Viral-Specific Adoptive Immunotherapy After Allo-SCT

REVIEW Viral-speciﬁc adoptive immunotherapy after allo-SCT: the role of multimer-based selection strategies

N Ramı´rez1 and E Olavarrı´a1,2

Recipients of hematopoietic SCT undergo a period of profound immunosuppression due to the chemotherapy and/or radiotherapy used for the conditioning and to the graft versus host reaction. SCT patients are highly susceptible to the development of viral infections such as CMV or EBV. The achievement of a competent immunological response, such as viral-specific T cells, is associated with a lower incidence of viral infections. Methods for direct identification of antigen-specific T cells have been based on the functional characteristics of these T cells. Techniques such as proliferation and ELISPOT assays, intracellular cytokine staining and IFN-g capture have been used to quantitate and obtain viral-specific T cells. Multimers are composed of several MHC molecules loaded with immunodominant peptides joined to a fluorescent molecule, which signal can be quantified by a flow cytometer. Multimer technology together with recent advances in flow cytometry, have facilitated the monitoring and selection of antigen- specific T cells without the need for in vitro cultures and manipulation. This has resulted in a better characterization of the function and phenotype of the different subpopulations of T cells involved in the immune recovery post allogeneic SCT. It is becoming a distinct possibility to isolate individual antigen-specific T cells, without long-term culture techniques, and potentially use them as adoptive immunotherapy in the SCT setting.

Bone Marrow Transplantation (2013) 48, 1265–1270; doi:10.1038/bmt.2012.262; published online 14 January 2013 Keywords: adoptive immunotherapy; cytotoxic T cells; multimer technology

INTRODUCTION influence on the long-time persistence of the viral-specific T cells During the initial months post transplant patients remain at a high in the recipients after cell transfer. At present, there is no standard risk of developing viral diseases due to a profound deficiency of process to isolate antigen-specific T cells that preserves the T-cell function induced partly by the immunoablative effect of the function of these cells before and after the infusion into a patient, conditioning regimen and partly by the complex host versus graft thus reducing the risk of viral infection during the initial phases of and graft versus host interactions that characterize the immediate the post-transplant period. post-transplant phase.1,2 Other factors contributing to this risk are following: the presence of clinically significant GvHD, steroid or other immunosuppressant use, low circulating CD4 counts, the CMV INFECTION, A MODEL OF VIRAL DISEASE use of virus-seronegative donors in seropositive patients, T-cell- IN HEMATOPOIETIC SCT PATIENTS depleted grafts and the use of donors other than HLA-identical CMV infection is by far the most extensively studied post SCT viral 1 siblings (unrelated, haploidentical and cord blood). infection. Riddell et al.4 have shown that CMV-specific CD8 þ The administration of antiviral drugs is the standard treatment cytotoxic T-cell clones could be obtained from BM donors using in cases where a viral infection is presumed or detected. However, in vitro co-culture of PBMCs from allogeneic donors together with antiviral drugs cause multiple side effects, mainly nephrotoxicity autologous CMV-infected fibroblasts for 5–12 weeks.5 This group and myelosuppression. Moreover, in the case of CMV infections, later adoptively transferred the CMV-specific CD8 þ cytotoxic the use of Ganciclovir has been associated with an increased rate T-cell clones to immunodeficient BMT recipients successfully. With 1,2 of bacterial sepsis, invasive fungal infections and viral resistance. this strategy, no toxicity was seen and the transferred clones 3 Therefore, there is an unmet need for alternative treatments. provided the same magnitude of lytic activity as that observed in Recently, the use of lymphocyte subpopulations from SCT donors the immunocompetent BM donors for at least 8 weeks after the is being considered as a potential new immunotherapy strategy completion of T-cell therapy. The rearranged Va and Vb genes for for SCT recipients with high risk of developing viral disease. Initial the TCR were used to evaluate the survival of the transferred approaches utilized long-time in vitro cultures to obtain T-cell clones in these patients.6 Similar approaches had also been clones or lines. However, these long-term cultures could performed to obtain and expand polyclonal CMV-specific T-cell potentially modify the functional characteristics of the T cells. lines for adoptive immunotherapy. In this setting, the group at NIH More recently, the use of new multimer-based technology as a has used monocyte-derived dendritic cells pulsed with CMV tool for cellular selection has shown promise in maintaining the antigens and co-cultured with PBLs from seropositive donors for function of viral-specific T cells. In this review, we focus on how CMV.7,8 Other groups have utilized virus lysate, recombinant these new techniques used to isolate viral-specific T cells may soluble CMVpp65 protein or pool of peptides to prepare CMV- affect the phenotype and function of T cells and their potential specific T-cell clones9 A different approach involves the use of

