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3. Cross‐presentation of dead‐cell‐associated antigens by DNGR‐1+ dendritic cells contributes to chronic allograft rejection in mice

Author

Balam, S., Kesselring, R., Eggenhofer, E., Blaimer, S., Evert, K., Evert, M., Schlitt, H.J., Geissler, E.K., Blijswijk, J. van, Lee, S., Reis e Sousa, C., Brunner, S.M., Fichtner-Feigl, S., and Publica

Abstract

The purpose of this study was to elucidate whether DC NK lectin group receptor‐1 (DNGR‐1)‐dependent cross‐presentation of dead‐cell‐associated antigens occurs after transplantation and contributes to CD8+ T cell responses, chronic allograft rejection (CAR), and fibrosis. BALB/c or C57BL/6 hearts were heterotopically transplanted into WT, Clec9a−/−, or Batf3−/− recipient C57BL/6 mice. Allografts were analyzed for cell infiltration, CD8+ T cell activation, fibrogenesis, and CAR using immunohistochemistry, Western blot, qRT2‐PCR, and flow cytometry. Allografts displayed infiltration by recipient DNGR‐1+ DCs, signs of CAR, and fibrosis. Allografts in Clec9a−/− recipients showed reduced CAR (p < 0.0001), fibrosis (P = 0.0137), CD8+ cell infiltration (P < 0.0001), and effector cytokine levels compared to WT recipients. Batf3‐deficiency greatly reduced DNGR‐1+ DC‐infiltration, CAR (P < 0.0001), and fibrosis (P = 0.0382). CD8 cells infiltrating allografts of cytochrome C treated recipients, showed reduced production of CD8 effector cytokines (P < 0.05). Further, alloreactive CD8+ T cell response in indirect pathway IFN‐g ELISPOT was reduced in Clec9a−/− recipient mice (P = 0.0283). Blockade of DNGR‐1 by antibody, similar to genetic elimination of the receptor, reduced CAR (P = 0.0003), fibrosis (P = 0.0273), infiltration of CD8+ cells (p = 0.0006), and effector cytokine levels. DNGR‐1‐dependent alloantigen cross‐presentation by DNGR‐1+ DCs induces alloreactive CD8+ cells that induce CAR and fibrosis. Antibody against DNGR‐1 can block this process and prevent CAR and fibrosis.

