Demonstration of strain-specific CD8 T cell responses to Theileria annulata

Abstract

The present study set out to examine the nature and specificity of the bovine CD8 T cell response at the clonal level in a group of eight animals immunized with a cloned population of Theileria annulata. The results demonstrated that immunized animals generated parasite-specific CD8 T cells that produced IFN in response to parasite stimulation but had highly variable levels of cytotoxicity for parasitized cells. The study also demonstrated that these parasite-specific CD8 T cells could be propagated and cloned in vitro from the memory T cell pool of cattle immunized with live T. annulata parasites. Within the small group of animals studied, there was evidence that responses were preferentially directed to antigens presented by an A10+ class I major histocompatibility complex (MHC) haplotype, suggesting that responses restricted by products of this haplotype may be dominant. The A10-restricted responses showed differential recognition of different parasite isolates and clones. By using a cloned population of parasites both for immunization of the animals and for in vitro analyses of the responses, we obtained unambiguous evidence that at least a proportion of CD8 T cells restricted by one MHC haplotype were parasite strain restricted.

Figure 1

(Top) FACS analysis of PBM from animal 744 after 2 rounds of re-stimulation with a T. annulata Ankara (C9) infected A10 homozygous cell line prior to enrichment of the CD8 component by mAb and complement mediated lysis. (Lower) FACS analysis of PBM after lysis of the CD4 and γδ populations showing enrichment of the CD8 population from 25 to 88%. The Y axis shows log of fluorescence height and the X-axis shows forward scatter. Percentage of positive cells for each antibody is shown in the upper left hand corner of each graph.

Figure 2

MHC restriction of CD8⁺ CTL clones 744·5 and 744·6. The cytotoxicity of two clones from animal 744 was analysed on autologous T. annulata and T. parva infected cell line targets, MHC – mismatched, and homozygous A10 T. annulata infected target cell lines.

Figure 3

IFNγ release assay of MHC restriction of CD8⁺ clone 663·14: Dot plot FACS profiles of bovine endothelial cells stained with class II MHC-specific mAb IL-A21 showing a representative analysis of IFNγ release by a CD8⁺ clone. From left to right, cells cultured with supernatant from clone 663·14 incubated with an MHC-mismatch target, the autologous target, an A18 homozygous target, and an A14 homozygous target. All targets were infected with the T. annulata Ankara C9 stock.

Figure 4

Cytotoxicity of a panel of 4 clones from animals 219, 744, and 1147 assayed on MHC –matched target cell lines infected with the cloned T. annulata stock C9 (TaA-C9) , Uncloned Ankara isolate (TaA) Gharb isolate (TaG) , or Hissar isolate (TaH) .

Figure 5

Cytotoxic activity of 3 CD8⁺ CTL clones tested on MHC-matched targets infected with the cloned T. annulata Ankara C9 parasite (C9) and two clones derived from a cell line infected with the Gharb isolate TaG.1 and TagG.5.