Establishment and Characterization of a Bank of Cytotoxic T Lymphocytes for Immunotherapy of Epstein-Barr Virus-Associated Diseases

Abstract

Adoptive immunotherapy using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) generated ex vivo can be an effective treatment of EBV-positive posttransplantation lymphoproliferative disease (PTLD). We describe the establishment of a cryopreserved repository of allogeneic virus-specific CTL lines, to our knowledge the first of its kind in the world. CTL lines were grown by weekly stimulation with autologous EBV immortalized lymphoblastoid cell lines (LCLs) from 96 EBV-seropositive blood donors. Analysis of 60 CTL lines grown continuously for 7 to 10 weeks showed an average proportional weekly increase in cell numbers of 1.4, with an overall increase ranging from 1.1 to 83.4. The greatest increase occurred during the early culture period. After four rounds of stimulation, killing of autologous LCLs was generally high (mean 48%); however, most lines required 9 or 10 stimulations to reduce the killing of nonspecific targets. Overall, 79% of CTLs generated showed acceptable levels of specific killing. Phenotypically, the CTL lines consisted of TCRalpha beta+, CD8+ T cells (medians 97% and 90% respectively) with a minority population of CD4+ T cells (median 2%). Most cells expressed the activation and differentiation markers, HLA-DR, CD26, CD45RO, CD69, and CD150. Favorable results have been obtained in an open trial using partially HLA-matched, allogeneic CTLs from this bank to treat PTLD patients. This now represents a single resource that can provide therapeutic CTLs rapidly on a countrywide basis, superseding the time-consuming, expensive practice of generating autologous CTLs from each patient requiring treatment. Additionally, other patient groups, such as those with EBV-positive Hodgkin disease, may benefit from CTL treatment.

EBV‐positive diffuse large B‐cell lymphoma of the elderly: 2016 update on diagnosis, risk‐stratification, and management

“…To reduce risk of GVHD, EBV-CTLs grown from peripheral blood healthy donors on a best HLA-match basis and banked (Edinburgh CTL bank). The most common method is to establish lymphoblastoid cell lines (LCLs) by in vitro EBV infection and used as stimulators for development of specific EBV-CTLs [60]. Best HLA-matched EBV-CTLs were administered to PTLD post SOT with a response rate of 50-60% [61].…”

CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus–transformed B cells

“…[6][7][8][9] Interestingly these same LCL-stimulated preparations also contain varying proportions of CD4 ϩ T cells. 12,13 Although poorly characterized, these CD4 ϩ T cells also appear to recognize and kill LCLs, 8,14 and, in a recent trial, their presence in adoptively transferred preparations has been linked with better clinical responses. 15 Furthermore, in that trial, LCL-stimulated T cells also appeared to be effective against rare cases of EBV-positive Burkitt (BL) and Hodgkin (HL) lymphomas arising in patients after receiving a transplant.…”

Rejection of Experimental Hodgkins Lymphoma by T-Cells Engineered with a CD19 Chimeric Antigen Receptor

“…Lymphoblastoid cell lines (LCLs) were derived by in vitro EBV infection of PBMCs from healthy donors. The Epstein Barr Virus (EBV)-specific CTL-A was produced as described [26] using PBMCs from a donor (HLA type: A1, A3, B35, B57). This CTL was chosen as it matched the HLA type of L591 cells (A1, A33, B8, B35) at two loci.…”