1Hemato-Oncology Research Group, Biomedical Research Center, Pamplona, Spain and 2Department of Hematology, BMT Unit, Complejo Hospitalario de Navarra, Pamplona, Spain. Correspondence: Dr E Olavarria, Department of Haematology, BMT Unit, Complejo Hospitalario de Navarra, Irunlarrea 3, Pamplona, 31008, Spain. Email: [email protected] Received 6 June 2012; revised 12 November 2012; accepted 17 November 2012; published online 14 January 2013 Viral-speciﬁc adoptive immunotherapy N Ramı´rez and E Olavarrı´a 1266

Design of a culture Naïve system for cellular T cell immunotherapy

Exogenous antigenic stimulus

Protein Pulsated Lysate APC Exogenous Peptide adittion Cytokines Effector Senescent TCR transgenic T cell T cell In vitrointernal production

Exogenous costimulatory molecules

Memory T cell

Figure 1. Different approaches have been used to obtain and expand polyclonal CMV-specific T-cell lines for adoptive immunotherapy. In this scheme, monocyte-derived dendritic cells can be pulsed with viral antigens and co-cultured with PBLs from seropositive donors for CMV. Viral lysate, recombinant soluble protein or pooled immunodominat peptides are also used to prepare CMV-specific T-cell clones. A different approach involves the use of gene therapy technology to obtain antigen-specific T cells: retroviral transfer of CMV-specific TCR genes to primary T cells from CMV-negative donors can be used to generate CMV-specific T-cell clones of several HLA restrictions with important effector functions. The common characteristic of these techniques is the extensive cell culture needed to generate virus-specific T-cell clones, resulting in alteration of the cellular differentiation status.

gene therapy technology to obtain antigen-specific T cells: differentiation status, different phenotypes and functional and retroviral transfer of CMV-specific TCR genes to primary T cells migratory capacities.16 Thus, Berger et al.17 showed that antigen- from CMV-negative donors has been able to generate CMV- specific CD8 þ TE derived from a subset of memory T cells named specific T-cell clones of several HLA restrictions with important effector memory (TEM) survive for only a short period after effector functions.10 The common characteristic of these adoptive transfer, and failed to home to lymph nodes or BM. techniques is the extensive cellular culture needed to generate However, TE clones derived from other different subset named virus-specific T-cell clones. TCM persist long term after adoptive transfer, migrate to memory The effectiveness of adoptive therapy as a treatment strategy T-cell niches and respond to antigen challenge. Therefore, for CMV infection post SCT may ultimately depend on generating selection of TCM cells would ensure that after adoptive transfer functional antigen-specific T cells in ways that mimic physiological the cellular product consisting of TE cells would be able to persist conditions of a competent immunological system. durably in vivo and revert to the memory pool.17,18 However, acquisition of terminal effector properties and an increase in cytotoxic activity in vitro generate less effective cells at triggering EXTENSIVE CULTURE CHANGES THE DIFFERENTIATION disease regression in vivo.19 Thus, the more-differentiated cells, STATUS OF ANTIGEN-SPECIFIC CD8 þ T SUBPOPULATIONS with superior in vitro antitumor properties, are less capable of Under physiological conditions, antigen exposure triggers the mediating tumor regression upon adoptive transfer. Gattinoni activation of naive CD8 þ T cells (CTLs), which results in et al.20 describe the relationship among the duration of in vitro proliferation and progressive differentiation through early, inter- T-cell culture, the functional and phenotypic characteristics mediate and late effector stages depending on signal strength.11 of T cells at various stages of differentiation and their 20,21 Effector T cells (TE) are derived from other different subset named immunocompetence upon adoptive transfer. central memory T cell (TCM). Antigen-specific memory T cells are The effectiveness of adoptive therapy to treat viral infection generated during this cellular process.12 These processes could be post SCT may ultimately depend on generating functional mimicked in vitro by long-term cultures (Figure 1) in which a memory cells in such a way that a competent immunological continuous contact with the specific antigen could induce system mimicking physiological conditions could be reproduced proliferation and differentiation of T cells into cells with different immediately after transplant (Figure 2). effector capacities.13 Lanzavecchia and Sallusto14 also considered the possibility that cytokines may drive T cells to an antigen- independent slow proliferation. This homeostatic T-cell IDENTIFICATION IN VITRO OF MEMORY VIRAL-SPECIFIC differentiation may occur in vitro during cellular expansion. TCR- T CELLS WITH HIGH EFFECTOR CAPACITY triggering stimulation by antigen exposure, leads to increasing For the detection of TCM among the viral-specific T-cell pool, it is division, altering the pattern of cytokines15 and thus changing the necessary to clarify the phenotype of these cells and a tool for its process of cellular differentiation. Stimulation of antigen-specific direct identification. Several authors have attempted to describe CD8 þ T cells using extensive cellular culture under specific the phenotype of the memory antigen-specific T-cell subpopula- antigenic stimulation does not induce a homogeneous cellular tion by means of a combination of extracellular and intracellular product but a high number of subpopulations with diverse molecules. In this sense, van Lier et al.22 defined two discrete

