dataesr/scientific-paragraphs-categorization huggingface dataset:
NanoString nCounter expression assay For Nanostring® experiment, 14 to 15 weeks-old NSG mice were humanized and treated with anti-HVEM or isotype. Day 28 post humanization, tumors were harvested and TIL were isolated as described above. To maximize mRNA recovery, TIL were pooled by treatment groups (4 mice in the anti-HVEM group and 5 in the isotype control group). Then, cells were stained with viability dye (eF506) and anti hCD45-APC (HI30, Biolegend). Live hCD45 + cells were sorted using Aria II cell sorter. After centrifugation, cells were suspended in RLT buffer (Qiagen®) before freezing at -80°C until analysis. Data were normalized through the use of NanoString's intrinsic negative and positive controls according to the normalization approach of the nSolver analysis software (Nanostring).
On the other hand, some genes belonging to immuno-suppressive pathways were clearly down-regulated in HVEM-treated TIL such as ENTPD1 (CD39), IL10 and the co-inhibitory receptors BTLA, TIGIT, LAG3 and HAVCR2 (TIM3), as well as the "don't eat me" receptor CD47 (Figure  S3 ). Other cytokines/chemokines were also negatively affected by the treatment, such as IL21, IL13, CXCL13, TNFSF10 (TRAIL) or TNFSF113B (BAFF). Enrichment analysis of the genes down regulated in the anti-HVEM group using the DisGeNET database showed that the "Immunosuppression" signature was highly enriched in this gene set (Figure  6C ). In addition, GSEA showed that genes belonging to the "immunoregulatory interactions between a lymphoid and a non lymphoid cell" signature was significantly depressed in the DEG signature (Figure  6D ).
Acknowledgments The authors would like to thank  Olivier Bregerie  and  Doriane Foret  for taking care of our mice,  Dr Pukar KC  for technical help,  Sylvaine Just-Landi  for preparing the 18. 10 mAb,  Dr Armanda Casrouge  and  Claude Baillou  for cell sorting, and  Dr Benoit Salomon  for critical reading of the manuscript.
Primer design Primer design was based on the ITS2 sequences of five out of six species available in GenBank (Table  2 ); the ITS2 sequence of An. campestris is not available. Primer names, sequences, size of the PCR products and respective melting temperatures (Tm) are presented in Table  3. Due to the close similarity of the ITS2 sequences of the six members, primer design was difficult with some species having similar individual size bands (bp), thus a dual PCR assay was developed in order to more reliably separate all species. Based on nucleotide alignment of the ITS2 region, nine primers were designed. A forward primer common to five species, except An. campestris (fBDSVW), then two species-specific forward primers (fBar, fCamp), and six reverse primers (rBar&Van, rVan&Dis, rCamp, rSaue1, rSaue2, rWej).
Ltd, Suzhou, Jiangsu, China), and then the membrane was scanned using the Tanon 4500 Gel Imaging System. Uncropped blotting scans were presented in the affiliated Source Data file. The antibodies were diluted with NCM universal antibody diluent (WB500D, New Cell & Molecular Biotech Co. Ltd. ). The antibodies used and the corresponding dilutions are listed in Supplementary Table  2. Ca 2+ flux. Sorted naive CD4 + T cells were labeled with 4 μg ml -1 Fluo4 (Invitrogen, New York, NY, USA) for 1 h at 37 °C, washed with ice-cold phosphatebuffered saline (PBS), and resuspended in PBS. The labeled cells were stimulated with ISO, Jo2, U73211, or Manoalide and immediately subjected to flow cytometry analysis. Mean fluorescence ratios were plotted after analysis with FlowJo software (TreeStar, Ashland, OR, USA).
After staining for surface markers, the cells were fixed and permeabilized according to the manufacturer's instructions (Cytofix/ Cytoperm Kit, Thermo Fisher Scientific) and then stained for intracellular products. The following monoclonal antibodies were used for the flow cytometric analyses: fixable viability dye eFluor TM 450-or phycoerythrin-conjugated anti-CD4 and allophycocyanin-conjugated anti-IFN-γ, anti-IL-4, anti-IL-9, anti-IL-17A, or anti-Foxp3. All events were acquired on a BeckmanCoulter DxFLEX flow cytometer equipped with CytExpert experiment-based software (BeckmanCoulter, Inc. ), and data were analyzed using FlowJo software (TreeStar). Gating strategies were presented in Supplementary Fig. 9. Retroviral infection of CD4 + T cells. Retroviruses were produced by transfecting Plat-E cells with 7.