Published
2020

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

Author

Cossarizza, A, Chang, H-D, Radbruch, A, Acs, A, Adam, D, Adam-Klages, S, Agace, WW, Aghaeepour, N, Akdis, M, Allez, M, Almeida, LN, Alvisi, G, Anderson, G, Andrae, I, Annunziato, F, Anselmo, A, Bacher, P, Baldari, CT, Bari, S, Barnaba, V, Barros-Martins, J, Battistini, L, Bauer, W, Baumgart, S, Baumgarth, N, Baumjohann, D, Baying, B, Bebawy, M, Becher, B, Beisker, W, Benes, V, Beyaert, R, Blanco, A, Boardman, DA, Bogdan, C, Borger, JG, Borsellino, G, Boulais, PE, Bradford, JA, Brenner, D, Brinkman, RR, Brooks, AES, Busch, DH, Buescher, M, Bushnell, TP, Calzetti, F, Cameron, G, Cammarata, I, Cao, X, Cardell, SL, Casola, S, Cassatella, MA, Cavani, A, Celada, A, Chatenoud, L, Chattopadhyay, PK, Chow, S, Christakou, E, Cicin-Sain, L, Clerici, M, Colombo, FS, Cook, L, Cooke, A, Cooper, AM, Corbett, AJ, Cosma, A, Cosmi, L, Coulie, PG, Cumano, A, Cvetkovic, L, Dang, VD, Dang-Heine, C, Davey, MS, Davies, D, De Biasi, S, Del Zotto, G, Dela Cruz, GV, Delacher, M, Della Bella, S, Dellabona, P, Deniz, G, Dessing, M, Di Santo, JP, Diefenbach, A, Dieli, F, Dolf, A, Doerner, T, Dress, RJ, Dudziak, D, Dustin, M, Dutertre, C-A, Ebner, F, Eckle, SBG, Edinger, M, Eede, P, Ehrhardt, GRA, Eich, M, Engel, P, Engelhardt, B, Erdei, A, Esser, C, Everts, B, Evrard, M, Falk, CS, Fehniger, TA, Felipo-Benavent, M, Ferry, H, Feuerer, M, Filby, A, Filkor, K, Fillatreau, S, Follo, M, Foerster, I, Foster, J, Foulds, GA, Frehse, B, Frenette, PS, Frischbutter, S, Fritzsche, W, Galbraith, DW, Gangaev, A, Garbi, N, Gaudilliere, B, Gazzinelli, RT, Geginat, J, Gerner, W, Gherardin, NA, Ghoreschi, K, Gibellini, L, Ginhoux, F, Goda, K, Godfrey, DI, Goettlinger, C, Gonzalez-Navajas, JM, Goodyear, CS, Gori, A, Grogan, JL, Grummitt, D, Gruetzkau, A, Haftmann, C, Hahn, J, Hammad, H, Haemmerling, G, Hansmann, L, Hansson, G, Harpur, CM, Hartmann, S, Hauser, A, Hauser, AE, Haviland, DL, Hedley, D, Hernandez, DC, Herrera, G, Herrmann, M, Hess, C, Hoefer, T, Hoffmann, P, Hogquist, K, Holland, T, Hollt, T, Holmdahl, R, Hombrink, P, Houston, JP, Hoyer, BF, Huang, B, Huang, F-P, Huber, JE, Huehn, J, Hundemer, M, Hunter, CA, Hwang, WYK, Iannone, A, Ingelfinger, F, Ivison, SM, Jaeck, H-M, Jani, PK, Javega, B, Jonjic, S, Kaiser, T, Kalina, T, Kamradt, T, Kaufmann, SHE, Keller, B, Ketelaars, SLC, Khalilnezhad, A, Khan, S, Kisielow, J, Klenerman, P, Knopf, J, Koay, H-F, Kobow, K, Kolls, JK, Kong, WT, Kopf, M, Korn, T, Kriegsmann, K, Kristyanto, H, Kroneis, T, Krueger, A, Kuehne, J, Kukat, C, Kunkel, D, Kunze-Schumacher, H, Kurosaki, T, Kurts, C, Kvistborg, P, Kwok, I, Landry, J, Lantz, O, Lanuti, P, LaRosa, F, Lehuen, A, LeibundGut-Landmann, S, Leipold, MD, Leung, LYT, Levings, MK, Lino, AC, Liotta, F, Litwin, V, Liu, Y, Ljunggren, H-G, Lohoff, M, Lombardi, G, Lopez, L, Lopez-Botet, M, Lovett-Racke, AE, Lubberts, E, Luche, H, Ludewig, B, Lugli, E, Lunemann, S, Maecker, HT, Maggi, L, Maguire, O, Mair, F, Mair, KH, Mantovani, A, Manz, RA, Marshall, AJ, Martinez-Romero, A, Martrus, G, Marventano, I, Maslinski, W, Matarese, G, Mattioli, AV, Maueroder, C, Mazzoni, A, McCluskey, J, McGrath, M, McGuire, HM, McInnes, IB, Mei, HE, Melchers, F, Melzer, S, Mielenz, D, Miller, SD, Mills, KHG, Minderman, H, Mjosberg, J, Moore, J, Moran, B, Moretta, L, Mosmann, TR, Mueller, S, Multhoff, G, Munoz, LE, Munz, C, Nakayama, T, Nasi, M, Neumann, K, Ng, LG, Niedobitek, A, Nourshargh, S, Nunez, G, O'Connor, J-E, Ochel, A, Oja, A, Ordonez, D, Orfao, A, Orlowski-Oliver, E, Ouyang, W, Oxenius, A, Palankar, R, Panse, I, Pattanapanyasat, K, Paulsen, M, Pavlinic, D, Penter, L, Peterson, P, Peth, C, Petriz, J, Piancone, F, Pickl, WF, Piconese, S, Pinti, M, Pockley, AG, Podolska, MJ, Poon, Z, Pracht, K, Prinz, I, Pucillo, CEM, Quataert, SA, Quatrini, L, Quinn, KM, Radbruch, H, Radstake, TRDJ, Rahmig, S, Rahn, H-P, Rajwa, B, Ravichandran, G, Raz, Y, Rebhahn, JA, Recktenwald, D, Reimer, D, Reis e Sousa, C, Remmerswaal, EBM, Richter, L, Rico, LG, Riddell, A, Rieger, AM, Robinson, JP, Romagnani, C, Rubartelli, A, Ruland, J, Saalmueller, A, Saeys, Y, Saito, T, Sakaguchi, S, Sala-de-Oyanguren, F, Samstag, Y, Sanderson, S, Sandrock, I, Santoni, A, Sanz, RB, Saresella, M, Sautes-Fridman, C, Sawitzki, B, Schadt, L, Scheffold, A, Scherer, HU, Schiemann, M, Schildberg, FA, Schimisky, E, Schlitzer, A, Schlosser, J, Schmid, S, Schmitt, S, Schober, K, Schraivogel, D, Schuh, W, Schueler, T, Schulte, R, Schulz, AR, Schulz, SR, Scotta, C, Scott-Algara, D, Sester, DP, Shankey, TV, Silva-Santos, B, Simon, AK, Sitnik, KM, Sozzani, S, Speiser, DE, Spidlen, J, Stahlberg, A, Stall, AM, Stanley, N, Stark, R, Stehle, C, Steinmetz, T, Stockinger, H, Takahama, Y, Takeda, K, Tan, L, Tarnok, A, Tiegs, G, Toldi, G, Tornack, J, Traggiai, E, Trebak, M, Tree, TIM, Trotter, J, Trowsdale, J, Tsoumakidou, M, Ulrich, H, Urbanczyk, S, van de Veen, W, van den Broek, M, van der Pol, E, Van Gassen, S, Van Isterdael, G, van Lier, RAW, Veldhoen, M, Vento-Asturias, S, Vieira, P, Voehringer, D, Volk, H-D, von Borstel, A, von Volkmann, K, Waisman, A, Walker, RV, Wallace, PK, Wang, SA, Wang, XM, Ward, MD, Ward-Hartstonge, KA, Warnatz, K, Warnes, G, Warth, S, Waskow, C, Watson, JV, Watzl, C, Wegener, L, Weisenburger, T, Wiedemann, A, Wienands, J, Wilharm, A, Wilkinson, RJ, Willimsky, G, Wing, JB, Winkelmann, R, Winkler, TH, Wirz, OF, Wong, A, Wurst, P, Yang, JHM, Yang, J, Yazdanbakhsh, M, Yu, L, Yue, A, Zhang, H, Zhao, Y, Ziegler, SM, Zielinski, C, Zimmermann, J, Zychlinsky, A, Cossarizza, A, Chang, H-D, Radbruch, A, Acs, A, Adam, D, Adam-Klages, S, Agace, WW, Aghaeepour, N, Akdis, M, Allez, M, Almeida, LN, Alvisi, G, Anderson, G, Andrae, I, Annunziato, F, Anselmo, A, Bacher, P, Baldari, CT, Bari, S, Barnaba, V, Barros-Martins, J, Battistini, L, Bauer, W, Baumgart, S, Baumgarth, N, Baumjohann, D, Baying, B, Bebawy, M, Becher, B, Beisker, W, Benes, V, Beyaert, R, Blanco, A, Boardman, DA, Bogdan, C, Borger, JG, Borsellino, G, Boulais, PE, Bradford, JA, Brenner, D, Brinkman, RR, Brooks, AES, Busch, DH, Buescher, M, Bushnell, TP, Calzetti, F, Cameron, G, Cammarata, I, Cao, X, Cardell, SL, Casola, S, Cassatella, MA, Cavani, A, Celada, A, Chatenoud, L, Chattopadhyay, PK, Chow, S, Christakou, E, Cicin-Sain, L, Clerici, M, Colombo, FS, Cook, L, Cooke, A, Cooper, AM, Corbett, AJ, Cosma, A, Cosmi, L, Coulie, PG, Cumano, A, Cvetkovic, L, Dang, VD, Dang-Heine, C, Davey, MS, Davies, D, De Biasi, S, Del Zotto, G, Dela Cruz, GV, Delacher, M, Della Bella, S, Dellabona, P, Deniz, G, Dessing, M, Di Santo, JP, Diefenbach, A, Dieli, F, Dolf, A, Doerner, T, Dress, RJ, Dudziak, D, Dustin, M, Dutertre, C-A, Ebner, F, Eckle, SBG, Edinger, M, Eede, P, Ehrhardt, GRA, Eich, M, Engel, P, Engelhardt, B, Erdei, A, Esser, C, Everts, B, Evrard, M, Falk, CS, Fehniger, TA, Felipo-Benavent, M, Ferry, H, Feuerer, M, Filby, A, Filkor, K, Fillatreau, S, Follo, M, Foerster, I, Foster, J, Foulds, GA, Frehse, B, Frenette, PS, Frischbutter, S, Fritzsche, W, Galbraith, DW, Gangaev, A, Garbi, N, Gaudilliere, B, Gazzinelli, RT, Geginat, J, Gerner, W, Gherardin, NA, Ghoreschi, K, Gibellini, L, Ginhoux, F, Goda, K, Godfrey, DI, Goettlinger, C, Gonzalez-Navajas, JM, Goodyear, CS, Gori, A, Grogan, JL, Grummitt, D, Gruetzkau, A, Haftmann, C, Hahn, J, Hammad, H, Haemmerling, G, Hansmann, L, Hansson, G, Harpur, CM, Hartmann, S, Hauser, A, Hauser, AE, Haviland, DL, Hedley, D, Hernandez, DC, Herrera, G, Herrmann, M, Hess, C, Hoefer, T, Hoffmann, P, Hogquist, K, Holland, T, Hollt, T, Holmdahl, R, Hombrink, P, Houston, JP, Hoyer, BF, Huang, B, Huang, F-P, Huber, JE, Huehn, J, Hundemer, M, Hunter, CA, Hwang, WYK, Iannone, A, Ingelfinger, F, Ivison, SM, Jaeck, H-M, Jani, PK, Javega, B, Jonjic, S, Kaiser, T, Kalina, T, Kamradt, T, Kaufmann, SHE, Keller, B, Ketelaars, SLC, Khalilnezhad, A, Khan, S, Kisielow, J, Klenerman, P, Knopf, J, Koay, H-F, Kobow, K, Kolls, JK, Kong, WT, Kopf, M, Korn, T, Kriegsmann, K, Kristyanto, H, Kroneis, T, Krueger, A, Kuehne, J, Kukat, C, Kunkel, D, Kunze-Schumacher, H, Kurosaki, T, Kurts, C, Kvistborg, P, Kwok, I, Landry, J, Lantz, O, Lanuti, P, LaRosa, F, Lehuen, A, LeibundGut-Landmann, S, Leipold, MD, Leung, LYT, Levings, MK, Lino, AC, Liotta, F, Litwin, V, Liu, Y, Ljunggren, H-G, Lohoff, M, Lombardi, G, Lopez, L, Lopez-Botet, M, Lovett-Racke, AE, Lubberts, E, Luche, H, Ludewig, B, Lugli, E, Lunemann, S, Maecker, HT, Maggi, L, Maguire, O, Mair, F, Mair, KH, Mantovani, A, Manz, RA, Marshall, AJ, Martinez-Romero, A, Martrus, G, Marventano, I, Maslinski, W, Matarese, G, Mattioli, AV, Maueroder, C, Mazzoni, A, McCluskey, J, McGrath, M, McGuire, HM, McInnes, IB, Mei, HE, Melchers, F, Melzer, S, Mielenz, D, Miller, SD, Mills, KHG, Minderman, H, Mjosberg, J, Moore, J, Moran, B, Moretta, L, Mosmann, TR, Mueller, S, Multhoff, G, Munoz, LE, Munz, C, Nakayama, T, Nasi, M, Neumann, K, Ng, LG, Niedobitek, A, Nourshargh, S, Nunez, G, O'Connor, J-E, Ochel, A, Oja, A, Ordonez, D, Orfao, A, Orlowski-Oliver, E, Ouyang, W, Oxenius, A, Palankar, R, Panse, I, Pattanapanyasat, K, Paulsen, M, Pavlinic, D, Penter, L, Peterson, P, Peth, C, Petriz, J, Piancone, F, Pickl, WF, Piconese, S, Pinti, M, Pockley, AG, Podolska, MJ, Poon, Z, Pracht, K, Prinz, I, Pucillo, CEM, Quataert, SA, Quatrini, L, Quinn, KM, Radbruch, H, Radstake, TRDJ, Rahmig, S, Rahn, H-P, Rajwa, B, Ravichandran, G, Raz, Y, Rebhahn, JA, Recktenwald, D, Reimer, D, Reis e Sousa, C, Remmerswaal, EBM, Richter, L, Rico, LG, Riddell, A, Rieger, AM, Robinson, JP, Romagnani, C, Rubartelli, A, Ruland, J, Saalmueller, A, Saeys, Y, Saito, T, Sakaguchi, S, Sala-de-Oyanguren, F, Samstag, Y, Sanderson, S, Sandrock, I, Santoni, A, Sanz, RB, Saresella, M, Sautes-Fridman, C, Sawitzki, B, Schadt, L, Scheffold, A, Scherer, HU, Schiemann, M, Schildberg, FA, Schimisky, E, Schlitzer, A, Schlosser, J, Schmid, S, Schmitt, S, Schober, K, Schraivogel, D, Schuh, W, Schueler, T, Schulte, R, Schulz, AR, Schulz, SR, Scotta, C, Scott-Algara, D, Sester, DP, Shankey, TV, Silva-Santos, B, Simon, AK, Sitnik, KM, Sozzani, S, Speiser, DE, Spidlen, J, Stahlberg, A, Stall, AM, Stanley, N, Stark, R, Stehle, C, Steinmetz, T, Stockinger, H, Takahama, Y, Takeda, K, Tan, L, Tarnok, A, Tiegs, G, Toldi, G, Tornack, J, Traggiai, E, Trebak, M, Tree, TIM, Trotter, J, Trowsdale, J, Tsoumakidou, M, Ulrich, H, Urbanczyk, S, van de Veen, W, van den Broek, M, van der Pol, E, Van Gassen, S, Van Isterdael, G, van Lier, RAW, Veldhoen, M, Vento-Asturias, S, Vieira, P, Voehringer, D, Volk, H-D, von Borstel, A, von Volkmann, K, Waisman, A, Walker, RV, Wallace, PK, Wang, SA, Wang, XM, Ward, MD, Ward-Hartstonge, KA, Warnatz, K, Warnes, G, Warth, S, Waskow, C, Watson, JV, Watzl, C, Wegener, L, Weisenburger, T, Wiedemann, A, Wienands, J, Wilharm, A, Wilkinson, RJ, Willimsky, G, Wing, JB, Winkelmann, R, Winkler, TH, Wirz, OF, Wong, A, Wurst, P, Yang, JHM, Yang, J, Yazdanbakhsh, M, Yu, L, Yue, A, Zhang, H, Zhao, Y, Ziegler, SM, Zielinski, C, Zimmermann, J, and Zychlinsky, A

