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

Immunodominance of islands, PE/PPE, Esx and T7SS proteins.

a<p>) Esx proteins include EsxA-W.</p>b<p>) T7SS includes the Esx proteins.</p

Antigens cluster in antigenic islands in the MTB genome.

<p>(A) All antigens recognized on the H37Rv genome map, % donors responding (black bars) and % of total response (red dotted line). (B) Antigenic islands identified by a 5-gene window spanning the entire MTB genome (top panel); Binomial distribution and Bonferroni correction, *, p<0.01. Proteins within each antigenic island, % donors responding (black bars) and % of total island response (grey bars) and the % of total (all antigens recognized) response per island (middle panel). Cartoons show relative length of proteins, direction of transcription and protein category of each protein. Esx proteins are part of the cell wall and cell processes category.</p

Cell wall/cell processes and PE/PPE specific CD4 T cells have a multifunctional phenotype.

<p>Epitope-specific IFN-γ, TNFα and IL-2 production by PBMCs from LTBI donors measured after 6 h peptide stimulation. (A, C) % of responding CD4⁺ expressing each of the seven possible combinations of IFN-γ, TNFα and IL-2 (A) cell wall and cell processes proteins, (C) PE/PPE proteins. Island proteins (black dots) and non-island (grey dots). Each dot represents one donor/epitope combination median ± interquartile range is indicated. (B, D) The fraction of the total cytokine response against (B) cell wall and cell processes, (D) PE/PPE proteins, expressing all 3, 2 or 1 cytokine. (E) Heat-map of each of the seven possible combinations of IFN-γ, TNFα and IL-2 for each individual donor and epitope tested grouped by protein category and island localization. Each column represents one donor. Colors indicate frequency of epitope-specific CD4 T cells, grey is considered negative for indicated cytokine production. Multiple proteins indicate that the peptide sequence is homologous in these proteins.</p

Summary of characteristics of novel CD4 T cell antigens recognized by more than 10% of LTBI donors, ranked according to response frequency.

<p>Summary of characteristics of novel CD4 T cell antigens recognized by more than 10% of LTBI donors, ranked according to response frequency.</p

The T cell library approach complements the ex vivo IFN-γ ELISPOT assay.

<p>CCR6⁺CXCR3⁺ T cell libraries were set up for 4 representative donors. The sorted T cells were polyclonally expanded and analyzed for the presence of antigen-specific T cells by stimulation with peptide pools and measurement of ³H-thymidine incorporation. Shown is proliferation (cpm) of individual cultures from 4 different donors. Dotted lines represent the cut-off value. Response to antigens within genomic islands is shown in yellow, orange, and red; response to antigens outside antigenic islands is shown in white. Antigenic islands are indicated by capped lines.</p

The T cell response to MTB is restricted to a CXCR3⁺CCR6⁺ memory subset.

<p>(A, B) Three CD45RA⁻CD25⁻CD4⁺ memory T cell subsets from four LTBI donors were sorted; 1) CCR6⁺CXCR3⁻; 2) CCR6⁺CXCR3⁺; and 3) CCR6⁻. (A) Representative dot plot from one donor; (B) Median percentages of the T cell subsets on total CD4⁺ memory T cells. Error bars indicate interquartile range (n = 4). (C) T cell libraries were set up from the sorted subsets by polyclonal stimulation and expansion for 3–4 weeks. Libraries were analyzed by stimulation with autologous monocytes with or without MTB whole cell lysate and proliferative response was measured by ³H-thymidine incorporation. Shown is the estimated frequency of MTB-specific T cells per 10⁶ CD4 memory T cells for LTBI donors. (D) Distribution of MTB-specific T cells in the three memory T cell subsets. Data represent median ± interquartile range from four donors. Mann Whitney test, *, <i>p</i><0.05.</p

Breadth and dominance at the epitope and antigen level.

<p>(A) Epitopes ranked on the basis of magnitude of response. LTBI (black line - % of total response, grey line – total SFC) and TB uninfected (grey dashed line – total SFC) donors. Black dashed lines indicate the top 80 and 175 epitopes. (B) Antigens ranked on the basis of the response frequency for LTBI donors. Black dashed line indicates antigens recognized by >10% of LTBI donors. (C) Antigens ranked on the basis of magnitude of response and response frequency (black line - % of total response, grey line – total SFC). Black dashed line indicates the top 82 antigens.</p

Memory phenotype of MTB-specific CD4 T cells using HLA class II tetramers.

<p>(A) HLA class II tetramer stained CD4-purified cells from LTBI donors. Tetramer⁺ cells were isolated following magnetic bead enrichment. Plots are gated on CD4⁺ T cells, and the numbers indicate the percentage of tetramer⁺ cells isolated from each of 4 representative donors CD4⁺ population. DPB1*04:01 AGCQTYKWETFLTSE n = 4 donors, DRB1*15:01 MHVSFVMAYPEMLAA n = 3, DRB1*15:01 MSQIMYNYPAMMAHA n = 5 and DRB1*01:01 GEEYLILSARDVLAV n = 2. (B) Memory phenotype of tetramer⁺ cells for one representative donor per tetramer. Plots are gated on total CD4⁺ T cells (black background) or epitope-specific CD4⁺ T cells (red dots). The numbers represent the percentages of tetramer⁺CD4⁺ T cells in the gate. (C) Scatter plot of the proportion of CCR7⁻CD45RA⁻ (effector memory), CCR7⁺CD45RA⁻ (central memory), CCR7⁺CD45RA⁺ (naïve), and CCR7⁻CD45RA⁺ (effector) CD4⁺ T cells for each tetramer. Each dot represents one donor/tetramer combination, median ± interquartile range is indicated.</p

Protein categories of identified antigens.

<p>The identified antigens (black bars) were divided into protein categories (TubercuList) and compared to the MTB genome (grey bars). Chi-square test, ***, p<0.001, ****, p<0.0001.</p