dataesr/scientific-paragraphs-categorization huggingface dataset:
S4F ), indicating disrupted binding. Similarly, in the SPA motif of UBE2E1 C loop 7 (uL7), a UBE2E1 C -A142D mutant entirely disrupts the complex formation as observed by NMR (Fig. S4G ), in agreement with the SPA region being critical for TRIM21-catalyzed conjugation activity  (25, 42). A, TRIM21 R -UBE2E1 C complex crystallized in space group C2 where 15 protomers make up the crystallographic ASU. Eight protomers of TRIM21 R form four homodimers (chains FD, EA, BC, and HG), whereas the remaining seven protomers comprise UBE2E1 C (chains I, J, K, L, M, N, and O). Altogether, three complete TRIM21 R :UBE2E1 C 2:2 assemblies are present in the ASU comprising chains MBCO, IFDL, and NEAK, and a 1:2 complex is present comprising chains JHG. B, bead models representing the solution structure of free TRIM21 R derived from the SAXS data using DAMMIF and assuming P2 symmetry (cyan) or no symmetry (blue dots) overlaid with the TRIM21 R dimer crystal structure.
3C  and S7C ); similar disorder is observed also in substrate-free states of full-length UBE2E1 (PDB code 3BZH), UBE2D1 (PDB code 2C4P), and ubiquitin-conjugated UBE2D1 (PDB code 4AP4). Together, this suggests that lysine-coordinating residues are ordered on substrate lysine coordination in the active site. We probed the nature of Lys 61 Љ as a possible target residue for autoubiquitination in several ways. First, a K61A mutation severely disrupts TRIM21 R autoubiquitination with both UBE2E1 and UBE2D1 (Fig. 4A ). A TRIM21 R -N62A mutation similarly disrupts ubiquitination, whereas TRIM21 R -N62R ubiquitination is close to WT, both by UBE2E1 and UBE2D1 (Fig. 4A ), in agreement with a structural role of an adjacent side-chain amide in supporting ubiquitination (Fig. 4A ). Ubiquitination at other sites in TRIM21 R (Lys 45 and Lys 77 ) is very weak or absent as judged by the very low residual ubiquitination in TRIM21-K61A (Fig.
Cloning of recombinant proteins Human TRIM21 (UniProt accession number P19474) constructs were subcloned by ligation-independent cloning (67) into pET28-MHL expression vectors (TRIM21 M1-R91 ) carrying an N-terminal, cleavable His 6 tag. Full-length UBE2E1 (UniProt accession number P51965) and UBE2E1 C (residues  36 -193)  were respectively subcloned into pET28b. Point mutations were introduced using the QuikChange II site-directed mutagenesis kit (Stratagene). In addition, the UBE2E1 C scaffold consistently included an S68R mutation to prevent noncovalent interactions between Ub and the backside of the E2's UBC domain, similar to UBE2D1-S22R  (60). The Ube1/ PET21d plasmid was a gift from Prof. Cynthia Wolberger (Addgene plasmid 34965)  (61).
NMR spectroscopy and data analysis UBE2E1 NMR samples were prepared in buffer containing 50 mM Tris-HCl, pH 8. 0, 100 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine, 0. 02 mM NaN 3 , and 90% H 2 0, 10% 2 H 2 0 (v/v). Triple-labeled [ 13 C, 2 H, 15 N]UBE2E1 (600 M) was prepared for the peptide backbone assignments. HNCA, HNCO, HNCOCA, HNCACO, HNCACB, and HNCOCACB triple-resonance experiments were collected at 30 °C on a Varian INOVA spectrometer operating at a proton frequency of 600 MHz (with cryoprobe). Due to poor stability at high concentrations for the UBE2E1 C construct, UBE2E1 CS (C131S) protein was prepared at a concentration of 400 M, and HNCA, HNCO, and HNCACB resonance experiments were recorded to confirm assignments in the shorter construct. Data were processed using NMRPipe/NMRDraw (63), visualized and analyzed with.