Abstract

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Published
2019

12. Phagocytosis of antigens by Langerhans cells

Author

Reis e Sousa, C, Reis e Sousa, Caetano, and Austyn, J

Subjects
Phagocytosis, Langerhans cells, Dendritic cells, Antigens, Immune response
Abstract

Mature dendritic cells (DC) isolated from lymphoid tissues initiate antigen-specific T-dependent responses even though they are non-phagocytic and weakly pinocytic, whereas Langerhans cells (LC; immature DC) can process protein antigens but are poorly immunostimulatory. Thus antigens may be acquired by cells of this lineage at an immature stage but, to our knowledge, there have been no studies on the phagocytic capacity of these cells in vitro. Using a newly-developed flow cytometric assay to measure the association between fluorescent markers and LC in epidermal cell cultures, and light and electron microscopy, we have observed phagocytosis of a variety of particles by freshly-isolated LC. The cells readily phagocytosed zymosan, heat-killed S. cerevisiae, bacteria (S. aureus and C. parvum) and fluorescent latex beads, but were unable to take up IgG- or complement-coated sheep erythrocytes, as opposed to MØ. Similarly, many freshly-isolated splenic DC had some phagocytic activity. However, the capacity of both LC and splenic DC to phagocytose zymosan, bacteria and fluorescent latex beads was markedly decreased after maturation in culture, consistently with the fact that mature DC are poorly phagocytic. Zymosan binding and uptake were much greater in fresh LC from C57BL/6 compared to BALB/c mice, and the loss of phagocytic capacity for zymosan during maturation followed different kinetics in the two strains. Two receptors mediating uptake of zymosan in LC were identified based on the effect of different inhibitors. Both of these receptors, recognising mannose and β-glucan residues, appear to be differentially regulated in the two mouse strains and during culture of LC. Our findings support the notion that DC are capable of acquiring particulate antigens for presentation at an immature stage, through recognition units for carbohydrate determinants common to a variety of potentially pathogenic organisms.