High-quality reads were aligned to a mouse reference genome (GRCm38) by Bowtie2. The expression levels of individual genes were normalized to the FKPM (fragments per kilobase million) reads by RNA-seq by an expectation maximization algorithm. Significant differential expression of a gene was defined as a >2-fold expression difference vs. the control with an adjusted P value <0. 05. A heat map was analyzed by Gene Ontology using Cluster software and visualized with Java Treeview. DEGs were analyzed by Gene Ontology using the AMIGO and DAVID software. The enrichment degrees of DEGs were analyzed using Kyoto Encyclopedia of Genes and Genomes annotations. Cell viability assay. LLC-OVA cell viability was measured using a CCK-8 assay according to the manufacturer's instructions. LLC-OVA cells were stimulated with dimethyl sulfoxide (DMSO) or 0.
15, 10, 20, or 30 μg ml -1 SB203580 for 72 h in a 96-well plate, and 10 μl of CCK-8 (MultiSciences, Hangzhou, Zhejiang, China) was added per well and incubated for 2 h at 37 °C. A multiplate reader was used to measure the absorbance at 450 nm. Cell viability was expressed as the absorbance at 450 nm. Chromatographic conditions and data acquisition. Mice received an intraperitoneal injection of DMSO or 0. 5 or 10 mg kg -1 SB203580. After 0. 5, 1, 3, or 6 h, plasma was collected and centrifuged at 12,000 × g for 10 min, and the supernatants were subjected to chromatographic detection. High-performance liquid chromatography analyses were carried out using Agilent 1100 series LC equipment. The elution system was set up as follows: an Inertil ODS-4 column (4. 6 × 250 mm 2 , 5 μm) was eluted using a water and methanol gradient at 40 °C with a constant flow rate of 1.
0 ml min -1 (0-20 min, 50-100% methanol; 20-25 min, 100% methanol). The fluorescence detector was set at 325 nm (excitation wavelength) and 408 nm (emission wavelength). Under these chromatographic conditions, the retention time of SB203580 was 13. 0 min. The acquired data were processed using the ChemStation for LC 3D software. To obtain chromatograms, data were extracted from the software and plotted in the Microsoft Excel 2010 program. Transfer of T H 9 cells into IBD mice. WT and Il9r -/-mice were randomized to produce groups with similar average body weights. Acute IBD was induced by administering 2. 5% (w/v) DSS (MP Biomedicals, Solon, OH, USA) with a molecular weight of 36,000-50,000 in acidified drinking water for 11 days. The day that the mice started to drink the DSS solution was defined as day 0.
Acknowledgements This study was supported by the  National Key R&D Program of China  ( 2016YFA0501800 ), the  National Key Basic Research Program of China  ( 2015CB943301 ), the  National Natural Science Foundation of China  ( 31770951  and  31670877 ), and the  Hangzhou Health Science and Technology Plan  ( OO20191170 ). We thank the  Core Facilities, Zhejiang University School of Medicine  for technical support.
Material and methods Medical records were reviewed for every consecutive canine patient with previously untreated multicentric Bcell lymphoma that presented to the Centro Oncologico Veterinario (Sasso Marconi, Italy) and Department of Veterinary Medicine (University of Milan, Italy) from 2013 to 2018. This study was prospectively conceived and clinical data on the efficacy and toxicity of treatments were analyzed retrospectively, thus the inclusion criteria were established a priori and all dogs were treated contemporaneously. To be eligible for inclusion, dogs were required to undergo a complete initial staging work-up and, whenever possible, surgical removal of a peripheral LN to obtain a histopathological diagnosis and material for the vaccine generation (Additional file 1). Based on histopathology and immunohistochemistry (CD20, CD3), tumors were classified according to the modified WHO criteria  [9].
Survival plots were generated according to the Kaplan-Meier product-limit method. Curves were compared with the log-rank test. The influence of potential prognostic variables on tumor progression and tumorrelated death was investigated with univariable and multivariable Cox's regression analysis. The survival advantage of vaccine administration was further tested upon stratification of cases according to the other considered variables. The variables with no survival advantage for one of the strata were selected for a scoring system to quantify the utility of vaccine administration. Dogs with the highest score were the best candidates for immunotherapy. Data were analyzed by use of commercial software programs (SPSS Statistics v19, IBM, Armonk, NY, USA, Prism v. 5. 0, GraphPad, San Diego, California).
