GM-CSF [35]. More recently, Keever et al. used an adenovirus-pp65 vector for episomal
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expression in mature non-dividing, CD141 -derived DC [36]. Interestingly, the T-cell clones generated by this approach had similar HLA-restriction patterns, T-cell receptor usage and specificity. A primary concern of viral transfer is to maintain good manufacturing conditions in the stem-cell processing laboratory.
Method of T-cell expansion, lines or clones? A more practical alternative is to generate ex vivo CMVspecific T-cell lines, instead of clones, from the donor. Virus-specific or protein-specific (as opposed to peptide-specific) T-cell lines have the advantage of containing both CD81 and CD41 T cells. EBV-transformed B lymphocytes (LCLs) have been used as stimulator cells for the raising of virus-specific T-cell lines. EBV-specific T-cell lines have already been raised successfully ex vitro and transfused to BMT patients with EBV-induced lymphoproliferative disorder post-allo-SCT [25]. Adoptive immunotherapy with EBV-specific CTLs has proved highly efficacious, and gene marking has demonstrated the long-term persistence of gene-marked CTLs that have the ability to respond to re-challenge with the virus [26]. LCLs express both HLA Class I and II molecules and a number of accessory molecules, and are highly efficient in stimulating immune responses that greatly facilitates the generation of EBV-specific T-cell lines. In one recent study, LCLs transduced with a retrovirus carrying the pp65 gene were used to modify autologous LCLs and present both CMV and EBV Ags, in order to elicit polyclonal, bispecific CTL responses [37]. However, this approach was not successful for the induction of responses from seronegative donors, which require DCs as professional APCs to initiate the response [38]. Similar approaches for the raising of bispecific T-cell lines have been developed, by infecting adenovirus into LCLs under the aegis of CD40 ligand, and mutated varieties of the human papilloma virus E7 protein, by lentivirus [38,39]. LCLs have a high proliferative rate and are ideally suited for modification with retrovirus.
fibroblast monolayers [40]. Only 5 of 10 cultures produced cultures that killed autologous CMV-infected fibroblasts or DCs at E:T ratios of 20:1. Some cultures were phenotyped, and consisted mostly of CD41 cells. The DCs were not matured with CD40L, which might have caused a relative lack of IL-12 [41,42]. However, preliminary results with these lines revealed that there is a broad T-cell repertoire, indicating that allo-reactive cells might not have been sufficiently deleted. Another disadvantage to this approach is that the lysates contain whole CMV, which can inhibit Ag processing and presentation [43–46]. The cultures contained only 107 cells at the end, which translates to a cell dose of only 1.4 3 105 T-cells/kg, which might not be enough, although the contribution in vivo to CMV specific cellular expansion is unknown. Definite advantages are that this method is not restricted to a certain HLA element; helper and cytotoxic T cells are provided, which should increase the longevity of the T cell; and that multiple Ags can be targeted [47]. More elegantly, DC can be pulsed with pp65 as an alternative to induce CMV-specific T-cell responses. However, pp65 protein is not commercially available, and synthesizing pp65 in the laboratory presents a significant problem due to the presence of a large hydrophobic epitope. In this issue of Cytotherapy, Santiago et al. describe how they overcome the problem of pp65 insolubility, by synthesizing a pp65–intermediate early-1 (IE-1) fusion protein, which is not only soluble but also contains additional antigenic sequences from the IE-1 protein.
Peptide Ag systems to induce CTL Difficulties with whole protein have led to current developments being made using peptides. CMV Ags, such as pp65, are processed into peptides by proteosomes, and subsequently transported to the endoplasmic reticulum for assembly of peptides with HLA Class I molecules, prior to expression at the cell surface. Many studies (including ours) have focused on the HLA-A*0201 restricted peptide pp65495–503, because of its identification as an immunodominant peptide and the high frequency of the HLA-A*0201 phenotype [37,48].