Published
2016

15. Syk Signaling in Dendritic Cells Orchestrates Innate Resistance to Systemic Fungal Infection

Author

Gaffen, S, Whitney, PG, Baer, E, Osorio, F, Rogers, NC, Schraml, BU, Deddouche, S, LeibundGut-Landmann, S, Reis e Sousa, C, Gaffen, S, Whitney, PG, Baer, E, Osorio, F, Rogers, NC, Schraml, BU, Deddouche, S, LeibundGut-Landmann, S, and Reis e Sousa, C

Abstract

Host protection from fungal infection is thought to ensue in part from the activity of Syk-coupled C-type lectin receptors and MyD88-coupled toll-like receptors in myeloid cells, including neutrophils, macrophages and dendritic cells (DCs). Given the multitude of cell types and receptors involved, elimination of a single pathway for fungal recognition in a cell type such as DCs, primarily known for their ability to prime T cell responses, would be expected to have little effect on innate resistance to fungal infection. Here we report that this is surprisingly not the case and that selective loss of Syk but not MyD88 in DCs abrogates innate resistance to acute systemic Candida albicans infection in mice. We show that Syk expression by DCs is necessary for IL-23p19 production in response to C. albicans, which is essential to transiently induce GM-CSF secretion by NK cells that are recruited to the site of fungal replication. NK cell-derived-GM-CSF in turn sustains the anti-microbial activity of neutrophils, the main fungicidal effectors. Thus, the activity of a single kinase in a single myeloid cell type orchestrates a complex series of molecular and cellular events that underlies innate resistance to fungal sepsis.

Published
2014

17. The dendritic cell receptor DNGR-1 controls endocytic handling of necrotic cell antigens to favor cross-priming of CTLs in virus-infected mice

Author

Zelenay, S, Keller, AM, Whitney, PG, Schraml, BU, Deddouche, S, Rogers, NC, Schulz, O, Sancho, D, Reis e Sousa, C, Zelenay, S, Keller, AM, Whitney, PG, Schraml, BU, Deddouche, S, Rogers, NC, Schulz, O, Sancho, D, and Reis e Sousa, C

Abstract

DNGR-1 (CLEC9A) is a receptor for necrotic cells required by DCs to cross-prime CTLs against dead cell antigens in mice. It is currently unknown how DNGR-1 couples dead cell recognition to cross-priming. Here we found that DNGR-1 did not mediate DC activation by dead cells but rather diverted necrotic cell cargo into a recycling endosomal compartment, favoring cross-presentation to CD8(+) T cells. DNGR-1 regulated cross-priming in non-infectious settings such as immunization with antigen-bearing dead cells, as well as in highly immunogenic situations such as infection with herpes simplex virus type 1. Together, these results suggest that DNGR-1 is a dedicated receptor for cross-presentation of cell-associated antigens. Our work thus underscores the importance of cross-priming in immunity and indicates that antigenicity and adjuvanticity can be decoded by distinct innate immune receptors. The identification of specialized receptors that regulate antigenicity of virus-infected cells reveals determinants of antiviral immunity that might underlie the human response to infection and vaccination.

Published
2012

25. Lipoxin-mediated inhibition of IL-12 production by DCs: a mechanism for regulation of microbial immunity.

Author

Aliberti, J., Hieny, S., Reis e Sousa, C., Serhan, C. N., and Sher, A.

Subjects
LIPOXINS, INTERLEUKIN-12, DENDRITIC cells
Abstract

Lipoxins are eicosanoid mediators that show potent inhibitory effects on the acute inflammatory process. We show here that the induction of lipoxin A4 (LXA4) accompanied the in vivo suppression of interleukin 12 (IL-12) responsiveness of murine splenic dendritic cells (DCs) after microbial stimulation with an extract of Toxoplasma gondii. This paralysis of DC function could not be triggered in mice that were deficient in a key lipoxygenase involved in LXA4 biosynthesis. In addition, DCs pre-treated with LXA4 became refractory to microbial stimulation for IL-12 production in vitro and mice injected with a stable LXA4 analog showed reduced splenic DC mobilization and IL-12 responses in vivo. Together, these findings indicate that the induction of lipoxins in response to microbial stimulation can provide a potent mechanism for regulating DC function during the innate response to pathogens. [ABSTRACT FROM AUTHOR]

Published
2002
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27. Clec9a-Mediated Ablation of Conventional Dendritic Cells Suggests a Lymphoid Path to Generating Dendritic Cells In Vivo

Author

Salvermoser, J, Van Blijswijk, J, Papaioannou, NE, Rambichler, S, Pasztoi, M, Pakalniškytė, D, Rogers, NC, Keppler, SJ, Straub, T, Reis E Sousa, C, and Schraml, BU

Subjects
dendritic cell, Immunology, CLEC9A/DNGR-1, fate mapping, lymphopoiesis, myelopoiesis, DC depletion, development, Original Research
Abstract

Conventional dendritic cells (cDCs) are versatile activators of immune responses that develop as part of the myeloid lineage downstream of hematopoietic stem cells. We have recently shown that in mice precursors of cDCs, but not of other leukocytes, are marked by expression of DNGR-1/CLEC9A. To genetically deplete DNGR-1-expressing cDC precursors and their progeny, we crossed Clec9a-Cre mice to Rosa-lox-STOP-lox-diphtheria toxin (DTA) mice. These mice develop signs of age-dependent myeloproliferative disease, as has been observed in other DC-deficient mouse models. However, despite efficient depletion of cDC progenitors in these mice, cells with phenotypic characteristics of cDCs populate the spleen. These cells are functionally and transcriptionally similar to cDCs in wild type control mice but show somatic rearrangements of Ig-heavy chain genes, characteristic of lymphoid origin cells. Our studies reveal a previously unappreciated developmental heterogeneity of cDCs and suggest that the lymphoid lineage can generate cells with features of cDCs when myeloid cDC progenitors are impaired.

