ProR-dependent, FKH-independent regulation of IL-2 upon activation.

<p>(A) Activation-induced expression of IL-2 in Foxp3 or Foxp3 mutant transduced cells measured by flow cytometry. Transduced cells were rested and re-activated with cell stimulation cocktail (PMA and ionomycin with protein transport inhibitor) before staining. Data are representative of three independent experiments. (B) Expression of <i>Il2</i> mRNA in Foxp3 or Foxp3 mutant transduced cells at resting or re-activation state was determined by quantitative RT-PCR relative to the expression of CD3ε. Circles indicate measurements from resting cells, square measurements from reactivated cells. Height of the bars indicates the mean of the two experiments.</p

Foxp3 interacts physically with class I HDACs to suppress IL-2.

<p>(A) Foxp3 associates with Class I HDACs within cells. HEK293T cells were co-transfected with vectors expressing FLAG-HDAC1, 2, 3 or FLAG-GFP and HA-Foxp3. Cell lysates were immunoprecipitated with anti-FLAG M2 agarose, followed by Western blotting with anti-HA (top), or anti-FLAG (middle) antibodies. The expression of Foxp3 in cell lysate was detected with anti-HA (bottom). (B) Foxp3 directly interacts with Class I HDACs. <i>In vitro</i> translated Foxp3 (left) was incubated with bacterially expressed GST-HDAC1, 2, 3 or GST alone. After pull down with GST-affinity beads, the eluent was analyzed by immunoblotting with anti-GST (middle) and anti-Foxp3 (right). Asterisk indicates corresponding GST-fused protein. (C) Interaction of Foxp3 with class I HDACs. (top) Schematic illustration of Foxp3 and the various deletion constructs. ProR: proline-rich domain; ZnF: zinc finger domain; CC: coiled-coil domain; FKH: forkhead domain. Immunoprecipitation of (bottom left) FLAG-HDAC1, (bottom middle) FLAG-HDAC2 and (bottom right) FLAG-HDAC3 co-transfected into 293T cells with HA-tagged Foxp3 or the respective Foxp3 mutants. The lysates were immunoprecipitated with anti-FLAG M2 agarose. Precipitated proteins were probed with anti-HA or anti-FLAG antibodies. (D) Intracellular IL-2 level in (top) T_H::Foxp3 cells and (bottom) T_H::control cells in the absence (green, black) or presence (red, purple) of the respective inhibitor (isotype control in grey). Primary T_H cells transduced, rested for 40 h and incubated 8 h with Class I HDAC inhibitor Entinostat (1.8 μM), Vii106 (2.4 μM), Class IIa HDAC inhibitor MC1568 (1.5 μM) or pan-HDAC inhibitor TSA (0.05 μM) respectively. The cells were re-activated in the presence of the corresponding HDAC inhibitor for 12 h. Data are representative of three independent experiments. (E) Mean percentage (± SD, n = 3) of cells producing IL-2 in total transduced rCD8a⁺ cells under the various conditions shown in (D). <i>P</i> values between control and treatment group were determined by ANOVA followed by Tukey’s post-hoc test (* indicates <i>P</i><0.05; ** indicates <i>P</i><0.01). (F) Intracellular Foxp3 expression level after HDAC inhibitor treatment. T_H::Foxp3 in the absence (green) or presence (red) of the respective inhibitor (isotype control in grey). For (A), (C), (D) and (F) are representative results of three independent experiments. Blots in (B) are representative of two independent experiments.</p

Separation of FKH-mediated DNA binding from the scaffolding function of Foxp3.