Non-peptide Ag systems to induce CTL CMV-specific CTLs have been generated by pulsing immature, donor monocyte-derived DCs with crude, inactivated, CMV Ag, consisting of lysates from human CMV-infected fibroblasts or human embryonic lung
Selecting Ag-specific T cells Unlike the affinity of an Ab for its Ag, the affinity of the T-cell receptor for a peptide presented by a single HLA molecule is very low. However, tetramers of HLA-Class
Adoptive transfer of Ag-specific T cells to prevent CMV disease
I molecules binding the relevant peptide have a much higher binding efficiency, and can be used to identify and select Ag-specific T cells [49]. Tetramers consist of four HLA Class I molecules, each folded around a restricted peptide ligand, enzymatically labeled with biotin, and bound together by an association with streptavidin. Fluorochrome labeling permits visualization of Ag-specific CTLs by flow cytometry [50]. Tetramer technology has been applied to characterize and isolate CTLs specific for influenza, HIV, simian immune deficiency virus, human T-cell lymphotropic virus and lymphocytic choriomeningitis virus [51–55]. Using tetramer technology, Ag-specific T cells labeled with HLA molecule/peptide complexes can be captured with immunomagnetic beads coated with antiPE Ab, or with immunomagnetic beads coated directly with HLA molecule/peptide complexes [56,57]. It is thus possible to directly isolate CMV-specific T-cells from leukapheresis products, with CMV peptide-HLA Class I tetramers [58]. Assuming a leukapheresis of 1–2 3 108 mononuclear cells/kg and 0.75–1.75% of CD81 cells from a seropositive donor are CMV-specific then, in theory, it should be possible to isolate around 1 3 105 CD81 cells/kg, CMV-specific T cells from a leukapheresis product (assuming some loss during processing). This amount of cells might be sufficient for adoptive immunotherapy purposes, but individual variation and the lack of circulating CMV-specific T cells in CMV seronegative donors would favor the use of an in vitro expansion step. Alternative methods of selection under evaluation are the use of an anti-IFN-c magnetic bead to select CTL stimulated by CMV to produce IFN-c . This method is elegant, however, it will require regulatory approval before clinical application. Since CMV-specific cells use the same Vb subtype in the context of a given HLA molecule, it would also be possible to sort expanded CMV-specific T cells to high purity using Abs to specific Vb families. However, we have found that Vb -selected T cells require time to recover function after selection. It may, in fact, be sufficient to exploit the attrition in culture of T cells not repeatedly stimulated by CMV-Ag, as a form of negative selection of non-CMV-specific T cells. In this issue of Cytotherapy, Einsele et al. describe the elimination of GvHD alloresponses, simply by a 3-week culture. In a similar manner, impure but non-alloreactive T cells expanded for CMV reactivity could be produced
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by first eliminating alloresponding T cells with immunotoxin, before expansion with CMV Ag [60].
A practical approach to the generation of CMV-specific T cells — a feasibility exercise We have established a system for generating sufficient CMV-specific CTLs for adoptive immunotherapy of HLA-A*0201 allo-SCT recipients using 200 mL of donor blood. Monocyte-derived DCs were cultured in media supplemented with autologous plasma (AP-DCs), IL-4, and GM-CSF. The use of FCS is still allowed by the FDA, although it is immunogenic and might be infectious. Several groups have demonstrated that AP-DCs and FCS-DCs from normal donors have similar morphology, immunophenotype, and functional activity. We have determined the optimal time for pulsing DCs cultured in autologous plasma with whole proteins, including the immunogenic CMV-derived pp65 protein. Furthermore, we have made CMV-specific cytotoxic T lymphocytes, using the immunodominant pp65 peptide 495–503. We confirmed the binding of this peptide to HLA-A*0201 and determined the appropriate peptide dose for generating CMV-specific cytotoxic T-cells. Donor monocytes are used to generate DCs in medium with autologous plasma (AP), IL-4, GM-CSF and CD40 ligand. The DCs are pulsed with the immunodominant HLA-A*0201 restricted CMV peptide pp65495–503, and incubated with donor T cells. These cultures were re-stimulated twice with peptide-pulsed lymphoblastoid cell lines (LCL) or CD40-ligated B cells, and purified with PElabeled pp65495–503/HLA-A* 0201 tetramers by flow sorting, or with anti-PE paramagnetic beads. The pure tetramer-positive population was then rapidly expanded to obtain sufficient cells for clinical immunotherapy. The expanded CTLs were . 80% pure, of memory phenotype, with a Tc1 cytokine profile. They efficiently killed CMV-infected fibroblasts, and peptidepulsed targets expressed the integrin VLA-4, suggesting that the CTLs could cross endothelial barriers [61]. This technique is reproducible and could be used for generating CMV-specific CTLs to prevent CMV disease after allogeneic blood and BM transplantation. The HLA tetramer also allows for the monitoring of the fate of the adoptively transferred CTLs, by staining peripheral blood samples with the CMV peptide/HLA tetramers for CMV-specific T cells.