Bone Marrow Transplantation (2013) 1265 – 1270 & 2013 Macmillan Publishers Limited Viral-speciﬁc adoptive immunotherapy N Ramı´rez and E Olavarrı´a 1267 Immunodominant peptide (antigen) α 1 α2 β Fluorescent label 2m Biotin α3

class I HLA TCR Steptavidin Anti-fluorescence antibody

Magnetic bead

Strep-tag

Multimerized Strep-Tactin

+ d-biotin

Figure 2. Shows a diagrammatic representation of the structure of three different multimers: (a) tetratmers, (b) pentamers and (c) streptamers and its mechanism of action. Only pentamers and streptamers are currently produced under good manufacturing practice conditions. primed CD8 þ T-cell subpopulations based on the expression of patients. Multimers do not use a phenotypic or functional cellular CD45 isoforms and CD27: CD45RO þ CD27 þ memory cells, which characteristic to identify viral-specific T cells, but recombinant self- lack immediate cytolytic function, and CD45RA þ CD27 À effector MHC molecules loaded with immunodominant peptides of cells with low proliferative capacity and high levels of perforin and antigen proteins that are recognized by specific CTLs. Multimers cytotoxicity; however, the heterogeneity of these memory are constituted by several of these MHC molecules joined to a subpopulations was revealed by Sallusto et al.23 with the fluorescent molecule that can be quantified by a flow cytometer. analysis of CCR7, a chemokine receptor for lymph node homing. The combination of multimers and flow cytometry has been for This new molecule defines the phenotype of the previously several years a very useful technique to identify and quantify described memory subpopulations: TCM (CCR7 þ CD45RA À ) and antigen-specific T cells. Direct visualization, enumeration, pheno- TEM (CCR7 À CD45RA À ). typic and functional characterization, isolation and cloning of Other authors have also tried to describe the correlation antigen-specific T cells are the most common applications that between phenotype and function. Scheinberg et al.24 assert that may be achieved with this new technology. the presence of great numbers of less-differentiated Distinct MHC multimer formats: dimers, tetramers, pentamers, CD27( þ )CD57( À )CD8( þ ) CMV-specific T cells in the donor streptamers, octamers and dextramers have been developed in appears to confer protection against viral reactivation in the the past few years. Differences between multimers are found recipient after hematopoietic SCT. Otherwise, Luo et al.25 observed in just three parameters: the valence of the multimeric complex, that high CMV-specific CTLs with terminally differentiated effector the expression system through which the MHC molecules are CD45RO À CD62L À cell phenotype in the allografts is associated produced (bacterial or eukaryotic expression systems) and the with a reduced risk of CMV reactivation in the presence of enough peptide-loading strategy used to achieve occupancy of the CD45RO þ CD62L À effector cell infusion. peptide-binding groove with the desired antigenic peptide.26 Although different groups have investigated the association The NIH Core Tetramer Facility (http://www.niaid.nih.gov/ between the phenotype of memory viral-specific T cells and viral reposit/tetramer/index.html) was established in 1999 with the disease protection, there is still a lack of a consensus panel of purpose of providing custom synthesis and distribution of soluble molecular markers that could define this phenotype. human and mouse class I and II MHC tetramers reagents for research. A tetramer is a recombinant molecule with four subunits (tetrameric) of MHC biotinylated. These subunits are folded with CLASS I MHC MULTIMERS the peptide of interest and tetramerized by a fluorescence-labeled The development of the multimer technology has brought a streptavidin molecule, which can join up to four biotin molecules. potential tool to detect antigen-specific T cells from the PB of SCT Tetramers can join up to three TCR molecules at once due to the