<p>(A) Model of complex formation and DNA binding of wild type, FKH deletion mutant with an SV40 NLS sequence (Foxp3^ΔFKHnls) and proline-rich region (exon 1 to exon 3) deletion mutant (Foxp3^ΔProR) of Foxp3. Spheres represent putative Foxp3 interaction partners with (purple) or without (blue) their own DNA binding capability. (B) Quantification of the similarity in the gene expression profiles of naïve T_H cells transduced with expression vectors for wild type (Foxp3), GFP (control) or mutant Foxp3 genes: Foxp3^ΔFKHnls, Foxp3^ΔFKH, Foxp3^ΔProR. Transcriptomes from two independent experiments are shown. Euclidean distances were calculated from regularized log-transformed read counts for Foxp3-regulated genes. (C) Principal-component analysis of the transcriptomes calculated from gene expression data using regularized log-transformed read counts. Color spheres represent individual experiments. PC1, PC2 and PC3 account for 55.4%, 21.1% and 7.22% of the variance, respectively. PC values were calculated using all samples used in this study, but only the samples relevant to this figure are shown here. (D) Venn diagrams showing sets of Foxp3-regulated genes which are differentially expressed (adjusted <i>P</i>< 0.05) in a comparison between T_H::Foxp3 cells and those transduced with other constructs. The set of all Foxp3-regulated genes is represented by a grey ellipse, while colored ellipses represent sets of differentially expressed genes. (E) Venn diagrams showing sets of Foxp3-regulated genes that retain their Foxp3-like control in cells transduced with the indicated constructs. The set of all Foxp3-regulated genes is represented by a grey ellipse, while colored ellipses represent sets of genes retaining Foxp3-like control. (F) Comparison of Foxp3-regulated gene expression in T_H::control cells and those transduced with other constructs. Each dot represents an individual Foxp3-regulated gene. Genes are separated into those shown to be upregulated by Foxp3 expression and those that are downregulated. Differential gene expression was calculated from the two independent experiments using Wald tests on moderated log2 fold changes as implemented by the DESeq2 R package with <i>P</i> values adjusted for multiple testing using the procedure of Benjamini and Hochberg [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005251#pgen.1005251.ref058" target="_blank">58</a>]. Numeric annotations on the graphs indicate the number of Foxp3-upregulated and Foxp3-downregulated genes that are dysregulated by each mutant.</p

Foxp3 recruits class I HDACs to modulate histone acetylation of the <i>Il2</i> promoter.

<p>(A) Schematic diagram showing binding sites of well-known transcription factors regulating the <i>Il2</i> promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005251#pgen.1005251.ref019" target="_blank">19</a>] and the regions analysed in (B to I). (B) Primary T_H cells were transduced with retrovirus carrying HA-Foxp3, HA-GFP and HA-tagged Foxp3 deletion mutants. Transduced cells were rested and re-activated by CD3/CD28 for 6 h. ChIP quantitative PCR (qPCR) was used to analyze the binding of HA-GFP, HA-Foxp3 and deletion mutants at the <i>Il2</i> promoter using anti-HA antibody. <i>Cd3ε</i> promoter was used as negative control. (C) Expression of the respective HA-tagged proteins. Western blot is representative of two independent experiments. (D to G) ChIP-qPCR analysis of the binding of Class I HDACs at different <i>Il2</i> promoter sites and <i>Cd3e</i> promoter in re-activated transduced T_H cells expressing HA-tagged GFP, Foxp3 or the respective deletion mutant—color code as in (B). (H) ChIP-qPCR analysis of <i>Il2</i> promoter and <i>Cd3e</i> promoter using anti-acetylated histone 3 antibody in primary T_H cells, with (dark red) or without (light red) CD3/CD28 activation. Normal rabbit IgG was used as control (white and yellow). (I) ChIP-qPCR analysis of <i>Il2</i> and <i>Cd3e</i> promoter in re-activated transduced T_H cells expressing HA-tagged GFP, Foxp3 or the respective deletion mutant using anti-acetylated histone 3 antibody—color code as in (B). Height of bars represent mean values ± SD. <i>P</i> values between T_H::HA-GFP and T_H::HA-Foxp3 or the respective Foxp3 mutant transduced cells were determined by one-way ANOVA followed by Tukey’s post-hoc test. P values in (H) between rested and activated T_H cells were determined by an unpaired, two-tailed t-test (* indicates <i>P</i><0.05; ** indicates <i>P</i><0.01; ***indicates <i>P</i><0.001). In all cases, data are representative of three independent experiments for each construct.</p

The role of nuclear localisation signals in the forkhead domain of Foxp3.

<p>(A) Schematic illustrations of nuclear localization signals and addition of SV40 NLS. (B) Expression of Foxp3, Foxp3^ΔFKH and Foxp3^ΔFKHnls and the rCD8 reporter in transduced primary T cells. (C) Expression of Foxp3, Foxp3^ΔFKH and Foxp3^ΔFKHnls and the rCD8 reporter in transfected HEK293 cells. (D) Subcellular localisation of the Foxp3, Foxp3^ΔFKH and Foxp3^ΔFKHnls stained with alexa488 anti-Foxp3 antibody in transfected HEK293 cells analysed by confocal microscope. Lower panel is a higher magnification of the boxed region in the upper panels with some cells having their nuclei outline with a dotted white line and the cell boundary with a dotted black line. (E) The effect of presence or absence of nls on expression of key molecules on the expression of key Foxp3 target genes. The expression of LAG3, CD25, CTLA4, GITR and CD62L was analysed by flow cytometry 48 h post-transduction in rested T_H::Foxp3, T_H::Foxp3^ΔFKH and T_H::Foxp3^ΔFKHnls cells. Plots were gated on transduced cells that were rCD8a⁺ and are representative of three independent experiments.</p

Mutational dissection of ProR of Foxp3 reveals distinct mechanisms of gene regulation.