TRIM21-UBE2E1 complex offers new insights in ubiquitination the Sparky program (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco). The backbone assignment was manually performed, assisted by the COM-PASS software  (64). Titrations experiments were carried out in a buffer containing 50 mM Tris-HCl, pH 8. 0, 100 mM NaCl, 10 mM ␤-mercaptoethanol, 500 M ZnCl 2 , and 90% H 2 0, 10% 2 H 2 0 (v/v). CSP data were collected at 30 °C in 1 H, 15 N heteronuclear single quantum coherence transverse relaxation optimized spectroscopy-based experiments on a Varian 500-MHz NMR spectrometer with uniformly 15 N-labeled UBE2E1 C (200 M) apo constructs and addition of 0. 25, 0. 5, 0. 75, 1, and 2 eq of unlabeled TRIM21 R constructs. CSPs were calculated with the formula ⌬␦ ϭ [(⌬␦ 1 H) 2 ϩ (⌬␦ 15 N ϫ 0.
1 Å) during the multiple anomalous dispersion phasing method. Our first model was built using the CCP4 (68) software Buccaneer  (69)  and completed by manual model building in Coot  (70). The molecules in the asymmetric unit were initially refined with local noncrystallographic symmetry (NCS) restraints in BUSTER and later with Phenix_Rosetta (71) that does not use NCS but improved the local geometry as judged by MolProbity. For final refinement, we uploaded the unmerged XDS_ASCII. HKL inflection point data set with the STARANISO web server (http://staraniso. global phasing. org/cgi-bin/staraniso. cgi)  7  (80) that performs an elliptical resolution cutoff for anisotropically diffracting crystals. Despite having a few diffraction spots to 2. 57-Å resolution in the best-diffracting direction, we decided to remove the data in the 2.
82-2. 57-Å interval because spherical completeness was only 15% in that interval. After removing that interval, the spherical/elliptical completeness was 50. 7/66. 5% in the highest-resolution shell (2. 91-2. 82 Å), and overall the spherical/elliptical completeness was 91. 2/93. 6% in the 47. 9 -2. 82-Å interval (Table  1 ). The final model was generated using local NCS restraints and jelly-body refinement in REFMAC5 (72) with 96. 6/3. 2/0. 2% of the amino acids in the preferred/allowed/disallowed regions of the Ramachandran plot. UBE2E1 C crystals were produced from the same material as in the UBE2E1 C -TRIM21 R crystals, and data were recorded at the same beam time. Diffraction data were collected at BL14. 1 beamline at BESSY Synchrotron. The structure was determined by molecular replacement in MOLREP using PDB code 3BZH (38) as a search model followed by manual model building in Coot and refinement in REFMAC5.
Model building and structural presentations The model of UBE2E1-Ub-TRIM21 R was generated by superimposing UBE2E1 C (module F) in the UBE2E1 C -TRIM21 R structure (PDB code 6FGA) onto the E2 module of the TRIM25-UBE2D1ϳUb ternary complex (PDB code 5FER; C␣ r. m. s. d. , 0. 54 Å). The resulting UBE2E1-Ub conjugate in which Ub is in a closed conformation shows essentially no clashes with UBE2E1 C or TRIM21 homodimers in the 6FGA structure, supporting its relevance in a ternary Ub-E2-E3substrate complex. All figures were generated using PyMOL Molecular Graphics System, Version 1. 2r3pre (Schro ¨dinger LLC).