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28. Structural and biochemical characterisation of the C-type lectin receptor DNGR 1 and its binding to F-actin

Author

Hanc, P. and Reis e Sousa, C.

Subjects
616.99
Abstract

DNGR-1 is a C-type lectin receptor that has been implicated in the regulation of endocytic trafficking and cross-presentation of dead cell-associated antigens. Dendritic cells deficient in DNGR-1 are impaired in priming effector T-cell responses against cytopathic viruses and other dead cell-associated antigens. The ligand for DNGR-1 is the polymerized form of actin (F-actin) revealed in dead cells upon loss of membrane integrity. In this study we set out to determine biophysical, biochemical, and structural properties of DNGR-1 and its interaction with F-actin. First, we describe a conformational change that occurs in the neck region of the receptor in a pH- and ionic strength-dependent manner. Notably, the conformational change happens between conditions corresponding to the extracellular environment and the environment present in the vesicles of the endosomal pathway respectively, suggesting a possible role in the spatial regulation of the DNGR-1 function. Second, in collaboration with Keichii Namba and Takashi Fujii (RIKEN Quantitative Biology Center, Osaka, Japan) we used electron cryomicroscopy to solve the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Interestingly, DNGR-1 binds into the groove between actin protofilaments, making contacts with three actin subunits that are helically arranged in the F-actin structure. We identify the residues directly involved in the interaction, confirm their contribution to the binding and demonstrate the importance of avidity of the multivalent interaction between DNGR-1 and F-actin. Additionally, in collaboration with David Sancho and Salvador Iborra (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain) we formally demonstrate that ligand recognition is prerequisite for the biological function of DNGR-1 in dendritic cells. Third, by using heterodimeric DNGR-1 proteins in which one half of the dimer is incapable of binding to ligand, we demonstrate that DNGR-1 can bind with both ligand binding domains to a single actin filament, suggesting an exceptional flexibility of the neck region, and demonstrating an absence of rigid dimerization interface between the ligand-binding domains. In summary, we provide a comprehensive description of the structural and biophysical properties of DNGR-1, offering novel insights into its function and shedding light into innate immune mechanisms involved in recognition of cell death.