<p>(A) Conservation plot (top) showing the average similarity score at individual amino acid positions from multiple sequence alignments of placental mammalian Foxp3 (red; seven species), non-placental mammalian Foxp3 (black; three species) and non-mammalian vertebrate Foxp3L (green; seven species). The ‘placental’ and ‘mammalian’ halves of the ProR were defined semi-arbitrarily based on the homology across different species. The plots were generated using EMBOSS plotcon with a window size of 20. The REL test as implemented in Datamonkey was used to perform a sliding window analysis (4 codons) of placental Foxp3s with calculated Bayes factors plotted in red (bottom). The average pairwise identity (averaged over 4 amino acids) between Foxp3s is plotted in green (dotted grey lines: exon boundaries). The positions of proline residues are denoted by purple bars. For orientation, a schematic representation of Foxp3 is shown at the bottom. m1-m5 denote regions of interest for which deletion mutants were generated. (B) Flow cytometry analysis of Foxp3 target gene expression in T_H cells transduced with wild type Foxp3 (blue), control vector (black) and Foxp3 mutants (red). Foxp3^Δe1: deletion of exon1; Foxp3^Δe1-2: deletion of exon 1–2, Foxp3^ΔProR: deletion of exon 1–3; Foxp3^Δm1: deletion of region m1; Foxp3^Δm2, Foxp3^Δm3, Foxp3^Δm4, Foxp3^Δm5: regions m1, m2, m3, m4, m5 were replaced by short stretches of alanines. Foxp3^Δm4.1 and Foxp3^Δm4.2 are outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005251#pgen.1005251.s002" target="_blank">S2A Fig</a>. Expression was measured in transduced cells rested 48 h post-transduction (plots were gated on rCD8a⁺ transduced cells and are representative of three independently transduced cell samples in parallel). For IL-2, resting cells were re-stimulated for 12 h with cell stimulation cocktail (PMA and ionomycin with protein transport inhibitor) before staining. (C) Summaries of normalized mean fluorescence intensity (MFI) in (B), shown as ratios of MFI (mean ± SD) of rCD8a⁺ cells/ rCD8a^- cells in each sample. Significant differences between T_H::Foxp3 and cells transduced with the other constructs were determined by one way analysis of variance (ANOVA) followed by Tukey’s post-hoc test(* indicates <i>P</i>≤0.05; ** indicates <i>P</i>≤0.01; *** indicates <i>P</i>≤0.001).</p

Mutational dissection of ProR of Foxp3 reveals distinct transcriptional programs.

