Human CD4+ Memory T Cells Can Become CD4+IL-9+ T Cells

Background: IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-β directs mouse CD4+CD25−CD62L+ T cells to commit to inflammatory IL-9 producing CD4+ T cells. Methodology/Principal Findings: Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-β induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4+CD25−CD45RO+ T cells as compared to naïve CD4+CD25−CD45RA+ T cells. In addition, as compared to pbCD3/sCD28 plus TGF-β stimulation, IL-4+TGF-β stimulated memory CD4+CD25−CD45RO+ T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4+IL-9+ T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNγ or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-β stimulated resting memory CD4+ T cells demonstrated that the addition of IL-1β, IL-12, and IL-21 further enhance IL-9 production. Conclusions/Significance: Taken together these data show both the differences and similarities between mouse and human CD4+IL9+ T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4+ T cells to antigen

The Role of TNF-α in Mice with Type 1- and 2- Diabetes

Background: Previously, we have demonstrated that short-term treatment of new onset diabetic Non-obese diabetic (NOD) mice, mice that are afflicted with both type 1 (T1D) and type 2 (T2D) diabetes with either Power Mix (PM) regimen or alpha1 antitrypsin (AAT) permanently restores euglycemia, immune tolerance to self-islets and normal insulin signaling. Methodology and Principal Findings: To search for relevant therapeutic targets, we have applied genome wide transcriptional profiling and systems biology oriented bioinformatics analysis to examine the impact of the PM and AAT regimens upon pancreatic lymph node (PLN) and fat, a crucial tissue for insulin dependent glucose disposal, in new onset diabetic non-obese diabetic (NOD) mice. Systems biology analysis identified tumor necrosis factor alpha (TNF-$\alpha$) as the top focus gene hub, as determined by the highest degree of connectivity, in both tissues. In PLNs and fat, TNF-$\alpha$ interacted with 53% and 32% of genes, respectively, associated with reversal of diabetes by previous treatments and was thereby selected as a therapeutic target. Short-term anti-TNF-$\alpha$ treatment ablated a T cell-rich islet-invasive and beta cell-destructive process, thereby enhancing beta cell viability. Indeed anti-TNF-$\alpha$ treatment induces immune tolerance selective to syngeneic beta cells. In addition to these curative effects on T1D anti-TNF-e33254 treatment restored in vivo insulin signaling resulting in restoration of insulin sensitivity. Conclusions: In short, our molecular analysis suggested that PM and AAT both may act in part by quenching a detrimental TNF-$\alpha$ dependent effect in both fat and PLNs. Indeed, short-term anti-TNF-$\alpha$ mAb treatment restored enduring euglycemia, self-tolerance, and normal insulin signaling

Resistance of Renal Cell Carcinoma to Sorafenib Is Mediated by Potentially Reversible Gene Expression

Purpose: Resistance to antiangiogenic therapy is an important clinical problem. We examined whether resistance occurs at least in part via reversible, physiologic changes in the tumor, or results solely from stable genetic changes in resistant tumor cells. Experimental Design: Mice bearing two human RCC xenografts were treated with sorafenib until they acquired resistance. Resistant 786-O cells were harvested and reimplanted into naïve mice. Mice bearing resistant A498 cells were subjected to a 1 week treatment break. Sorafenib was then again administered to both sets of mice. Tumor growth patterns, gene expression, viability, blood vessel density, and perfusion were serially assessed in treated vs control mice. Results: Despite prior resistance, reimplanted 786-O tumors maintained their ability to stabilize on sorafenib in sequential reimplantation steps. A transcriptome profile of the tumors revealed that the gene expression profile of tumors upon reimplantation reapproximated that of the untreated tumors and was distinct from tumors exhibiting resistance to sorafenib. In A498 tumors, revascularization was noted with resistance and cessation of sorafenib therapy and tumor perfusion was reduced and tumor cell necrosis enhanced with re-exposure to sorafenib. Conclusions: In two RCC cell lines, resistance to sorafenib appears to be reversible. These results support the hypothesis that resistance to VEGF pathway therapy is not solely the result of a permanent genetic change in the tumor or selection of resistant clones, but rather is due to a great extent to reversible changes that likely occur in the tumor and/or its microenvironment

Mapping Differentiation under Mixed Culture Conditions Reveals a Tunable Continuum of T Cell Fates

