Human <i>Mycobacterium tuberculosis</i> CD8 T Cell Antigens/Epitopes Identified by a Proteomic Peptide Library

<div><p>Identification of CD8⁺ T cell antigens/epitopes expressed by human pathogens with large genomes is especially challenging, yet necessary for vaccine development. Immunity to tuberculosis, a leading cause of mortality worldwide, requires CD8⁺ T cell immunity, yet the repertoire of CD8 antigens/epitopes remains undefined. We used integrated computational and proteomic approaches to screen 10% of the <i>Mycobacterium tuberculosis</i> (Mtb) proteome for CD8 Mtb antigens. We designed a weighting schema based upon a Multiple Attribute Decision Making:framework to select 10% of the Mtb proteome with a high probability of containing CD8⁺ T cell epitopes. We created a synthetic peptide library consisting of 15-mers overlapping by 11 aa. Using the interferon-γ ELISPOT assay and Mtb-infected dendritic cells as antigen presenting cells, we screened Mtb-specific CD8⁺ T cell clones restricted by classical MHC class I molecules (MHC class Ia molecules), that were isolated from Mtb-infected humans, against this library. Three novel CD8 antigens were unambiguously identified: the EsxJ family (Rv1038c, Rv1197, Rv3620c, Rv2347c, Rv1792), PE9 (Rv1088), and PE_PGRS42 (Rv2487c). The epitopes are B5701-restricted EsxJ_24–34, B3905-restricted PE9_53–67, and B3514-restricted PE_PGRS42_48–56, respectively. The utility of peptide libraries in identifying unknown epitopes recognized by classically restricted CD8⁺ T cells was confirmed, which can be applied to other intracellular pathogens with large size genomes. In addition, we identified three novel Mtb epitopes/antigens that may be evaluated for inclusion in vaccines and/or diagnostics for tuberculosis.</p></div

Number of genes according to functional category.

1<p>Functional categories assigned by TubercuList.</p

TubercuList Functional Score.

<p>TubercuList Functional Score.</p

Summary of CD8⁺ T cell epitopes identified.

1<p>Number of sister clones is in parentheses.</p>2<p>TubercuList accession numbers are EsxJ (Rv1038c), PE9 (Rv1088), and PE_PGRS42 (2487c).</p>3<p>IFN-γ spot forming units per 250,000 CD8⁺ T cells. IND, indeterminate.</p

Esx J family members.

<p>Esx J family members.</p

Epitope mapping of EsxJ-specific CD8⁺ T cell clones.

<p>To map the minimal epitopes of CD8⁺ T cell clones, A) D504 F9, B) 432 D8, and C) D432 H8, autologous LCL (20,000 cells/well) were pulsed with peptide at the concentrations indicated and co-cultured with T cells (1000 cells/well) in duplicate wells. IFN-γ was assessed by ELISPOT after 18 h co-culture. Each point represents the mean of duplicate determinations.</p

Screen of CFP10-specific CD8⁺ T cell clone, D432 A5, against peptide library.

<p>A) D432 A5, D432 D2, D432 E7, D432 E8, D432 H8 and D432 A11 T cells (5000 cells of each clone/well) were incubated with DC (20,000 cells/well) in the presence of the peptide pools (5 µg/ml, individual peptides) and IL-2 (0.5 ng/ml) in single wells in the IFN-γ ELISPOT assay. Positive well is plate 4 well A11. B) To identify the epitope recognized by T cell clone, D432 A5, T cells (5,000 cells/well) were incubated in single wells with autologous LCL (20,000 cells/well) and individual 15 aa peptides from Rv0377, Rv3763 and CFP10 (5 µg/ml) that together constitute the peptide pool from Plate 4 well A11. IFN-γ was assessed by ELISPOT after 18 hours of co-culture. Pictures of ELISPOT wells are shown.</p

Additional components of Composite Evidence Score.

<p>Additional components of Composite Evidence Score.</p

Mtb infection alters the HLA-E host ligand repertoire and results in a minor Mtb-specific ligand pool.

<p>Percentage of total ion intensity as determined in the materials and methods section of indicated HLA-E ligand groups from uninfected (A) or Mtb infected cells (B).</p

Mtb ligands are recognized by human Mtb-reactive CD8⁺ T cells.

<p>CD8⁺ T cells from four healthy, eight LTBI, and four Mtb-infected donors were incubated in an IFN-γ ELISPOT plate with autologous DC pulsed with each of the 28 validated Mtb peptides. Shown are the IFN-γ spot forming units for the 12 peptides for which at least one donor responded.</p