SAXS sample preparation, data acquisition, analysis, and modeling SAXS data were acquired for TRIM21 R using the ESRF BM29 SAXS beamline  (74, 75)  with a robotic sample changer (76) and a Pilatus 1M detector (Dectris). SAXS data were also acquired using the Anton Paar SAXSess at Linko ¨ping University for TRIM21 R. SAXS samples were prepared by extensive dialysis against their buffer: 50 mM Tris-HCl, pH 8. 0, 150 mM NaCl, 10 mM ␤-mercaptoethanol, and 500 M ZnCl 2. For SAXSess measurements only, 10% glycerol was added to the buffer solution. Exact solvent blanks for all measurements were obtained from the last dialysis step. Table  S3  provides the SAXS data acquisition parameters, sample parameters, and software used for data reduction to I(q) versus q, where I(q) ϭ 4sin/, 2 is the angle between the incident and scattered X-rays, and is their wavelength), analysis, and interpretation.
All data were placed on an absolute scale using the scattering from pure H 2 O (SAXSess) or incident beam flux (BM29). I(q) versus q for the protein was obtained by subtraction of the solvent scattering from that of protein ϩ solvent. For the BM29 data, solvent measurements taken immediately before and after the protein ϩ solvent measurement were averaged to optimize solvent subtraction. As there was no discernible concentration dependence to I(q) for TRIM21 R , SAXSess data from the highest concentration samples were averaged to improve signal to noise. Molecular weights for the proteins were estimated using the method of Orthaber and Glatter  (77). Values for contrast and partial specific volumes were determined using the MULCh program  (78)  with the known chemical compositions of samples and solvent.
SAXS data analysis and modeling were performed using the tools of the ATSAS program package  (79). The online interface and software used are listed in Table  S3. Default software parameters were employed unless otherwise specified. The ESRF and SAXSess data overlay well (Fig. S3D ), and a Kratky (Fig. S3E ) plot of the ESRF data shows the expected bell shape for a globular, mostly folded protein with a rising profile at high q-values, indicating some degree of flexibility in the structure. The Guinier results for the ESRF and SAXSess data were the same within error (Table  S4 ), but the SAXSess data were measured to lower minimum q-values and hence are more reliable for d max determination in P(r) calculations compared with the ESRF data. Indeed, the latter consistently showed a similar shape to that obtained with the SAXSess data with a weak tail to longer r values of indeterminate length that increased the apparent R g values.
E, same as B, where the acceptor lysines Lys 61 Љ from TRIM21 R Љ and Lys 524 of RanGAP1 from UBC9 -RanGAP1 substrate complex (PDB code2GRN) and residue analog Arg 720 of CUL1 from E2-NEDD8 -RBL1-CUL1 (PDB code 4P5O) complex are shown.
This work was supported by funding from the  Swedish Research Council  (to M. S. , J. T. , and M. W. -H. ); The  Swedish Rheumatism Association , The  Swedish Heart-Lung Foundation , The  Stockholm County Council , the King Gustaf the Vth 80-year foundation, and  Karolinska Institutet  (to M. W. -H. ); and the  Swedish Foundation for International Cooperation in Research and Higher Education , the  Swedish Child Cancer Foundation , the  Swedish Cancer Foundation , the  Carl Trygger Foundation , and  Linko ¨ping University  (to M. S. ). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S7 and Tables  S1-S5. The atomic coordinates and structure factors (codes  5LBN  and  6FGA ) have been deposited in the Protein Data Bank (http://wwpdb.
We thus established a comprehensive list of ISGs of zebrafish larvae, and performed a detailed comparison with the human ISG repertoire. Our comparative analysis was facilitated by a compilation of human ISGs made to perform a systematic screen  (30) , and by the specialized database Interferome  (31). We identify about 70 orthology groups that include ISG in both species and thus approximate the ISG repertoire of the common ancestor of all Osteichthyes. As ISGs typically evolve fast, with frequent duplications and gene loss, we also identify many families of fish-specific ISGs, which represent a rich resource for seeking new antiviral mechanisms. Our study provides a broad overview of the evolutionary patterns of genes belonging to the type I IFN pathway, and identifies gene modules induced by a viral infection independently of IFN.
