Extensive major histocompatibility complex class I binding promiscuity for Mycobacterium tuberculosis TB10.4 peptides and immune dominance of human leucocyte antigen (HLA)-B*0702 and HLA-B*0801 alleles in TB10.4 CD8 T-cell responses

Abstract

The molecular definition of major histocompatibility complex (MHC) class I-presented CD8(+) T-cell epitopes from clinically relevant Mycobacterium tuberculosis (Mtb) target proteins will aid in the rational design of T-cell-based diagnostics of tuberculosis (TB) and the measurement of TB vaccine-take. We used an epitope discovery system, based on recombinant MHC class I molecules that cover the most frequent Caucasian alleles [human leucocyte antigen (HLA)-A*0101, A*0201, A*0301, A*1101, A*2402, B*0702, B*0801 and B*1501], to identify MHC class I-binding peptides from overlapping 9-mer peptides representing the Mtb protein TB10.4. A total of 33 MHC class I-binding epitopes were identified, spread across the entire amino acid sequence, with some clustering at the N- and C-termini of the protein. Binding of individual peptides or closely related peptide species to different MHC class I alleles was frequently observed. For instance, the common motif of xIMYNYPAMx bound to six of eight alleles. Affinity (50% effective dose) and off-rate (half life) analysis of candidate Mtb peptides will help to define the conditions for CD8(+) T-cell interaction with their nominal MHC class I-peptide ligands. Subsequent construction of tetramers allowed us to confirm the recognition of some of the epitopes by CD8(+) T cells from patients with active pulmonary TB. HLA-B alleles served as the dominant MHC class I restricting molecules for anti-Mtb TB10.4-specific CD8(+) T-cell responses measured in CD8(+) T cells from patients with pulmonary TB.

Figure 1

Major histocompatibility complex (MHC) class I-binding peptides identified by binding to recombinant MHC class I molecules. Peptide binding to (a) human leucocyte antigen (HLA)-A molecules and (b) HLA-B molecules. The x-axis shows the peptide ID 1–88 (the aa sequence for the positive binders can be found in Table 1) and the z-axis shows the per cent binding for the respective peptides compared with an allele-specific positive control peptide. In (a), yellow colour indicates binding to HLA-A*0101, light blue binding to A*0201, dark blue binding to A*0301, light green binding to A*1101 and dark green binding to A*2402. In (b), yellow indicates binding to B*0702, red binding to B*0801 and brown binding to B*1501.

Figure 2

Alignment of the major histocompatibility complex (MHC) class I-binding peptides within the amino acid sequence of TB10.4. Peptides identified for HLA-A*0101 are shown in light blue, peptides for A*0201 are in dark blue, peptides for A*0301 are in purple, peptides for A*1101 are in pink, peptides for A*2402 are in yellow, peptides for B*0702 are in light green and peptides for B*1501 are in dark green. Clustering of peptides around the N- and C-termini and extensive ‘cross-binding’ of single peptides to different MHC class I alleles can be seen.

Figure 3

Affinity and off-rate curves for a set of TB10.4 candidate peptides and human leucocyte antigen (HLA) HLA-A*0201. Raw data for (a) affinity and (b) off-rate are shown for a representative major histocompatibility complex (MHC) class I allele (HLA-A*0201) with five individual candidate peptides. The affinity graph shows individual peptide binding for different peptide concentrations (in m) compared with a positive control. The 50% effective dose (ED₅₀) value can be calculated using sigmoidal curve-fitting. The off-rate graph shows in a similar manner individual peptide off-rates (in hours) compared with an allele-specific positive control; the half life (t_½) value was calculated using sigmoidal curve-fitting. Curves were fitted using Graphpad prism software.