Generation of bovine cytotoxic cell lines, specific for cells infected with the protozoan parasite Theileria parva and restricted by products of the major histocompatibility complex

Abstract

Cytotoxic cell lines specific for cells infected with the protozoan parasite Theileria parva were generated in vitro by repeated stimulation of peripheral blood mononuclear cells (PBMC) from immune animals with autologous parasitized cells from continuously growing cell lines. In PBMC of 5/7 immune animals tested, maximal levels of killing (80-100%) were obtained at effector to target ratios of 10/1 following 3 to 5 stimulations. No killing of uninfected lymphoblasts was observed. Cytotoxic activity was contained in the BoT4- lymphocyte population. The results of analyses of cytotoxicity on panels of target cells of diverse major histocompatibility (MHC) phenotypes indicated that the effectors were restricted by class I MHC antigens. Only autologous targets and allogenic targets sharing one or other bovine lymphocyte antigen A locus-encoded specificity with the cytotoxic cell line were killed. In addition, two of the three cytotoxic cell lines, when tested on targets infected with different parasite stocks, exhibited a high degree of specificity for the stock used for immunization and stimulation of the cultures. The capacity to generate highly cytotoxic, MHC-restricted effectors from peripheral blood was dependent on the immune status of the donor animals. This was confirmed by comparing cytotoxicity generated from the same animal before and after immunization using the same autologous infected cell line as a stimulator. These results indicate that these effector cells are analogous to the cytotoxic cells detected in vivo in cattle undergoing immunization or challenge with T. parva.

Techniques for the generation, cloning, and characterization of bovine cytotoxic T cells specific for the protozoan Theileria parva

“…These stocks were selected on the basis of cross-protection experiments, in which it has been shown that the Marikebuni stock protected agginst challenge with the Muguga stock (S. Morzaria, unpublished data), whereas the Muguga stock did not always protect against challenge with the Marikebuni stock I12). Cytotoxic cell lines generated from animals that had 108 been immunized with the Marikebuni stock of the parasite were not restricted to the immunizing strain (16), whereas cytotoxic T cell lines generated from animals immunized with the Muguga stock were, depending on the animal, either restricted (9) or not restricted to the immunizing stock 19, unpublished data). A major problem in interpreting these results is that with uncloned cell lines, the response detected is a combined result of different cells, possibly having different specificities.…”

“…First, in contrast to Theileria-specific cytotoxic T cells generated in vivo, which are detected in the blood only for a few days after immunization or challenge of immune animals {7, 8,17), the capacity to generate effector cells in vitro weeks or months after immunization permits repeated access to the same animal, which enables in depth analyses of the response to be carried out. Second, due to the increase in the frequency of the cytotoxic T cells achieved during restimulation, higher levels of eytotoxicity are attained, thus providing greater sensitivity in experiments directed at analyzing the specificity of the response (9,18) (Fig. 1).…”

“…The cytotoxic activity of the Theileria-specific cytotoxic cell lines resided in the BoT4-BoT8 ÷ population and was restricted to autologous and BoLA-A-matched targets (9). Most of the killing was restricted to one of the BoLA haplotypes in each animal.…”

Escape from CD8+ T Cell Response by Natural Variants of an Immunodominant Epitope from Theileria parva Is Predominantly Due to Loss of TCR Recognition

“…Cattle that naturally recover from infection or are immunized by infection and treatment develop long-lasting immunity to the homologous parasite isolate, but show incomplete immunity to heterologous isolates (22). Observations on the kinetics of the CD8 + T cell response, coupled with the demonstration that immunity can be adoptively transferred from immune to naive twin calves by highly enriched populations of CD8 + T cells, indicate that immunity is mediated by CD8 + T cells specific for the intralymphocytic schizont stage of the parasite (23)(24)(25). Moreover, the parasite strain specificity of CD8 + T cell responses induced by immunization with a single parasite strain has been shown to correlate with protection against challenge with a heterologous parasite strain (26,27).…”

Theileria parva : the nature of the immune response and its significance for immunoprophylaxis

“…This effect was interpreted as being a reflection of a hierarchy in dominance among the BoLA products which restrict the response. A subsequent study of the phenotype of the cells which mediate this class I MHC-restricted cytolytic activity (41) has demonstrated that they are CD8+ T lymphocytes.…”