EBV and CMV-specific CD8 T cells express significantly more CD160 than Flu-specific CD8 T cells.

<p>Flow cytometric profiles of Flu, EBV and CMV-specific CD8 T cells expressing PD-1, CD160 and 2B4 <i>ex vivo</i>. The expression profiles of CD8 T cells were performed in 22 individuals using polychromatic flow cytometry. <b>(A)</b> 2B4, CD160 and PD-1 expression profiles of Flu, EBV and CMV-specific CD8 T cells detected by multimer staining (red) as compared to total CD8 T cells (black/grey) of three representative individuals (#KEL12 FLU HLA-A*02 GILGFVFTL, #652 EBV HLA-B*08 RAKFKQLL and #KEL12 CMV HLA-B*07 TPRVTGGGAM, respectively). <b>(B)</b> Frequencies of Flu, EBV and CMV-specific CD8 T cells expressing 2B4, CD160, PD-1. Red bars correspond to mean ± SEM. Statistical significance (<i>P</i> values) in panel <b>B</b> were obtained using One-way ANOVA (Kruskal-Wallis test) followed by a unpaired Student's t-test. <b>(C)</b> Expression profiles of 2B4, CD160, PD-1 of CMV, EBV and Flu-specific CD8 T cells. All the possible combinations of 2B4, CD160 and PD-1 expression are shown on the x axis and frequencies of 2B4, CD160 and PD-1 expression on CD8 and T-cell populations are shown on the y axis. Combinations of expression are grouped and color-coded on the basis of the number of molecules expressed. The pie chart summarizes the data, and each slice corresponds to the fraction of T cells expressing a given combination of molecules within the CD8 T-cell populations. Bars correspond to the fractions of distinct T-cell populations within the total T cells. Stars indicate statistical significance (*:<i>P</i><0.025; **:<i>P</i><0,005; ***:<i>P</i><0.0001) and were calculated using the SPICE software.</p

CD160, but not PD-1 and/or 2B4 expression inversely correlates with CD8 T-cell proliferative capacity.

<p><b>(A–B)</b> Representative flow cytometric profiles of 2B4, PD-1 and CD160 expression of CMV (#700 CMV HLA-A*02 NLVPMVATV) and Flu (#304 FLU HLA-A*02 GILGFVFTL) specific CD8 T cells detected by multimer staining (red) as compared to total CD8 T cells (black/grey). <b>(C–D)</b> Representative CMV (#700 CMV HLA-A*02 NLVPMVATV) and Flu (#MP FLU HLA-A*02 GILGFVFTL) specific CD8 T-cell proliferation capacity assessed by CFSE based assay. <b>(E)</b> Proliferation index (CFSElow CD8 T-cell frequency/Multimer-specific CD8 T-cell frequency) CMV, EBV and FLU-specific CD8 T cells. Red bars correspond to mean ± SEM. <b>(F)</b> Correlations between proliferation index (CFSElow CD8 T-cell frequency/Multimer-specific CD8 T-cell frequency; x axes) and the virus-specific CD8 T-cell subsets distribution (percentage of Multimer-specific CD8 T cells expressing the different combinations of 2B4, PD1 and CD160; y axes). Statistical significance (<i>P</i> values) in panel <b>E</b> were obtained using One-way ANOVA (Kruskal-Wallis test) followed by a Student's t-test. <i>P</i> values in <b>F</b> were obtained using Spearman's rank correlations.</p

Kinetic of CD160 or PD-1 expression on CD8 T cells stimulated upon T-cell stimulation.

<p><b>(A)</b> Representative flow cytometric profile of CD160 and PD-1 expression on CD8 T cells after 0, 24, 48, 72 and 120 hours of stimulation with anti-CD3/anti-CD28 magnetic beads. CD8 T-cell proliferation was also measured by CFSE-based assay at 48, 72 and 120 hours after stimulation. <b>(B)</b> Cumulative data of CD160 or PD-1-expressing CD8 T-cell frequencies at distinct time points after stimulation. Red bars correspond to mean ± SEM.</p

Sequences of peptides used for CD8 T-cell stimulations and multimer staining.

<p>Sequences of peptides used for CD8 T-cell stimulations and multimer staining.</p

Restoration of CD8 T-cell proliferation by CD160/CD160-ligand blockade directly correlates with the level of the <i>ex vivo</i> CD160 expression.

