PD-1 Inhibitory Receptor Downregulates Asparaginyl Endopeptidase and Maintains Foxp3 Transcription Factor Stability in Induced Regulatory T Cells.

CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.

Pro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises.

Recurring episodes of acute pain, also referred to as vaso-occlusive crises (VOC), are characteristic of sickle cell disease (SCD), during which pro-inflammatory cytokines, chemokines, adhesion markers and white cell count, some already elevated at steady state, increase further. Hydroxyurea (HU) is licensed by the FDA for reducing frequency of VOCs in SCD; increased fetal hemoglobin (HbF) together with reduction of the neutrophil count and circulating inflammatory markers, contribute to its clinical efficacy. Here, using paired plasma samples from HbSS patients (in steady-state and VOC) we determined that despite HU treatment, the SCD environment remained highly inflammatory and particularly at VOC, triggered neutrophil activity. While neutrophil extracellular traps (NETs) induction by the steady state plasmas were comparable to that of plasma from healthy donors, the NETs response triggered by crisis plasmas was significantly increased over that of the steady state (P = 0.0124*). Levels of IL-6 and IL-1α, IL-1ra/IL1F3 and adhesion molecule P-selectin were significantly increased in the VOC plasma when compared with steady state plasma. Higher levels of IL-6 and IL-1ra were also found in the crises samples that yielded an increased NETs response suggesting that increased NETs production associated with increased levels of the inflammatory products of the IL-6 family and regulators of IL-1 family of cytokines during sickle VOCs.

Mature enteroendocrine cells contribute to basal and pathological stem cell dynamics in the small intestine.

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here, we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage-tracing studies identified a subset of EECs that reside at +4 position for more than 2 wk, most of which were BrdU-label-retaining cells. Under basal conditions, cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally, the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium, and in the development of EC-derived small intestinal tumors. NEW & NOTEWORTHY The current manuscript demonstrating that a subset of mature enteroendocrine cells (EECs), predominantly enterochromaffin cells, dedifferentiates to fully functional intestinal stem cells (ISCs) is novel, timely, and important. These cells dedifferentiate to ISCs not only in response to injury but also under basal homeostatic conditions. These novel findings provide a mechanism in which a specified cell can dedifferentiate and contribute to normal tissue plasticity as well as the development of EEC-derived intestinal tumors under pathologic conditions.

Masks in imaging flow cytometry.

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis.

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4+ T cells.

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.

Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL.

T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. ß2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.

Restricted mitochondrial protein acetylation initiates mitochondrial autophagy.

Because nutrient-sensing nuclear and cytosolic acetylation mediates cellular autophagy, we investigated whether mitochondrial acetylation modulates mitochondrial autophagy (mitophagy). Knockdown of GCN5L1, a component of the mitochondrial acetyltransferase machinery, diminished mitochondrial protein acetylation and augmented mitochondrial enrichment of autophagy mediators. This program was disrupted by SIRT3 knockdown. Chronic GCN5L1 depletion increased mitochondrial turnover and reduced mitochondrial protein content and/or mass. In parallel, mitochondria showed blunted respiration and enhanced 'stress-resilience'. Genetic disruption of autophagy mediators Atg5 and p62 (also known as SQSTM1), as well as GCN5L1 reconstitution, abolished deacetylation-induced mitochondrial autophagy. Interestingly, this program is independent of the mitophagy E3-ligase Parkin (also known as PARK2). Taken together, these data suggest that deacetylation of mitochondrial proteins initiates mitochondrial autophagy in a canonical autophagy-mediator-dependent program and shows that modulation of this regulatory program has ameliorative mitochondrial homeostatic effects.

Bone marrow mesenchymal stromal cells to treat tissue damage in allogeneic stem cell transplant recipients: correlation of biological markers with clinical responses.

Bone marrow mesenchymal stromal cells (BMSCs) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage BMSCs for patients with steroid-refractory GVHD, tissue injury, or marrow failure following SCT to investigate safety and efficacy. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were serially monitored for plasma GVHD biomarkers, cytokines, and lymphocyte phenotype. Ten subjects were infused a fixed dose of 2 × 10(6) BMSCs/kg intravenously weekly for three doses. There was no treatment-related toxicity (primary endpoint). Eight subjects were evaluable for response at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved a complete response, two of two patients with tissue injury (pneumomediastinum/pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines were observed. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD or tissue injury. The GVHD complete responders survived significantly longer and had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts. Cytokine changes also segregated with survival. These results confirm that BMSCs are associated with rapid clinical and biomarker responses in GVHD and tissue injury. However, BMSCs were ineffective in patients with prolonged GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSCs requires a relatively intact immune system.

