Distribution of the answers at the second and last Delphi round.

<p>*Letters A, B, C, D, or E are codes corresponding to the following answers: A, absolutely required; B, important; C, of minor interest; D, useless; E, not assess(ed/able) in my routine practice environment.</p>‡<p>Choice of answer E was unavailable, absent from the menu list of answers.</p

CD38 and HLA-DR expression on bulk and HIV-specific CD8⁺ T cells.

<p>(A) Proportions of bulk CD8⁺ T cells from HD (n = 16, light gray bars), HAART-treated patients (n = 19, mid gray bars), viremic patients (n = 21, dark gray bars) and HIC (n = 79, black bars) expressing CD38 and HLA-DR. (C) Proportions of HIV-specific CD8⁺ T cells from HAART-treated patients (n = 13, mid gray bars), viremic patients (n = 39, dark gray bars) and HIC (n = 80, black bars) expressing CD38 and HLA-DR. Pie charts representing CD38⁻/HLA-DR⁻ (white), CD38⁻/HLA-DR⁺ (black), CD38⁺/HLA-DR⁻ (light gray) and CD38⁺/HLA-DR⁺ (dark gray) cells among bulk (B) and HIV-specific CD8⁺ T cells (D). Statistical differences shown in the pie charts are based on the difference in the frequency of the CD38⁻/HLA-DR⁺ subset between the different groups. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

High antigen sensitivity is associated with a high frequency of CD38⁻/HLA-DR⁺ cells in HIC.

<p>The antigen sensitivity of HIV-specific CD8⁺ T cells was measured in ELISpot assays with serial limiting dilutions of antigenic peptides (from 10⁻⁵ to 10⁻¹¹ M) and was expressed as the log molar concentration of peptide yielding 50% of the maximum response (EC_50%). Correlations between the proportion of CD38⁻/HLA-DR⁺ (A) or CD38⁺/HLA-DR⁺ (B) and the antigen sensitivity of HIV-specific CD8⁺ T cells from HIC. Correlations were evaluated using the Spearman rank correlation coefficient. The Spearman r correlation and the Pearson correlation curve are indicated for significant correlations (n = 47).</p

Stimulation with a low antigen concentration induces the CD38⁻/HLA-DR⁺ phenotype on specific CD8⁺ T cells.

<p>(A) Graphs representing the frequency of (A) CD38⁻/HLA-DR⁺ cells (dark histograms) and (B) CD38⁺/HLA-DR⁺ cells (dark gray histograms) among activated EBV-specific healthy donor CD8⁺ T cells (i.e. those expressing CD38 and/or HLA-DR) after a four-day culture period (n = 8).</p

Study population.

<p>Median values [1^st–3^rd interquartile range] are shown for age, time since diagnosis, CD4⁺ and CD8⁺ T cell counts, and HIV RNA viral loads.</p>a<p>Viremic and HAART-treated patients' RNA viral loads were measured using an assay with a quantification limit of <50 copies/ml, while values in HIC were obtained with ultrasensitive assays.</p

Influence of stimulatory conditions on the activation phenotype of specific CD8⁺ T cells.

<p>(A) Representative dot plots of HLA-DR and CD38 expression in unstimulated conditions (upper graph), after IFN-α stimulation (middle graph) or peptide stimulation (2 µM, lower graph) among specific (dark dots) and non-specific (gray dots) CD8⁺ T cells from healthy donors after a four-day culture period. CD38 and HLA-DR expression on bulk (C left panel) and specific CD8⁺ T cells (B and C right panel). (B) Flow cytometry histograms showing representative results for cells from one individual in unstimulated conditions (dark lines), after IFN-α stimulation (light gray histograms) or peptide stimulation (dark gray histograms). (C) Surface expression of CD38 (white bars) and HLA-DR (gray bars) on bulk and specific CD8⁺ T cells (n = 4).</p

Qualitative features of CD38⁻/HLA-DR⁺ and CD38⁺/HLA-DR⁺ HIV-specific CD8⁺ T cell subsets in HIC.