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Towards simpler strategies for adoptive Tcell transfer Besides the immediate goal of protecting patients from CMV disease, the development of adoptive T-cell transfer has two important, but more distant goals. Firstly, the goal of devising simple economical ways to stimulate, expand and select Ag-specific T cells for the clinic could be applied to many viruses that cause post-transplant problems — CMV, VZV, HSZ adenovirus and viruses responsible for hemorrhagic cystitis. Secondly, the approach serves as a model for developing practical techniques for generating tumor-specific T cells for the clinic. Here, the problem remains to identify useful tumorspecific Ags but, in principle, the lessons learned from generating CMV-specific T cells should be widely applicable.
References 1
2
3
4
5
6
7
8
9
Gerberderling JL. Incidence and prevalence of human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and cytomegalovirus among health care personnel at risk for blood exposure: final report from a longitudinal study. J Infect Dis 1994;170:1410–17. Bevan IS, Daw RA, Day PJ et al. Polymerase chain reaction for detection of human cytomegalovirus infection in a blood donor population. Br J Haematol 1991;78:94–9. Bolovan-Fritts C, Mocarski ES, Wiedeman JA. Peripheral blood CD141 cells from healthy subjects carry a circular conformation of latent cytomegalovirus genome. Blood 1999;93:394–8. Hahn G, Jores R, Mocarski ES. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci USA 1998;95:3937–42. Reusser P, Riddell SR, Meyers JD et al. Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease. Blood 1991;78:1373–80. Meyers JD, Flournoy N, Thomas ED. Risk factors after human bone marrow transplantation. J Infect Dis 1986; 153:478–88. Enright H, Haake R, Weisdorf D et al. Cytomegalovirus pneumonia after bone marrow transplantation: risk factors and response to therapy. Transplantation 1993;55:1339–46. Reed EC, Bowden RA, Dandliker PS et al. Treatment of cytomegalovirus pneumonia with ganciclovir and intravenous immunoglobulin in patients with bone marrow transplant. Ann Intern Med 1988;109:783–8. Reusser P, Attenhofer R, Hebart H et al. Cytomegalovirusspecific T-cell immunity in recipients of autologous peripheral blood stem cell or bone marrow transplants. Blood 1997;89:3873–9.
10 Goodrich JM, Mori M, Gleaves CA et al. Early treatment with ganciclovir to prevent cytomegalovirus disease after allogeneic bone marrow transplantation. N Engl J Med 1991;325:1601–7. 11 Schmidt GM, Horak DA, Niland JC et al. A randomized, controlled trial of prophylactic ganciclovir for cytomegalovirus pulmonary infection in recipients of allogeneic bone marrow transplants [The City of Hope–Stanford–Syntex CMV Study Group]. N Engl J Med 1991;324:1005–11. 12 Winston DJ, Ho WG, Bartoni RN et al. Ganciclovir prophylaxis of cytomegalovirus infection and disease in allogeneic bone marrow transplant recipient. Results of a placebo-controlled, double blind trial. Ann Intern Med 1993;118:179–84. 13 Boeckh M, Gooley TA, Myerson D et al. Cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus ganciclovir at engraftment: a randomized doubleblind study. Blood 1996;88:4063–71. 14 Goodrich JM, Bowden RA, Fisher L et al. Ganciclovir prophylaxis to prevent cytomegalovirus infection after allogeneic marrow transplant. Ann Intern Med 1993;118:173–8. 15 Salzberger B, Bowden RA, Hackman RC et al. Neutropenia in allogeneic marrow transplant recipients receiving ganciclovir for prevention of cytomegalovirus disease: risk factors and outcome. Blood 1997;90:2502–8. 16 Knox KK, Drobyski WR, Carrigan DR. Cytomegalovirus isolate resistant to gancoclovir and foscarnet from a marrow transplant patient. Lancet 1991;337:1292–3. 