Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata

Abstract

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.

Fig. 1.

Identification of a CD8 T cell target antigen by screening with T. annulata-specific T cells. COS-7 cells cotransfected with the N*02101 and N*02201 MHC class I heavy-chain cDNAs, along with 60 individual T. annulata cDNAs, were screened with an MHC A31-restricted CD8⁺ T cell line. Supernatants harvested after incubation of the T cells with the transfected cells for 24 h were screened for IFN-γ release by assaying induction of class II MHC expression on a bovine endothelial cell line. Levels of class II expression, measured by flow cytometry using a DR-specific monoclonal antibody (IL-A21), are expressed as percentages of positive cells based on gates set with unstimulated endothelial cells stained with the same antibody. A positive result was obtained with cDNA number 5 (TA17545). Two IFN-γ-positive control wells were included (*). A further 10 parasite cDNAs encoding orthologues of defined T. parva CD8 T cell antigens, screened separately, did not yield any positive results.

Fig. 2.

Localization of the Ta9 epitope-encoding region to a 130bp fragment. COS cells cotransfected with full-length cDNA or two overlapping cDNA fragments of TA15705 (Ta9), along with the N*01201 class I heavy chain cDNA, were screened for recognition by an A10-restricted CD8⁺ T cell line from animal 744, using an IFN-γ release assay. The results indicate that the epitope(s) is encoded within the 5′-terminal 130 bp.

Fig. 3.

Identification of the A10-restricted Ta9 epitope. A minimal length peptide representing the A10-restricted Ta9 epitope presented by the N*00201 class I gene product was identified by screening A10⁺ T. parva-infected cells pulsed with a range of concentrations of different length peptides, using an uncloned Tp9-specific CD8 T cell line in an ¹¹¹In release cytotoxicity assay.

Fig. 4.

Alignment of nucleotide sequences of alleles of Ta9 and Tp9. Comparison of aligned sequences of the genes encoding Ta9 and Tp9 reveals that the signal peptide sequence (black) is conserved among all sequences while gaps in the alignment (dark gray) were identified throughout the hypervariable central region.

Fig. 5.

Ta9 alleles display extensive polymorphism in amino acid sequence. (A) Amino acid alignment of the conserved N-terminal region of the genes encoding Ta9 and Tp9 showing the signal peptide, polymorphic sites within each species (gray), and alignment gaps (−). Sites conserved among T. annulata alleles are marked with an asterisk (*), and those with evidence of positive selection are marked with a plus symbol (+). The A10 and A14 epitopes are indicated in boxes. (B) Allelic variants of the two CD8 T cell epitopes. Residues differing from the C9 sequence are indicated in boldface.