Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers

Abstract

Cytomegalovirus (CMV) reactivation in immunocompromised recipients of allogeneic stem cell transplantation is a cause of morbidity and mortality from viral pneumonitis. Antiviral drugs given to reactivating patients have reduced the mortality from CMV but have toxic side effects and do not always prevent late CMV disease. Cellular immunotherapy to prevent CMV disease is less toxic and could provide prolonged protection. However, a practical approach to generating sufficient quantities of CMV-specific cytotoxic T cells (CTLs) is required. This study describes a system for generating sufficient CMV-specific CTLs for adoptive immuno-

Immunosuppressive effects of radiation on human dendritic cells: reduced IL-12 production on activation and impairment of naïve T-cell priming

“…CTL represent a key end point in many protocols which currently employ irradiated DC as APC, both in vitro and in vivo (Gong et al, 1997;Celluzzi and Falo, 1998;Fields et al, 1998;Szmania et al, 2001). Using MART-1 presented by HLA-A2 as a model antigen, we have shown that irradiated human DC prime fewer T cells which are less effective at lysing peptide-pulsed targets ( Figure 5).…”

“…Both in vitro applications of DC and in vivo immunotherapy strategies can involve treatment of DC with ionising radiation, often to high dose. The rationale for ex vivo DC irradiation is not always clear, but may be designed to prevent continued DC division or the outgrowth of tumour cell components of vaccine preparations (Gong et al, 1997;Celluzzi and Falo, 1998;Szmania et al, 2001). In vivo irradiation of DC may be incidental during clinical radiotherapy, or part of sequencing combined modality treatment with immunotherapy (TeitzTennenbaum et al, 2003).…”

“…There is a wealth of pre-clinical data to support such strategies, and early studies have shown some promise (Timmerman and Levy, 1999). Alternatively, antigenloaded DC can be used ex vivo to expand specific effector T-cell populations for subsequent adoptive transfer back into patients (Yee et al, 2000;Szmania et al, 2001;Ho et al, 2003). During many of these applications, DC are irradiated with X-rays or gamma sources, often to high doses (typically 30 Gy) in a single fraction.…”

“…In some cases, such as irradiation of DC/tumour cell co-cultures or hybrids (Gong et al, 1997;Celluzzi and Falo, 1998), this treatment is rationally designed to prevent any outgrowth of viable tumour cells from within the proposed vaccine. In others, such as vaccination with tumour lysate-loaded DC (Fields et al, 1998), and priming of T cells in vitro for subsequent adoptive transfer (Szmania et al, 2001), the purpose of DC irradiation is less clear and potentially deleterious. Regardless of application, irradiation of DC is often assumed to have no bearing on subsequent DC/Tcell interactions or immune priming and to be immunologically neutral.…”

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

“…CMV-infected fibroblasts as APC [6] have generally been replaced either by unseparated PBMC [5] or by defined professional APC populations such as dendritic cells (DC) [9][10][11][12] or activated B cells [13,14]. The CMV antigenic stimulus has been provided by infection of APC with recombinant viral vectors introducing relevant CMV antigens [9,10,13,14], loading with epitope peptides [12] or CMV protein lysates [5,11].…”