17 Li C-R, Greenberg PD, Gilbert MJ et al. Recovery of HLArestricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood 1994;83:1971–9. 18 Slavin MA, Bindra RR, Gleaves CA et al. Ganciclovir sensitivity of cytomegalovirus at diagnosis and during treatment of cytomegalovirus pneumonia in marrow transplant recipients. Antimicrob Agents Chemother 1993;37:1360–3. 19 Reusser P, Cordonnier C, Einsele H et al. for the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation (EBMT). European Survey of herpes virus resistance to antiviral drugs in bone marrow transplant recipients. Bone Marrow Transplant 1996;17:813–19. 20 Griffiths RI, Bass EB, Powe NR et al. Factors influencing third party payer cost for allogeneic BMT. Bone Marrow Transplant 1993;12:43–8. 21 Borysiewicz LK, Graham S, Hickling S et al. Human cytomegalovirus-specific T-cytotoxic T cells: their precursor frequency and stage specificity. Eur J Immunol 1988;18:269–75. 22 Cwynarski K, Ainsworth J, Cobbold M et al. Direct visualization of cytomegalovirus-specific-T-cell reconstitution after allogeneic stem cell transplantation. Blood 2001; 97:1232–40.
Adoptive transfer of Ag-specific T cells to prevent CMV disease
23 Quinnan GV, Kirmani NG, Rook AH et al. Cytotoxic T cells in cytomegalovirus infection. N Engl J Med 1982;307:6–13. 24 Walter EA, Greenberg PD, Gilbert MJ et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow transplantation. N Engl J Med 1995;333:1038–44. 25 Rooney C, Colton-Smith C, Loftin S et al. Use of gene modified virus specific T lymphocytes to control Epstein–Barr virus related lymphoproliferation. Lancet 1995;345:9–13. 26 Heslop HE, Ng YC, Li C et al. Long-term restoration of immunity against Epstein–Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes. Nat Med 1996;2:551–5. 27 McLauglin-Taylor E, Pnade H, Forman SJ et al. Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD81 virus-specific cytotoxic T lymphocytes. J Med Virol 1994;43:103–10. 28 Beninga J, Kropff B, Mach M et al. Comparative analysis of fourteen individual human cytomegalovirus proteins for helper T cell response. J Virol 1995;76:153–60. 29 van Zanten J, Harmsen MC, van der Giessen M et al. Proliferative T cell responses to four human cytomegalovirus (HCMV)-specific proteins in healthy subjects and solid organ transplant recipients. J Infect Dis 1995;172:872–9. 30 Boppana SB, Britt WJ. Recognition of human cytomegalovirus gene products by HCMV-specific cytotoxic T cells. Virology 1996;222:293–6. 31 Wills MR, Carmichael AJ, Mynard K et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65–specific CTL. J Immunol 1996;70:7569–79. 32 Weekes MP, Wills MR, Mynard K, Carmichael AJ. The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J Virol 1999;73:2099–108. 33 Diamond DJ, York J, Sun JY et al. Development of a candidate HLA-A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997;90:1751–67. 34 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;163:5512–18. 35 Reeves ME, Royal RE, Lam JS et al. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res 1996;56:5672–7. 36 Keever-Taylor CA, Margolis D, Sandford G et al. Generation of cytomegalovirus-specific cytolytic T cell lines and clones using pp65 transfected dendritic cells. Blood 1999:94[abstract 667,52a]. 37 Qi S, Pollok KE, Burton RL et al. Simultaneous ex vivo expansion of cytomegalovirus and Epstein–Barr virusspecific cytotoxic T lymphocytes using B-lymphoblastoid
38
39
40
41
42
43
44
45
46
47
48
49
50
51
9