In T. lanuginosus, annexinC7 is involved in conidia formation and resistance to oxidative stress  [17]. In amoeba Dictyostelium discoideum, one annexin gene is found and its deletion led to a delay in growth in low Ca 2+ conditions  [18]. Cryptococcus neoformans is the causative agent of cryptococcosis, a life-threatening infection that often results in high mortality and morbidity  [19]. The cryptococcal genome contains one predicted annexin gene, CNAG_02415, annotated as AnnexinXIV. A literature search for references to AnnexinXIV, fungal annexin or CNAG_02415 found no reports on the function of this gene. Exploration of the available full datasets from high-throughput experiments gave us some clues into the possible roles of AnnexinC1. A genetic screen showed that CNAG_02415 deletion led to altered susceptibility to a range of small molecule compounds  [20].
Further, CNAG_02415 (AnnexinC1) is dependent on Crz1, a transcription factor of the calcineurin pathway, which is essential for virulence in C. neoformans  [20] [21] [22]. Chipsequencing data identified AnnexinC1 as a direct target of Crz1  [23]. This finding posits a role for AnnexinC1 as a component of the Crz1-calcineurin response, involved in high-temperature growth, cell-wall stability and heavy-metal susceptibility  [22, 24]. Available gene and protein expression datasets in C. neoformans detected no alterations in expression of AnxC1 expression upon infection  [25] , nor was the protein detected as secreted into the extracellular milieu  [26, 27]. In a transcriptomic dataset, absence of Rim101 led to an increase in expression of ANXC1 when placed for 3 h in Dulbecco's Modified Eagle Medium  [28, 29] , which suggests ANXC1 is additionally regulated by Rim101  [28] , an important cell morphology regulator.
Phylogenetic and protein modelling analysis Conserved domains were detected with NCBI Conserved Domains  [29]. Fasta files were downloaded and aligned using muscle  [30]  with Mega7 software. Phylogenetic analysis was constructed with a maximum likelihood tree, neighbour-join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model. A model of AnnexinC1 was built using Swiss-Model workspace (https:// swissmodel. expasy. org/ interactive# structure)  [31, 32] , using AnnexinA5 from Homo sapiens as a template. FungiDB (http:// fungidb. org/ fungidb/) was used to look for gene expression patterns of CNAG_02415  [33].
6855)]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 17 amino acid sequences. All positions with less than 95 % site coverage were eliminated. That is, fewer than 5 % alignment gaps, missing data and ambiguous bases were allowed at any position. There were a total of 250 positions in the final dataset. Evolutionary analyses were conducted in mega7  [54]. increase in cell body size compared to its parental strain. We detected a significant increase in the number of titan cells produced by anxC1Δ strains in vitro  [44]  when compared to the parental strain H99, in all experiments performed. These data implicate AnxC1 as a negative regulator of titan cell formation. We note the magnitude of this effect is small and therefore its biological relevance is difficult to ascertain.
dISCuSSIon The importance of annexins to mammalian and plant biology led us to wonder about the role of fungal annexins, specifically in C. neoformans. The scarcity of knowledge regarding fungal annexins and our preliminary searches implicating annexin in responses to two major regulators of fungal virulence, the calcineurin and the Rim101 pathways, encouraged us to pursue a characterization of the cryptococcal annexin. Based on the most recent annexin nomenclature classifications, we propose that the name annexinXIV should be replaced by AnnexinC1. Our initial search through available datasets suggested that in C. neoformans, annexin could be involved in the calcineurin pathway, given that it was previously identified as a target of Crz1 and upregulated in high temperature  [22]. When testing for phenotypes indicative of deficiencies in calcineurin/Crz1 pathway, AnxC1 is not an effector of the known functions of calcineurin/Crz1.
's dataset (the only RNAseq dataset available to date for titan cell formation), generated after 7 and 18 h of titan cell induction as compared to normal sized cells  [60]. A role in membrane dynamics would also fit within the generalized function of annexins as a bridge between phospholipid structures, given the dynamic membrane remodelling needed during cellular replication. Future studies should define the cellular localization and regulation of annexinC1 protein to complement the transcriptomic data already available and better define these hypotheses. Given the scarcity of studies focusing on fungal annexins and shortage of (dramatic) phenotypic insights from deletion strains, the role of cryptococcal annexins remains cryptic. In summary, C. neoformans possesses one single annexin gene as evident from sequence homology analysis.
Funding information A. C. was supported by  National Institutes of Health (NIH)  awards  5R01HL059842 ,  5R01AI033774 ,  5R37AI033142 , and  5R01AI052733. E. C. was supported by a  Postdoctoral Fellowship  from the  Johns Hopkins Malaria Research Institute. E. E. F. was supported by  RISE  grant number:  1R25GM113748-01. The fungal deletion libraries were funded by  NIH   R01AI100272  to  Hiten D. Madhani  and deposited in the FGSC (http:// www. fgsc. net/).
