DataSheet_1_Inflammatory perturbations in early life long-lastingly shape the transcriptome and TCR repertoire of the first wave of regulatory T cells.pdf

The first wave of Foxp3+ regulatory T cells (Tregs) generated in neonates is critical for the life-long prevention of autoimmunity. Although it is widely accepted that neonates are highly susceptible to infections, the impact of neonatal infections on this first wave of Tregs is completely unknown. Here, we challenged newborn Treg fate-mapping mice (Foxp3eGFPCreERT2xROSA26STOP-eYFP) with the Toll-like receptor (TLR) agonists LPS and poly I:C to mimic inflammatory perturbations upon neonatal bacterial or viral infections, respectively, and subsequently administrated tamoxifen during the first 8 days of life to selectively label the first wave of Tregs. Neonatally-tagged Tregs preferentially accumulated in non-lymphoid tissues (NLTs) when compared to secondary lymphoid organs (SLOs) irrespective of the treatment. One week post challenge, no differences in the frequency and phenotypes of neonatally-tagged Tregs were observed between challenged mice and untreated controls. However, upon aging, a decreased frequency of neonatally-tagged Tregs in both NLTs and SLOs was detected in challenged mice when compared to untreated controls. This decrease became significant 12 weeks post challenge, with no signs of altered Foxp3 stability. Remarkably, this late decrease in the frequency of neonatally-tagged Tregs only occurred when newborns were challenged, as treating 8-days-old mice with TLR agonists did not result in long-lasting alterations of the first wave of Tregs. Combined single-cell T cell receptor (TCR)-seq and RNA-seq revealed that neonatal inflammatory perturbations drastically diminished TCR diversity and long-lastingly altered the transcriptome of neonatally-tagged Tregs, exemplified by lower expression of Tigit, Foxp3, and Il2ra. Together, our data demonstrate that a single, transient encounter with a pathogen in early life can have long-lasting consequences for the first wave of Tregs, which might affect immunological tolerance, prevention of autoimmunity, and other non-canonical functions of tissue-resident Tregs in adulthood.</p

B cells and macrophages fail to induce transcriptional enhancer activity of the TSDR.

<p>Dual luciferase assays were performed after transfecting reporter plasmids carrying the indicated inserts or an empty pGL3 vector (EV) into RLM-11 cells (T cell line), A20 cells (B cell line) or RAW 264.7 cells (macrophage cell line). Three hrs (RLM-11, A20) or 20 hrs (RAW 264.7) after transfection, cells were stimulated for 16 hrs with PMA/iono (RLM-11, A20) or for 14 hrs with LPS/IFN-γ (RAW 264.7), followed by measurement of luciferase activities (mean±SD, n = 3). Data are representative of two to four independent experiments.</p

c-Rel^−/− Tregs show a stable phenotype.

<p>(<b>A</b>) CD4⁺CD25^hi Tregs and CD4⁺CD25⁻ Tconv were isolated from wild-type (WT) or c-Rel^−/− mice. Genomic DNA was isolated and subjected to bisulfite sequencing in order to determine the methylation status of CpG dinucleotides within the TSDR. (<b>B</b>) CD4⁺CD8⁻CD62L^hiCD25^hi Tregs from spleen and lymph nodes of c-Rel^−/− or WT mice were sorted and an aliquot was analyzed for Foxp3 expression by flow cytometry (top panel). Cells were cultured in the presence of IL-2 and stimulated by plate-bound α-CD3/CD28 for six days followed by flow cytometric analysis of Foxp3 expression. Cells depicted were pregated to viable CD4⁺ T cells. Results represent one out of two independent experiments.</p

Degradation of IκBα is not required for TSDR enhancer activity.

<p>Luciferase plasmids integrating either the NF-κB-RE or TSDR-FoxPro were co-transfected with either an empty vector or with a vector encoding the super-repressor, a non-degradable form of IκBα, into RLM-11 cells. Dual luciferase assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a> and unstimulated cells served as controls. Luciferase activities are shown as percent of empty vector controls and standard deviations of performed triplicates are shown. One representative experiment out of at least two independent experiments is depicted.</p

Kinase activity of IKKα and IKKβ is largely dispensable for TSDR enhancer activity.

<p>(<b>A</b>) Luciferase plasmids encoding NF-κB-RE or TSDR-FoxPro were co-transfected with plasmids encoding kinase dead (KD) or wild-type (WT) forms of IκB kinase α and β (IKKα and IKKβ) into RLM-11 cells. Cells were cultured for one day allowing efficient protein expression before cells were stimulated overnight with PMA/iono and dual luciferase assays were performed. Luciferase activities are given as percent of luciferase activity of WT samples and standard deviations were calculated from three replicates. (<b>B</b>) Dual luciferase assays as described in (A) were performed co-transfecting the indicated luciferase constructs with a plasmid encoding the constitutively active form of IKKβ (IKK-CA) or empty vector as control (mean±SD, n = 3). One representative out of three independent experiments is shown.</p

Kinase-selective proteomics provided comprehensive insight into the kinase complement of human Tregs and Teffs.

<p>(<b>A</b>) Enrichment of kinases. Percentage of kinases and non-kinases (others: proteins without kinase activity) after VI16743/Purvalanol-B-mediated affinity purification is shown. (<b>B</b>) Quality of kinase identification. Mean Mascot Mowse score of kinases <i>vs</i>. non-kinases (others). The Mascot score is reflecting the probability of the match between the mass spectrometry spectra/data and the protein sequence given in the database. Data shown in A and B are representative for four independently performed LC-MS/MS experiments. (<b>C</b>) Kinase complement of human Tregs and Teffs. In total, 185 kinases were identified. Kinases from nearly all groups of the human kinome were detected: AGC, PKA/PKG/PKC-family kinases; CAMK, calcium/calmodulin-dependent kinases; CK1, casein kinases; CMGC, CDK/MAPK/GSK3/CLK-family kinases; RCG, receptor guanylate cyclases; STE, sterile homologue kinases; TK, tyrosine kinases; TKL, tyrosine kinase-like kinases; atypical protein kinases; Other, kinases belonging to non of the mentioned groups; NPK, non-protein-kinases (kinases having non protein substrates). The kinase dendrogram was adapted with permission from Cell Signaling Technology, Inc. (<a href="http://www.cellsignal.com" target="_blank">www.cellsignal.com</a>).</p

The postulated NF-κB binding site of the TSDR is not transcriptionally responsive to activated NF-κB.

<p>(<b>A</b>) RLM-11 cells were stimulated with PMA/iono for indicated time periods and applied to subcellular fractionation. Nuclear and cytoplasmic extracts were analyzed by Western blotting using the indicated antibodies. p44/42 and lamin B served as loading and purity controls for cytoplasmic and nuclear fractions, respectively. (<b>B</b>) A luciferase reporter plasmid integrating the NF-κB-RE was transfected into RLM-11 cells and dual luciferase assays were performed in triplicates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a>. Mean luciferase activity is shown as fold increase to unstimulated control. Results are representative of four independent experiments. (<b>C</b>) A schematic view on the first part of the <i>Foxp3</i> gene locus is depicted. White boxes indicate untranslated exons, the first translated exon is indicated in black. Evolutionary conserved non-coding sequences (CNS) are indicated in grey. The distended region of the TSDR includes the previously described NF-κB binding site (black frame), which is flanked by the seventh CpG motif (underlined) of the TSDR. (<b>D</b>) A tandem of five repetitive sequences of the putative NF-κB binding site was inserted into the pCpGL luciferase reporter plasmid upstream of the <i>EF</i> promoter (tandem-EFPro). Dual luciferase assays were performed as described in (B) using pCpGL-TSDR-EFPro as a positive control. Data represent one out of two independent experiments.</p

iTRAQ-based quantification and statistical evaluation of relative kinase expression in Tregs and Teffs.

<p>iTRAQ-based quantification and statistical analysis of differential kinase expression in Tregs and Teffs is exemplarily shown using the example of cyclin-dependent kinase 6 (CDK6). (<b>A</b>) MS/MS fragmentation spectrum of a tryptic peptide derived from cyclin-dependent kinase 6 (CDK6). Ions used for peptide sequencing and protein identification are indicated in red (b ions series) or blue (y ions series). The resulting peptide sequence (GSSDVDQLGK) is shown in the upper part of the diagram. The box embedded in the graph displays a magnification of the low molecular mass range where the iTRAQ reporter intensities could be observed. The intensity of the iTRAQ reporter 115 is correlating with the peptide abundance in Tregs. The peak at 114 Da represents the amount of the respective peptide in Teffs. (<b>B</b>) Statistical analysis of relative CDK6 peptide abundances. Analyses were performed by iTRAQassist as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone.0040896-Hundertmark1" target="_blank">[40]</a>. Most likely (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone-0040896-t001" target="_blank">Table 1</a>) and further possible peptide regulations were calculated and depicted as likelihood curves for every peptide. All peptides derived from CDK6 were significantly less abundant in Tregs. All peptide regulations calculated for individual CDK6 peptides were used to determine the overall regulation of the CDK6 protein (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone-0040896-t001" target="_blank">Table 1</a>). Here CDK6 peptide data are shown obtained from one representative LC-MS/MS experiments.</p

Differential kinase abundances in Tregs and Teffs.

<p>Kinases regarded as expressed differentially in both T cell subsets (RF = Treg/Teff) had to met two criteria: −1.5>RF>1.5 at least in two of the conducted experiments and −1.5>RF_[median] >1.5 (¹median RF over all performed experiments).</p

Kinase signaling network specificities of human Tregs.

<p>Comparative kinome analysis revealed differential kinase expression in Tregs and Teffs. Altered kinase expression in Tregs (RF = Treg/Teff) is indicated by green (increased expression in Tregs) and red boxes (reduced expression in Tregs). Kinases regarded as expressed differentially had to met two criteria: −1.5>RF>1.5 at least in two of the conducted experiments and −1.5>RF_[median] >1.5 (median RF over all performed experiments). The figure provides an overview of possible kinase functions in Tregs. Median RF values and references for indicated kinase functions are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone-0040896-t002" target="_blank">Table 2</a>.</p