& 2013 Macmillan Publishers Limited Bone Marrow Transplantation (2013) 1265 – 1270 Viral-specific adoptive immunotherapy N Ramı´rez and E Olavarrıá 1268 tetrahedral disposition of the complex.27 This is a limiting factor As described above, the influence of tetramer and pentamer given that all subunits cannot bind at the same time to a specific T staining over the function of the T cell remains an important cell. This problem has been solved by the pentamers that contain unresolved issue. This is a problem that potentially could limit five class I MHC–peptide complexes that are multimerized by a substantially the clinical applicability of the multimer technology. self-assembling coiled coil domain. In this case, all five MHC– To address this problem, a reversible human MHC/peptide peptide complexes face in the same direction resulting in a very multimer (streptamer) was constructed by Neudorfer et al.34 The high avidity. Low frequency specific T-cell subpopulations are main characteristic of this multimer is the reversible binding better detected by pentamers due to this high avidity. However, between the streptamer and the TCR of the antigen-specific T this difference has not increased the sensibility of the technique. cells. The streptamer monomerizes in the presence of a In fact, Yao et al.28 did not find any differences in CMV competitor molecule, resulting in rapid release of the staining phosphoprotein 65-specific CD8( þ ) T-cell frequencies using reagent. Following dissociation, the streptamer-treated CTLs are tetramers and pentamers in CMV-seropositive healthy volunteers functionally indistinguishable from untreated T cells.31 Wang and CMV-seropositive patients before and after allogeneic PBSC et al.35 demonstrated that the in vitro treatment of the T cells with transplantation. Tetramers have been evaluated by the European peptide-loaded OT-I-streptamers induced an increased Working Group on Clinical Cell Analysis, and efforts have been proliferation and strong expression of CD69 and CD25 activation made towards a reproducible assay for routine enumeration of markers. It also induced a sustained phosphorylation of Akt (a antigen-specific CD8 þ T cells.29 serine threonine kinase, which is critically involved in T-cell survival) and ERK1/2 (serine/threonine kinase important for cellular proliferation and survival), a weak activation of caspase- ROLE OF THE MULTIMER IN THE REGULATION OF FUNCTION 3, a weak anexin V and propidium iodide expression and OF ANTIGEN-SPECIFIC T CELL expression of Bcl-xL (that prevents TCR-mediated apoptosis and Most methods for direct selection of T cells require in vitro promotes T-cell survival). stimulation of the T cells, which significantly changes their This expression of molecular markers after streptamer engage- differentiation and functional status as has already been ment supports the idea that streptamer could trigger positive described. Up until recently, it was accepted that multimer signals for activation and proliferation, in the specific CD8 þ T cells technology allowed identification and purification of antigen- and no functional disruption or tolerance as it has been observed specific T cells without altering their differentiation or functional with tetramers. Moreover, streptamers have comparable sensitivity status. However, recent studies with tetramers have provided and specificity to conventional tetramers and pentamers,28,31 and further evidence that MHC multimer binding interferes with the the streptamer technology additionally offers the advantage of functional status of epitope-specific T-cell populations in vivo, selecting specific T cells under good manufacturing practice showing evidence that the usage of MHC multimers can induce conditions for adoptive T-cell transfer. This later aspect has so far epitope-specific tolerance in a dose-dependent manner. After the proved a significant challenge for translational researchers. tetramer and TCR have engaged, a strong ‘signal 1’ is delivered to antigen-specific T-cell populations and causes an initial expansion of reactive CD8 þ T cells. However, multiple