Systematic Determination of TCR–Antigen and Peptide–MHC Binding Kinetics among Field Variants of a Theileria parva Polymorphic CTL Epitope

Nicholas Svitek,Lucilla Steinaa,Rosemary Saya,Houshuang Zhang,Vishvanath Nene

doi:10.4049/jimmunol.2100400

Abstract

CTLs are known to contribute to immunity toward Theileria parva, the causative agent of East Coast fever. The Tp967-75 CTL epitope from the Muguga strain of T. parva is polymorphic in other parasite strains. Identifying the amino acids important for MHC class I binding, as well as TCR recognition of epitopes, can allow the strategic selection of Ags to induce cellular immunity toward T. parva In this study, we characterized the amino acids important for MHC class I binding and TCR recognition in the Tp967-75 epitope using alanine scanning and a series of variant peptide sequences to probe these interactions. In a peptide-MHC class I binding assay, we found that the amino acids at positions 1, 2, and 3 were critical for binding to its restricting MHC class I molecule BoLA-1*023:01. With IFN-γ ELISPOT and peptide-MHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 5-8 in the epitope were required for TCR recognition. Only two of eight naturally occurring polymorphic Tp9 epitopes were recognized by both CTLs. Finally, using a TCR avidity assay, we found that a higher TCR avidity was associated with a stronger functional response toward one of two variants recognized by the CTL. These data add to the growing knowledge on the cross-reactivity of epitope-specific CTLs and specificities that may be required in the selection of Ags in the design of a wide-spectrum vaccine for East Coast fever.

Highlights

We had defined by ELISPOT and Tet staining a 10-mer peptide from the T. parva (Muguga) Tp9 Ag (67AKFPGMKKSK76) that is recognized by a CTL line from animal 495 vaccinated by infection and treatment method (ITM) that kills autologous schizontinfected cells
The binding assay clearly shows that deletion of 1 or 2 aa at the N-terminal end (Tp96876 and Tp96976) completely prevents peptide binding to this bovine leukocyte antigen” (BoLA) molecule because no folding can be measured, but that deletion of 1 or 2 aa at the C-terminal end does not (Tp96774 and Tp96775)
Because it was difficult to determine which was the authentic minimal epitope recognized by this cell line, a peptideMHC class I staining was done using combinations of Tets harboring the different 8, 9, and 10-mer Tp9 peptides, which were positive in the IFN-g ELISPOT assay

Summary

Introduction

With IFN-g ELISPOT and peptideMHC class I Tet staining assays on two parasite-specific bovine CTL lines, we showed that amino acids at positions 58 in the epitope were required for TCR recognition. We assessed the capacity of two Tp9Muguga-specific CTL lines, generated from cattle of the A14 BoLA serotype immunized with the T. parva Muguga strain with specificity for Tp9 to cross-react with naturally occurring Tp9 epitope variants.

Results

Conclusion