Published
2015

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

Author

Lara Gibellini, Sussan Nourshargh, Susanna Cardell, Wlodzimierz Maslinski, Mar Felipo-Benavent, Florian Mair, Hans-Martin Jäck, Lilly Lopez, Klaus Warnatz, John Trowsdale, Diana Ordonez, Marcus Eich, William Hwang, Anne Cooke, Dirk Mielenz, Alberto Orfao, Winfried F. Pickl, Vladimir Benes, Alice Yue, T. Vincent Shankey, Maria Tsoumakidou, Virginia Litwin, Gelo Victoriano Dela Cruz, Andrea Cavani, Sara De Biasi, Larissa Nogueira Almeida, Jonathan J M Landry, Claudia Haftmann, Charlotte Esser, Ana Cumano, Anneke Wilharm, Francesco Dieli, Rudi Beyaert, Alessio Mazzoni, Burkhard Ludewig, Carlo Pucillo, Dirk H. Busch, Joe Trotter, Stipan Jonjić, Marc Veldhoen, Josef Spidlen, Aja M. Rieger, Dieter Adam, Srijit Khan, Todd A. Fehniger, Giuseppe Matarese, Maximilien Evrard, Christian Maueröder, Steffen Schmitt, Kristin A. Hogquist, Barry Moran, Raghavendra Palankar, Markus Feuerer, S Schmid, Susann Rahmig, Amy E. Lovett-Racke, James V. Watson, Megan K. Levings, Susanne Melzer, Dinko Pavlinic, Christopher M. Harpur, Christina Stehle, A. Graham Pockley, Toshinori Nakayama, Attila Tárnok, Juhao Yang, Michael Lohoff, Paulo Vieira, Francisco Sala-de-Oyanguren, Christian Kurts, Anastasia Gangaev, Alfonso Blanco, Hans Scherer, Regine J. Dress, Bruno Silva-Santos, Kiyoshi Takeda, Bimba F. Hoyer, Ilenia Cammarata, Daryl Grummitt, Isabel Panse, Günnur Deniz, Bianka Baying, Friederike Ebner, Esther Schimisky, Leo Hansmann, Thomas Kamradt, Edwin van der Pol, Daniel Scott-Algara, Anna Iannone, Giorgia Alvisi, Sebastian R. Schulz, Francesco Liotta, Irmgard Förster, Beatriz Jávega, Hans-Peter Rahn, Caetano Reis e Sousa, Livius Penter, Xuetao Cao, David P. Sester, Keisuke Goda, Peter Wurst, Iain B. McInnes, Ricardo T. Gazzinelli, Federica Piancone, Gerald Willimsky, Yotam Raz, Pärt Peterson, Wolfgang Fritzsche, Yvonne Samstag, Martin Büscher, Thomas Schüler, Susanne Hartmann, Robert J. Wilkinson, Anna E. S. Brooks, Steven L. C. Ketelaars, Catherine Sautès-Fridman, Anna Rubartelli, Petra Bacher, Katja Kobow, Marco A. Cassatella, Andrea Hauser, Henrik E. Mei, Kilian Schober, Silvia Della Bella, Graham Anderson, Michael D. Ward, Garth Cameron, Sebastian Lunemann, Katharina Kriegsmann, Katarzyna M. Sitnik, Brice Gaudilliere, Chantip Dang-Heine, Marcello Pinti, Paul Klenerman, Frank A. Schildberg, Joana Barros-Martins, Laura G. Rico, Hanlin Zhang, Christian Münz, Thomas Dörner, Jakob Zimmermann, Andrea M. Cooper, Jonni S. Moore, Andreas Diefenbach, Yanling Liu, Wolfgang Bauer, Tobit Steinmetz, Katharina Pracht, Leonard Tan, Peter K. Jani, Alan M. Stall, Petra Hoffmann, Christine S. Falk, Jasmin Knopf, Simon Fillatreau, Hans-Dieter Volk, Luis E. Muñoz, David L. Haviland, William W. Agace, Jonathan Rebhahn, Ljiljana Cvetkovic, Mohamed Trebak, Jordi Petriz, Mario Clerici, Diether J. Recktenwald, Anders Ståhlberg, Tristan Holland, Helen M. McGuire, Sa A. Wang, Christian Kukat, Thomas Kroneis, Laura Cook, Wan Ting Kong, Xin M. Wang, Britta Engelhardt, Pierre Coulie, Genny Del Zotto, Sally A. Quataert, Kata Filkor, Gabriele Multhoff, Bartek Rajwa, Federica Calzetti, Hans Minderman, Cosima T. Baldari, Jens Geginat, Hervé Luche, Gert Van Isterdael, Linda Schadt, Sophia Urbanczyk, Giovanna Borsellino, Liping Yu, Dale I. Godfrey, Achille Anselmo, Rachael C. Walker, Andreas Grützkau, David W. Hedley, Birgit Sawitzki, Silvia Piconese, Maria Yazdanbakhsh, Burkhard Becher, Ramon Bellmas Sanz, Michael Delacher, Hyun-Dong Chang, Immanuel Andrä, Hans-Gustaf Ljunggren, José-Enrique O'Connor, Ahad Khalilnezhad, Sharon Sanderson, Federico Colombo, Götz R. A. Ehrhardt, Inga Sandrock, Enrico Lugli, Christian Bogdan, James B. Wing, Susann Müller, Tomohiro Kurosaki, Derek Davies, Ester B. M. Remmerswaal, Kylie M. Quinn, Christopher A. Hunter, Andreas Radbruch, Timothy P. Bushnell, Anna Erdei, Sabine Adam-Klages, Pascale Eede, Van Duc Dang, Rieke Winkelmann, Thomas Korn, Gemma A. Foulds, Dirk Baumjohann, Matthias Schiemann, Manfred Kopf, Jan Kisielow, Lisa Richter, Jochen Huehn, Gloria Martrus, Alexander Scheffold, Jessica G. Borger, Sidonia B G Eckle, John Bellamy Foster, Anna Katharina Simon, Alicia Wong, Mübeccel Akdis, Gisa Tiegs, Toralf Kaiser, James McCluskey, Anna Vittoria Mattioli, Aaron J. Marshall, Hui-Fern Koay, Eva Orlowski-Oliver, Anja E. Hauser, J. Paul Robinson, Jay K. Kolls, Luca Battistini, Mairi McGrath, Jane L. Grogan, Natalio Garbi, Timothy Tree, Kingston H. G. Mills, Stefan H. E. Kaufmann, Wolfgang Schuh, Ryan R. Brinkman, Tim R. Mosmann, Vincenzo Barnaba, Andreas Dolf, Lorenzo Cosmi, Bo Huang, Andreia C. Lino, Baerbel Keller, René A. W. van Lier, Alexandra J. Corbett, Paul S. Frenette, Pleun Hombrink, Helena Radbruch, Sofie Van Gassen, Olivier Lantz, Lorenzo Moretta, Désirée Kunkel, Kirsten A. Ward-Hartstonge, Armin Saalmüller, Leslie Y. T. Leung, Salvador Vento-Asturias, Paola Lanuti, Alicia Martínez-Romero, Sarah Warth, Zhiyong Poon, Diana Dudziak, Andrea Cossarizza, Kovit Pattanapanyasat, Konrad von Volkmann, Jessica P. Houston, Agnès Lehuen, Andrew Filby, Pratip K. Chattopadhyay, Stefano Casola, Annika Wiedemann, Hannes Stockinger, Jürgen Ruland, Arturo Zychlinsky, Claudia Waskow, Katrin Neumann, Ari Waisman, Lucienne Chatenoud, Sudipto Bari, Kamran Ghoreschi, David W. Galbraith, Yvan Saeys, Hamida Hammad, Andrea Gori, Miguel López-Botet, Gabriel Núñez, Sabine Ivison, Michael Hundemer, Dorothea Reimer, Mark C. Dessing, Günter J. Hämmerling, Rudolf A. Manz, Tomas Kalina, Jonas Hahn, Holden T. Maecker, Hendy Kristyanto, Martin S. Davey, Henning Ulrich, Michael L. Dustin, Takashi Saito, Yousuke Takahama, Milena Nasi, Johanna Huber, Jürgen Wienands, Paolo Dellabona, Andreas Schlitzer, Michael D. Leipold, Kerstin H. Mair, Christian Peth, Immo Prinz, Chiara Romagnani, José M. González-Navajas, Josephine Schlosser, Marina Saresella, Matthias Edinger, Dirk Brenner, Nicole Baumgarth, Rikard Holmdahl, Fang-Ping Huang, Guadalupe Herrera, Malte Paulsen, Gergely Toldi, Luka Cicin-Sain, Reiner Schulte, Christina E. Zielinski, Thomas Winkler, Christoph Goettlinger, Philip E. Boulais, Jennie H M Yang, Antonio Celada, Heike Kunze-Schumacher, Julia Tornack, Florian Ingelfinger, Jenny Mjösberg, Andy Riddell, Leonie Wegener, Thomas Höfer, Christoph Hess, James P. Di Santo, Anna E. Oja, J. Kühne, Willem van de Veen, Mary Bebawy, Alberto Mantovani, Bart Everts, Giovanna Lombardi, Laura Maggi, Anouk von Borstel, Pia Kvistborg, Elisabetta Traggiai, A Ochel, Nima Aghaeepour, Charles-Antoine Dutertre, Matthieu Allez, Thomas Höllt, Wenjun Ouyang, Regina Stark, Maries van den Broek, Shimon Sakaguchi, Paul K. Wallace, Silvano Sozzani, Francesca LaRosa, Annette Oxenius, Malgorzata J. Podolska, Ivana Marventano, Wilhelm Gerner, Oliver F. Wirz, Britta Frehse, Gevitha Ravichandran, Martin Herrmann, Carl S. Goodyear, Gary Warnes, Helen Ferry, Stefan Frischbutter, Tim R. Radstake, Salomé LeibundGut-Landmann, Yi Zhao, Axel Schulz, Angela Santoni, Pablo Engel, Daniela C. Hernández, Andreas Acs, Cristiano Scottà, Francesco Annunziato, Thomas Weisenburger, Wolfgang Beisker, Sue Chow, Fritz Melchers, Daniel E. Speiser, Immanuel Kwok, Florent Ginhoux, Dominic A. Boardman, Natalie Stanley, Carsten Watzl, Marie Follo, Erik Lubberts, Andreas