<p>(A) Bcl-2 expression on CD38⁻/HLA-DR⁺ and CD38⁺/HLA-DR⁺ HIV-specific CD8⁺ T cells (n = 11). (B) Proportion of CD38⁻/HLA-DR⁺ and CD38⁺/HLA-DR⁺ HIV-specific CD8⁺ T cells producing both IFN-γ and IL-2 among HIV-specific CD8⁺ T cells producing IFN-γ or IL-2 (n = 35). (C) Fold increase in CD38⁻/HLA-DR⁺ and CD38⁺/HLA-DR⁺ HIV-specific CD8⁺ T cell numbers after 5 days of culture with HIV peptides (2 µM) (n = 7). (D–E) Proportion of CD38⁻/HLA-DR⁺ and CD38⁺/HLA-DR⁺ HIV-specific CD8⁺ T cells producing perforin (D) and granzyme B (E) (n = 35). (F) Graphs representing percentage cytotoxicity (measured as granzyme-B-mediated intracellular cleavage of a fluorogenic substrate) of CD38⁻/HLA-DR⁺ and CD38⁺/HLA-DR⁺ HIV-specific CD8⁺ T cells (n = 11). * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Scatterplot with overlaid linear prediction plot between the log₁₀ ADCC titers and (A) the log₁₀ RNA HIV-1 viral load (r = 0.37 and p = 0.03 in B57-, r = 0.43 and p = 0.02 in B57+), (B) the number of HIV-specific CD8 T cells (r = 0.21 and p = 0.31 in B57-, r = 0.44 and p = 0.02 in B57+), or (C) the ability of CD8 T cell to control viral replication quantified by the log₁₀ p24 decrease (r = 0.02 and p = 0.93 in B57-, r = 0.60 and p = 0.0008 in B57+), respectively in HLA B57+ (red) and HLA B57- controllers (blue).

<p>Scatterplot with overlaid linear prediction plot between the log₁₀ ADCC titers and (A) the log₁₀ RNA HIV-1 viral load (r = 0.37 and p = 0.03 in B57-, r = 0.43 and p = 0.02 in B57+), (B) the number of HIV-specific CD8 T cells (r = 0.21 and p = 0.31 in B57-, r = 0.44 and p = 0.02 in B57+), or (C) the ability of CD8 T cell to control viral replication quantified by the log₁₀ p24 decrease (r = 0.02 and p = 0.93 in B57-, r = 0.60 and p = 0.0008 in B57+), respectively in HLA B57+ (red) and HLA B57- controllers (blue).</p

Univariate and multivariate bootstrap linear regression analysis of the role of age, sex, HLA B57, Delay since diagnosis, and the CD4 T cell counts or HIV-1 RNA levels in log₁₀ ADCC in the 67 HIV-infected patients enrolled in the ANRS CO18 cohort.

<p>The multivariate analysis included all variables.</p>*<p>per one year increase; ** per a 1 log copies/mL increase, ***per a 100-CD4 increase;. Weak responders were compared to Strong responders; females were compared to males, HLA B57 positive patients to HLA B57 negative patients, White patients were compared with others, HLA B27 positive patients to HLA B27 negative patients. **** The number of HIV-specific CD8 T cells was not included in the multivariate analysis because there was a strong link with the Weak/Strong Responders status: all the Weak Responders had SFC below the median (less than 1960 SFC).</p

ADCC responses in HIV-1 Viremic and Controller (HIC) patients.

<p>The whisker plots represent the distribution of Log₁₀ ADCC Ab titers among the 40 viremic and the 67 HIC patients. ADCC activity was detected according to our previously described ADCC-GranToxiLux (GTL) procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074855#pone.0074855-Pollara1" target="_blank">[13]</a>.</p