Esther Bandala-Sanchez

T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10

Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52hiCD4+ T cells were distinct from CD4+CD25+Foxp3+ regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor–associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52hiCD4+ T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52hi cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.

The polycomb repressive complex 2 governs life and death of peripheral T cells

Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.

Revisiting regulatory T cells in type 1 diabetes.

Purpose of review Regulatory T cells (Treg) maintain immune homeostasis and prevent autoimmune disease. This review summarizes the recent advances in Treg knowledge relevant to type 1 diabetes, focusing on Treg signature, antigen specificity and development and function in the face of inflammation. Recent findings Thymus-derived natural regulatory T cells (nTreg) programmed by the transcription factor forkhead box P3 (FOXP3) and peripheral-induced regulatory T cells (iTreg) have largely nonoverlapping T-cell receptor repertoires to self-antigens and jointly contribute to immune homeostasis. Initial reports that CD4+CD25+ (FOXP3+) Treg were impaired in frequency or function in type 1 diabetes have not been confirmed. The Treg-specific demethylated region in the FOXP3 locus in nTreg is, in contrast, methylated in iTreg and conventional T cells (Tconv) and is the only feature that reliably distinguishes activated human nTreg and Tconv. Inflammatory cytokines regulate extrathymic differentiation of nTreg but can also reprogram nTreg into Th17 or Th1 effectors and prevent the differentiation of iTreg. Summary The methylation status of the FOXP3 locus provides a means to re-examine Treg in autoimmune disease. nTreg and iTreg recognize different self-antigens. Shaping of Treg by the cytokine milieu has implications for the application of Treg cell-based immune therapies.

Antigen-based vaccination and prevention of type 1 diabetes

Insulin-dependent or type 1 diabetes (T1D) is a paradigm for prevention of autoimmune disease: Pancreatic β-cell autoantigens are defined, at-risk individuals can be identified before the onset of symptoms, and autoimmune diabetes is preventable in rodent models. Intervention in asymptomatic individuals before or after the onset of subclinical islet autoimmunity places a premium on safety, a requirement met only by lifestyle–dietary approaches or autoantigen-based vaccination to induce protective immune tolerance. Insulin is the key driver of autoimmune β-cell destruction in the nonobese diabetic (NOD) mouse model of T1D and is an early autoimmune target in children at risk for T1D. In the NOD mouse, mucosal administration of insulin induces regulatory T cells that protect against diabetes. The promise of autoantigen-specific vaccination in humans has yet to be realized, but recent trials of oral and nasal insulin vaccination in at-risk humans provide grounds for cautious optimism.

Stored red blood cell susceptibility to in vitro transfusion-associated stress conditions is higher after longer storage and increased by storage in saline-adenine-glucose-mannitol compared to AS-1.

Biochemical changes induced in red blood cells (RBCs) during storage may impair their function upon transfusion. Transfusion‐associated stresses may further amplify storage lesion effects including increased phosphatidylserine (PS) exposure at the RBC membrane, microparticle (MP) release, and adhesion to endothelial cells (ECs). RBC stress susceptibility in vitro was investigated in relation to storage time and additive solution.

Forward light scatter is a simple measure of T-cell activation and proliferation but is not universally suited for doublet discrimination

Following activation by antigen, T cells enter the cell cycle in stages that can be defined with flow cytometric markers. We show that these markers include the increase of forward light scatter width (FSC‐W) signal and the ratio of FSC area/peak. This change in light scatter precedes the first cell division and may reflect blast transformation. We show that the FSC‐W parameter can be used, alone or in combination with other activation markers, to monitor the relative and absolute numbers of T cells responding to a proliferative stimulus. In contrast to dye dilution assays, the FSC‐W method does not allow discrimination between consecutive cell divisions, but it has several advantages and could complement the dye dilution assay. Our findings also show that the routine elimination of doublets based on FSC signals may exclude proliferating T cells from the analysis.

CD52 inhibits Toll-like receptor activation of NF-κB and triggers apoptosis to suppress inflammation.

Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-κB and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.

Localization of dipeptidyl peptidase-4 (CD26) to human pancreatic ducts and islet alpha cells

AIM DPP-4/CD26 degrades the incretins GLP-1 and GIP. The localization of DPP-4 within the human pancreas is not well documented but is likely to be relevant for understanding incretin function. We aimed to define the cellular localization of DPP-4 in the human pancreas from cadaveric organ donors with and without diabetes. METHODS Pancreas was snap-frozen and immunoreactive DPP-4 detected in cryosections using the APAAP technique. For co-localization studies, pancreas sections were double-stained for DPP-4 and proinsulin or glucagon and scanned by confocal microscopy. Pancreata were digested and cells in islets and in islet-depleted, duct-enriched digests analyzed for expression of DPP-4 and other markers by flow cytometry. RESULTS DPP-4 was expressed by pancreatic duct and islet cells. In pancreata from donors without diabetes or with type 2 diabetes, DPP-4-positive cells in islets had the same location and morphology as glucagon-positive cells, and the expression of DPP-4 and glucagon overlapped. In donors with type 1 diabetes, the majority of residual cells in islets were DPP-4-positive. CONCLUSION In the human pancreas, DPP-4 expression is localized to duct and alpha cells. This finding is consistent with the view that DPP-4 regulates exposure to incretins of duct cells directly and of beta cells indirectly in a paracrine manner.

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis.

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6–24 h, before transfer and storage at −80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.

Interferon-gamma released from omental adipose tissue of insulin-resistant humans alters adipocyte phenotype and impairs response to insulin and adiponectin release

Background:Inflammatory factors derived from adipose tissue have been implicated in mediating insulin resistance in obesity. We sought to identify these using explanted human adipose tissue exposed to innate and adaptive immune stimuli.Methods:Subcutaneous and omental adipose tissue from obese, insulin-resistant donors was cultured in the presence of macrophage and T-cell stimuli, and the conditioned medium tested for its ability to inhibit insulin-stimulated glucose uptake into human Simpson–Golabi–Behmel Syndrome (SGBS) adipocytes. The nature of the inhibitory factor in conditioned medium was characterized physicochemically, inferred by gene microarray analysis and confirmed by antibody neutralization.Results:Conditioned medium from omental adipose tissue exposed to a combination of macrophage- and T-cell stimuli inhibited insulin action and adiponectin secretion in SGBS adipocytes. This effect was associated with a pronounced change in adipocyte morphology, characterized by a decreased number of lipid droplets of increased size. The bioactivity of conditioned medium was abolished by trypsin treatment and had a molecular weight of 46 kDa by gel filtration. SGBS adipocytes exposed to a bioactive medium expressed multiple gene transcripts regulated by interferon-gamma (IFN-γ). Recombinant human IFN-γ recapitulated the effects of the bioactive medium and neutralizing antibody against IFN-γ but not other candidate factors abrogated medium bioactivity.Conclusions:IFN-γ released from inflamed omental adipose tissue may contribute to the metabolic abnormalities seen in human obesity.