Krueger, Susanne Ziegler, Göran K. Hansson, David Voehringer, Antonia Niedobitek, Eleni Christakou, Lai Guan Ng, Sabine Baumgart, Nicholas A Gherardin, Antonio Cosma, Orla Maguire, Jolene Bradford, Daniel Schraivogel, Linda Quatrini, Stephen D. Miller, Rheumatology, Università degli Studi di Modena e Reggio Emilia (UNIMORE), Deutsches Rheuma-ForschungsZentrum (DRFZ), Deutsches Rheuma-ForschungsZentrum, Swiss Institute of Allergy and Asthma Research (SIAF), Universität Zürich [Zürich] = University of Zurich (UZH), Institut de Recherche Saint-Louis - Hématologie Immunologie Oncologie (Département de recherche de l’UFR de médecine, ex- Institut Universitaire Hématologie-IUH) (IRSL), Université de Paris (UP), Ecotaxie, microenvironnement et développement lymphocytaire (EMily (UMR_S_1160 / U1160)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Department of Internal Medicine, Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI)-DENOTHE Center, Institute of Clinical Molecular Biology, Kiel University, Department of Life Sciences [Siena, Italy], Università degli Studi di Siena = University of Siena (UNISI), Institut Pasteur, Fondation Cenci Bolognetti - Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Réseau International des Instituts Pasteur (RIIP), Dulbecco Telethon Institute/Department of Biology, Caprotec Bioanalytics GmbH, International Occultation Timing Association European Section (IOTA ES), International Occultation Timing Association European Section, European Molecular Biology Laboratory [Heidelberg] (EMBL), VIB-UGent Center for Inflammation Research [Gand, Belgique] (IRC), VIB [Belgium], Fondazione Santa Lucia (IRCCS), Department of Immunology, Chinese Academy of Medical Sciences, FIRC Institute of Molecular Oncology Foundation, IFOM, Istituto FIRC di Oncologia Molecolare (IFOM), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Department of Physiopatology and Transplantation, University of Milan (DEPT), University of Milan, Monash University [Clayton], Institut des Maladies Emergentes et des Thérapies Innovantes (IMETI), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institute of Cellular Pathology, Université Catholique de Louvain = Catholic University of Louvain (UCL), Lymphopoïèse (Lymphopoïèse (UMR_1223 / U1223 / U-Pasteur_4)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Experimental Immunology Unit, Dept. of Oncology, DIBIT San Raffaele Scientific Institute, Immunité Innée - Innate Immunity, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Department of Biopharmacy [Bruxelles, Belgium] (Institute for Medical Immunology IMI), Université libre de Bruxelles (ULB), Charité Hospital, Humboldt-Universität zu Berlin, Agency for science, technology and research [Singapore] (A*STAR), Laboratory of Molecular Immunology and the Howard Hughes Institute, Rockefeller University [New York], Kennedy Institute of Rheumatology [Oxford, UK], Imperial College London, Theodor Kocher Institute, University of Bern, Leibniz Research Institute for Environmental Medicine [Düsseldorf, Germany] ( IUF), Université Lumière - Lyon 2 (UL2), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), University of Edinburgh, Integrative Biology Program [Milano], Istituto Nazionale Genetica Molecolare [Milano] (INGM), Singapore Immunology Network (SIgN), Biomedical Sciences Institute (BMSI), Universitat de Barcelona (UB), Rheumatologie, Cell Biology, Department of medicine [Stockholm], Karolinska Institutet [Stockholm]-Karolinska University Hospital [Stockholm], Department for Internal Medicine 3, Institute for Clinical Immunology, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Delft University of Technology (TU Delft), Medical Inflammation Research, Karolinska Institutet [Stockholm], Department of Photonics Engineering [Lyngby], Technical University of Denmark [Lyngby] (DTU), Dpt of Experimental Immunology [Braunschweig], Helmholtz Centre for Infection Research (HZI), Department of Internal Medicine V, Universität Heidelberg [Heidelberg], Department of Histology and Embryology, University of Rijeka, Freiburg University Medical Center, Nuffield Dept of Clinical Medicine, University of Oxford [Oxford]-NIHR Biomedical Research Centre, Institute of Integrative Biology, Molecular Biomedicine, Berlin Institute of Health (BIH), Laboratory for Lymphocyte Differentiation, RIKEN Research Center, Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Immunité et cancer (U932), Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Department of Surgery [Vancouver, BC, Canada] (Child and Family Research Institute), University of British Columbia (UBC)-Child and Family Research Institute [Vancouver, BC, Canada], College of Food Science and Technology [Shangai], Shanghai Ocean University, Institute for Medical Microbiology and Hygiene, University of Marburg, King‘s College London, Erasmus University Medical Center [Rotterdam] (Erasmus MC), Centre d'Immunophénomique (CIPHE), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Brustzentrum Kantonsspital St. Gallen, Immunotechnology Section, Vaccine Research Center, National Institutes of Health [Bethesda] (NIH)-National Institute of Allergy and Infectious Diseases, Heinrich Pette Institute [Hamburg], Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI), Department of Immunology and Cell Biology, Mario Negri Institute, Laboratory of Molecular Medicine and Biotechnology, Don C. Gnocchi ONLUS Foundation, Institute of Translational Medicine, Klinik für Dermatologie, Venerologie und Allergologie, School of Biochemistry and Immunology, Department of Medicine Huddinge, Karolinska Institutet [Stockholm]-Karolinska University Hospital [Stockholm]-Lipid Laboratory, Università di Genova, Dipartimento di Medicina Sperimentale, Department of Environmental Microbiology, Helmholtz Zentrum für Umweltforschung = Helmholtz Centre for Environmental Research (UFZ), Department of Radiation Oncology [Munich], Ludwig-Maximilians-Universität München (LMU), Centre de Recherche Publique- Santé, Université du Luxembourg (Uni.lu), William Harvey Research Institute, Barts and the London Medical School, University of Michigan [Ann Arbor], University of Michigan System, Centro de Investigacion del Cancer (CSIC), Universitario de Salamanca, Molecular Pathology [Tartu, Estonia], University of Tartu, Hannover Medical School [Hannover] (MHH), Centre d'Immunologie de Marseille - Luminy (CIML), Monash Biomedicine Discovery Institute, Cytometry Laboratories and School of Veterinary Medicine, Purdue University [West Lafayette], Data Mining and Modelling for Biomedicine [Ghent, Belgium], VIB Center for Inflammation Research [Ghent, Belgium], Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, RIKEN Research Center for Allergy and Immunology, Osaka University [Osaka], Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome], Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Institute of Medical Immunology [Berlin, Germany], FACS and Array Core Facility, Johannes Gutenberg - Universität Mainz (JGU), Otto-von-Guericke University [Magdeburg] (OVGU), SUPA School of Physics and Astronomy [University of St Andrews], University of St Andrews [Scotland]-Scottish Universities Physics Alliance (SUPA), Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), General