HBD1 is expressed by circulating monocytes and gut epithelial cells.

<p>(A, B) Cells were sorted from 5 different bulk colon excess tissue (n = 5) or PBMCs (n = 5), RNA extracted and HBD1 transcription quantified by qPCR. For colon, epithelial cells were identified using the surface marker CD326 and CD45 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). For PBMCs, monocytes were identified as CD14+, DCs as CD11c+ (mDCs) or CD123+ (pDCs) and lymphocytes as CD3+CD19+ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). Epithelial cells were the main producers of HBD1 in colon whereas CD14+ cells were the main producers in PBMCs. (C, D) The frequency of HBD1 producing cells is not altered during acute infection. Frequency of monocytes (C) and DC (D) populations in PMBCs from HIV-1- (n = 9) or acutely (Acute; n = 8) infected individuals was determined by flow cytometry. Conventional monocytes were identified as CD14+ CD16-, inflammatory monocytes as CD14+ CD16+ and mDCs as well as pDCs as described above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). No significant difference in cell frequencies was found between HIV-1- and sample from subjects with acute HIV-1 infection for any of the analyzed cell populations (Kruskal-Wallis and Dunn’s multiple post comparison test). (A-D) Data points are presented with median and interquartile range.</p

IFN-α is a potent inducer of HBD1 in monocytes <i>in vitro</i> and correlates with HBD1 transcription <i>in vivo</i>.

<p>(A, B) CD14+ monocytes (A n = 11 and B n = 5) were isolated from PBMCs and incubated with different concentrations of recombinant IFN-α2 or left untreated. Where indicated cells were treated with either an anti-IFN-α or an isotype control antibody 30 min before stimulation with 50 pg/ml recombinant IFN-α2. Relative expression of HBD1 and ISG15 was assessed using quantitative PCR. IFN-α2 was found to significantly upregulate HBD1 and ISG15. Each dot represents one independent experiment from a different healthy control subject. (C, D) Human PBMCs were isolated from whole blood from HIV-1 uninfected (HIV-) (C n = 5, D n = 9), HIV-1 untreated chronic progressors (PG) (C n = 9, D n = 14) and acutely HIV-1 infected (Acute) individuals (C n = 12, D n = 15). IFNα (IFNA) transcription of PBMCs was assessed by qPCR. IFNA and ISG15 were both significantly upregulated in acutely but not chronically infected subjects compared to HIV-uninfected control subjects (A-D) Kruskal-Wallis and Dunn’s multiple comparison test with graphs showing median and interquartile range. (E, F) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.g001" target="_blank">Fig 1A</a>) was plotted against the corresponding transcription of IFNA (E) or ISG15 (F) with each dot representing one subject. ISG15 and IFNA were found to significantly correlate with HBD1 transcription in acutely infected individuals (Spearman r correlation).</p

HIV-1 induces HBD1 <i>in vitro</i> but is unlikely to be sufficient <i>in vivo</i>.

<p>(A-C) CD14+ monocytes isolated using Miltenyi MACS technology were incubated with either HIV-1 R5, poly I:C (TLR3 ligand), 5’ppp-dsRNA (RIG-I ligand), R837 (TLR7 ligand), LPS (TLR4 ligand) or FLA-ST (TLR5 ligand), or left untreated. After 24 h cells were harvested and RNA extracted. Relative expression of HBD1 (A, C) or HBD2 (B) was assessed using quantitative PCR. HBD1 expression was found to be significantly increased in monocytes when incubated with HIV-1 R5, poly I:C or 5’pppdsRNA (A), whereas LPS and FLA-ST induced HBD2 (B). (C) Significant upregulation of HBD1 by HIV-1 was only achieved at 1x10^6 infection HIV-1 particles (i.p.) (Kruskal-Wallis and Dunn’s multiple post comparison test). Data points are presented with median and interquartile range which each dot representing an independent experiment from a different healthy control subject. (D) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.g001" target="_blank">Fig 1A</a>) was plotted against the corresponding viral load which each dot representing one subject. No significant correlation was found between viral load and HBD1 transcription (Spearman r correlation). (E, F) CD4+ T cells or CD14+ monocytes were isolated from PBMCs and incubated with either β-lactamase packaged HIV-1 X4 (CXCR4 tropic)/ HIV-1 R5 (CCR5 tropic) or left untreated. Where indicated cells were pre-treated with the CCR5 antagonist maraviroc (Mav) for 30min before HIV-1 treatment. All virus contained Vpr-BlaM which allowed assessment of HIV-1 entry into cells 12h after exposure. The highest entry was observed in CD4+ T cells by HIV-1 X4, whereas HIV-1 R5 entered CD4+ T cells as well CD14+ monocytes at low frequency. The graph shows the median and interquartile range with each dot representing one independent experiment from a different healthy control subject (F).</p

HBD1 is upregulated in PBMCs during acute HIV-1 infection.