A way to explain our results is that larvae with higher hemocytes counts are in an "inflammatory-like" state that is deleterious to the host. Indeed, chronic activation of the immune system has been associated with precocious lethality in Drosophila  [31, 32]. Therefore, we monitored in these larvae the level of activation of the Imd, Toll and JAK-STAT immune pathways using appropriate read-out genes (Diptericin, Drosomycin, and upd3 respectively). We did not observe any chronic activation of these pathways nor defect in immune inducibility in larvae with higher hemocyte count (Figure  S1A-C ). Moreover, hemocytes from larvae over-expressing pvf2 or ras V12 retain the ability to phagocyte Gram-negative and Gram-positive bacteria (Figure  S1D, E ). These data indicate that hemocytes from larvae with higher blood cell count are fully functional and the lethality observed on a poor diet cannot be explained by the chronic activation of the immune system.
Cell area measurement upon spreading To measure hemocyte area upon spreading, we proceeded as for hemocyte counting. hmlΔDsRed. nls hemocytes were let to spread on a glass slide for 45 minutes in Schneider medium supplemented with PTU. After 45 min, cells were fixed with PFA 4%/Triton 0,1% for 15 min, stained with Alexa Fluor TM 488 phalloidin for 3h at a dilution of 1/100 (Thermo Fisher Scientific, A12379) and DAPI at a dilution of 1/80000 for 10min. Cells were imaged under a Zeiss AxioImager Z. 1 with a 20X objective and images were analyzed with the Cell profiler software. We design software to select DAPI positive and hmlΔDsRed. nls positive cells and to measure their respective GFP area staining. All settings were designed by visual inspection. About 1000 cells per genotype pooled from three independent experiments were analysed.
Acknowledgements We thank the  VDCR in Vienna and the Bloomington Stock Center  ( NIH P40OD018537 ) at  Indiana University  for the fly stocks. We thank the  BIOP platform (EPFL)  for help with using the confocal microscope and design of Cell profiler analysis. We thank the  BioEM platform (EPFL)  for help with electron microscopy experiments. We thank  Marie Meister ,  Claudine Neyen  and  Mark Hanson  for critical reading of the manuscript.
Validation The 13 genes statistically differentially expressed were validated on the datasets of two other UMs: a set of 110 tumors from Genoa and Paris and another set of 80 UMs of The Cancer Genome Atlas (TCGA) project  [42]. In each validation set, median expression levels for every gene were calculated to establish two groups of tumors for Kaplan-Meier analyses. The occurrence of metastases was the event of interest in the tumors from Genoa and Paris (taken together), while death due to UM metastases was the endpoint of analysis for the TCGA tumors. In Genoa and Paris sets, more than one p-value is presented for some genes, since several probes were available for these genes. The association of the expression of a gene with survival was considered 'validated' if a significant association was observed in all three sets (LUMC set and the two validation sets).
Study Population Our 'training set' contained 64 UMs obtained by primary enucleation at the Leiden University Medical Center (LUMC), Leiden, The Netherlands, between 1999 and 2008. Patient and tumor characteristics are shown in Table  1. Sufficient frozen material of these tumors was available and DNA of adequate quality could be retrieved. Survival data was retrieved from the patients' charts and from the Netherlands Comprehensive Cancer Organisation (https://iknl. nl/over-iknl/about-iknl), and updated in March 2017. In The Netherlands, general physicians report every cancer patient to the Netherlands Comprehensive Cancer Organisation, which collects and registers information on the survival status by contacting the general physicians yearly. The follow-up in The Netherlands is not intensive because of a lack of effective treatments for UM metastases and patients are often referred back to their general physician after treatment of the primary tumor.
The median follow-up time was 62 months and no patient was lost to follow-up. Validation of the data was performed using two independent cohorts of post enucleation surgery patients: microarray datasets from Genoa and Paris, and RNAseq data of The Cancer Genome Atlas (TCGA) project  [42, 75]. Sixty-three untreated uveal melanoma provided by the Biological Resource Centre of Institut Curie (GSE2213840)  [86]  and 48 UM samples from the Genoa cohort (GSE2783141 and GSE5188042)  [41, 87]  were obtained from the Gene Expression Omnibus (www. ncbi. nlm. nih. gov/geo/). The datasets were combined and normalized as described. The study followed the tenets of the Declaration of Helsinki (World Medical Association of Declaration 1964; ethical principles for medical research involving human subjects) and the Medical Ethics Committee of the LUMC, Leiden, The Netherlands, had no objection regarding this research (G16.
