Comparison between ArchiLD and Haploview.

<p>(A) Gene-centered visualization for the gene LAMP3 in CEU as provided by ArchiLD1k. (B) Analogous visualization in Haploview. The large number of variants analyzed makes it difficult, for example, to identify the position of the SNPs in perfect LD with rs76730564.</p

Comparison between ArchiLD and SNAP.

<p>(A) SNP-centered visualization for the variant rs76730564 in CEU as provided by ArchiLD1k. Clusters are ordered by descending values of r² with the reference SNP. (B) Analogous visualization in SNAP. The plot does not contain any SNP label nor provide any information on the location of exons.</p

An example of hierarchical clustering.

<p>On the left the hierarchical clustering of all SNP blocks associated to a particular gene. On the right a graphical representation of a gene-centered architecture. The first track contains the names and positions of all SNPs considered in the analysis. The following tracks are ordered as they appear in the hierarchical clustering plot.</p

An example of SNP-centered architecture.

<p>The first track contains the names and positions of all SNPs considered in the analysis. The second track contains the reference SNP and its linked variants. The following tracks are ordered by descending r² with respect to the reference SNP.</p

An example of chromosome-centered architecture.

<p>The first track contains the names and positions of all SNPs considered in the analysis. The second track contains all clusters located on the chromosome.</p

Expression of FOXP3fl, FOXP3Δ2 and FOXP3Δ2Δ7 in human Treg cells.

<p>(A) Protein expression levels of FOXP3 isoforms in human Treg cells. CD25+CD4+ T cells were isolated from PBMC by MACS using α-CD25 Treg isolation kit. FOXP3-specific eBio7979 antibody was used after Western blotting. The number of CD25+ cells loaded per lane is indicated. (B) RT-PCR analysis of human CD4+CD25+ cells. Relative transcription levels of FOXP3 isoforms in five healthy donors were investigated.</p

All FOXP3 isoforms interact with NFAT, NF-kB and RUNX1.

<p>(A) FOXP3 isoforms repressed NFAT-mediated transcription of luciferase in reporter assays. NIH3T3 cells were co-transfected with either control vector, FOXP3fl, FOXP3Δ2 or FOXP3Δ2Δ7 and NFAT luciferase reporter plasmid. Transcription was normalized to <i>Renilla</i> luciferase activity; the average induction in absence of FOXP3 expression was set 100%. Reporter gene assay shown is representative for three independent experiments. Error bars represent SD; statistical analysis was performed with 2-tailed unpaired student's t-test. (B) FOXP3 isoforms repressed NF-kB-mediated transcription of luciferase. Experiments were carried out as above except that a NF-kB luciferase reporter plasmid was used. Reporter gene assay shown is representative for three independent experiments. Error bars represent SD; statistical analysis was performed with 2-tailed unpaired student's t-test. (C) FOXP3 isoforms bind RUNX1 in co-immunoprecipitation experiments. HEK293T cells were co-transfected with FLAG-tagged RUNX1 and either control vector, FOXP3fl, FOXP3Δ2 or FOXP3Δ2Δ7. Co-immunoprecipitation experiments (IP) were performed with α-FLAG agarose and complexes were analyzed in immunoblots (IB) with the FOXP3-specific antibody eBio7979 (upper panels). Expression of non-FLAG-tagged constructs was confirmed by immunoblotting of total cell lysates (lower panels). Co-immunoprecipitation shown is representative for three independent experiments.</p

FOXP3Δ2Δ7 can act as dominant negative inhibitor of FOXP3fl.

<p>Mouse CD4+CD25− T cells were transduced with retroviral vector pairs encoding for FOXP3fl-IRES-CFP / IRES-GFP or FOXP3fl-IRES-CFP / FOXP3Δ2Δ7-IRES-GFP. The relative increase in CD25 expression was measured by determining the increase in mean fluorescence intensity (MFI) from GFP+ single transduced cells to GFP+CFP+ double transduced cells. The FOXP3fl-induced CD25 expression was significantly lower in the presence of FOXP3Δ2Δ7. Data represent the mean of four individual experiments; p-value was determined by using the paired student's t-test.</p

Suppression of CFSE labelled responder cells by FOXP3-transduced T cells.

<p>(A) Suppressive capacity of FOXP3 isoforms. Freshly isolated murine CD4+CD25− cells were labelled with carboxy-fluorescein diacetate succinimidylester (CFSE). Lymphoproliferation was detected in co-culture experiments with FACS-sorted FOXP3-transduced CD4+ cells or freshly isolated CD4+CD25+ regulatory mouse T cells in the presence of plate bound α-CD3 and irradiated APC. Ratio of suppressor cells to responder cells was 1∶3. CFSE dilutions were analysed by flow cytometry after 4 days, gating on live CD4+ T cells. Proliferation was suppressed by freshly isolated CD4+CD25+ regulatory T cells (Treg) and CD4+CD25− T cells transduced with murine Foxp3 (mFoxp3), with human FOXP3fl or FOXP3Δ2 but not with T cells transduced with empty control vector (Control) or with FOXP3Δ2Δ7. Suppression assay shown is representative for three independent experiments. (B) Titration of FOXP3-transduced suppressor T cells (Tsupp) and CFSE labelled responder T cells (Tresp). Experiment was carried out as described above except that T cells transduced with FOXP3fl, FOXP3Δ2 or FOXP3Δ2Δ7 were titrated to indicated ratios. The experiment was carried out in triplicates; error bars represent the SD. Inhibition is expressed as relative suppression in reference to the proliferation of T cells transduced with the empty vector.</p

Ektopic expression of FOXP3 isoforms.

<p>(A) HEK293T cells were transfected with plasmids encoding for FOXP3fl, FOXP3Δ2 and FOXP3Δ2Δ7 isoforms. Protein levels of splice variants were analyzed by Western blots using isoform-non specific α-FOXP3 antibody eBio7979. FOXP3 protein detection shown is representative for three independent experiments. (B) HEK293T cells were transfected with plasmids encoding for N-terminal GFP fusion proteins of FOXP3fl, FOXP3Δ2, FOXP3Δ2Δ7 and FOXP3ΔFKH. GFP-FOXP3, GFP-FOXP3Δ2 and GFP-FOXP3Δ2Δ7 were translocated into the nucleus, whereas GFP-FOXP3ΔFKH remained in the cytoplasm. Distribution of GFP-FOXP3 fusion proteins shown is representative for three independent experiments.</p