doses of relevant SOLUBLE FACTORS AND CELLULAR MICROENVIRONMENT tetramer can reverse the priming effect, suggesting that there is CHANGE THE FUNCTION OF THE THERAPEUTIC ANTIGEN- elimination of the tetramer-reactive T cells by clonal exhaustion or SPECIFIC CTLS IN SCT PATIENTS anergy.30 Other authors have observed the unusual functional Homeostatic conditions and the presence of specific lymphocyte status of the specific T cells after tetramer selection, given that subpopulations could alter both the function of circulating lytic function and proliferation of the specific T cells was impaired therapeutic CTLs after adoptive transfer and the long-term after joining with conventional tetramers.31 However, this persistence of these cells in the SCT patients. phenomenon of functional disruption has not been observed The lymphopenic environment associated with the early post in vivo conditions.32 SCT period is likely to be favorable for T-cell proliferation. It is This group found that CMV-specific CTLs selected with tetramer possible that T cells obtained directly from the donor may be able technology and transferred to SCT recipients efficiently contrib- to expand more efficiently in vivo than in vitro. It has been reported uted to the control of viral replication.32 In this study, the authors that exposure to viral antigens during the period of profound could demonstrate the persistence of CMV-specific CTLs at least lymphopenia results in a significant boost in cellular immu- 100 days after the infusion, which suggested expansion of the nity.32,36,37 In this respect, it has been shown that lympho- infused cells, not compatible with an anergic status of the cellular depletion before adoptive T-cell transfer-based immunotherapies product. The same phenomenon was observed in a study in which can enhance antitumor responses by depressing the numbers of eight patients received CTLs specific for three different viruses regulatory T cells, by increasing access to homeostatic cytokines (CMV, adenovirus and EBV) obtained from HLA-identical and and by augmenting innate immunity, so that the lymphopenic haploidentical donors with pentamer technology. Donor-derived- environment could induce qualitative improvements in the specific CTLs were detected using chimerism analysis in five out of adoptively transferred T cells.38 This phenomenon was first shown six evaluable patients. In addition, six patients showed a decrease by Pittet et al.,39 who observed that both intratumoral CTL in viral titers within 2 weeks of CTl infusion.33 accumulation and tumor regression improved when CTLs were With respect to tetramer multimers, in vitro and in vivo studies injected to lymphopenic animals. Furthermore, regulatory T cells show conflicting results when analyzing the functional status of can suppress the activity of effector T cells in different ways: virus-specific CTLs after tetramer selection. On the other hand, expression of negative costimulatory molecules, induction of anti- there is no in vitro study addressing whether pentamer staining inflammatory biochemical pathways in effector T cells and APCs, and selection could induce anergy in specific T cells. Pentamer direct or indirect killing of effector cells and APCs, the consumption technology uses the same approach that tetramer technology to of proinflammatory cytokines such as IL-2 or the production of identify antigen-specific T cells: an interaction between the TCR immunoregulatory cytokines, such as IL-10, transforming growth and MHC–peptide complexes. Therefore, it is theoretically possible factor-b or IL-35.40 Hence, their eradication in a lymphopenic that a similar impairment of the T-cell phenotype and function environment could potentially help the engraftment, expansion could occur in vitro with pentamers. However, the previously and function of the transferred T cells. mentioned study shows functional EBV-specific CTLs that are able Functional status and survival of transferred antigen-specific to remove the EBV-driven lymphoma.33 CTLs depend on specific circulating CD4 þ T cells. One example of