Pathology and Immunology (GPI), University of Brescia, Université de Lausanne (UNIL), Terry Fox Laboratory, BC Cancer Agency (BCCRC)-British Columbia Cancer Agency Research Centre, Department of Molecular Immunology, Medizinische Universität Wien = Medical University of Vienna, Dept. Pediatric Cardiology, Universität Leipzig [Leipzig], Universitaetsklinikum Hamburg-Eppendorf = University Medical Center Hamburg-Eppendorf [Hamburg] (UKE), Center for Cardiovascular Sciences, Albany Medical College, Dept Pathol, Div Immunol, University of Cambridge [UK] (CAM), Department of Information Technology [Gent], Universiteit Gent, Department of Plant Systems Biology, Department of Plant Biotechnology and Genetics, Universiteit Gent = Ghent University [Belgium] (UGENT), Division of Molecular Immunology, Institute for Immunology, Department of Geological Sciences, University of Oregon [Eugene], Centers for Disease Control and Prevention [Atlanta] (CDC), Centers for Disease Control and Prevention, University of Colorado [Colorado Springs] (UCCS), FACS laboratory, Cancer Research, London, Cancer Research UK, Regeneration in Hematopoiesis and Animal Models of Hematopoiesis, Faculty of Medicine, Dresden University of Technology, Barbara Davis Center for Childhood Diabetes (BDC), University of Colorado Anschutz [Aurora], School of Computer and Electronic Information [Guangxi University], Guangxi University [Nanning], School of Materials Science and Engineering, Nanyang Technological University [Singapour], Max Planck Institute for Infection Biology (MPIIB), Max-Planck-Gesellschaft, Work in the laboratory of Dieter Adam is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 125440785 – SFB 877, Project B2.Petra Hoffmann, Andrea Hauser, and Matthias Edinger thank BD Biosciences®, San José, CA, USA, and SKAN AG, Bale, Switzerland for fruitful cooperation during the development, construction, and installation of the GMP‐compliant cell sorting equipment and the Bavarian Immune Therapy Network (BayImmuNet) for financial support.Edwin van der Pol and Paola Lanuti acknowledge Aleksandra Gąsecka M.D. for excellent experimental support and Dr. Rienk Nieuwland for textual suggestions. This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO‐TTW), research program VENI 15924.Jessica G Borger, Kylie M Quinn, Mairi McGrath, and Regina Stark thank Francesco Siracusa and Patrick Maschmeyer for providing data.Larissa Nogueira Almeida was supported by DFG research grant MA 2273/14‐1. Rudolf A. Manz was supported by the Excellence Cluster 'Inflammation at Interfaces' (EXC 306/2).Susanne Hartmann and Friederike Ebner were supported by the German Research Foundation (GRK 2046).Hans Minderman was supported by NIH R50CA211108.This work was funded by the Deutsche Forschungsgemeinschaft through the grant TRR130 (project P11 and C03) to Thomas H. Winkler.Ramon Bellmàs Sanz, Jenny Kühne, and Christine S. Falk thank Jana Keil and Kerstin Daemen for excellent technical support. The work was funded by the Germany Research Foundation CRC738/B3 (CSF).The work by the Mei laboratory was supported by German Research Foundation Grant ME 3644/5‐1 and TRR130 TP24, the German Rheumatism Research Centre Berlin, European Union Innovative Medicines Initiative ‐ Joint Undertaking ‐ RTCure Grant Agreement 777357, the Else Kröner‐Fresenius‐Foundation, German Federal Ministry of Education and Research e:Med sysINFLAME Program Grant 01ZX1306B and KMU‐innovativ 'InnoCyt', and the Leibniz Science Campus for Chronic Inflammation (http://www.chronische-entzuendung.org).Axel Ronald Schulz, Antonio Cosma, Sabine Baumgart, Brice Gaudilliere, Helen M. McGuire, and Henrik E. Mei thank Michael D. Leipold for critically reading the manuscript.Christian Kukat acknowledges support from the ISAC SRL Emerging Leaders program.John Trowsdale received funding from the European Research Council under the European Union's Horizon 2020 research and innovation program (Grant Agreement 695551)., European Project: 7728036(1978), Università degli Studi di Modena e Reggio Emilia = University of Modena and Reggio Emilia (UNIMORE), Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Università degli Studi di Firenze = University of Florence (UniFI)-DENOTHE Center, Università degli Studi di Milano = University of Milan (UNIMI), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Humboldt University Of Berlin, Leibniz Research Institute for Environmental Medicine [Düsseldorf, Germany] (IUF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Danmarks Tekniske Universitet = Technical University of Denmark (DTU), Universität Heidelberg [Heidelberg] = Heidelberg University, Universitäts Klinikum Freiburg = University Medical Center Freiburg (Uniklinik), University of Oxford-NIHR Biomedical Research Centre, Universität Bonn = University of Bonn, Università degli Studi di Firenze = University of Florence (UniFI), Università degli studi di Genova = University of Genoa (UniGe), Universidad de Salamanca, Università degli Studi di Roma 'La Sapienza' = Sapienza University [Rome] (UNIROMA), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Otto-von-Guericke-Universität Magdeburg = Otto-von-Guericke University [Magdeburg] (OVGU), Université de Lausanne = University of Lausanne (UNIL), Universität Leipzig, Universiteit Gent = Ghent University (UGENT), HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany., Cossarizza, A., Chang, H. -D., Radbruch, A., Acs, A., Adam, D., Adam-Klages, S., Agace, W. W., Aghaeepour, N., Akdis, M., Allez, M., Almeida, L. N., Alvisi, G., Anderson, G., Andra, I., Annunziato, F., Anselmo, A., Bacher, P., Baldari, C. T., Bari, S., Barnaba, V., Barros-Martins, J., Battistini, L., Bauer, W., Baumgart, S., Baumgarth, N., Baumjohann, D., Baying, B., Bebawy, M., Becher, B., Beisker, W., Benes, V., Beyaert, R., Blanco, A., Boardman, D. A., Bogdan, C., Borger, J. G., Borsellino, G., Boulais, P. E., Bradford, J. A., Brenner, D., Brinkman, R. R., Brooks, A. E. S., Busch, D. H., Buscher, M., Bushnell, T. P., Calzetti, F., Cameron, G., Cammarata, I., Cao, X., Cardell, S. L., Casola, S., Cassatella, M. A., Cavani, A., Celada, A., Chatenoud, L., Chattopadhyay, P. K., Chow, S., Christakou, E., Cicin-Sain, L., Clerici, M., Colombo, F. S., Cook, L., Cooke, A., Cooper, A. M., Corbett, A. J., Cosma, A., Cosmi, L., Coulie, P. G., Cumano, A., Cvetkovic, L., Dang, V. D., Dang-Heine, C., Davey, M. S., Davies, D., De Biasi, S., Del Zotto, G., Dela Cruz, G. V., Delacher, M., Della Bella, S., Dellabona, P., Deniz, G., Dessing, M., Di Santo, J. P., Diefenbach, A., Dieli, F., Dolf, A., Dorner, T., Dress, R. J., Dudziak, D., Dustin, M., Dutertre, C. -A., Ebner, F., Eckle, S. B. G., Edinger, M., Eede, P., Ehrhardt, G. R. A., Eich, M., Engel, P., Engelhardt, B., Erdei, A., Esser, C., Everts, B., Evrard, M., Falk, C. S., Fehniger, T. A., Felipo-Benavent, M., Ferry, H., Feuerer, M., Filby, A., Filkor, K., Fillatreau, S., Follo, M., Forster, I., Foster, J., Foulds, G. A., Frehse, B., Frenette, P. S., Frischbutter, S., Fritzsche, W., Galbraith, D. W., Gangaev, A., Garbi, N., Gaudilliere, B., Gazzinelli, R. T., Geginat, J., Gerner, W., Gherardin, N. A., Ghoreschi, K., Gibellini, L., Ginhoux, F., Goda, K., Godfrey, D. I., Goettlinger, C., Gonzalez-Navajas, J. M., