Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

Background: Regulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood.Results: To gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset.Conclusion: The data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases

T-bet and GATA3 orchestrate Th1 and Th2 differentiation through lineage-specific targeting of distal regulatory elements

T-bet and GATA3 regulate the CD4+ T cell Th1/Th2 cell fate decision but little is known about the interplay between these factors outside of the murine Ifng and Il4/Il5/Il13 loci. Here we show that T-bet and GATA3 bind to multiple distal sites at immune regulatory genes in human effector T cells. These sites display markers of functional elements, act as enhancers in reporter assays and are associated with a requirement for T-bet and GATA3. Furthermore, we demonstrate that both factors bind distal sites at Tbx21 and that T-bet directly activates its own expression. We also show that in Th1 cells, GATA3 is distributed away from Th2 genes, instead occupying T-bet binding sites at Th1 genes, and that T-bet is sufficient to induce GATA3 binding at these sites. We propose these aspects of T-bet and GATA3 function are important for Th1/Th2 differentiation and for understanding transcription factor interactions in other T cell lineage decisions

Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: implications for immune cell biobanking.

Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum), biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC) and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9): at a distal location (shipped overnight) and in the central laboratory (processed immediately). PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might be that despite changes due to overnight shipment, highly standardized central processing (and analysis) could be superior to multicentric de-central processing with more difficult standardization

The effects of overnight shipping on lymphocyte subpopulations.

<p>PBMC were analyzed post cryo preservation by flow cytometry, to determine the <i>ex vivo</i> frequencies of CD3⁺CD4⁺ T cell, CD3⁺CD8⁺ T cell, CD3⁺CD127⁻CD25⁺CD4⁺ regulatory T cell, CD3⁻CD19⁺ B cell and CD3⁻ CD56⁺ NK cell populations. A) The gating strategy is presented on dot plots from one representative donor. Abbreviations: EM-effector memory cells, CM-central memory cells B) Bar graphs indicate the average frequencies for all tested donors (n = 9) in non-shipped vs. shipped samples. All graphs are shown with SEM values. Before-after graphs show the same data broken down for each individual.</p

Antibody composition of staining master mix (basic panel and T_reg panel) applied for multicolor flow cytometry.

<p>Antibody composition of staining master mix (basic panel and T_reg panel) applied for multicolor flow cytometry.</p

The effects of overnight shipping on PBMC yield and survival.

<p>The cell number of blood derived PBMC was determined based on Trypan blue exclusion. A) Blood was collected in S-Monovette (Sarsted) or K2E Vacutainer (BD) tubes from 4 donors on site or at another location. Shipped samples were transported in the collection tube. PBMC yield was calculated per volume (10⁶ living PBMC/ml blood). B) Yield (10⁶ living PBMC/ml blood) is shown for PBMC derived from non-shipped vs. shipped blood (n = 9) prior to freezing. C) Viability (% of living cells/total cell count) is shown for non-shipped vs. shipped samples (n = 9), prior to freezing. D) Post-cryopreservation recovery is calculated for thawed samples (non-shipped vs. shipped, n = 9) and is provided as percentage of living cells from 10⁷ total (frozen amount/vial).</p

The effect of overnight shipment on signal transduction machinery.

<p>PBMC were stimulated with anti-CD3 (OKT3) + anti-CD28 antibodies for 5 min (A) or with rhIL-6 for 30 min (B) at 37°C in RPMI medium and lysed immediately after stimulation. Post-nuclear lysates were subjected to SDS-PAGE and proteins were transferred onto nitrocellulose membrane. Subsequently, membranes were probed with anti-pERK (A) or with anti-p-STAT3 (Y705) (B) antibody. Equal protein loading was controlled by probing the membranes against β-actin. Results are shown in a form of scanned blots for 2 representative donors out of 4 tested individuals (for both signaling pathways).</p

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function. Ullah et al. find that HIC1 is induced during human iTreg cell differentiation. HIC1 binds to and regulates the expression of key genes during iTreg differentiation. Several autoimmune-disease-associated SNPs are enriched near HIC1 ChIP-seq peaks.Peer reviewe

Time-resolved transcriptome and proteome landscape of human regulatory T cell (Treg) differentiation reveals novel regulators of FOXP3

Background: Regulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first-in-man trials of Treg transfer have achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood. Results: To gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by a targeted shRNA screen confirming a functional role in FOXP3 induction, discriminant analyses classifying iTregs accordingly, and comparable expression in an independent novel iTreg RNA-Seq dataset. Conclusion: The data generated by this novel approach facilitates understanding of the molecular mechanisms underlying iTreg generation as well as of the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune, and inflammatory diseases.Peer reviewe