Characterization of immune cells in psoriatic adipose tissue.

BACKGROUND: Adipose tissue normally contains immune cells that regulate adipocyte function and contribute to metabolic disorders including obesity and diabetes mellitus. Psoriasis is associated with increased risk for metabolic disease, which may in part be due to adipose dysfunction, which has not been investigated in psoriasis. There is currently no standardized method for immunophenotyping human adipose tissue. In prior studies, characteristic phenotypic markers of immune cell populations identified in animal models or in other human tissues have been applied in a similar manner to human adipose tissue. Rarely have these populations been verified with confirmatory methodologies or functional studies. Thus, we performed a comprehensive phenotypic and functional analysis of immune cell populations in psoriatic adipose tissue. METHODS: Conventional and imaging flow cytometry were used to define immune cell populations in biopsy specimens of psoriatic adipose tissue (n = 30) including T cells, B cells, NK cells, NKT cells, neutrophils, and macrophages. Relationships between adipose immune cell types and body mass index were determined using Spearman regression analysis, and multivariate linear regression analysis was performed to adjust for cardiometabolic disease risk factors. RESULTS: These analyses revealed a wide range of cell surface receptors on adipose tissue macrophages, which may serve a dual purpose in immunity and metabolism. Further, both CD16+CD56(Lo) and CD16-CD56(Hi) NK cells were found to correlate inversely with body mass index. The relationship between the predominant CD16+CD56(Lo) NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use. CONCLUSIONS: Together, these studies enhance our understanding of adipose immune cell phenotype and function, and demonstrate that examination of adipose tissue may provide greater insight into cardiometabolic pathophysiology in psoriasis.

Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development.

Thymic stromal lymphopoietin is produced by dendritic cells.

Thymic stromal lymphopoietin (TSLP) is a type 1 cytokine that contributes to lymphopoiesis and the development of asthma and atopic dermatitis. TSLP acts on multiple lineages, including dendritic cells (DCs), T cells, NKT cells, eosinophils, and mast cells, mediating proliferation and survival and linking innate and adaptive immune responses. TSLP is produced by a range of cells, including epithelial cells, fibroblasts, stromal cells, and keratinocytes. DCs are important primary targets of TSLP, and we unexpectedly demonstrated that DCs also produce TSLP in response to TLR stimulation and that this is augmented by IL-4. Moreover, we demonstrated that when mice were challenged with house dust mite extract, lung CD11c(+) DCs expressed TSLP mRNA at an even higher level than did epithelial cells. These data suggested that DCs not only respond to TSLP but also are a source of TSLP during pathogen and/or allergen encounter.

Apparent mtDNA sequence heterogeneity in single human blood CD34+ cells is markedly affected by storage and transport.

Single CD34(+) cells from adult human peripheral blood show mtDNA sequence heterogeneity. In this study, we compared mtDNA sequence variation in single CD34(+) cells from peripheral blood (PB) mononuclear cells (MNCs) from the same donors but under different conditions of storage and transport: group I, MNCs from heparinized PB that inadvertently required six days to be transported to the testing laboratory; group II, MNCs which were isolated from PB within a day of phlebotomy and frozen prior to transportation and storage. We observed more cell death for MNCs of group I than group II. Concordantly, group I CD34(+) cells had a very low potential for hematopoietic colony formation in vitro compared with group II cells. CD34(+) cells of group II showed an unexpectedly higher level of mtDNA sequence heterogeneity than was present in group I cells. These observations suggest that reduced mtDNA sequence heterogeneity in single CD34(+) cells of group I was likely due to elimination of cells harboring mutations. CD34(+) cells that survive stress ex vivo may be more enriched in quiescent primitive hematopoietic stem cells, with fewer mtDNA mutations than are present in committed progenitors. Technically, attention is required to conditions of preparation of human blood samples for single cell mtDNA analysis.

Cytokine signature profiles in acquired aplastic anemia and myelodysplastic syndromes.

