Clinical Characteristic of Patients at the Time of Blood Sampling.

<p>^apatients with >200 CD4 cells/mm³;</p><p>^bND: Not Detected;</p><p>^cpatients with <200 CD4 cells/mm³.</p><p>Clinical Characteristic of Patients at the Time of Blood Sampling.</p

CD107a-, IFNγ- and MIP 1β-producing Vγ9Vδ2 T-cell subsets decrease in HIV+ patients.

<p>According to the type of function, Vγ9Vδ2 T-cell response was analyzed in the healthy donors and compared to the HIV+ patients by flow cytometry after Picostim stimulation. (A) The absolute number of different Vγ9Vδ2 T-cell subsets according to the type of functions expressed (CD107a, IFNγ and MIP-1β). The results are shown as median and inter-quartile range (box plot), and vertical lines show the minimum and maximum values. (B) Summary pie chart; each sector of the pie chart is matched to the colored squares shown below the bar graphand shows the median percentages of responding Vγ9Vδ2 T-cells grouped according to the type of function analyzed. The Mann–Whitney U test was used to compare group medians and a permutation test based on χ² test to compare pie charts. A p value less than 0.05 was considered statistically significant, ***p<0.001; **p<0,01; *p<0.05; HD = healthy donors, black bar; HIV+ = HIV-infected individuals, white bar.</p

Multi-parameter flow cytometric analysis of the functional profile of Vγ9Vδ2 T-cells.

<p>Representative dot plots of functional parameters (MIP-1β, CD107A, IFNγ panels B and D) and the respective Isotype control of antibodies (panels A and C) were shown in healthy donors (HD) and HIV-infected patients (HIV+) after stimulation with Picostim. For the gating strategy, after the lymphocyte population was gated sequentially, CD3+ and Vγ9Vδ2 T-cell events were defined, and gates for each respective function were made (panels B and D, light blue gates) using combinations that provided optimal separation. Single function gates were set based on the negative control (unstimulated) samples and were placed consistently across samples. We used the Boolean gate platform to create the full array of possible combinations, equating to 8 (2³) response patterns when testing three functions. Data are reported after background correction. Numbers on the light blue gates indicate the percentage of functional Vγ9Vδ2 T-cells among the total Vγ9Vδ2 T-cells (black boxes).</p

Total and functional Vγ9Vδ2 T-cells correlate with CD4 cell count.

<p>Total (A) and functional (B) Vγ9Vδ2 T-cells from the HIV+ patients were correlated with CD4 cell count. Correlation statistics were analyzed using the Spearman R test and a p value less than 0.05 was considered statistically significant, ***p<0.001; **p<0,01; *p<0.05.</p

Responding Vγ9Vδ2-individuals decreases among HIV-infected patients who present lower Vγ9Vδ2 T-cell number than HD.

<p>Flow cytometer analyses were made to show (A) the absolute number of Vγ9Vδ2 T-cells from <i>ex vivo</i> HIV-infected patients (HIV+) and healthy donors (HD). The results are shown as median and interquartile range (box plot), and vertical lines show the minimum and maximum values; (B) the percentage of individuals among the HIV+ patients and HD having responding Vγ9Vδ2 T-cells after Picostim stimulation. Responding individuals, grey bar; Non-responding individuals, white bar. The Mann–Whitney U test was used to compare group medians. A p value less than 0.05 was considered statistically significant, ***p<0.001.</p

Modulation of unique Vγ9Vδ2 T-cell subsets during HIV infection.

<p>Seven unique Vγ9Vδ2-cell subsets derived from all possible combinations of the three parameters were analyzed from the whole blood of healthy donors and compared to the HIV+ patients by flow cytometry after Picostim stimulation. The graph shows the absolute number of different unique Vγ9Vδ2T-cell subsets according to the particular combination of functions expressed. The results are shown as median and inter quartile range (box plot), and vertical lines show the minimum and maximum values. The Mann–Whitney U test was used to compare group medians. A p value less than 0.05 was considered statistically significant, ***p<0.001; **p<0,01; *p<0.05; HD = healthy donors, black bar; HIV+ = HIV-infected individuals, white bar.</p

Three-, bi- and mono-functional Vγ9Vδ2 T-cell subsets decrease in HIV+ patients.

<p>According to the number of functions, Vγ9Vδ2 T-cell response was analyzed in the healthy donors and compared to the HIV+ patients by flow cytometry after Picostim stimulation. The graph shows the absolute number of different Vγ9Vδ2 T-cell subsets according to the number of simultaneous functions expressed. The results are shown as median and inter-quartile range (box plot), and vertical lines show the minimum and maximum values. The Mann–Whitney U test was used to compare group median. A p value less than 0.05 was considered statistically significant, ***p<0.001; **p<0,01; *p<0.05; HD = healthy donors, black bar; HIV+ = HIV-infected individuals, white bar.</p

Low-CD4-Patients showed a lower number of two Vγ9Vδ2 T-cell subsets expressing MIP-1β.

<p>Patients were divided into two groups according to CD4 cell count and Vγ9Vδ2 T-cell function was analyzed by flow cytometry after Picostim stimulation. (A) Vγ9Vδ2 T-cell total response. (B) The absolute number of different Vγ9Vδ2 T-cell subsets according to the to the type of functions expressed (CD107a, IFNγ and MIP-1β). (C) The absolute number of seven different unique Vγ9Vδ2 T-cell subsets according to the particular combination of functions expressed.</p

NK cells in survivors and in fatalities.

<p>The frequency of NK (<b>A</b>), and of NK subsets (<b>B:</b> CD56^bright, CD56^dim, and CD56^neg) was analyzed in HD (n = 14), EBOV-survivors (n = 7) and EBOV-fatalities (n = 6). Statistical analysis was performed by using Mann Whitney test and differences were considered significant with a p<0.05, and highlighted with an asterisk. *: p<0.05; **: p<0.01; ***: p<0.001.</p

Frequency of Vδ2 T-cells in survivors and in fatalities.

<p>The frequency of Vδ2 (<b>A</b>), Vδ2^posCD95^pos (<b>B</b>), of Vδ2^posCCR7^neg (<b>C</b>) and Vδ2^posCTLA-4^pos (<b>D</b>) was analyzed in HD (n = 14), EBOV-survivors (n = 10) and EBOV-fatalities (n = 6). Statistical analysis was performed by using Mann Whitney test and differences were considered significant with a p<0.05 and highlighted with an asterisk.*: p<0.05; **: p<0.01; ***: p<0.001.</p