IL-33 Receptor-Expressing Regulatory T Cells Are Highly Activated, Th2 Biased and Suppress CD4 T Cell Proliferation through IL-10 and TGFβ Release

Immunomodulatory Foxp3+ regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2+ Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2+ Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 –especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2+ Tregs are superior to ST2− Tregs in suppressing CD4+ T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2+ Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2+ Tregs useful targets for immunomodulatory therapies

Impaired p53-Mediated DNA Damage Response Contributes to Microcephaly in Nijmegen Breakage Syndrome Patient-Derived Cerebral Organoids

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within nibrin (NBN), a DNA damage repair protein. Hallmarks of NBS include chromosomal instability and clinical manifestations such as growth retardation, immunodeficiency, and progressive microcephaly. We employed induced pluripotent stem cell-derived cerebral organoids from two NBS patients to study the etiology of microcephaly. We show that NBS organoids carrying the homozygous 657del5 NBN mutation are significantly smaller with disrupted cyto-architecture. The organoids exhibit premature differentiation, and Neuronatin (NNAT) over-expression. Furthermore, pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. After exposure to bleomycin, NBS organoids undergo delayed p53-mediated DNA damage response and aberrant trans-synaptic signaling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders

Circulating and Tissue-Resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function Is Altered During Inflammation.

BACKGROUND & AIMS: Interactions between commensal microbes and the immune system are tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We measured the abundance of T cells in circulation and intestinal tissues that respond to intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation. METHODS: We collected samples of peripheral blood mononuclear cells and intestinal tissues from healthy individuals (controls, n = 13-30) and patients with inflammatory bowel diseases (n = 119; 59 with ulcerative colitis and 60 with Crohn's disease). We used 2 independent assays (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species (Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium, and Clostridium difficile) to quantify, expand, and characterize microbe-reactive CD4+ T cells. We sequenced T-cell receptor Vβ genes in expanded microbe-reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and gene expression patterns were measured by flow cytometry and quantitative polymerase chain reaction. RESULTS: Circulating and gut-resident CD4+ T cells from controls responded to bacteria at frequencies of 40-4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T cells were mainly of a memory phenotype, present in peripheral blood mononuclear cells and intestinal tissue, and had a diverse T-cell receptor Vβ repertoire. These cells were functionally heterogeneous, produced barrier-protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A, interferon gamma, and tumor necrosis factor. In patients with inflammatory bowel diseases, microbiota-reactive CD4+ T cells were reduced in the blood compared with intestine; T-cell responses that we detected had an increased frequency of interleukin 17A production compared with responses of T cells from blood or intestinal tissues of controls. CONCLUSIONS: In an analysis of peripheral blood mononuclear cells and intestinal tissues from patients with inflammatory bowel diseases vs controls, we found that reactivity to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire, and does not necessarily indicate disrupted interactions between immune cells and the commensal microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing barrier-protective cytokines and providing a large pool of T cells that react to pathogens

DCE-MRI in Glioma, Infiltration Zone and Healthy Brain to Assess Angiogenesis: A Biopsy Study

Purpose: To explore the focal predictability of vascular growth factor expression and neovascularization using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in glioma. Methods: 120 brain biopsies were taken in vital tumor, infiltration zone and normal brain tissue of 30 glioma patients: 17 IDH(isocitrate dehydrogenase)-wildtype glioblastoma (GBM), 1 IDH-wildtype astrocytoma °III (together prognostic group 1), 3 IDH-mutated GBM (group 2), 3 anaplastic astrocytomas IDH-mutated (group 3), 4 anaplastic oligodendrogliomas and 2 low-grade oligodendrogliomas (together prognostic group 4). A mixed linear model evaluated the predictabilities of microvessel density (MVD), vascular area ratio (VAR), mean vessel size (MVS), vascular endothelial growth factor and receptors (VEGF-A, VEGFR‑2) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) expression from Tofts model kinetic and model-free curve parameters. Results: All kinetic parameters were associated with VEGF‑A (all p < 0.001) expression. K trans, k ep and v e were associated with VAR (p = 0.006, 0.004 and 0.01, respectively) and MVS (p = 0.0001, 0.02 and 0.003, respectively) but not MVD (p = 0.84, 0.74 and 0.73, respectively). Prognostic groups differed in K trans (p = 0.007) and v e (p = 0.004) values measured in the infiltration zone. Despite significant differences of VAR, MVS, VEGF‑A, VEGFR‑2, and VE-PTP in vital tumor tissue and the infiltration zone (p = 0.0001 for all), there was no significant difference between kinetic parameters measured in these zones. Conclusion: The DCE-MRI kinetic parameters show correlations with microvascular parameters in vital tissue and also reveal blood-brain barrier abnormalities in the infiltration zones adequate to differentiate glioma prognostic groups

Stroke-derived neutrophils demonstrate higher formation potential and impaired resolution of CD66b + driven neutrophil extracellular traps

Abstract Background Recent evidence suggests a merging role of immunothrombosis in the formation of arterial thrombosis. Our study aims to investigate its relevance in stroke patients. Methods We compared the peripheral immunological profile of stroke patients vs. healthy controls. Serum samples were functionally analyzed for their formation and clearance of Neutrophil-Extracellular-Traps. The composition of retrieved thrombi has been immunologically analyzed. Results Peripheral blood of stroke patients showed significantly elevated levels of DNAse-I (p < 0.001), LDG (p = 0.003), CD4 (p = 0.005) as well as the pro-inflammatory cytokines IL-17 (p < 0.001), INF-γ (p < 0.001) and IL-22 (p < 0.001) compared to controls, reflecting a TH1/TH17 response. Increased counts of DNAse-I in sera (p = 0.045) and Neutrophil-Extracellular-Traps in thrombi (p = 0.032) have been observed in patients with onset time of symptoms longer than 4,5 h. Lower values of CD66b in thrombi were independently associated with greater improvement of NIHSS after mechanical thrombectomy (p = 0.045). Stroke-derived neutrophils show higher potential for Neutrophil-Extracellular-Traps formation after stimulation and worse resolution under DNAse-I treatment compared to neutrophils derived from healthy individuals. Conclusions Our data provide new insight in the role of activated neutrophils and Neutrophil-Extracellular-Traps in ischemic stroke. Future larger studies are warranted to further investigate the role of immunothrombosis in the cascades of stroke. Trial registration DRKS, DRKS00013278, Registered 15 November 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS0001327

ST2⁺ Tregs suppress CD4⁺ T cell proliferation more effectively than ST2⁻ Tregs <i>in vitro</i>.

<p><i>(A)</i> Proliferation profiles of CellTrace-labelled WT CD25^- CD62L^hi CD4⁺ responder T cells (Tresp) co-cultured with WT ST2⁺ (black) and ST2⁻ (grey) CD25⁺ Tregs during an <i>in vitro</i> suppression assay at day 4 of culture. T cells were stimulated by APCs and anti-CD3 antibody with <i>(right column)</i> or without <i>(left column)</i> the addition of recombinant IL-33. Treg:Tresp ratios are indicated <i>(left)</i>. Percentage of divided cells and the division index (number in brackets) are shown in each histogram in the respective color. <i>(B)</i> Proliferation profile of Tresp cultured under the same conditions as in 3A but without Tregs, either with (grey) or without (black) the addition of anti-CD3 antibody. <i>(C)</i> MFI of the ST2 staining on all Tregs recovered from the cultures described in 3A. Data are representative of 2–3 independent experiments.</p

ST2⁺ Tregs preferentially home outside of secondary lymphoid organs and exhibit a highly activated phenotype.

<p>Flow cytometric analysis of the phenotype and frequency of WT ST2⁺ and ST2⁻ Foxp3⁺ Tregs in spleen, pLN, blood, lung, lamina propria of the small intestine (siLP) and colon (coLP): <i>(A)</i> Frequency of ST2⁺ Tregs <i>(left)</i> and MFI of the ST2 staining on the ST2⁺ Treg fraction <i>(right)</i>. <i>(B)</i> MFI of chemokine receptor and α4β7 staining on ST2⁺ and ST2⁻ Tregs. <i>(C)</i> KLRG1 and CD103 expression in ST2⁺ <i>(top)</i> and ST2⁻ <i>(bottom)</i> Tregs from spleen; quantified frequencies from indicated organs <i>(right)</i>. <i>(D)</i> Frequency of CD44^hi, CD62L^lo and CTLA-4⁺ T cells within ST2⁺ and ST2⁻ Treg populations. <i>(E)</i> MFI of the Foxp3 staining <i>(left)</i> and geometric mean index of GATA-3 <i>(right)</i> in ST2⁺ and ST2⁻ Tregs. <i>(F)</i> Quantification of mRNA expression of the indicated genes from FACS-sorted ST2⁺ and ST2⁻ CD25⁺ Tregs from spleen and pLN <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(G)</i> Frequency of ST2⁺ and ST2⁻ Tregs with IL-10 production capability as detected by GFP expression from <i>B6</i>.<i>Foxp3</i>^<i>hCD2</i> <i>xIl10</i>^<i>gfp</i> reporter mice. Fig <i>2A</i>: Data are representative of at least 2 independent experiments. Bar graphs show the mean ± SD of at least 5 biological replicates. Fig <i>2B</i>: pooled data from 2 independent experiments with 3–5 biological replicates each. Bar graphs show the mean ± SD. Fig <i>2C–2E</i> and <i>2G</i>: Data are representative of at least 2 independent experiments. Scatter plots depict one mouse as individual dot with mean ± SD. Fig <i>2F</i>: pooled data from 2 independent experiments. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p

ST2⁺ Tregs express Th2 cytokines and suppress CD4⁺ T cell proliferation via IL-10 and TGFβ.

<p><i>(A-D)</i> ST2⁺ and ST2⁻ Tregs from spleen and lymph nodes of WT mice activated <i>in vitro</i> by plate-bound anti-CD3/anti-CD28 antibodies in the presence of IL-2 with or without recombinant IL-33 for 60–70 hours: <i>(A)</i> Fold change in the number of viable Tregs upon IL-33 treatment. <i>(B) Tgfb1</i> mRNA expression normalized to <i>Hprt</i> endogenous control. <i>(C)</i> Cytokine concentration in the supernatants as determined by cytometric bead array. <i>(D)</i> Geometric mean index of GATA-3 in stable ST2⁺ and ST2⁻ Tregs at the end of culture. <i>(E) In vitro</i> suppression assay with ST2⁺ and ST2⁻ Tregs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161507#pone.0161507.g003" target="_blank">Fig 3</a> (Treg:Tresp ratio 1:5) with addition of blocking anti-IL-10R antibody or TGFβRI inhibitor. The relative division index indicates the fold increase in division of Tresp upon treatment. Division index of untreated Tresp was set to 1 in each group. <i>(F)</i> Quantification of mRNA expression of the indicated genes from sorted ST2⁺ and ST2⁻ CD25⁺ Tregs <i>ex vivo</i>. mRNA expression normalized to <i>Hprt</i> endogenous control. Fig <i>4A</i>–<i>4C</i>, <i>4E</i> and <i>4F</i>, data pooled from 2–3 independent experiments each performed with 2 replicates per condition. Fig <i>4D</i> is representative of 2 independent experiments with at least 2 replicates per condition each. Bar graphs show the mean ± SD. Significance was tested using unpaired Student’s t test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; non-significant (ns) p > 0.05.</p