Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection

Neutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.Peer reviewe

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression

Endogenous IL-1 receptor antagonist restricts healthy and malignant myeloproliferation.

Here we explored the role of interleukin-1β (IL-1β) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1β monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1β/IL-1rn levels under steady-state, and that loss of repression of IL-1β signaling may underlie pre-leukemic lesion and AML progression.We thank K. Tasken, J. Saarela and the NCMM at the University of Oslo (UiO), S. Kanse (UiO) and B. Smedsrød (UiT), for access to facilities. We acknowledge Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital (Bergen, Norway) and R. Hovland for karyotyping, FISH, translocation and DNA analyses of AML and MDS patients included in this study, and Department of Pathology, Oslo University Hospital (Oslo, Norway) and S. Spetalen for deep sequencing. L.M. Gonzalez, L.T. Eliassen, X. Zhang, M. Ristic and other members of L. Arranz group, O.P. Rekvig, R. Doohan, L.D. Håland, M.I. Olsen, A. Urbanucci, J. Landskron, K.B. Larsen, R.A. Lyså and UiT Advanced Microscopy Core Facility, UiO and UiT Comparative Medicine Units, for assistance. P. Garcia and S. Mendez-Ferrer for providing NRASG12D and Nes-gfp mice, respectively. P. Garcia and L. Kurian for careful reading of the manuscript. E. Tenstad (Science Shaped) for artwork in schematics. We would also like to thank the AML and MDS patients, and healthy volunteers, who donated biological samples. Our work is supported by a joint meeting grant of the Northern Norway Regional Health Authority, the University Hospital of Northern Norway (UNN) and UiT (Strategisk-HN06-14), Young Research Talent grants from the Research Council of Norway, (Stem Cell Program, 247596; FRIPRO Program, 250901), and grants from the Norwegian Cancer Society (6765150), the Northern Norway Regional Health Authority (HNF1338-17), and the Aakre-Stiftelsen Foundation (2016/9050) to L.A. Vav-Cre NRASG12D experiments were supported by NIH grant R01CA152108 to J.Z.S

Incongruence between transcriptional and vascular pathophysiological cell states

Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice.S

NLRC4 -mediated activation of CD1c+ dendritic cells contributes to perpetuation of synovitis in rheumatoid arthritis.

The individual contribution of specific myeloid subsets such as CD1c+ conventional dendritic cells (cDC) to perpetuation of Rheumatoid Arthritis (RA) pathology remains unclear. In addition, the specific innate sensors driving pathogenic activation of CD1c+ cDCs in RA patients and their functional implications have not been characterized. Here, we assessed phenotypical, transcriptional and functional characteristics of CD1c+ and CD141+ cDCs and monocytes from the blood and synovial fluid of RA patients. Increased levels of CCR2 and the IgG receptor CD64 on circulating CD1c+ cDC associated with the presence of this DC subset in the synovial membrane in RA patients. Moreover, synovial CD1c+ cDCs are characterized by increased expression of proinflammatory cytokines and high abilities to induce pathogenic IFNγ+IL-17+ CD4+ T cells in vitro. Finally, we identified the crosstalk between Fcγ Receptors and NLRC4 as a new potential molecular mechanism mediating pathogenic activation, CD64 upregulation and functional specialization of CD1c+ cDCs in response to dsDNA-IgG in RA patients.E.M.G. was supported by Comunidad de Madrid Talento Program (2017-T1/BMD5396), Ramón y Cajal Program (RYC2018-024374-I), the MINECO RETOS program (RTI2018-097485-A-I00), La Caixa Foundation (HR20-00218), CIBERINF (CB21/13/00107) and the TV3 Marató (REDINCOV). C.D.A. was supported by Comunidad de Madrid Talento Program (2017-T1/BMD-5396). M.C.M was supported by the NIH (R21AI140930). HR17-00016 grant from “La Caixa Banking Foundation to F.S.M also supported the study. D.C-F. is supported by the Fellowship “la Caixa” Foundation LCF/BQ/DR19/11740010. A.T.M. was supported by a PhD fellowship from the Autonomous Region of Madrid (PEJD-2019-PRE/BMD-16851) and the RD21/0002/0027 grant. R.L. and G.H.B. were supported by PI18/00261 AND PI20/00349 grants from Ministerio de Ciencia e Innovación and Instituto de Salud Carlos III, respectively. O.P. was supported by the TV3 Marató (REDINCOV) and I.T. was funded by grant for the promotion of research studies master-UAM 2021. S.C. was supported by PI21/01474 grant from the Instituto de Salud Carlos III and co-funded by The European Regional Development Fund (ERDF). I.G.A was supported by RD21/0002/0027 and PI21/00526 grants from the Spanish MINECO and Instituto de Salud Carlos III and co-funded by The European Regional Development Fund (ERDF) “A way to make Europe.S

MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.S

Regulación de la actividad tirosina quinasa del receptor del factor de creciminento epidérmico por la calmodulina y la fosfocalmodulina

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 13-07-1993Una de las primeras respuestas celulares a la estimulación por el factor de crecimiento epidérmico (EGF) es el aumento de la concentración citoplásmica de Caz+. Por tanto, cabe suponer que la principal proteina receptora de este ión, calmodulina, se active durante este proceso, siendo capaz de unirse a los diferentes enzimas diana y regular su actividad. Así, hemos estudiado el papel de la calmodulina en la regulación de la actividad tirosina quinasa del receptor del EGF. Hemos demostrado que el receptor del EGF de la membrana plasmática de hígado de rata se puede aislar mediante cromatografía de afinidad en calmodulina-agarosa uniéndose en presencia de Ca2+ y eluyéndose con EGTA, lo que sugiere que el receptor tiene un lugar de unión para esta proteína reguladora. La unión del receptor del EGF a la calmodulina es específica y eficiente. La calmodulina inhibe tanto la autofosforilación del receptor del EGF en tirosina, como su actividad tírosina quinasa hacia sustratos exógenos. Dicha inhibición es parcialmente dependiente de Caz+ y no es revertida por concentraciones altas de EGF. Estos resultados sugieren que la calmodulina podría inducir la desensibilización del receptor del EGF, y por tanto, podría ser capaz de parar la señal mitogénica. También hemos demostrado que la calmodulina se fosforila en serina, treonina y tirosina por una(s) proteína quinasa(s) de la membrana plasmática. Además, el receptor del EGF, aislado mediante cromatografía en calmodulina-agarosa, fosforila fuertemente a la calmodulina en la tirosina-99. La fosforilación de la calmodulina se inhibe por Caz+ y es dependiente de ciertos cofactores básicos como la poli-L-lisina y la histona. En las condiciones óptimas, la estequiometría de la fosforilación de la calmodulina por el receptor del EGF es aproximadamente de 1 mol de fosfato unido por mol de calmodulina, lo que indica que el 100 % de las moléculas de calmodulina se fosforilan. La fosfocalmodulina, fosforilada por el receptor del EGF, estimula aparentemente la actividad tirosina quinasa del receptor del EGF, por lo que presenta un efecto contrario a su forma no fosforilada y por tanto, podría amplificar la señal mitogénica. Estos resultados sugieren que la calmodulina, tanto fosforilada como no fosforilada, está ínvolucrada en la regulación de los procesos iniciales de la transducción de señales inducidos por el EGF

Sir2p suppresses recombination of replication forks stalled at the replication fork barrier of ribosomal DNA in Saccharomyces cerevisiae

In the ribosomal DNA (rDNA) of Saccharomyces cerevisiae replication forks progressing against transcription stall at a polar replication fork barrier (RFB) located close to and downstream of the 35S transcription unit. Forks blocked at this barrier are potentially recombinogenic. Plasmids bearing the RFB sequence in its active orientation integrated into the chromosomal rDNA in sir2 mutant cells but not in wild-type cells, indicating that the histone deacetylase silencing protein Sir2 (Sir2p), which also modulates the aging process in yeast, suppresses the recombination competence of forks blocked at the rDNA RFB. Orientation of the RFB sequence in its inactive course or its abolition by FOB1 deletion avoided plasmid integration in sir2 mutant cells, indicating that stalling of the forks in the plasmid context was required for recombination to take place. Altogether these results strongly suggest that one of the functions of Sir2p is to modulate access of the recombination machinery to the forks stalled at the rDNA RFB