Characterization of CD4 T cell responses using epitope pools.

<p>(A) Gating strategy for ICS assay. SSC-A; Side-scatter area, FSC-A; Forward-scatter area, SSC-W; Side-scatter width, FSC-W; Forward-scatter width, LD; Live/Dead discrimination. (B) Percentage cytokine detected from CD3⁺CD4⁺ T cells in response to the pool of 66, 125 and 300 epitopes, as well as heat killed H37Rv Mtb lysate. Each dot represents one donor (n = 34) median ± interquartile range is indicated. (C) Percentage Epitope pool-specific (125 epitopes) IFNγ, TNFα, IL-2 and IL-22 production by CD3⁺CD4⁺ T cells expressing each of the fifteen possible combinations. Each dot represents one donor (n = 34) median ± interquartile range is indicated. (D) The fraction of the total cytokine response against each stimuli expressing each combination of cytokines (pie chart) and all 4, 3, 2 or 1 cytokine (outer circle).</p

Breadth and dominance of T cell epitopes in Mtb antigens.

<p>(A) All epitopes (n = 125) as a percentage of the total magnitude of response ranked on the basis of magnitude of T cell response and the 66 epitopes identified from TB vaccine and IGRA antigens and previously described epitopes. The dotted line indicates the 66 most immunodominant epitopes. (B) Proportion of the 63 donors who respond to the indicated number of epitopes of the top 66 identified epitopes.</p

HLA restriction and penetrance of dominant epitopes.

<p>HLA restriction and penetrance of dominant epitopes.</p

Hierarchy in T cell reactivity against TB Vaccine and IGRA antigens.

<p>Magnitude of responses, expressed as the total magnitude of response (black bars, left y-axis) or frequency of donors responding (grey bars, right y-axis), amongst the 63 donors. Rv number and synonyms for each antigen are indicated on the x-axis. Antigens were divided into protein categories as defined by Tuberculist [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005760#ppat.1005760.ref036" target="_blank">36</a>]. All five antigens that are part of the cell wall and cell processes category are involved in the type VII secretion system [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005760#ppat.1005760.ref037" target="_blank">37</a>].</p

Promiscuity of HLA restrictions.

<p>Number of epitopes identified (left y-axis) and the corresponding number of restricting HLA alleles per epitope (x-axis). Percentage of epitopes (right y-axis) restricted by ≥ indicated number of HLA alleles. All restrictions identified (open bars and circles). Restrictions in epitopes tested in ≥2 subjects (black bars and squares).</p

Demographic characteristics of enrolled participants.

<p>Demographic characteristics of enrolled participants.</p

HLA genotype and phenotype frequencies, and proportions of identified epitope restrictions.

<p>HLA genotype and phenotype frequencies, and proportions of identified epitope restrictions.</p

Responses to TB Vaccine and IGRA antigens are highly IFNγ polarized.

<p>Responses to TB Vaccine and IGRA antigens are highly IFNγ polarized.</p

Determination of HLA restriction of Mtb epitopes using HLA-transfected cell lines.

<p>Representative examples showing determination of HLA restriction for two epitopes in three donors. (A-C) PBMCs were incubated with peptide-pulsed cell lines transfected with each individual HLA molecule that matched the HLA alleles of the PBMC donor. IFNγ release was measured by ELISPOT. Positive responses (black bars, p<0.05), negative responses (white bars). N/A indicates cell line not available for the HLA allele.</p

Memory T Cells in Latent <em>Mycobacterium tuberculosis</em> Infection Are Directed against Three Antigenic Islands and Largely Contained in a CXCR3⁺CCR6⁺ Th1 Subset

<div><p>An understanding of the immunological footprint of <em>Mycobacterium tuberculosis</em> (MTB) CD4 T cell recognition is still incomplete. Here we report that human Th1 cells specific for MTB are largely contained in a CXCR3⁺CCR6⁺ memory subset and highly focused on three broadly immunodominant antigenic islands, all related to bacterial secretion systems. Our results refute the notion that secreted antigens act as a decoy, since both secreted proteins and proteins comprising the secretion system itself are targeted by a fully functional T cell response. In addition, several novel T cell antigens were identified which can be of potential diagnostic use, or as vaccine antigens. These results underline the power of a truly unbiased, genome-wide, analysis of CD4 MTB recognition based on the combined use of epitope predictions, high throughput ELISPOT, and T cell libraries using PBMCs from individuals latently infected with MTB.</p> </div