Use of "one-pot, mix-and-read" peptide-MHC class I tetramers and predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle

Abstract

Peptide-major histocompatibility complex (p-MHC) class I tetramer complexes have facilitated the early detection and functional characterisation of epitope specific CD8+ cytotoxic T lymphocytes (CTL). Here, we report on the generation of seven recombinant bovine leukocyte antigens (BoLA) and recombinant bovine β2-microglobulin from which p-MHC class I tetramers can be derived in ~48 h. We validated a set of p-MHC class I tetramers against a panel of CTL lines specific to seven epitopes on five different antigens of Theileria parva, a protozoan pathogen causing the lethal bovine disease East Coast fever. One of the p-MHC class I tetramers was tested in ex vivo assays and we detected T. parva specific CTL in peripheral blood of cattle at day 15-17 post-immunization with a live parasite vaccine. The algorithm NetMHCpan predicted alternative epitope sequences for some of the T. parva CTL epitopes. Using an ELISA assay to measure peptide-BoLA monomer formation and p-MHC class I tetramers of new specificity, we demonstrate that a predicted alternative epitope Tp229-37 rather than the previously reported Tp227-37 epitope is the correct Tp2 epitope presented by BoLA-6*04101. We also verified the prediction by NetMHCpan that the Tp587-95 epitope reported as BoLA-T5 restricted can also be presented by BoLA-1*02301, a molecule similar in sequence to BoLA-T5. In addition, Tp587-95 specific bovine CTL were simultaneously stained by Tp5-BoLA-1*02301 and Tp5-BoLA-T5 tetramers suggesting that one T cell receptor can bind to two different BoLA MHC class I molecules presenting the Tp587-95 epitope and that these BoLA molecules fall into a single functional supertype.

Figure 1

Tp1_214-224-BoLA-6*01301 p-MHC class I tetramer staining of bovine CTL line BB007. The CTL line was stained with buffer alone (panel A), p-MHC class I tetramers made with the control peptide, YMYRVWSPL (panel B), p-MHC class I tetramers made with the peptide Tp2_96-104, which is restricted by another BoLA molecule [BoLA-T2c] (panel C), p-MHC class I tetramers made in the absence of peptide (panel D) and p-MHC class I tetramers made with peptide Tp1_214-224 (panel E). All tetramers were generated with PE-streptavidin.

Figure 2

Assay of the panel of p-MHC class I tetramers on different antigen specific CTL lines. In each panel the left graph represents unstained negative control cells and the right graph p-MHC class I tetramer stained cells. Panel A: Tp1_214-224-BoLA-6*01301 tetramers on cell line BV115; Panel B: Tp2_49-59-BoLA-2*01201tetramers on cell line 592; Panel C: Tp2_96-104-BoLA-T2c tetramers on cell line Bx196; Panel D: Tp5_87-95-BoLA-T5 tetramers on cell line BV50; Panel E: Tp8_379-387-BoLA-3*00101 tetramers on cell line Bx63; Panel F: Tp9_67-76-BoLA-1*02301 tetramers on cell line 495. All tetramers were generated with PE-streptavidin.

Figure 3

Definition of an alternative Tp2 epitope that binds to BoLA-6*04101. Panel A: p-MHC class I monomer formation ELISA assay using Tp2_27-37 and Tp2_29-37 peptides. Panel B: ELISpot performed on two clones (5 and 7) of the cell line BW02 with cell media alone, Tp1_214-224, Tp2_27-37 or Tp2_29-37 peptides. Data are presented as spot forming units (SFU) and each well contained 2.5 × 10⁴ CTLs. Asterisk (*) indicates saturated wells. Panel C: Two clones of the cell line BW02 was stained with anti-bovine CD8 (PerCP) and different BoLA-6*04101 tetramers. The unstained control or stained with the unfolded (no peptide) BoLA-6*04101 tetramer are depicted on the left. Tetramer labelling was measured on the CD8-gated cell population.

Figure 4

Multiple staining of Tp1_214-224 peptide-MHC tetramer positive cells. In each panel the left graph represents unstained negative control cells. The cell line BB007 was stained with Tp1_214-224-BoLA-6*01301 tetramer in conjunction with anti-bovine CD8 (panel A), anti-Fas-ligand (panel B), or anti-perforin (panel C). Panel D: BB007 CTL line stained with Tp1_214-224-BoLA-6*01301 tetramer and with anti-bovine CD8 and anti-perforin. Data in Panels A, B and C, and data in Panel D are from two independent experiments.

Figure 5

Identification of Tp1_214-224 peptide-MHC tetramer positive cells in ex vivo assays. PBMC were isolated from three BoLA-A18 cattle vaccinated with the live T. parva (Muguga 3308 strain) and stained with Tp1_214-224-BoLA-6*01301 tetramers at day 8 and day 15-17 post infection (pi). Tetramer labelling was measured on the CD8-gated population.

Figure 6

MHCcluster prediction of the functional similarity of BoLA-T5 and BoLA-1*02301. Panel A: UPGMA tree-based clustering of the relationship between the seven BoLA molecules (sequence logo representations of the predicted binding motifs of BoLA-T5 and BoLA-1*02301 were generated by MHCcluster using Seq2Logo [52]); Panel B; heat-map visualization of the degree of functional relationship between the seven BoLA molecules, red colour shows closer functional relationship.

Figure 7

The Tp5_87-95 epitope is presented by two BoLA molecules (BoLA-T5 and BoLA-1*02301). Panel A: predicted binding affinity of Tp5_87-95 peptide epitope for BoLA-1*02301 and BoLA-T5; Panel B: The cell line BV50 stained with anti-bovine CD8 (PerCP) and Tp5_87-95 p-MHC class I tetramers made with BoLA-1*02301 or BoLA-T5. Panel C: The cell line BV50 co-stained with anti-bovine CD8 (PerCP), Tp5_87-95-BoLA-1*02301 tetramers (PE) and Tp5_87-95-BoLA-T5 tetramers (APC). Tetramer labelling was measured on the CD8-gated population.

Figure 8

Sequence alignment of BoLA-T5 and BoLA-1*02301 molecules and identification of amino acid differences in the peptide-binding groove. Panel A: MUSCLE alignment of BoLA-T5 and BoLA-1*02301 amino acid sequence; differences in amino acid sequence is highlighted by the red letters; asterisks (*) in red indicate amino acids that map to the pseudo-sequence of 34 amino acids that were defined as in contact with peptide; sequence highlighted in yellow indicate the signal peptide; sequence highlighted in grey indicate the α1 domain; sequence highlighted in blue indicate the α2 domain; sequence highlighted in purple indicate the α3 domain. Panel B: Ribbon drawing of the BoLA-2*01801 [43] molecule with position of all amino acid differences in the α2 (highlighted in blue) and α3 (highlighted in purple) domains between BoLA-T5 and BoLA-1*02301. Amino acids that map to the pseudo-sequence are illustrated in red; amino acids that do not map to the pseudo-sequence are illustrated in green. The β2m molecule is illustrated in orange and the peptide in black.