<p><b>(A–B)</b> Representative flow cytometric profile of 2B4, CD160 and PD-1 expression of CMV (#215 CMV HLA-A*02 NLVPMVATV) and EBV (#762 EBV HLA-B*07 RPPIFIRRL)-specific CD8 T cells detected by multimer staining (red) compared with total CD8 T cells (black/grey). <b>(C–D)</b> Representative examples of CMV (#215 CMV HLA-A*02 NLVPMVATV) and EBV (#762 EBV HLA-B*07 RPPIFIRRL)-specific CD8 T-cell proliferation in presence or not of anti-CD160 mAbs assessed by CFSE-based assay. <b>(E)</b> Impact of CD160 expression on the restoration of CD8 T-cell proliferation by CD160/CD160-ligand blockade. <b>(F–G)</b> Correlation between fold increase in proliferation capacity (y axes) upon CD160 blockage and percentage of 2B4⁺CD160⁺PD-1⁺ or 2B4⁺CD160⁺PD-1⁻ virus-specific CD8 T cells. <b>(H–J)</b> Correlation between fold increase in proliferation capacity (y axes) upon PDL-1/2 blockage and percentage of 2B4⁺CD160⁺PD-1⁺, 2B4⁺CD160⁻PD-1⁺ or 2B4⁻CD160⁻PD-1⁺ virus-specific CD8 T cells (x axes). Statistical significance (<i>P</i> values) in <b>B</b>–<b>I</b> were obtained using Spearman's rank correlations.</p

Relative capacity of CD8 T-cell subsets defined by 2B4, CD160 and PD-1 expression to produce IL-2 and IFN-γ.

<p>CD8 T-cell responses were analyzed within six CD8 T-cell subsets defined by 2B4, CD160 and/or PD-1 expression. <b>(A)</b> Representative flow cytometric profiles of CD8 T cells producing IL-2 and/or IFN-γ following stimulation of total blood mononuclear cells with SEB in Subject #1096. Unstimulated cells (negative control) are also shown. <b>(B)</b> Frequencies of IL-2 and IFN-γ-producing CD8 T-cell populations. Red bars correspond to mean ± SEM. Red stars indicate statistical significance (<i>P</i><0.05). NS: not significant. Statistical significance (<i>P</i> values) in panels <b>A</b> and <b>B</b> were obtained using One-way ANOVA (Kruskal-Wallis test) followed by a paired Student's t-test.</p

Expression levels of perforin and granzyme B in CD8 T-cell subsets defined by the expression of 2B4, CD160 and PD-1.

<p>CD8 T-cell responses were analyzed within six CD8 T-cell subsets defined by 2B4, CD160 and/or PD-1 expression. <b>(A)</b> Representative flow cytometric profiles of CD8 T cells expressing perforin and/or granzyme B <i>ex vivo</i>. <b>(B)</b> Frequencies of CD8 T cells expressing perforin and granzyme B. <b>(C)</b> Representative flow cytometric profiles of differentiated CD8 T-cell subsets defined by the CCR7 and CD45RA expressing perforin, 2B4, CD160 and PD-1. (D) Cumulative analyses (n = 10) representing the expression of perforin differentiated CD8 T-cell subsets in function of CD160 expression. Red bars correspond to mean ± SEM. Red stars indicate statistical significance (<i>P</i><0.05). NS: not significant. Statistical significance (<i>P</i> values) in panels <b>B</b> and <b>D</b> were obtained using One-way ANOVA (Kruskal-Wallis test) followed by a paired Student's t-test.</p

Proliferative capacity virus-specific CD8 T-cell populations is influenced by co-inhibitory molecule expression.

<p>CD8 T-cell populations were sorted on the basis of 2B4, PD-1 and CD160 expression, labeled with CFSE and stimulated with (viral) peptides in the presence of autologous irradiated CD8-depleted PBMCs (ratio CD8/feeder cells 1∶10) for 6 days (n = 11). <b>(A)</b> Representative CMV (#1260 CMV HLA-A*01 VTEHDTLLY) and CMV (#1261 CMV HLA-A*02 NLVPMVATV) specific CD8 T-cell proliferation capacity assessed by CFSE based assay. <b>(B)</b> Proliferation index (CFSElow CD8 T-cell frequency/Multimer-specific CD8 T-cell frequency) of virus-specific CD8 T cells. Red bars correspond to mean ± SEM. Red stars indicate statistical significance (<i>P</i><0.05). Statistical significance (<i>P</i> values) in panel B was obtained using One-way ANOVA (Kruskal-Wallis test) followed by a Student's t-test.</p

Combined assessment of co-inhibitory molecule expression and differentiation state.

<p>CD8 T-cell differentiation and CD8 T-cell expression of 2B4, CD160 and/or PD-1 were analyzed. <b>(A)</b> Representative example of flow cytometric profile of CD8 T-cell subsets defined by CCR7 and CD45RA expressing 2B4 and/or CD160. <b>(B)</b> Cumulative analyses (n = 10) of co-inhibitory molecules expression within 3 distinct memory CD8 T-cell subsets. <b>(C)</b> Cumulative analyses (n = 10) of the differentiation within 6 distinct CD8 T-cell population defined by 2B4, PD-1 and CD160 expression. NS: not significant. Statistical significance (<i>P</i> values) was obtained using One-way ANOVA (Kruskal-Wallis test) followed by a paired Student's t-test. Red bars correspond to mean ± SEM. Red stars indicate statistical significance (<i>P</i><0.05).</p