Cell differentiation is typically directed by external signals that drive opposing regulatory pathways. Studying differentiation under polarizing conditions, with only one input signal provided, is limited in its ability to resolve the logic of interactions between opposing pathways. Dissection of this logic can be facilitated by mapping the system's response to mixtures of input signals, which are expected to occur in vivo, where cells are simultaneously exposed to various signals with potentially opposing effects. Here, we systematically map the response of naïve T cells to mixtures of signals driving differentiation into the Th1 and Th2 lineages. We characterize cell state at the single cell level by measuring levels of the two lineage-specific transcription factors (T-bet and GATA3) and two lineage characteristic cytokines (IFN-γ and IL-4) that are driven by these transcription regulators. We find a continuum of mixed phenotypes in which individual cells co-express the two lineage-specific master regulators at levels that gradually depend on levels of the two input signals. Using mathematical modeling we show that such tunable mixed phenotype arises if autoregulatory positive feedback loops in the gene network regulating this process are gradual and dominant over cross-pathway inhibition. We also find that expression of the lineage-specific cytokines follows two independent stochastic processes that are biased by expression levels of the master regulators. Thus, cytokine expression is highly heterogeneous under mixed conditions, with subpopulations of cells expressing only IFN-γ, only IL-4, both cytokines, or neither. The fraction of cells in each of these subpopulations changes gradually with input conditions, reproducing the continuous internal state at the cell population level. These results suggest a differentiation scheme in which cells reflect uncertainty through a continuously tuneable mixed phenotype combined with a biased stochastic decision rather than a binary phenotype with a deterministic decision

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

Background: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. Methodology/Principal Findings: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis. Conclusions: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes

CD4+CD25+T regulatory cells in multiple sclerosis

Multiple sclerosis (MS) is a chronic, inflammatory, and demyelinating disease of the central nervous system (CNS), which is likely to be mediated by autoaggressive immunity. Treatments of MS that may beneficially influence the disease course consist of Avonex (interferon-beta; IFN-beta), and Copaxone (glatiramer acetate; GA). Studies in experimental autoimmune encephalomyelitis (EAE), a commonly used experimental model of human MS, have demonstrated a crucial role of CD4+CD25+ T regulatory (Tr) cells in suppressing autoreactive T cells and promoting peripheral tolerance. Recent studies have shown that Tr cells inhibiting the proliferation and cytokine secretion by CD4+ T cells, are enriched in the CD25high cells among CD4+ T cells. It is not yet known if circulating CD4+CD25 high Tr cells are involved in MS. The aims of this study were to: 1. identify and quantify CD4+CD25+ and CD4+CD25high Tr cells among blood CD4+ T cells, and proportions of CD4+CD25high Tr cells expressing cell surface and intracellular molecules in patients with untreated MS vs MS treated with either IFN-beta-1a or GA or the combination of IFN-beta-1a+GA vs healthy controls (HC); 2. to correlate the properties of circulating CD4+CD25+ Tr cells with lesion load (LL) and gadolinium (Gd) enhancement detected by magnetic resonance imaging (MRI) of the brain in untreated MS patients. 3. analyse the in-vitro influence of GA upon CD4+CD25 high Tr cells from MS patients vs from HC. 4. examine whether a functional deficit inherent to CD4+CD25+ Tr cells occurred in untreated MS patients vs HQ in terms of the ability of Tr cells to suppress the production of pro-inflammatory cytokines by blood mononuclear cells (MNC) upon auto- or foreign-antigenic stimulation. These studies demonstrated that i) there are no alterations of frequency of circulating CD4+CD25+ and CD4+CD25high Tr cells nor of the proportions of CD4+CD25 high Tr cells expressing CD45RO, HLADR, CD95, CTLA-4 or IL-10 in MS patients compared to HQ ii) the frequency of CD4+CD25+ Tr cells, and the proportions of IL-10 expressing CD4+CD25+ Tr cells are elevated in MS patients with Gdenhancing lesions; iii) the proportion of CXCR3+ CD4+ T cells is lower in the group of patients with Gdenhancing lesions on T1weighted images, as compared to the group with no Gd-enhancing lesions; iv) GA, in vitro elevates the levels of IL-10 producing blood CD4+CD25 high Tr cells in both MS patients and HC, and; v) CD4+CD25+ Tr cells augments the secretion of Th1 cytokines IFN-gamma and IL-2, but not of Th.2 cytokine IL-13 in response to MBP, equally in MS patients and HC, suggesting that the functional activity of CD4+CD25+ Tr cells in terms of controlling Th1 and Th2 cytokine secretions in response to MBP, is not significantly altered in MS

IL-1β amplifies IL-4+TGF-β induced IL-9 production by memory CD4⁺CD25⁻CD45RO⁺ T cells.

<p>Resting memory CD4⁺CD25⁻CD45RO⁺ T cells (2.0×10⁵/ml in 96 well plates) activated with pbCD3/sCD28 alone or with IL-4+TGF-β, in the presence or absence of IL-1β, IL-2, IL-6, IL-12, and IL-21 for 96hrs and supernatants were collected. Influence of IL-1β, IL-2, IL-6, IL-12, and IL-21 on IL-9 production of IL-4+TGF-β treated CD4⁺CD25⁻CD45RO⁺ T cells was examined. IL-1β, IL-12 or IL-21 significantly elevated IL-4+TGF-β induced IL-9 production, but IL-1β had significantly higher influence compared to IL-12 or IL-21 (<i>n = 3</i>). Data is expressed as the mean±SD.</p

IL-4+TGF-β in presence of pbCD3/sCD28 activation induce generation of CD4⁺IL-9⁺ T cells.

<p><b>A)</b> CD4⁺CD25⁻ T cells (1.0×10⁶/ml in 24 well plates) were activated with plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) in presence or absence of IL-4 or TGF-β or IL-4+TGF-β for 96hrs and analyzed by flow cytometry for IL-9 expression. IL-4+TGF-β in combination induced significantly higher percentage of CD4⁺ T cells positive for IL-9, as compared to IL-4, TGF-β, or neither (<i>n = 14</i>). Data is expressed as the mean±SD. <b>B)</b> CD4⁺CD25⁻ T cells, CD4⁺CD25⁻CD45RA⁺ T cells (naïve T cells), CD4⁺CD25⁻CD45RO⁺ T cells (resting memory T cells) (2.0×10⁵/ml in 96 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β in a 96 well plate for 96hrs. Cells were surface stained for CD4 PerCP-Cy5.5 and intracellular stained for IL-9 PE. IL-4+TGF-β in combination induced IL-9 expression by both naïve and memory T cells, but memory T cells expressed high levels of IL-9. Data are representative of six independent experiments (six different donors).</p

Cytokine and transcription factor profile of memory CD4⁺CD25⁻CD45RO⁺ T cells activated with IL-4+TGF-β.

<p><b>A)</b> Log-transformed quantities of cytokines (pg/ml) are shown. CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4+TGF-β. Supernatants were collected at 96hrs post activation and IFNγ, IL-2, IL-5, IL-9, IL-10, IL-13, and IL-17 were quantified by ELISA. IL-4+TGF-β treated CD4⁺ T cells produced significantly high IL-2 and IL-9, but significantly low IFNγ, IL-13, and IL-17, as compared to CD4⁺CD25⁻CD45RO⁺ T cells not treated with IL-4+TGF-β (<i>n = 3</i>). Data is expressed as the mean±SD. <b>B)</b> Log-transformed ratios of mRNA copies to GAPDH mRNA copies for GATA3, RORC, IL-9, and Tbet are shown. CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were harvested and single cell sorted. IL-9 transcripts were quantified by qt-RT-PCR. 10,000 cells comprising of total cell population (TC) was also taken and the gene expression was averaged for single cell for reference. Cells positive for IL-9 transcripts were further quantitated for GATA3, RORC, and Tbet (<i>n</i> = 3). As IL-9 is expressed in only 10% of all CD4⁺ T cells activated with pbCD3/sCD28 in presence of IL-4+TGF-β, average IL-9 mRNA copies of TC are always lower than that of a single IL-9⁺ cell. CD4⁺IL-9⁺ T cells expressed GATA3 and RORC, but not Tbet. <b>C)</b> CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence of IL-4+TGF-β. Cells were surface stained for CD4 and intracellular stained for IL-9 and FOXP3. Cells were gated for CD4 and then IL-9⁺ or/and FOXP3⁺ cells were analyzed. 25% of CD4⁺IL-9⁺ T cells were also FOXP3⁺. Data are representative of seven independent experiments. <b>D)</b> CD4⁺CD25⁻CD45RO⁺ T cells (1.0×10⁶/ml in 24 well plates) were activated with pbCD3/sCD28 in presence or absence of IL-4 or TGF-β or IL-4 plus TGF-β for 96hrs and analyzed by flow cytometry for FOXP3 expression. IL-4 significantly inhibited TGF-β induced FOXP3 expression (<i>n = 3</i>). Data is expressed as the mean±SD.</p