Bone Marrow Transplantation (2013) 1265 – 1270 & 2013 Macmillan Publishers Limited Viral-specific adoptive immunotherapy N Ramı´rez and E Olavarrıá 1269 the limiting role of the antigen-specific CD4 þ T cells in a chronic Both approaches represent a new perspective with respect viral infection is during the pathogenesis of AIDS. HIV-1-infected to the applicability of streptamer-selected CTLs in adoptive persons who have no evidence of virus-related clinical disease and immunotherapy. low to undetectable viral loads, show a high virus-specific CD4 þ proliferative response and an extremely vigorous CTL response.41 On the other hand, the resurgence of HIV-infected CD4 þ T cells 7 FUTURE PROSPECTS OF THE MULTIMER TECHNOLOGY days after adoptive therapy with HIV-1-specific CTLs correlates The simultaneous study of extracellular and intracellular molecules with the disappearance of transferred CTLs from the blood, in T cells is possible thanks to the advance of the multicolor flow indicating that the transient nature of the antiviral effect reflects cytometric technology provided by the new generation of flow the limited in vivo persistence of the transferred CTLs.42 cytometers and new fluorescence molecules that have been The exact mechanisms whereby CD4 þ cells contribute to the commercialized in recent years. Furthermore, multimer staining maintenance of effective antiviral immunity are not known but could be combined with the study of functional cellular markers to could be related to an enhanced CTL precursor activity, increased provide a multiparameter phenotypic and/or functional analysis of production of antiviral cytokines and augmentation of humoral an antigen-specific T-cell subpopulation. Depending on the immune responses.41 Pourgheysari et al.43 found a significant researcher’s interest, these markers may analyze the activation correlation between the CMV-specific CD4 þ and CMV-specific status (CD69, CD25, CD38, HLA-DR and so on), T-cell subset CD8 þ T-cell count. They have shown a clear association between markers that have been associated with the differentiation cell reconstitution of the CD4 þ CMV-specific T-cell immune response status (CCR7, CD45RA, CD62L and so on) or expression of other within the first 50 days post transplantation and protection against functional molecules (perforin, granzyme, IFN-g, IL-7, TNF-a and so CMV reactivation. However, CD8 þ CMV-specific T-cell response was on). The incorporation of these sophisticated cytometers will allow not predictive of protection from a single episode of reactivation, the study of the phenotype and function of specific cells in daily suggesting that reconstitution of CD4 þ CMV-specific T cells has a routine samples. more important role in the early period post transplantation. Another major development that could help in the detection of To examine the central role of CMV-specific CD4 þ T cells new epitopes is the method for high-throughput parallel during the post-transplant period, the development of class II detection of antigen-specific T cells by combinatorial encoding MHC multimers, to measure CD4 þ T cells, is likely to represent a of MHC multimers described by Andersen et al.49 Using this major step forward.44 However, there are still a number of aspects method, a large number of different T-cell populations can be to improve regarding the applicability of these reagents, in detected in a single sample, and therefore it can be used for T-cell particular the strength of the TCR/pMHC II interactions.45,46 epitope mapping and for the monitoring of CD8-positive immune responses during cancer or infectious disease immunotherapy. The usual limit of detection of flow cytometry assays is 10 À 4 TRANSLATIONAL APPLICABILITY OF THE MULTIMER cells. Recently, it has been shown that sensitivity might be TECHNOLOGY increased by 1-log by simply acquiring 10 times more cells There are many strategies to select antigen-specific CD8 þ T cells compared with standard acquisition conditions without compro- for adoptive transfer; however, multimer-based technology has mising the specificity.50 Thus, the development of multimer the potential to select antigen-specific T cells from healthy staining in conjunction with the continuing advances in flow seropositive donors and transfer them directly to the patient cytometry represents an excellent platform to study antigen- without any further manipulation. At present, there are only two specific T-cell subpopulations, which until now have eluded published studies that report on adoptive immunotherapy conventional methods in hematological transplant recipients. strategies based on tetramers and pentamers. Both describe a Monitoring of these cells together with the possibility of successful protocol using multimers and magnetic beads to select simultaneously studying 2–10 cellular markers may help the CMV- and EBV-specific T cells from healthy donors and transfer quantitative and qualitative characterization of the immune them to recipients diagnosed with hematological malignancies.32 response of these patients. Given that both techniques have obtained excellent results, multimer technology should be considered as a great opportunity CONFLICT OF INTEREST to expand adoptive therapy strategies. In addition, streptamer technology removes the previously The authors declare no conflict of interest. raised doubt of the prolonged TCR signaling and subsequently altered T-cell function caused by the irreversible union between ACKNOWLEDGEMENTS the TCR of specific T cell and the multimer. Whether such reversible binding improves the in vivo activity of adoptively This work was supported by a research grant (PI10/00136) from the Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III. transferred T cells remains to be studied. Two other approximations have recently been presented. The first one used streptamer-based selection available on a good REFERENCES manufacturing practice level to generate Wilms tumor gene 1 1 Zaia J, Baden L, Boeckh MJ, Chakrabarti S, Einsele H, Ljungman P et al. Viral (WT1)-specific effector T cells. The primary objective was to disease prevention after hematopoietic cell transplantation. Bone Marrow Trans- generate an improved GVL effect and to minimize the risk of a plant 2009; 44: 471–482. GvHD WT1-specific CD8 þ T cells were evaluated in a standard 2 Winston DJ, Ho WG, Bartoni K, Du Mond C, Ebeling DF, Buhles WC et al. 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