Accessing Epstein-Barr virus-specific T-cell memory with peptide-loaded dendritic cells.

The conventional means of studying Epstein-Barr virus (EBV)-induced cytotoxic T-lymphocyte (CTL) memory, by in vitro stimulation with the latently infected autologous lymphoblastoid cell line (LCL), has important limitations. First, it gives no information on memory to lytic cycle antigens; second, it preferentially amplifies the dominant components of latent antigen-specific memory at the expense of key subdominant reactivities. Here we describe an alternative approach, based on in vitro stimulation with epitope peptide-loaded dendritic cells (DCs), which allows one to probe the CTL repertoire for any individual reactivity of choice; this method proved significantly more efficient than stimulation with peptide alone. Using this approach we first show that reactivities to the immunodominant and subdominant lytic cycle epitopes identified by T cells during primary EBV infection are regularly detectable in the CTL memory of virus carriers; this implies that in such carriers chronic virus replication remains under direct T-cell control. We further show that subdominant latent cycle reactivities to epitopes in the latent membrane protein LMP2, though rarely undetectable in LCL-stimulated populations, can be reactivated by DC stimulation and selectively expanded as polyclonal CTL lines; the adoptive transfer of such preparations may be of value in targeting certain EBV-positive malignancies.

Inhibitory effect of calf fetuin on the cytotoxic activity of LAK cell-derived factors and tumor necrosis factor.

A protein-inhibiting LAK cell-mediated cytotoxicity was isolated from a LAK cell-conditioned medium. The N-terminal amino acid sequence of this protein appeared to be identical to fetal calf fetuin. Pure isolated fetuin, as well as commercially available preparations of this protein, were shown to inhibit cytotoxic activity of both cytotoxic proteins released by LAK cells and TNF.

Contribution of membrane-associated cytotoxic proteins to cytolysis of L929 and K562 target cells mediated by LAK cells with different phenotypes.

We have demonstrated that the relative contribution of secreted and membrane-associated proteins to the cytotoxicity of LAK cells depended on LAK cell phenotype: the cytotoxicity of CD16+ CD8+ CD3- LAK cells was associated mainly with membrane-bound proteins, and the activity of CD3+ CD8+ CD16- LAK cells was due mainly to secreted soluble proteins. The cytotoxic activity of membrane fractions of LAK cells against cell line L929 was determined by 40-, 32- and 21-kDa proteins for LAK cells bearing NK-specific markers and due to proteins of 34 and 21 kDa in the case of 'CTL-like' LAK-cells.

Time-dependent changes of LAK cell phenotypes correlate with the secretion of different cytotoxic proteins.

Human lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) of normal volunteers by interleukin-2 (IL-2) stimulation for 1-8 days. During the first 3 days the surface marker CD16 characteristic for natural killer (NK) cells was expressed and later the CD3 marker characteristic for cytotoxic T cells became predominant. The conditioned media of LAK cells collected after interaction of LAK cells with K562 target cells was chromatographically separated into two cytotoxic fractions: F1 and F2. It was demonstrated that fraction F1 contained cytotoxic proteins having molecular weights of 30 and 40 kDa, and fraction F2 contained cytotoxic proteins having molecular weights of 22, 38 and 75 kDa. The presence of the proteins in each of these two fractions correlated with the phenotype changes of LAK cells: the F2 cytotoxic proteins were characteristic for NK-like cells, and the F1 proteins for cytotoxic T-lymphocyte (CTL)-like phenotypes.