cell lines expressing cytomegalovirus pp65. Blood 1999; 94:3242. Regn S, Raffegerst S, Chen X et al. Ex vivo generation of cytotoxic T lymphocytes specific for one or two distinct viruses for the prophylaxis of patients receiving an allogeneic bone marrow transplant. Bone Marrow Transplant 2001;27:53–64. Spencer HT, Galloway A, Pirisi-Creek L et al. Generating cytotoxic T lymphocytes against cervical cancer with lentivirus-transduced antigen-presenting cells. Third Annual Meeting of the American Society of Gene Therapy. Mol Ther 2000;1:S262 [Abstract 718]. Peggs K, Verfuerth S, Mackinnon S. Induction of cytomegalovirus (CMV)-specific T-cell responses using dendritic cells pulsed with CMV antigen: a novel culture system free of live CMV virions. Blood 2001;97:994–1000. Cella BM, Scheidegger D, Palmer-Lehmann K et al. Ligation of CD40 on dendritic cells triggers production of high level of interleukin-12 and enhances T cell stimulatory capacity: T:T help via APC activation. J Exp Med 1996;184:747–52. Bhardwaj N, Seder RA, Reddy A et al. IL-12 in conjunction with dendritic cells enhances antiviral CD81 CTL responses in vitro. J Clin Invest 1996;98:715–22. Ahn K, Angulo A, Ghazal P et al. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci USA 1996;93:10990–5. Koszinowski UH, Reddehase MJ, Del Val M. Principles of cytomegalovirus antigen presentation in vitro and in vivo. Semin Immunol 1992;4:71–9. Wiertz EJHJ, Jones TR, Sun L et al. The human cytomegalovirus US11 gene product dislocates MHC Class 1 heavy chains from the endoplasmic reticulum to the cytosol. Cell 1996;84:769–79. Hengel H, Flohr T, Hammerling GJ et al. Human cytomegalovirus inhibits peptide translocation into the endoplasmic reticulum for MHC class assembly. J Gen Virol 1996;77:2287–96. Kern F, Surel IP, Faulhaber N et al. Target structures of the CD1 -T-cell response to human cytomegalovirus: the 72kilodalton major immediate-early protein revisited. J Virol 1999;73:8179–84. Ellis JM, Henson V, Slack R et al. Frequencies of HLA-A2 alleles in five US population groups. Hum Immunol 2000;61:334–40. Corr M, Slanetz AE, Boyd LF et al. T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity. Science 1994;265:946–9. Altman JD, Moss PAH, Goulder PJR et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996; 274:94–6. Dunbar PR, Ogg GS, Chen et al. Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood. Curr Biol 1998;8:413–16.
10
F van Rhee and J Barrett
52 Ogg GS, Jin X, Bonhoeffer S et al. Quantitation of HIV1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998;279:2103–6. 53 Kuroday MJ, Schmitz JE, Barouch DH et al. Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected Rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J Exp Med 1998;187:1373–81. 54 Bieganowska K, Hollsberg P, Buckle GJ et al. Direct analysis of viral-specific CD81 T cell with soluble HLA-A2/ Tax11-19 tetramer complexes in patients with human T cell lymphotropic virus-associated myelopathy. J Immunol 1999;162:1765–71. 55 Gallimore A, Glithero A, Godkin A et al. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J Exp Med 1998;187:1383–93. 56 Clave E, Jin SWA, Bradstock K et al. Use of a CMV pp65specific HLA tetramer to select and expand CMV-specific CD81 CTL. Blood 1999;94:155a.
57 Mavroudis DM, Barrett J. Separating GvHD and GvL reactions. In: Barrett AJ and Jiang Y-Z, editors. Graft versus leukemia. New York: Marcel Dekker Publishers, 2000:435–46. 58 Luxembourg AT, Borrow P, Teytom L et al. Biomagnetic isolation of antigen-specific CD81 T cells usable in immunotherapy. Nature Biotechnol 1998;16:281–5. 59 Brosterhus H, Brings S, Leyendeckers H, Manz RA. Enrichment and detection of live antigen-specific CD4(1 ) and CD8(1 ) T cells based on cytokine secretion. Eur J Immunol 1999;29:4053–9. 60 Asemissen AM, Nagorsen D, Keilholz U. Flow cytometric determination of intracellular or secreted IFNc for the quantification of antigen reactive T cells. J Immunol Methods 2001;251:101–8. 61 Keenan R, Ainsworth J, Assenmacher M et al. Magnetic selection of cytomegalovirus-specific CTLs for use in antigen-specific donor leukocyte infusion. Blood 1999;94:320a. 62 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;98:505–12.