Identification of differentially expressed genes (DEG) Differentially expressed nuclear genes between larvae treated with IFNj1 and controls, between larvae infected by CHIKV and controls, or between crfb1+2 and control morphants all infected by CHIKV, were identified. DEG were identified using DESeq 1. 18. 0 (BioConductor) (Love et al. , 2014) and R: 3-1-2 (R core team, 2017). Briefly, raw counts of genes were subjected to a minimal pre-filtering step: genes for which the count sum, per group of samples, was equal or higher than 10, in at least one group, were kept. Raw counts were normalized for library size and normalized data were fitted using a negative binomial general linear model. Data were adjusted for multiple testing using the Benjamini-Hochberg procedure (adjusted p value). Genes with an adjusted p value less than 0.
Identification of human orthologues and ISGs Orthology analysis was primarily based on data from the Ensembl database (www. ensembl. org), the zfin database (zfin. org), and the literature, notably our previous analysis of zebrafish orthologues of human ISGs  (26). Data were systematically curated manually, and conflicts resolved using a combination of literature search, synteny analysis, and sequence homology analysis (two-way protein BLAST). When human genes were present on the list compiled by Schoggins et al. (30).
Human orthologues of zebrafish ISGs are enriched in ISGs We then searched for the human orthologues of the 337 identified protein-coding zebrafish ISGs (Table  S2 ). All types of orthology relationships between zebrafish and human were observed, from none to "many-to-many". One-to-one orthology was found for 77 genes (Figure  1C ). We identified one or several human orthologues for 200 zebrafish ISGs. This proportion (200/337, 59%) is significantly lower than the 71% reported for the whole genome (29) (Fisher's exact test, p<0. 0001). We then searched which of these human genes were themselves ISGs. We found 61 ISGs present in the list of 446 human ISGs compiled by Schoggins et al. (30) ; by querying the Interferome database  (31) , we identified 11 additional human ISGs (Table  S2 ). In total, 97 zebrafish IFNf1-inducible genes were orthologous to at least one human ISGs (Figure  1C ).
IFN-downregulated genes Two of the most strongly IFNj1-downregulated genes (Table  S1 , bottom) were orthologous to human genes downregulated by type I IFNs, according to the Interferome database: plin1 (perilipin 1) and acox1 (palmytoil acyl-CoA oxidase 1). This suggests that downregulation of fatty acid oxidation pathway is an ancient feature of the IFN system. Many IFNj1-downregulated genes were orthologous to a human gene in a 1-to-2 manner, with the two zebrafish paralogues having arisen during the teleost specific whole genome duplication (ohnologues). Systematically, only one of the two paralogues was downregulated. Remarkably, no gene was downregulated by both IFNj1 injection and CHIKV infection (Figure  3b , Table  S3 ).
Discussion The zebrafish has become an important model to study host-pathogen interactions, particularly at its early life stages which are the most prone to live imaging and genetically tractable. Although its antiviral interferon genes and receptors are now well identified, knowledge of IFN-induced genes, or ISGs, was only partial. In this work, we used deep sequencing to characterize the transcriptomic response of the 3dpf zebrafish larva to recombinant IFNj1, the first type I IFN identified in zebrafish and the most highly inducible one. We analyzed in parallel the response to an alphavirus inducing a strong type I IFN induction, ant the impact of IFN receptor knock-down on this response. From these different datasets, we established a comprehensive list of zebrafish ISGs. This list was compared to the human ISG repertoire, and a phylogenetic analysis was performed to approach the ancestral ISG repertoire of early vertebrates.
Acknowledgements We thank  Jean-Yves Coppee  and  Caroline Proux  from the transcriptomics platform of  Institut Pasteur , for generation and sequencing of RNA libraries. We are indebted to  Rune Hartmann  ( Aarhus, Denmark ) for recombinant zebrafish interferon and stimulating discussion. We thank  Emma Colucci  and  Pedro Hernandez-Cerda  for critical reading of the manuscript.
, they were labelled as ISGs. If absent from the list, gene names were further queried on the Interferome website (http://www. interferome. org/interferome/search/showSearch. jspx) which compiles the results of many transcriptomic studies on human and mouse samples after IFN stimulation (31). We postulated that human genes present in Interferome could be considered as ISG when being significantly induced at least 2-fold with a stimulation for no more 12 hours by type I IFN in at least 4 datasets. qRTPCR RNA was extracted from individual larvae using RNeasy® Mini Kit (Qiagen). cDNA was obtained using M-MLV H-reverse-transcriptase (Promega) with a dT17 primer. Quantitative PCR was then performed on an ABI7300 thermocycler (Applied Biosystems) using Takyon™ ROX SYBR© 2x MasterMix (Eurogentec) in a final volume of 25 µl.
For example, MoDCs do not migrate efficiently to lymph nodes and are particularly prone to develop immunosuppressive functions, whereas cDC1 excel in the activation of CTLs, which are critical effector cell types for antitumor immunity  (17). Thus, major efforts have been conducted in the last 10 years to investigate whether cDC1 might be critical for defense against cancer, and how. Here, we are reporting on studies addressing this issue in mice, under experimental conditions of spontaneous immune rejection of tumor grafts in syngeneic recipients, or in preclinical models of immunotherapies. We also summarize human studies that mined large datasets of tumor gene expression profiles to investigate correlations between clinical outcome and digital deconvolution of the tumor immune infiltrate.
State-of-the-Art in Assessing cDC1 Infiltration From Whole Tumor Tissue Gene Expression Profiles Several public datasets are available with gene expression profiles of whole tumor tissue from large cohorts of patients with well documented clinical characteristics. Increasing numbers of teams are querying this gold mine to test whether higher expression in tumors of gene signatures specific for various cell types or biological pathways are associated with a better or worse clinical outcome. Such analyses could allow high throughput testing of the possible relationship between overall survival and tumor infiltration by specific cell types in a given activation state. Such analyses would then allow focusing further studies on the most promising observations, to test whether they are confirmed by using immunohistofluorescence or flow cytometry to directly measure the frequency of specific combinations of immune cell types and activation states in the tumors.
FUNDING This work was supported by grants from the  European Research Council  under the European Community's  Seventh Framework Program  ( FP7/2007-2013  grant agreement number  281225  to MD), from the  Agence Nationale de la Recherche  ( ANR ;  XCR1-DirectingCells  to KC), from the  Fondation pour la Recherche Médicale  (label Equipe FRM 2011, project number  DEQ20110421284  to MD), from the  Fondation ARC pour la recherche sur le cancer  (to KC) and from the  Institut National du Cancer (INCa   PLBIO 2018-152 ). This work also benefited from institutional funding from  CNRS  and  INSERM. J-CC was supported by doctoral fellowship from  La Ligue Nationale Contre le Cancer. RM was supported by doctoral fellowships from the  Biotrail Ph. D. program  ( Fondation A * MIDEX ) and from  Fondation ARC  pour la recherche sur le cancer.
2009 A study of CDX-1401 Advanced NCT00948961 CDX-1401 I/II Induction of humoral and cellular (185) (DEC205/NY-ESO-1) in malignancies + Resiquimod immunity to NY-ESO-1. patients with refractory to ± Poly(IC:LC) Disease stabilization in 13 of 45 malignancies known to available therapies patients. express NY-ESO-1 Tumor regression in 2 patients. Objective tumor regression in 6 of 8 patients who received ICB after CDX-1401. 2014 CDX-1401 Resected NCT02129075 CDX-1401 II Higher tumor-specific immune (188, 189) (DEC205/NY-ESO-1) melanoma + Poly(IC:LC) responses observed in subjects and Poly(IC:LC) ± rhuFLT3-L who received FLT3-L vaccine therapy with or (CDX-301) without CDX-301 in pre-treatment treating patients with stage IIB-IV melanoma SLN, Sentinel lymph nodes.
