Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus‐infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) ‐A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1‐specific cytotoxic T‐lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT‐specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA‐A2‐restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT‐specific CTL clones did not lyse EBV‐carrying lymphoblastoid cell lines and Burkitt’s lymphoma cell lines nor EBNA1‐transfected Burkitt’s lymphoma cells but specifically released interferon‐γ upon stimulation with HLA‐matched EBNA1‐expressing cells and this response was enhanced by deletion of the Gly‐Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.