<p>Samples from HIV-1 uninfected (HIV-), HIV-1 untreated chronic progressors (PG), HIV-1 chronic ART treated (CT) and acutely HIV-1 infected (Acute) individuals were analyzed for HBD1 expression. HBD1 transcription in PBMCs (A, B) or gut pinch biopsies (C, D) was determined by qPCR. Grey color indicates subjects with matched gut biopsy samples. (A) HBD1 expression in blood of acutely infected subjects (n = 32) was found to be significantly higher compared to HIV-1 individuals from PG (n = 8), CT (n = 9) or HIV- (n = 17) subjects (Kruskal-Wallis and Dunn’s multiple comparison test with error bars indicate min and max of boxplots). (B) Longitudinal sampling of individuals (n = 10) showed significant downregulation of HBD1 in chronic HIV-1 infection compared to acute infection (Wilcoxon matched-pairs signed rank test). (C, D) No significant differences in HBD1 expression in ileum (C) or colon (D) was found between HIV- (n = 5), PG (n = 4), CT (n = 9) and HIV acute infected subjects (n = 4), (Kruskal-Wallis and Dunn’s multiple comparison test and median with interquartile range).</p

HLA-G⁺ Treg reduce bystander activation of T cells.

<p>(A–B): Representative flow cytometry dot plots reflecting the surface expression of CD38 on Vβ13.1⁺ and Vβ13.1⁻ responder T cells following exposure to indicated CD4 (A) or CD8 (B) Treg cell populations or negative control CD4 or CD8 T cells from HIV-seronegative donors. (C–D): Cumulative data representing relative suppression of CD38, HLA-DR and CD69 on Vβ13.1⁺ and Vβ13.1⁻ responder T cells following exposure to indicated CD4 (C) or CD8 (D) Treg cell populations. Mean and standard deviation from n = 8 HIV-1 negative study subjects are shown. Significance was tested by paired T test.</p

Diminished proportions of HLA-G⁺ CD4 and CD8 T cells in progressive HIV-1 infection.

<p>(A): Representative flow cytometry dot plots reflecting the proportions of HLA-G⁺ CD4 and CD8 T cells in indicated study subjects. FMO control reflects “fluorescence minus one” control condition without addition of HLA-G antibodies. (B): Box and Whisker plots summarizing the relative proportions and absolute numbers of HLA-G⁺ CD4 and CD8 T cells in indicated study cohorts. ANOVA followed by post-hoc analysis with Tukey's Multiple Comparison Test was used to determine significance. (C): Correlations between frequencies of HLA-G⁺ T cells and total CD4 T cell counts in controllers (n = 23), progressors (n = 27) and HIV seronegative individuals (n = 15). (D): Correlations between proportions of HLA-G⁺ Treg and CD8 T cell immune activation determined by surface expression of CD38 and HLA-DR in controllers (n = 19), progressors (n = 20), and HIV seronegative individuals (n = 15). (C/D): Spearman's correlation coefficient is shown.</p

HLA-G⁺ Treg minimally inhibit proliferative activities of antigen-specific T cells.

<p>(A–B): Representative dot plots reflecting proliferative activities of HIV-1-, CMV-, or PHA-stimulated CD4 (A) or CD8 (B) T cells from HIV controllers following incubation with indicated autologous Treg subsets or HLA-G⁻ CD25⁻ control cells. (C–D): Cumulative data reflecting the Treg-mediated suppression of HIV-1-specific CD4 (C) or CD8 (D) T cell proliferation from n = 3 HIV-1 controllers. Significance was tested by paired T test.</p

Stable LILRB1 expression on T cells after “bystander activation”.

<p>(A) Representative dot plots demonstrating proportions of LILRB1-expressing Vβ13.1⁺ and Vβ13.1⁻ T cells after activation with SEB over indicated time course. (B): Fold change of LILRB1 expression on Vβ13.1⁺ or Vβ13.1⁻ T cells at indicated time points. Mean and standard error from 7 different HIV-1 negative donors are shown. Significance was tested by paired T test.</p

Maturation phenotype of HIV-1 specific CD8+ T cells by HLA type and epitope specificity.

<p>Panel A shows the percentage of CD45RA+/CCR7− CD8+ T cells of 46 HLA-A2, -A3, -B14, -B27 and -B57-restricted epitopes tested in the study cohort. Panel B depicts the percentage of CD45RA+/CCR7− CD8+ T cells specific for six specific HIV-1 epitopes tested (restricting HLA allele and peptide sequence are shown). Data reflective of a total of 21 study subjects, who had responses to the specific HLA-matched optimal epitopes tested (14 Controllers and 5 Progressors).</p

The effector phenotype of an individual CD8+T cell response may not be adequately reflected in a peptide pool.

<p>Panels A and C show IFN-γ production upon stimulation with the Pol peptide pool (A) and an individual Pol peptide (C). Panels B and D are gated on the IFN-γ producing cells and show differentiation phenotype of these HIV-1 specific cells as defined by CCR7 and CD45RA isoform. The terminally differentiated response to the Pol peptide 356–374 (31.9% CCR7−/CD45RA+ of gated cells) is diluted in the response to the entire Pol peptide pool (only 8.8% gated cells are CCR7−/CD45RA+).</p