The generation of LMP2a-specific cytotoxic T lymphocytes for the treatment of patients with Epstein-Barr virus-positive Hodgkin disease

Abstract

Based upon the success of using polyclonal, Epstein‐Barr virus (EBV)‐specific CTL lines for the prophylaxis and treatment of patients with post‐transplant lymphoproliferative disease (PTLPD), there is now considerable incentive to develop CTL directed against the sub‐dominant EBV antigens EBNA1, LMP1 and LMP2, which are expressed by the tumor cells of Hodgkin disease and nasopharyngeal carcinoma. To develop a system for generating LMP2a‐specific CTL in vitro, we transfected autologous immature dendritic cells (DC), which had been grown in the absence of serum, with LMP2a RNA in the presence of the cationic lipid DOTAP. This transfection method did not adversely affect the DC in terms of immunophenotyping and they expressed high levels of HLA class I and II and critical costimulatory molecules. These LMP2a+ DC, as compared to DC which had been transfected with irrelevant RNA, were shown to be highly immunostimulatory in autologous mixed lymphocyte reactions and, importantly, could stimulate the generation of CD8+ and CD4+ CTL which exclusively recognized LMP2a‐expressing targets. This specific cytotoxicity was confirmed using antibody blocking experiments and cytotoxicity assays of the separated T cell subsets. Using this DC‐based system we could also reactivate LMP2a‐specific memory in EBV‐seropositive donors whose polyclonal CTL response to LCL stimulation did not contain a LMP2a‐specific component.

Virally targeted therapies for EBV-associated malignancies

“…Clinical trials of EBV-CTL therapy for such tumors are ongoing Chua et al, 2001). CTL responses to LMP-1/2 epitopes can be generated in vitro (Meij et al, 2002), and several groups have addressed in vitro the presumed requirement to enhance CTL response to LMP-2 and/or LMP-1 (Gahn et al, 2001;Su et al, 2001). Nevertheless, the fact that such tumors can develop in hosts with apparently intact CTL function suggests that tumor paracrine effects, such as secreted TGF-b and IL-10, may limit the function of EBV-specific CTLs.…”

Epstein-Barr Virus-Associated Classical Hodgkin Lymphoma and Its Therapeutic Strategies

“…However, unlike the in vivo experimental animal settings, the induction of effective CTL responses against those proteins has showed diffi culties per se due to unique characteristics of HRS cells and their microenvironment as described (Marshall et al, 2003). For example, in vitro HRS cells that present epitopes from LMP1 and LMP2A are subject to lysis by EBV-specifi c CTLs, but EBV-infected HRS cells survive in vivo (Sing et al, 1997;Chapman et al, 2001;Su et al, 2001). Thus, although EBV-associated HL patients initially achieve an effective anti-EBV response after the period of fi rst EBV infection, they do not completely eradicate EBV-infected HRS cells.…”

Immunotherapies for Hodgkin's lymphoma

“…The question arises as to whether CTL specific for LMP2 might be more effective than polyclonal EBV-CTL in the adoptive immunotherapy of HL [35]. CTL specific for LMP1 [36] or for LMP2 [37,38] have been successfully generated. One approach is to stimulate patient T cells using autologous dendritic cells and LCLs, both overexpressing LMP2A through an adenoviral vector [34].…”