Statistical Analysis For data analysis, we used the statistical programming language R version 3. 0. 1 (R: A Language and Environment for Statistical Computing, R Core Team, R foundation for Statistical Computing, Vienna, Austria, 2014, http://www. R-project. org) supplemented with specialized packages for SNP and RNA analysis. The main package used for SNP analysis was aroma. affymetrix, supported by 'DNAcopy' (Venkatraman E. Seshan and Adam Olshen, DNAcopy: DNA copy number data analysis. R package version 1. 34. 0), 'sfit' (Henrik  Bengtsson and Pratyaksha Wirapati (2013) , sfit: Multidimensional simplex fitting. R package version 0. 3. 0/r185, http://R-Forge. R-project. org/projects/matrixstats/), and 'R. utils' (Henrik Bengtsson (2014), R. utils: Various programming utilities, R package version 1.
29. 8, http://CRAN. R-project. org/package=R. utils). The 'Aroma. Affymetrix' package made it possible to use the information from the SNP microarrays to determine copy number values  [94, 95]. The packages used for RNA microarray analysis were 'limma' version 3. 16. 8, and the specific packages for Illumina microarrays: 'lumi' version 2. 12. 0, 'annotate' (R. Gentleman, annotate: Annotation for microarrays, R package version 1. 38. 0), and the database package 'IlluminaHumanv4. db' (Mark Dunning, Andy Lynch and Matthew Eldridge, IlluminaHumanv4. db: Illumina HumanHT12v4 annotation data (chip IlluminaHumanv4), R package version 1. 18. 0). The statistical software package SPSS v. 20. 0. 0 (IBM SPSS Statistics for Windows, IBM Corp. , Armonk, NY, USA) was used for data analysis. Population characteristics were described using medians and percentages.
1b  and Additional file 1: Figure  S1f ). The functional avidity of CD8 + T cells, also called antigen sensitivity, is measured as the peptide concentration able to mobilize 50% of the maximal response (EC 50 ). The functional avidity of T cells is independent of the magnitude of a response measured at saturating antigen concentrations  [26]  and is usually associated with superior control of virus replication or tumor growth  [22, 27]. We evaluated the functional avidity of a panel of virusspecific polyclonal CD8 + T cells exposed to decreasing peptide concentrations. We observed that Ado significantly reduced the functional avidity of all virus-specific CD8 + T cells (Fig. 1c-d ), with the strongest effect observed in the T CM subset (Fig. 1c-d ). These data indicate that Ado limits the ability of T cells to respond to targets expressing cognate antigens.
We also measured A2AR and A2BR expression with antibodies by flow cytometry in CD8 + T cells and found a correlation with A2AR expression measured by PrimeFlow RNA (Additional file 1: Figure  S2f-g ). Moreover, we confirmed the lower/undetectable expression of A2BR by CD8 + T cells (Additional file 1: Figure  S2f, h ). We then evaluated AdoR expression in total CD8 + T cells and in different memory subsets ex vivo. Of interest, A2AR was expressed at a higher percentage than other AdoRs in total CD8 + T cells (Fig. 2c ). Among the memory subsets, it was predominantly expressed by CD8 + T CM cells, similar to naïve CD8 + T cells (Fig. 2d ). These data strongly suggest that the high sensitivity of CD8 + T CM cells to Ado is associated with their high expression of A2AR. In addition, our results not only support the role of A2AR as a critical player in Adomediated suppressive effects in CD8 + T cells but also indicate that A2AR expression quantitatively dictates sensitivity to Ado.
The preventive action of A2AR-or A2BR-selective Representative example and d cumulative data of the virus-specific CD8 + T cell functional avidity measured in the presence or absence of Ado in total (n = 12) and in distinct memory CD8 + T cell subsets (T CM , T EM , and T EMRA ) (n = 6). Wilcoxon test inhibitors was equal in all the differentiation subsets evaluated (data not shown). The blockade of A2AR or A2BR alone did not affect cytokine production significantly (Additional file 1: Figure  S3c ). Finally, selective A2AR blockade also efficiently prevented Ado-mediated immunosuppression of virus-specific CD8 + T cells (Fig. 2g ). Taken together, these data not only confirm A2A as the main receptor responsible for Ado-mediated immunosuppression but also highlight the relevance of A2AR expression levels in quantitatively determining Ado suppressive effects in primary human CD8 + T cells.