CD8+ T-cell responses to Theileria parva are preferentially directed to a single dominant antigen: Implications for parasite strain-specific immunity

Abstract

Although immunodominance of CD8(+) T-cell responses is a well-recognised feature of viral infections, its role in responses to more antigenically complex pathogens is less clear. In previous studies we have observed that CD8(+) T-cell responses to Theileria parva exhibit different patterns of parasite strain specificity in cattle of different MHC genotypes. In the current study, we demonstrated that animals homozygous for the A10 and A18 MHC haplotypes have detectable responses to only one of 5 T. parva antigens. Over 60% of the responding T cells from the A18(+) and A10(+) animals recognised defined epitopes in the Tp1 and Tp2 antigens, respectively. Comparison of T-cell receptor beta chain expression profiles of CD8(+) T-cell lines and CD8(+) T cells harvested ex vivo confirmed that the composition of the T-cell lines was representative of the in vivo memory CD8(+) T-cell populations. Analysis of the Tp1 and Tp2 antigens revealed sequence polymorphism, which was reflected by differential recognition by T-cell lines. In conclusion, we have demonstrated a profound immunodominance in the CD8(+) T-cell response to T. parva, which we propose is a major determinant of the parasite strain specificity of the response and hence immune protection.

Figure 1

Recognition of the Tp1_214–224 epitope by CD8⁺ T-cell clones derived from animal 641 (A18/A18): The clones were tested using a 4-h ¹¹¹In-release assay with MHC class I A18⁺, T. annulata-infected target cells alone or incubated with 1 μg/mL of Tp1_214–224 peptide. Results are shown for target cells pulsed with Tp1_214–224 peptide. The level of killing of unpulsed T. annulata-infected target cells was <1% for all clones (data not shown).

Figure 2

Recognition of the Tp2_49–59 and Tp2_98–106 epitopes by CD8⁺ T-cell clones derived from animal 1011 (A10/A10): The clones were tested using a 4-h ¹¹¹In-release assay with MHC class I A10⁺, T. annulata-infected target cells incubated with 1 μg/mL of peptide. Results are shown for targets pulsed with Tp2_98–106 peptide (A) or with Tp2_49–59 peptide (B).

Figure 3

Tp2-specific CD8⁺ T-cell clones show different levels of cytolytic activity for target cells incubated with Tp2_49–59-peptide: Four T-cell clones (solid symbols) were tested in a 4-h ¹¹¹In-release assay using MHC class I A10⁺, T. annulata-infected target cells incubated with 1 μg/mL Tp2_49–59 peptide. Results obtained using the clones at a range of effector to target ratios are shown (error bars – 95% confidence intervals). Results are also shown for one of the clones tested against the same target cells without added peptide (open squares); similar negative results were obtained with the other clones tested on unpulsed target cells. These assays have been repeated utilising a narrower range of effector to target ratios (2:1 to 0.5:1) and, although the maximal levels of cytotoxicity showed small differences, the relative ranking of levels of cytotoxicity of the clones was similar.

Figure 4

TCR CDR3β heteroduplex analysis of bovine CD8⁺ T-cell responses to T. parva: Results obtained for each Vβ subgroup are shown for CD8⁺ T cells harvested ex vivo 10 days after parasite challenge of animal 592 (A) and with a CD8⁺ T-cell line derived from the same animal prior to challenge (B). Homoduplexes formed by re-annealing of the carrier DNA strands are represented by a prominent band towards the bottom of each lane. Additional more slowly migrating bands in the upper part of the gel represent heteroduplexes formed from cDNA of expanded clonotypes expressing Vβ genes of the respective subgroup.

Figure 5

TCR CDR3β heteroduplex analysis of bovine CD8⁺ T-cell responses to T. parva: Results are shown for CD8⁺ T cells from animal 641 (A) analysed for subgroups Vβ3 and Vβ16, and for CD8⁺ T cells from animal 011 (B) analysed for subgroups Vβ14 and Vβ28. Lanes labelled Pr represent resting CD8⁺ memory T cells harvested ex vivo prior to parasite challenge, those labelled Po represent CD8⁺ T cells harvested ex vivo 10 days following challenge and those labelled IV represent a CD8⁺ T-cell line derived immediately prior to challenge. Lanes labelled Ca show the respective heteroduplex carriers loaded with dH20 as a control for bands not related to T-cell populations. Homoduplexes formed by re-annealing of the carrier DNA strands are represented by a prominent band towards the bottom of each lane. Additional more slowly migrating bands in the upper part of the gel represent heteroduplexes formed from cDNA of expanded clonotypes expressing Vβ genes of the respective subgroup.

Figure 6

Differential recognition of Tp1 alleles, expressed by the Muguga and Marikebuni isolates of T. parva, by a cloned parasite-specific CD8⁺ T-cell line from animal BV115 (A10/A18): (A). A 4-h ⁵¹Cr-release cytotoxicity assay was used to test reactivity with different parasitised cell lines. Target cells consisted of Muguga-infected cell lines derived from the autologous animal (BV115 Tp Mug), an A18-homozygous animal (4229 Tp Mug) and a class I MHC-mismatched (−/−) animal (BV57 Tp Mug), and a Marikebuni-infected cell line from the autologous animal (BV115 Tp Mar). (B). An IFN-γ ELISpot assay was used to examine recognition of Tp1 cDNA expressed in COS-7 cells. Results (error bars – 95% confidence intervals) are shown for cells co-transfected with the N^*01301 class I heavy chain together with Muguga Tp2 cDNA (COS Tp2Mug), Muguga Tp1 cDNA (COS TP1Mug) or Marikebuni Tp1 cDNA (COS Tp1Mar). Results obtained using cells infected with T. parva (Muguga) (TpMug) as targets are shown for comparison. Data are representative of two independent experiments.

Figure 7

Differential recognition of alleles of the Tp2_49–59 epitope by two specific CD8⁺ T-cell clones derived from animal 592 (A10/A10): The clones were tested in a 4-h ¹¹¹In-release cytotoxicity assay using autologous T. annulata-infected target cells incubated with 1 μg/mL peptide. Results are shown (error bars – 95% confidence intervals) for peptides representing the Muguga Tp2_49–59 epitope (squares) and the two allelic variants identified in the Nyairo BR187 (diamonds) and Kakuzi 498 (triangles) T. parva isolates (see Table 4). Data are representative of two independent experiments. Similar results have also been obtained with additional T-cell clones.