HIV-specific CD4⁺ T-cell responses were amplified by IL-2 treatment and inversely correlate with VL after treatment interruption.

<p>(A) Example of gating strategy used for detecting antigen-specific CD4⁺CD25⁺CD134⁺ cells after in vitro stimulation with p24 or none (OX40 assay), at wk0, wk16 and wk36. (B and D) Representation of HIV- and CMV- specific CD4⁺CD25⁺CD134⁺ percentages in patients at wk0, wk16 and wk36 using the gating strategy shown in (A). (C and E) Correlations between Í4⁺CD25⁺CD134⁺ and viral load at resuming ART, at wk36 for p24 and CMV, respectively. Correlations were calculated using spearman correlation coefficients (Prism 5.0, version 5.0d). P values were considered significant when < 0.05.</p

Subcutaneous injections of IL-2 expand peripheral CD4⁺CD25⁺CD127^lowFoxp3⁺ naïve and total memory Tregs.

<p>(A) Gating strategy for naïve CD4⁺ CD45RO^- CD25⁺ CD127^low FoxP3⁺, total memory CD4⁺ CD45RO⁺ CD25⁺ CD127^low FoxP3⁺ and memory CD4⁺ CD45RO⁺ CD25⁺ CD127^low FoxP3⁺ CD39⁺. (B) Proportions of total CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs, (C) naïve CD45RO^-CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs, (D) total memory CD45RO⁺CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs and (E) memory CD39⁺Tregs, were measured in eighteen patients at wk0, wk16 and wk36 after vaccination and IL-2 injections. Each patient can be distinguished by a colored symbol in the figures. (F) Pie chart showing comparison for compartments of Tregs subsets expansion at wk0, wk16 and wk36. Prism 5.0, version 5.0d, (GraphPad Software, Inc.) was used for statistical analyses. P values were considered significant when < 0.05.</p

Correlation between total memory or naïve Tregs with PD1⁺ CD95⁺ cells.

<p>At week36% total memory CD45RO⁺CD25⁺CD127^lowFoxP3⁺ (A) or naïve CD45RO^-CD25⁺CD127^lowFoxP3⁺ (B) were measured by flow cytometry and then correlated with %PD-1⁺CD95⁺CD4⁺. Correlations were calculated using spearman correlation coefficients (Prism 5.0, version 5.0d). P values were considered significant when < 0.05.</p

Ab reactivities against the MAP3865c protein are inhibited by MAP3865c_125–133 and MAP3865c_133–141 peptides.

<p>Ab+ and Ab-negative sera from T1D patients were pre-incubated overnight with saturating concentrations (5.5 µM) of MAP3865c_125–133, MAP3865c_133–141, the two peptides in combination, MAP3865c-MBP fusion protein and control or no peptide. Their reactivity on MAP3865c-MBP-coated ELISA plates was subsequently tested. Bars depict means ± SEM of triplicate wells and results are representative of three separate experiments.</p

Prevalence of Abs against homologous MAP3865c and ZnT8 epitopes in T2D patients.

<p>Prevalence of Abs against MAP3865c_125–133 (A) and its homologous ZnT8_178–186 (B); and against MAP3865c_133–141 (C) and its homologous ZnT8_186–194 (D) in T2D and healthy subjects. Data representation is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026931#pone-0026931-g001" target="_blank">Fig. 1</a>.</p

HIV-specific Tregs decrease after IL-2 treatment and positively correlate with viral load after treatment interruption.

<p>(A) Gating strategy used for detecting HIV-specific CD4⁺CD25⁺CD134⁺CD39⁺FoxP3⁺ Tregs after p24 stimulation using the “OX40 assay”. (B, D and F) HIV-specific CD4⁺CD25⁺CD134⁺CD39⁺FoxP3⁺ Tregs percentages in patients at wk0 and wk36 using the gating strategy shown in (A). (C, E and G) Correlations between Í4⁺CD25⁺CD134⁺CD39⁺FoxP3⁺ Tregs at wk36 and viral load at resuming ART. Correlations were calculated using spearman correlation coefficients (Prism 5.0, version 5.0d). P values were considered significant when < 0.05.</p

Correlations between Í4⁺CD25⁺CD134⁺CD39⁺FoxP3⁺ Tregs and p24 IFNγ-ELISpot at wk36.

<p>IFN-γ producing cells (SFC) were measured by ELISpot as described in the methods. Correlations were calculated using spearman correlation coefficients (Prism 5.0, version 5.0d). P values were considered significant when < 0.05.</p

T1D duration and age at T1D diagnosis in Ab+ and Ab-negative T1D patients.

<p>T1D patients whose Ab reactivities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026931#pone-0026931-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026931#pone-0026931-g005" target="_blank">5</a> were compared using the Mann-Whitney U test. Mean ± SD are shown.</p

Prevalence of anti-MAP3865c Abs in Sardinian T1D and T2D patients.

<p>Sera were tested for their reactivity against plate-coated MAP3865c-MBP fusion protein. Ab distribution is shown for T1D (A) and T2D (B) patients compared to healthy controls. Dotted lines indicate the cut-off for positivity used in each assay, as calculated by ROC analysis. The percent fraction of Ab+ sera is indicated on top of each distribution, while bars indicate the corresponding median ± interquartile range. AUC and <i>p</i> values are given in the top right corner. Figures show representative experiments out of three performed.</p

CD25+CD134+ CD39+FoxP3+ cells have bona fide Tregs phenotype.

<p>(A) Gating strategy used for detecting CMV-specific CD4+CD25+CD134+ CD39+FoxP3+ Tregs, and IFN-γ producing CD25+CD134+CD39-FoxP3- and CD25+CD134+ CD39-FoxP3+. PBMCs from CMV+ individuals were stimulated with CMV lysate for 44hrs. Flow cytometry was used to detect CMV-specific cells as described in the methods. (B) Mean fluorescence intensity (MFI) of Helios, CD15s, CTLA-4, ICOS, PD-1 and T-bet molecules were measured by flow cytometry in 8 CMV+ individuals to detect CMV-specific subsets (CD134+CD25+Foxp3+CD39+, CD134+CD25+Foxp3+CD39- and CD134+CD25+Foxp3-CD39-). P values were considered significant when < 0.05.</p