Viral-Specific Adoptive Immunotherapy After Allo-SCT

Bone Marrow Transplantation (2013) 48, 1265–1270 & 2013 Macmillan Publishers Limited All rights reserved 0268-3369/13 www.nature.com/bmt REVIEW Viral-specific adoptive immunotherapy after allo-SCT: the role of multimer-based selection strategies N Ramı´rez1 and E Olavarrıá1,2 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 Olavarrıá 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

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