Associations between functional avidity of HIV-specific CD8 T-cell responses and other immunological parameters.

<p>Associations between the functional avidity of HIV-specific CD8 T-cell responses and the frequency of HIV-specific CD8 T cells co-expressing CD28 and CD27 (<b>A</b>) or PD-1, 2B4 and CD160 (<b>B</b>).</p

Qualitative changes of HIV-specific CD8 T cells in patients experiencing a virus rebound following treatment interruption.

<p><b>A.</b> Longitudinal analysis of the CD4 T-cell counts and HIV viremia of 2 representative HIV-infected patients (#1017 and #1023) treated with ART since acute infection. Patient #1017 remained on ART, whereas patient #1023 spontaneously interrupted ART after 119 weeks (orange box). <b>B.</b> Functional profiles of B*0702-_GPGHKARVL-specific CD8 T cells from patients #1017 and #1023 analyzed at two distinct time-points corresponding to weeks 48 and 300 (identified by the arrows in A). <b>C.</b> Cumulative analysis of the functional profile of HIV-specific CD8 T-cell responses on the basis of the expression of IFN-γ, IL-2 and perforin in patients with acute (PHI) infection after one year of ART (-T1Y) and after either treatment interruption (-ATI) or after five years of ART (-T5Y). Matched-paired HIV-specific CD8 T-cell responses are considered for the comparison between T1Y and ATI or between T1Y and T5Y. Although TNF-α was also detected, analysis is restricted to the expression of IFN-γ, IL-2 and perforin for clarity. All possible combinations of IFN-γ, IL-2 and perforin are shown on the <i>x</i> axis, whereas the percentages of the distinct cell subsets within HIV-specific CD8 T cells are shown on the <i>y</i> axis. The pie charts summarize the data, and each slice corresponds to a certain combination of functions. Colors in the pie charts are based on the colored boxes at the bottom of the panel. <b>D.</b> PD-1 expression in B*0702-_GPGHKARVL-specific CD8 T cells in patients #1017 and #1023 measured at the two time-points identified with arrows in A. <b>E.</b> Mean fluorescence intensity (MFI) of PD-1 expression in HIV-specific CD8 T cells measured in patients with acute (PHI) HIV infection after 1 year of ART (-T1Y) and after either treatment interruption (-ATI, left panel) or after five years of ART (-T5Y, right panel). <b>F.</b> Proportion of PD-1⁺2B4⁺CD160⁺ cells in B*0702-_GPGHKARVL-specific CD8 T cells in patients #1017 and #1023 measured at the two time-points identified with arrows in A. <b>G.</b> Proportion of PD-1⁺2B4⁺CD160⁺ cells in HIV-specific CD8 T cells measured in patients with acute (PHI) HIV infection after 1 year of ART (-T1Y) and after either treatment interruption (-ATI, left panel) or after five years of ART (-T5Y, right panel).</p

Functional profile and expression of co-stimulatory molecules and of co-inhibitory receptors of HIV-specific CD8 T-cell responses during acute and chronic HIV infections.

<p>Analysis of the functional profile (<b>A</b>), of the expression of CD27 and CD28 (<b>B</b>) and of the expression of 2B4, CD160 and PD-1 (<b>C</b>) in HIV-specific CD8 T cells in patients with acute (PHI-B), untreated chronic progressive (CP-B) or non-progressive (LTNP) HIV infection. Representative examples of the distinct flow cytometry panels are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003423#ppat.1003423.s001" target="_blank">Fig. S1B</a>-D. Regarding the functional profile (<b>A</b>), although TNF-α was detected, analyses are restricted to the expression of IFN-γ, IL-2 and perforin for clarity. All possible combinations of the distinct markers are shown on the <i>x</i> axis, whereas the percentages of the distinct cell subsets within virus-specific CD8 T cells are shown on the <i>y</i> axis. The pie charts summarize the data, and each slice corresponds to a certain combination of molecules. Colors in the pie charts are based on the colored boxes at the bottom of the panel.</p

Functional avidity of HIV-specific CD8 T-cell responses.

<p><b>A.</b> Representative examples of functional avidity of HIV-specific CD8 T-cell responses in patients with acute (PHI-B-09; circles), chronic progressive (CP-B-08; squares) or chronic non-progressive (LTNP-2082; triangles) infection after stimulation with decreasing concentrations of B*0801-_EIYKRWII peptides. The dashed line corresponds to half of the maximal response allowing the extrapolation of the 50% effect concentration (EC₅₀). <b>B.</b> Cumulative analysis of the functional avidity of HIV-specific CD8 T cells during acute (PHI-B), untreated chronic progressive (CP-B) and non-progressive infection (LTNP). Medians and interquartile ranges are shown and each point represents one HIV-specific CD8 T-cell response. <b>C.</b> Cumulative analysis of the frequency of HIV-specific CD8 T cells during acute (PHI-B), untreated chronic progressive (CP-B) and non-progressive infection (LTNP). <b>D.</b> Functional avidity of HIV-specific CD8 T-cell responses against common optimal epitopes recognized by patients with acute (PHI-B), chronic progressive (CP-B) or not progressive (LTNP) infections. The red line (right panel) shows the functional avidity of B*2705-_KRWIILGLNK (KK10)-specific CD8 T-cell response.</p

Increased CDR3 renewal of HIV-specific CD8 T cells following treatment interruption and association with functional avidity.

<p><b>A.</b> Percentage of CDR3 renewal of HIV-specific CD8 T cells before (under treatment) and after treatment interruption (TI). CDR3 diversity and renewal were determined as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003423#ppat.1003423-Miconnet1" target="_blank">[32]</a>. Example of TRBV usage and CDR3 size pattern analysis of B*0702-_GPGHKARVL-specific CD8 T cells in patient #1023 at week 18, 96 and 125 are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003423#ppat.1003423.s003" target="_blank">Fig. S3</a>. <b>B.</b> Association between the percentage of CDR3 renewal and changes in the functional avidity of HIV-specific CD8 T cells.</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

Identification of antigenic regions within the TB Vaccine and IGRA antigens.

<p>Magnitude of responses, expressed as the total magnitude of response (black dots, solid line, left y-axis) or number of responding donors (squares, dashed line, right y-axis) identified for Rv0288 (A), Rv3619c (B), Rv3620c (C), Rv3874 (D), Rv3875 (E), Rv0125 (F), Rv1886 (G), Rv3804c (H), Rv1196 (I), and Rv2608 (J). X-axes indicate peptide number of overlapping peptides spanning the entire protein. Dashed horizontal lines indicates 2 responding donors, and arrows indicate non-redundant antigenic regions. Cell wall and cell processes (A-E), Intermediary metabolism and respiration (F), Lipid metabolism (G, H) and PE/PPE antigens (I, J). Previously defined antigenic regions [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005760#ppat.1005760.ref035" target="_blank">35</a>] (also expressed as total magnitude of response) are indicated by black triangles and dotted lines (left y-axis) for Rv3874 (D) and Rv3875 (E).</p

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

Epitope pool responses are highly polarized towards IFNγ production.

<p>Frequencies of cytokine-expressing CD3⁺CD4⁺ memory T cells (A) or CD3⁺CD4⁺ naïve T cells (B) in response to the pool of 66, 125 and 300 epitopes. Each dot represents one donor, median ± interquartile range is indicated. (C) Gating strategy for memory versus naïve CD4 T cells. Plots are gated on total CD3⁺CD4⁺ T cells.</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