<p>(A) A representation of the Foxp3 mutants’ effects on the expression of target genes measured by flow cytometry. Each box maps the region that has been deleted/replaced by stretches of alanines with the fill in color denoting the effect on the examined gene. (grey: no loss of function; light red: partial loss of gene regulation; red: complete loss of gene regulation) A representation of the full length Foxp3 is shown above with the dotted lines marking the exon boundaries. (B) Suppressive activity of Foxp3 or Foxp3 mutant transduced cells were normalized to that of control transduced cells, which were measured as the relative proliferation of co-cultured 'target' CD4⁺CD25^- T_H cells. Data are shown as means ± SD of three independent experiments. (C) FACS sorted CD4⁺CD25^-CD45RB^hi T_H cells were co-transferred with empty vector (control), Foxp3 or Foxp3 mutant transduced CD4⁺CD25^- T_H cells into weight-matched female C.B.-17 SCID mice. The mean weight of the mice (n≥5 mice per group) was set to 100% the day before the control mice started to lose weight. Each symbol represents the mean weight ± SEM value for each group on each measurement day. <i>P</i> values were determined by one-way ANOVA (ns: not significant, *** <i>P</i>≤0.001). (D) Quantification of the similarity in gene expression profiles of T_H cells transduced with wild type (Foxp3), GFP (control), Foxp3^ΔProR, Foxp3^Δe1, Foxp3^Δm4.2 or Foxp3^Δm5. Transcriptomes from two independent experiments are shown. Euclidean distances were calculated from regularized log-transformed read counts for Foxp3-regulated genes. (E) Principal-component analysis of the transcriptomes calculated from gene expression data using regularized log-transformed read counts for each Foxp3-regulated gene. Color spheres represent individual experiments. PC1, PC2 and PC3 account for 55.4%, 21.1% and 7.22% of the variance, respectively. PC values were calculated using all samples used in this study, but only the samples relevant to this figure are shown here. (F) Venn diagram showing sets of Foxp3-regulated genes that retain their Foxp3-like control in cells transduced with the indicated constructs. The set of all Foxp3-regulated genes is represented by a grey ellipse while the blue ellipse indicates the set of genes that retain their regulation in T_H::Foxp3^ΔProR and the brown ellipse those genes that retain their regulation in T_H::Foxp3^Δm5. (G) Venn diagram showing the set of all Foxp3-regulated genes dysregulated by deletion of the ProR (T_H::Foxp3^ΔProR; blue ellipse). The pink ellipse represents those genes that are dysregulated in TH::Foxp3Δm4.2 and the green ellipse those dysregulated in T_H::Foxp3^Δe1. (H) Heat-map of log-transformed fold-changes in expression of Foxp3-regulated genes between T_H::Foxp3 cells and cells transduced with the various Foxp3 mutants. The dendrogram above the plot indicates hierarchical clustering performed using Euclidean distance. Differential gene expression in (F), and (G) was calculated from the two independent transduction experiments shown in (D) and (E) using Wald tests on moderated log2 fold changes as implemented by the DESeq2 R package with <i>P</i> values adjusted for multiple testing using the procedure of Benjamini and Hochberg [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005251#pgen.1005251.ref059" target="_blank">59</a>].</p

Analysis of immunology-related genes and enriched GO terms within gene lists.

<p>^a<i>P</i> values calculated by Fisher’s exact test in comparison with all genes in mouse genome.</p><p>^b<i>P</i> values calculated by Binomial test in comparison with distribution of immunology-related and non-immunology-related terms enriched in set of 2,407 Foxp3-regulated T_Reg-relevant genes.</p><p>Analysis of immunology-related genes and enriched GO terms within gene lists.</p

Ontogeny of the B- and T-cell response in a primary Zika virus infection of a dengue-naïve individual during the 2016 outbreak in Miami, FL

<div><p>Zika virus (ZIKV) is a mosquito-borne flavivirus of significant public health concern. In the summer of 2016, ZIKV was first detected in the contiguous United States. Here we present one of the first cases of a locally acquired ZIKV infection in a dengue-naïve individual. We collected blood from a female with a maculopapular rash at day (D) 5 and D7 post onset of symptoms (POS) and we continued weekly blood draws out to D148 POS. To establish the ontogeny of the immune response against ZIKV, lymphocytes and plasma were analyzed in a longitudinal fashion. The plasmablast response peaked at D7 POS (19.6% of CD19⁺ B-cells) and was undetectable by D15 POS. ZIKV-specific IgM was present at D5 POS, peaked between D15 and D21 POS, and subsequently decreased. The ZIKV-specific IgG response, however, was not detected until D15 POS and continued to increase after that. Interestingly, even though the patient had never been infected with dengue virus (DENV), cross-reactive IgM and IgG binding against each of the four DENV serotypes could be detected. The highest plasma neutralization activity against ZIKV peaked between D15 and D21 POS, and even though DENV binding antibodies were present in the plasma of the patient, there was neither neutralization nor antibody dependent enhancement (ADE) of DENV. Interestingly, ADE against ZIKV arose at D48 POS and continued until the end of the study. CD4⁺ and CD8⁺ T-cells recognized ZIKV-NS2A and ZIKV-E, respectively. The tetramer positive CD8⁺ T-cell response peaked at D21 POS with elevated levels persisting for months. In summary, this is the first study to establish the timing of the ontogeny of the immune response against ZIKV.</p></div

Phylogenetic tree of ZIKV isolated from Hu0015 compared to previously sequenced ZIKV genomes.

<p>A detailed maximum likelihood phylogenetic analysis of published ZIKV genomes from the Pacific and Americas (Asian genotype, from 2013–2016). The pink branches represent isolates from the 2016 ZIKV outbreak in Florida. Hu0015 is one of the first ZIKV sequences from autochthonous transmission in the contiguous US and clades with one of four ZIKV lineages detected during the outbreak in Florida. The scale of 0.005 represents nucleotide substitutions per site in the viral genome.</p