Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease

Abstract

The treatment of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (PTLD) in EBV seronegative solid organ transplant recipients who acquire their EBV infection after engraftment poses a considerable challenge because of underlying immunosuppression that inhibits the virus-specific cytotoxic T cell (CTL) response in vivo. We have developed a protocol for activating autologous EBV-specific CTL lines from these patients and show their potential use for immunotherapy against PTLD in solid organ transplant patients. Peripheral blood mononuclear cells from a panel of solid organ transplant recipients with and without active PTLD were used to assess EBV-specific memory CTL responses. The activation protocol involved cocultivation of peripheral blood mononuclear cells with an autologous lymphoblastoid cell line under conditions that favored expansion of virus-specific CTL and hindered the proliferation of allospecific T cells. These CTL consistently showed (i) strong EBV-specificity, including reactivity through defined epitopes in spite of concurrent immunosuppressive therapy, and (ii) no alloreactivity toward donor alloantigens. More importantly, adoptive transfer of these autologous CTLs into a single patient with active PTLD was coincident with a very significant regression of the PTLD. These results demonstrate that a potent EBV-specific memory response can be expanded from solid organ recipients who have acquired their primary EBV infection under high levels of immunosuppressive therapy and that these T cells may have therapeutic potential against PTLD.

Figure 1

EBV specificity of T cell lines from five different solid organ transplant patients with or without PTLD. A detailed description of these patients is presented in Table 1. A–E show the CTL recognition of autologous and HLA-matched and -unmatched allogeneic LCLs while F–J show peptide epitope specificity of these CTL lines tested on PHA blasts. Also illustrated in F and H is the CTL recognition of PHA blasts from the organ donor. The HLA class I restriction and antigen location for the peptide epitopes used in the CTL assays is as follows: YLQQNWWTL (HLA A2; LMP1; ref. 10), YLLEMLWRL (HLA A2; LMP1; ref. 10), RPPIFIRRL (HLA B7; EBV nuclear antigen 3; ref. 11), GLCTLVAML (HLA A2; BamH1 fragment on left forward 1; ref. 12), LLDFVRFMGV (HLA A2; EBV nuclear antigen 6; ref. 4). Data from patient KVM is shown in A and F, from patient MM in B and G, from patient LF in C and H, from patient TT in D and I, and patient MD in E and J. The data is presented as percent specific lysis.

Figure 2

(A) CT scans of liver before initial CTL therapy showing two of the three lymphomas. (B) CT scan of liver 2 weeks after first CTL infusion. (C) CT scan of liver 20 weeks after the first infusion and before the fourth CTL infusion revealed complete regression of PTLD.

Figure 3

Quantitation of EBV-specific T cells from patient KVM pre- and postinfusion. The frequency of LMP1-specific CTLs in the peripheral blood of patient KVM was determined by LDA. The precursor frequency for two HLA A2-restricted CTL epitopes (YLLEMLWRL and YLQQNWWTL) is presented in A. Also illustrated in B is the precursor frequency for the epitopes in a healthy EBV seropositive donor, SB.