A broad profile of co-dominant epitopes shapes the peripheral Mycobacterium tuberculosis specific CD8+ T-cell immune response in South African patients with active tuberculosis

Abstract

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.

Figure 1. Alignment of the major histocompatibility complex (MHC) class I-binding epitopes with the amino acid sequence of Rv1886c.

Epitopes identified for HLA-A*02:01 are shown in light blue, for A*24:02 in dark blue, for A*30:01 in dark green, peptides for A*30:02 in light green, for A*68:01 in yellow, for B*07:02 in orange, for B*58:01 in brown and for C*07:01 in red. Alignments of MHC class II restricted epitopes (HLA-DR1 – dark grey, DR2 – medium grey and DR4 – light grey) that have previously been reported in reference are included as well. The signal sequence of Rv1886c is shown in red and the rest of the protein in black.

Figure 2. Percentage multimer positive CD8+ T-cells divided into different compartments.

(A) Percentage antigen-specific recognition of the 45 individual multimers used in this study, each dot represents the staining in PBMCs from one patient and the different restricting alleles are shown in different colors (HLA-A*02:01 – black, A*24:02 – red, A*30:01 – orange, A*30:02 – green, A*68:01 – blue, B*07:02 – purple, B*58:01 – pink and C*07:01 – turquoise), each individual multimer within an allele is shown by different shapes of dots. (B) Average detection in all patients of antigen-specific T-cells divided per antigenic protein and per restricting allele (A*02:01 – black squares, A*24:02 – red triangles, A*30:01/A*30:02 – green triangles, A*68:01 – blue diamonds, B*07:02 – purple circles, B*58:01 – pink crosses and C*07:01 – turquoise crosses). (C) Individual detection of antigen-specific T-cells specific for the ‘super-epitope’ (QI)MYNYPAM(LG), each dot represents the staining in an individual patient, different colors represents the different restricting allele (A*02:01 – black, A*24:02 – red, A*30:01 – orange and A*30:02 – green).

Figure 3. Frequencies of total CD8+ T-cells as well as different compartments of antigen-specific CD8+ T-cells expressing differentiation and maturation markers.

(A) Total CD8+ T-cells (grey) vs. antigen-specific CD8+ T-cells (black) belonging to the different phenotypic compartments; naïve/precursor (CD45RA+CCR7+) (circles), central memory (CD45RA−CCR7+) (triangles), effector memory (CD45RA−CCR7−) (diamonds) and terminally differentiated cells (CD45RA+CCR7−) (squares). (B) Total CD8+ and antigen-specific T-cells belonging to the terminally differentiated compartment (CD45RA+CCR7−) and (C) effector memory compartment (CD45RA−CCR7−) divided per restricting MHC class I allele (Total CD8+ T-cells – filled circles, A*02:01 – filled squares, A*24:02 – filled triangles, A*30:01/A*30:02 – filled diamonds, A*68:01 – open circles, B*07:02 – open squares, B*58:01 – open triangles and C*07:01 – open diamonds). (D) Expression of the degranulation marker CD107a in total CD8+ T-cells (grey) vs. antigen-specific CD8+ T-cells (black). (E) Total CD8+ and antigen-specific T-cells expressing CD107a divided per restricting MHC class I allele (total CD8+ T-cells – filled circles, A*02:01 – filled squares, A*24:02 – filled triangles, A*30:01/A*30:02 – filled diamonds, A*68:01 – open circles, B*07:02 – open squares, B*58:01 – open triangles and C*07:01 – open diamonds). (F) Expression of the survival marker CD127 in total CD8+ T-cells (grey) vs. antigen-specific CD8+ T-cells (black). (G) Total CD8+ and antigen-specific T-cells expressing CD127 divided per restricting MHC class I allele (total CD8+ T-cells – filled circles, A*02:01 – filled squares, A*24:02 – filled triangles, A*30:01/A*30:02 – filled diamonds, A*68:01 – open circles, B*07:02 – open squares, B*58:01 – open triangles and C*07:01 – open diamonds). Each dot represents an individual multimer in one individual TB patient. Student's two-sided t-test was performed and significant values were calculated based on the following p-values: *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Antigen-specific recognition of the immunogenic epitopes from the H37Rv reference strain as well as the same epitopes containing naturally occurring amino acid substitutions.

(A) Percent antigen specific CD8+ T-cell recognizing the A*02:01 restricted epitopes (AML(G/D)HAGDM) (circles) and (ML(G/D)HAGDMA) (squares) and the A*24:02 restricted epitope (MYNYP(A/T)ML(G/D) (diamonds), wild-type epitopes (closed symbols) and variant epitopes (open symbols). (B) Representative figures of multimer staining regarding the A*24:02 restricted epitope (MYNYP(A/T)ML(G/D)). The x-axis shows the staining of the wt epitope and the y-axis staining the variant epitopes. Numbers of events for the wt/mut epitopes regarding patient 6088 were +– 162, –+ 181 and ++ 2. A similar T-cell distribution could be detected regarding patient 5905 (+– 17, –+ 21 and ++ 1).

Figure 5. Heat-map comparing epitope-MHC affinity, epitope-MHC dissociation rate and antigen-specific CD8+ T-cell recognition.

The affinity was divided into the following groups <10 nM (dark red), 10–70 nM (light red), 70–500 nM (orange), 500–1000 nM (dark yellow) and >1 µM (light yellow). The dissociation-rate (off-rate) were divided according to >10 h (dark red), 6–10 h (light red), 3–6 h (orange), 1.5–3 h (dark yellow) and <1.5 h (light yellow). Finally, the T-cell recognition was grouped as follows; >1% antigen-specific CD8+ T-cells recognizing the epitope (dark red), 0.6–1% (light red), 0.4–0.6% (orange), 0.2–0.4% (dark yellow) and <0.2% (light yellow).