De novo transcriptome profiling of highly purified human lymphocytes primary cells

To help better understand the role of long noncoding RNAs in the human immune system, we recently generated a comprehensive RNA-seq data set using 63 RNA samples from 13 subsets of T (CD4(+)\u2009naive, CD4(+)\u2009TH1, CD4(+)\u2009TH2, CD4(+)\u2009TH17, CD4(+)\u2009Treg, CD4(+)\u2009TCM, CD4(+)\u2009TEM, CD8(+)\u2009TCM, CD8(+)\u2009TEM, CD8(+)\u2009naive) and B (B naive, B memory, B CD5(+)) lymphocytes. There were five biological replicates for each subset except for CD8(+)\u2009TCM and B CD5(+)\u2009populations that included 4 replicates. RNA-Seq data were generated by an Illumina HiScanSQ sequencer using the TruSeq v3 Cluster kit. 2.192 billion of paired-ends reads, 2 7100\u2009bp, were sequenced and after filtering a total of about 1.7 billion reads were mapped. Using different de novo transcriptome reconstruction techniques over 500 previously unknown lincRNAs were identified. The current data set could be exploited to drive the functional characterization of lincRNAs, identify novel genes and regulatory networks associated with specific cells subsets of the human immune system

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells

Whether human IL-10-producing regulatory T cells (“Tr1”) represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4 + T-cell subsets, including conventional cytotoxic CD4 + T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4 + T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes + GzmK + T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4 + Eomes + T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes + Tr1-like cells are effector cells of a unique GzmK-expressing CD4 + T-cell subset

An Efficient Strategy to Induce and Maintain In Vitro Human T Cells Specific for Autologous Non-Small Cell Lung Carcinoma

BACKGROUND: The efficient expansion in vitro of cytolytic CD8+ T cells (CTLs) specific for autologous tumors is crucial both for basic and translational aspects of tumor immunology. We investigated strategies to generate CTLs specific for autologous Non-Small Cell Lung Carcinoma (NSCLC), the most frequent tumor in mankind, using circulating lymphocytes. PRINCIPAL FINDINGS: Classic Mixed Lymphocyte Tumor Cultures with NSCLC cells consistently failed to induce tumor-specific CTLs. Cross-presentation in vitro of irradiated NSCLC cells by autologous dendritic cells, by contrast, induced specific CTL lines from which we obtained a high number of tumor-specific T cell clones (TCCs). The TCCs displayed a limited TCR diversity, suggesting an origin from few tumor-specific T cell precursors, while their TCR molecular fingerprints were detected in the patient's tumor infiltrating lymphocytes, implying a role in the spontaneous anti-tumor response. Grafting NSCLC-specific TCR into primary allogeneic T cells by lentiviral vectors expressing human V-mouse C chimeric TCRalpha/beta chains overcame the growth limits of these TCCs. The resulting, rapidly expanding CD4+ and CD8+ T cell lines stably expressed the grafted chimeric TCR and specifically recognized the original NSCLC. CONCLUSIONS: This study defines a strategy to efficiently induce and propagate in vitro T cells specific for NSCLC starting from autologous peripheral blood lymphocytes

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

NEPHROTIC RANGE PROTEINURIA AND ALBUMINURIA IN DOGS: A RETROSPECTIVE EVALUATION OF 338 CASES

In dogs, persistent renal proteinuria with Urinary Protein to Creatinine ratio (UPC) 652.0 usually is due to glomerular renal disease. In humans, Nephrotic-Range Proteinuria (NRP) is defined as UPC 65 3.5, while Overt Albuminuria (OA) is defined as Urinary Albumin to Creatinine ratio (UAC) > 0.3. Analogous cut-off values are lacking for dogs. The aim of this study was to characterize clinical and clinicopathological features associated with 'glomerular proteinuria' in dogs, with particular regard to UAC.Retrospective analysis included a total of 338 dogs admitted to our Teaching Hospital, between January 2002 and September 2011, with glomerular proteinuria defined by UPC 652.0. Data collected were signalment, clinical and clinicopathological findings including Blood Pressure, CBC, serum Albumin (Alb), Total Protein, Cholesterol, Creatinine (Crea), Urea, Electrolytes, CRP, Total Iron, Total Iron Binding Capacity (TIBC), Antithrombin%, D-Dimers, Fibrinogen, and urinalysis comprehensive of UPC and UAC (immunoturbidimetric method). Data were analyzed with descriptive and non parametric statistics. A difference was considered significant for p <0.05. The number of dogs included in the statistical analyses was not uniform because some results were not available for all dogs.Median age was 8.4 years (range 0.25-17.4) with a 58.8% of male and 32.5% of mixed-breed. An underlying inflammatory disease was recognized in 61% of dogs (24.2% Leishmaniasis). The Median UPC was 4.9 (range 2.0-40.1) and according to the human cut-off (UPC 653.5) 65% of dogs was classified as having NRP. UAC was available in 309/388 patients (Median 2.24; range 0.001-25.391; RI <0.024) and 92% of dogs (287/309) presented OA (UAC >0.3). Hypoalbuminemia (Alb 642.5 g/dl) was detected in 57.2% of cases (Median 2.35; range 0.74-4.29). A significant negative correlation between UPC, UAC and Alb (r=-0.43 and r=-0.25, respectively), was detected. Crea concentration was above the reference interval in 53% of dogs (Median 1.85; range 0.47-22.07; RI 0.65- 1.35). Median Fibrinogen concentration was 5.28 g/l (range 0.64-10.63; RI 1.45-3.85), Median D-Dimers concentration was 0.26 \u3bcg/ml (range 0.01-8.84; RI <0.26). Median CRP concentration was 3.22 mg/dl (range 0.01-40.38; RI <0.5) and 76% of dogs presented elevated CRP. NRP dogs showed a significant increase in CRP, Phosphorus, Urea and D-Dimers and a significant decrease in Alb, HCT and TIBC.These findings suggest that NRP dogs have higher inflammatory state and potential hemostatic imbalance on clinical pathology. Further studies are required to investigate the potential role of UAC in the diagnosis of protein-losing glomerulopathies in dogs

The effect of inter-laboratory variability on the protein: Creatinine (UPC) ratio in canine urine

Quantification of proteinuria is a fundamental step in staging dogs with chronic kidney disease and in monitoring the course of disease or the efficacy of anti-proteinuric treatments. Analytical precision and accuracy of the proteinuria assessment could be affected by several factors such as biological variability, different operators and quality control materials. The aim of this study was to assess whether inter-laboratory variability could affect the urinary protein to creatinine (UPC) ratio and whether this variability may affect patient classification according to the International Renal Interest Society (IRIS) sub-staging system. The same urine samples were analysed in three different laboratories using different instruments and different reagent brands.The results of the three laboratories were highly correlated to each other although urinary protein (UP), urinary creatinine (UC) and the UPC ratio of one laboratory were found to be significantly higher than those of the other two. No significant differences between the other two laboratories were recorded. The concordance in classifying dogs according to the IRIS guidelines was good if all three proteinuria categories were analysed separately or if borderline proteinuric (BP) dogs were included in the proteinuric group, and very good if BP dogs were merged into the non-proteinuric group. The inter-laboratory variability in UPC ratio measurement was not so great as to impede the identification of proteinuric dogs, but may influence the estimation of the magnitude of proteinuria