Although aplastic anemia and myelodysplasia have been extensively investigated, little is known about their circulating cytokine patterns. We compared plasma soluble cytokines in 33 aplastic anemia, 57 myelodysplasia patients, and 48 healthy controls. High levels of thrombopoietin and granulocyte colony-stimulating factor, with low levels of CD40 ligand, chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand 5, chemokine (C-X-C motif) ligand 11, epidermal growth factor, vascular endothelial growth factor, and chemokine (C-C motif) ligand 11 were a signature profile for aplastic anemia. High levels of tumor necrosis factor-α, interleukin-6, chemokine (C-C motif) ligand 3, interleukin-1 receptor antagonist, and hepatocyte growth factor were a cytokine signature for myelodysplasia. Despite similar clinical presentations, distinct cytokine profiles were observed between aplastic anemia and hypocellular myelodysplasia. Future studies focusing on cytokines that better discriminate these two entities such as thrombopoietin and chemokine (C-C motif) ligand 3 may be useful tools in clinical practice.

Inflammation, TNFα and endothelial dysfunction link lenalidomide to venous thrombosis in chronic lymphocytic leukemia.

Patients receiving lenalidomide are at an increased risk for deep venous thrombosis (DVT). Here, we prospectively investigated the DVT risk in patients with relapsed chronic lymphocytic leukemia (CLL) treated with lenalidomide (n = 32). Five patients developed six incidents of DVT over 1 year for an annual incidence of 16%. Three of these were considered drug-related. Median time to DVT was 105 days (range 56-259 days). No pulmonary embolism was detected. Hypercoagulability screen before study entry was negative in all patients who subsequently developed DVTs. Compared to normal volunteers CLL patients had increased baseline levels of D-dimer, thrombin-antithrombin, soluble vascular endothelial adhesion molecule 1 (sVCAM-1), and thrombomodulin (p < 0.001). After 1 week on lenalidomide D-dimer, thrombomodulin, sVCAM-1, factor VIII, TNFα, and C-reactive protein were significantly increased while protein C was decreased (p < 0.001). In patients with lenalidomide-related DVTs, TNFα, and sVCAM-1 were more strongly upregulated than in all other patients (p < 0.05) and TNFα and sVCAM-1 levels were significantly correlated (r = 0.65, p < 0.001). These data link lenalidomide associated DVTs with TNFα upregulation and endothelial cell dysfunction and suggest that aspirin may have a role for DVT prophylaxis in these patients.

Phenotypic characterization of disseminated cells with TSC2 loss of heterozygosity in patients with lymphangioleiomyomatosis.

RATIONALE: Lymphangioleiomyomatosis (LAM), occurring sporadically (S-LAM) or in patients with tuberous sclerosis complex (TSC), results from abnormal proliferation of LAM cells exhibiting mutations or loss of heterozygosity (LOH) of the TSC genes, TSC1 or TSC2. OBJECTIVES: To identify molecular markers useful for isolating LAM cells from body fluids and determine the frequency of TSC1 or TSC2 LOH. METHODS: Candidate cell surface markers were identified using gene microarray analysis of human TSC2⁻(/)⁻ cells. Cells from bronchoalveolar lavage fluid (BALF), urine, chylous effusions, and blood were sorted based on reactivity with antibodies against these proteins (e.g., CD9, CD44v6) and analyzed for LOH using TSC1- and TSC2-related microsatellite markers and single nucleotide polymorphisms in the TSC2 gene. MEASUREMENTS AND MAIN RESULTS: CD44v6(+)CD9(+) cells from BALF, urine, and chyle showed TSC2 LOH in 80%, 69%, and 50% of patient samples, respectively. LAM cells with TSC2 LOH were detected in more than 90% of blood samples. LAM cells from different body fluids of the same patients showed, in most cases, identical LOH patterns, that is, loss of alleles at the same microsatellite loci. In a few patients with S-LAM, LAM cells from different body fluids differed in LOH patterns. No patients with S-LAM with TSC1 LOH were identified, suggesting that TSC2 abnormalities are responsible for the vast majority of S-LAM cases and that TSC1-disease may be subclinical. CONCLUSIONS: Our data support a common genetic origin of LAM cells in most patients with S-LAM, consistent with a metastatic model. In some cases, however, there was evidence for genetic heterogeneity between LAM cells in different sites or within a site.

Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. The long isoform is generated via intra-exon splicing creating a frame-shift to expose a novel upstream translation start site. Mitochondrial localization is significantly more robust following transfection of the long compared with the short isoform. Insertion of this alternatively spliced novel 5' sequence upstream of a GFP-reporter plasmid shows greater than 80% enrichment in mitochondria, confirming this region as a legitimate mitochondrial localization sequence. Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.

Serum IL-6: a candidate biomarker for intracranial pressure elevation following isolated traumatic brain injury.

BACKGROUND: Increased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patient's risk for developing this pathology. METHODS: In the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCSor= 25 mm Hg had significantly higher IL-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained 128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained