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Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor (NGF) receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis. we apologize to those authors whose work could not be cited due to size restrictions. We thank M. S. Soengas and the members of her laboratory for melanoma cells, primary melanocyte preparations and helpful discussions. We thank M. Detmar and S. Proulx for the mouse B16-F1R2 cell line. We are grateful to M. Yañez-Mo and M. Valés for antibodies against sEV markers. We thank D. Grela and A. Escobar from IESMAT for their support with the Zetasizer analysis. We thank G. Roncador, L. Maestre and J. L. Martinez Torrecuadrada for their help with the development and characterization of anti-NGFR antibodies and C. Villarroya Beltri for her help in flow cytometry analysis. This work was funded by the Starr Cancer Consortium (B.J.M., D.L. and H.P.), the US NIH (R01-CA169416), the Nancy C. and Daniel P. Paduano Foundation, the Children’s Cancer and Blood Foundation (H.P. and D.L.), the Melanoma Research Alliance, the Feldstein Foundation, RETOS SAF2017-82924-R (AEI/10.13039/501100011033/FEDER-UE), the Fundación Ramón Areces, the Fundación Bancaria ‘la Caixa’ (HR18-00256), ATRES-MEDIA AXA Foundation (CONSTANTES Y VITALES, una iniciativa de laSexta y Fundación AXA) and the Fundación Científica AECC (LABAE19027PEIN, GCB15152978SOEN-HP) (H.P.), the Malcolm Hewitt Wiener Foundation, the AHEPA Fifth District Cancer Research Foundation, the Hartwell Foundation and the Manning Foundation (D.L.). We are also grateful for the support of the Translational Network for the Clinical Application of Extracellular Vesicles (TeNTaCLES), RED2018-102411-T (AEI/10.13039/501100011033), the Ramón y Cajal Programme, the FERO Foundation, Comunidad of Madrid 2017-T2/BMD6026 (L.N.) and La Caixa Foundation (ID100010434, fellowship LCF/BQ/ES17/11600007) (A.H.-B.). The CNIO, certified as a Severo Ochoa Excellence Centre, is supported by the Spanish government through the ISCIII. No

García-Silva et al. show for the first time that extracellular vesicles isolated from the exudative seroma obtained from the lymphatic drainage implanted in melanoma patients after lymphadenectomy can be interrogated for melanoma markers and BRAF mutations. Profiling the BRAFV600E mutation in this biofluid is a novel approach to predict disease relapse., Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse., Graphical Abstract

EMILIN-1; Melanoma; Metàstasi EMILIN-1; Melanoma; Metástasis EMILIN-1; Melanoma; Metastasis Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis. The authors gratefully acknowledge the support of the following sources of funding: Fundación Ramon Areces, MINECO (SAF2014-54541-R), Ramón y Cajal Programme, Asociación Española Contra el Cáncer, Constantes y Vitales (ATRES MEDIA/AXA Foundation) and FERO Foundation. We are also grateful for the support of the support of the Translational NeTwork for the CLinical application of Extracellular VesicleS, TeNTaCLES. RED2018-102411-T (AEI/10.13039/501100011033) and MINECO-Severo Ochoa predoctoral program to support A.A.L thesis and short term stay in Italy to perform this study. The CNIO, certified as Severo Ochoa Excellence Centre, is supported by the Spanish Government through the Instituto de Salud Carlos III (ISCIII).

Individuals exhibiting an intermediate alcohol drinking pattern in conjunction with signs of metabolic risk present clinical features of both alcohol‐associated and metabolic‐associated fatty liver diseases. However, such combination remains an unexplored area of great interest, given the increasing number of patients affected. In the present study, we aimed to develop a preclinical DUAL (alcohol‐associated liver disease plus metabolic‐associated fatty liver disease) model in mice. C57BL/6 mice received 10% vol/vol alcohol in sweetened drinking water in combination with a Western diet for 10, 23, and 52 weeks (DUAL model). Animals fed with DUAL diet elicited a significant increase in body mass index accompanied by a pronounced hypertrophy of adipocytes, hypercholesterolemia, and hyperglycemia. Significant liver damage was characterized by elevated plasma alanine aminotransferase and lactate dehydrogenase levels, extensive hepatomegaly, hepatocyte enlargement, ballooning, steatosis, hepatic cell death, and compensatory proliferation. Notably, DUAL animals developed lobular inflammation and advanced hepatic fibrosis. Sequentially, bridging cirrhotic changes were frequently observed after 12 months. Bulk RNA‐sequencing analysis indicated that dysregulated molecular pathways in DUAL mice were similar to those of patients with steatohepatitis. Conclusion: Our DUAL model is characterized by obesity, glucose intolerance, liver damage, prominent steatohepatitis and fibrosis, as well as inflammation and fibrosis in white adipose tissue. Altogether, the DUAL model mimics all histological, metabolic, and transcriptomic gene signatures of human advanced steatohepatitis, and therefore serves as a preclinical tool for the development of therapeutic targets., DUAL model perfectly mimics all histological, metabolic and transcriptomic gene signatures of human advanced steatohepatitis, and thus serve as a preclinical tool for the development of therapeutic targets.

Tissue-resident macrophages display varying phenotypic and functional properties that are largely specified by their local environment. One of these functions, phagocytosis, mediates the natural disposal of billions of cells, but its mechanisms and consequences within living tissues are poorly defined. Using a parabiosis-based strategy, we identified and isolated macrophages from multiple tissues as they phagocytosed blood-borne cellular material. Phagocytosis was circadianally regulated and mediated by distinct repertoires of receptors, opsonins, and transcription factors in macrophages from each tissue. Although the tissue of residence defined the core signature of macrophages, phagocytosis imprinted a distinct antiinflammatory profile. Phagocytic macrophages expressed CD206, displayed blunted expression of Il1b, and supported tissue homeostasis. Thus, phagocytosis is a source of macrophage heterogeneity that acts together with tissue-derived factors to preserve homeostasis., This study was supported by Ministerio de Economia, Competitividad e Industria (MINECO) grants SAF2015-65607 and SAF2013-49662-EXP to A. Hidalgo; SVP-2014-068595 to J.A. Nicolás-Ávila; BES-2013–065550 to J.M. Adrover; JCI-2012-12659 to N. A-Gonzalez; and SAF2014-56819-R and SAF2015-71878-REDT to A. Castrillo. Grant CAM S2010/BMD-2350 RAPHYME to A. Castrillo; SAF2015-64287-R, SAF2015-71878-REDT (MINECO), and CardioNext-ITN-608027 (European Commission FP7) to M. Ricote; and R01 AI089824 (National Institutes of Health) to C.V. Rothlin. H.P. is supported by grants from MINECO (SAF2014-54541-R), ATRES-MEDIA – AXA, Asociación Española Contra el Cáncer, WHRI Academy, and Worldwide Cancer Research. The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).

Objective To characterize the alterations, as well as their mechanisms, induced in the HLA–B27–bound peptidome expressed in live cells by the natural ERAP1 polymorphisms predisposing to ankylosing spondylitis (AS): R528K and N575D/Q725R. Methods HLA–B*27:05–bound peptides were isolated from 3 human lymphoid cell lines expressing distinct ERAP1 variants differing at residues 528 and/or 575/725. The high-performance liquid chromatography–fractionated peptide pools were compared by mass spectrometry based on identity of molecular mass and chromatographic retention time. The relative amount of each shared peptide in any given cell line pair was estimated from the respective ion peak intensities. Peptide sequencing was also carried out by mass spectrometry. Results HLA–B27–bound ligands predominant in the context of the ERAP1 variant with K528 collectively showed higher molecular mass, higher frequency of N-terminal residues resistant to ERAP1, and bulkier residues downstream of the N-terminus, relative to peptides predominant in the R528 context. None of these differences were observed with ERAP1 variants differing at positions 575/725, but not at residue 528. Neither R528K nor N575D/Q725R altered the mean length of B*27:05-bound ligands. Conclusion The R528K, but not the N575D/Q725R, polymorphism alters the expression levels of many HLA–B*27:05–bound peptides, depending on the susceptibility of their N-terminal residues to trimming and depending on the size of the amino acid side chains at multiple positions downstream of the N-terminus. The significant alterations in the B*27:05 peptidome and the structural features of the peptides that determine their differential expression in distinct ERAP1 contexts account for the association of the R528K polymorphism with AS.

Heterotrimers composed of B cell CLL/lymphoma 10 (BCL10), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and caspase recruitment domain-containing (CARD) family adaptors play a role in NF-κB activation and have been shown to be involved in both the innate and the adaptive arms of immunity in murine models. Moreover, individuals with inherited defects of MALT1, CARD9, and CARD11 present with immunological and clinical phenotypes. Here, we characterized a case of autosomal-recessive, complete BCL10 deficiency in a child with a broad immunodeficiency, including defects of both hematopoietic and nonhematopoietic immunity. The patient died at 3 years of age and was homozygous for a loss-of-expression, loss-of-function BCL10 mutation. The effect of BCL10 deficiency was dependent on the signaling pathway, and, for some pathways, the cell type affected. Despite the noted similarities to BCL10 deficiency in mice, including a deficient adaptive immune response, human BCL10 deficiency in this patient resulted in a number of specific features within cell populations. Treatment of the patient's myeloid cells with a variety of pathogen-associated molecular pattern molecules (PAMPs) elicited a normal response; however, NF-κB-mediated fibroblast functions were dramatically impaired. The results of this study indicate that inherited BCL10 deficiency should be considered in patients with combined immunodeficiency with B cell, T cell, and fibroblast defects.

To characterize the alterations, as well as their mechanisms, induced in the HLA-B27-bound peptidome expressed in live cells by the natural ERAP1 polymorphisms predisposing to ankylosing spondylitis (AS): R528K and N575D/Q725R.HLA-B*27:05-bound peptides were isolated from 3 human lymphoid cell lines expressing distinct ERAP1 variants differing at residues 528 and/or 575/725. The high-performance liquid chromatography-fractionated peptide pools were compared by mass spectrometry based on identity of molecular mass and chromatographic retention time. The relative amount of each shared peptide in any given cell line pair was estimated from the respective ion peak intensities. Peptide sequencing was also carried out by mass spectrometry.HLA-B27-bound ligands predominant in the context of the ERAP1 variant with K528 collectively showed higher molecular mass, higher frequency of N-terminal residues resistant to ERAP1, and bulkier residues downstream of the N-terminus, relative to peptides predominant in the R528 context. None of these differences were observed with ERAP1 variants differing at positions 575/725, but not at residue 528. Neither R528K nor N575D/Q725R altered the mean length of B*27:05-bound ligands.The R528K, but not the N575D/Q725R, polymorphism alters the expression levels of many HLA-B*27:05-bound peptides, depending on the susceptibility of their N-terminal residues to trimming and depending on the size of the amino acid side chains at multiple positions downstream of the N-terminus. The significant alterations in the B*27:05 peptidome and the structural features of the peptides that determine their differential expression in distinct ERAP1 contexts account for the association of the R528K polymorphism with AS.

Resumen Las inmunodeficiencias humanas del TCR son enfermedades autosomicas recesivas con baja prevalencia, caracterizadas por un defecto de expresion del TCR asociado a una linfopenia T selectiva (mas leve en el caso de CD3γ, TCRα o CD247, o grave en el caso de CD3δ o CD3ɛ). La ausencia congenita de componentes del TCR tiene un impacto diferencial en el desarrollo y funcion de los linfocitos T, que depende de la cadena del TCR afectada y de la especie, siendo en algunos casos diferente en los pacientes humanos en comparacion con los modelos en ratones. El estudio del inmunofenotipo mediante citometria de flujo, junto con los estudios moleculares, proporciona informacion esencial para el diagnostico y el tratamiento, que continua siendo a dia de hoy el trasplante de progenitores hematopoyeticos en los casos asociados a inmunodeficiencia grave.