TCD4 + Lymphocytes analysis by flow cytometry The analyses were carried out using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). The TCD4 + lymphocyte subpopulations were analyzed using the anti CD4 + mouse rat monoclonal antibody, marked with PE-Cy5 (BD Bioscience Pharmingen). The samples were characterized according to the intensity of fluorescence radiated by the fluorochrome used in the markings as well as by rugosity and size of the cells. The obtained data were analyzed using the Cell Quest Program for McIntosh.
The volume of the induration was determined measuring the width of the paws, using a digital Vernier caliper; the values obtained from these measures were correlated with the volume data (mL) of the inflammation taken from the limbs, using an automatic Plesthysmometer, (Ugo Basile, Italy), (data on comparison not shown). Both formulations based on nPL and nCh administered through each of the immunization routes generated HSR responses (Figure  3 ). All groups IM immunized (Figure  3A ) were able to induce a similar response among them and with respect to the VA-MENGOC-BC® vaccine used as the trial positive control. In all groups, the %IP was statistically higher than the CN group (p<0. 05). In the group inoculated with PD-Tp, the %IP values were higher (p<0. 05), not only with regard to CN, but also compared to the rest of the groups.
Acknowledgements Authors address special thanks to  Dr. Juan Ayala  and  Dr. Manuel Fresno  from the  Center of Molecular Biology "Severo Ochoa ", for their support in the implementation of the flow cytometry trials. The cooperation agreements between  Universidad Autonoma de Madrid, Spain, Universidad de La Habana ,  Cuba , and  University of Santiago de Compostela , Spain have been very competitive in terms of technical support (the electron microscopy studies). Dr. Vet. Med. Virgilio Bourg ,  Tec. Damaris Torralba  from the  Center for Biologicals and Lic. Viviana Falcon  from  Finlay Institute  rendered valuable contributions to this work. We express our appreciation to the editor,  Dr. Damià Barceló , and to the anonymous reviewers for helping to improve our paper.
Data Collection Patients age >50 years with AML who underwent their first or second allo-HSCT between 2004 and 2014 using a matched sibling donor (MSD) or a matched unrelated donor (MUD) were included. All patients were reported to the European Society for Blood and Marrow Transplantation (EBMT), a group of >500 centers that reports consecutive HSCTs. The validation and quality control include cross-checking with the national registries and onsite visits. Each patient provided written informed consent.
This was not seen for patients in CR1 and with advanced AML, who composed 85% of the patients. Overall, there appears to be conflicting data regarding whether or not chronic GVHD is further increased in patients age >65 years  [29, 33, 34]. In the CR patients age 70 years had higher NRM, lower survival, and lower LFS compared with younger patients. A similar outcome has been reported for patients with AML age 60 to 64 years compared with those age 65 years  [34]. In patients who received an NMC regimen for acute leukemia, outcomes were similar in different age-stratified groups between 60 and 75 years  [35]. Chevallier et al  [33]  also reported that age was not an adverse factor for outcome in patients age 60 years with AML. We included many more patients and patients of older age. NRM, survival, and LFS are similar to those in a recent report from the CIBMTR in patients age >70 years  [36].
ACKNOWLEDGMENTS The authors thank all the  EBMT Centers  for submitting data to the  Acute Leukemia Working Party (ALWP)  (see list in Supplementary Table 1). They also thank all the members of staff at the various departments for excellent patient care, all the volunteer donors and the staff at the  ALWP  for data collection, and  Gunilla Tillinger  for typing the manuscript. Financial disclosure: This study was supported by grants from the  EBMT. O. R. was supported by grants from the  Swedish Cancer Society , the  Swedish Research Council , the  Cancer Society in Stockholm , and the  Karolinska Institute. Conflict of interest statement: There are no conflicts of interest to report. Authorship statement: Study concept and design: O. R. ,  B. Sadeghi ,  A. Boumendil ,  M. L. , and A. N. ; financial support: O.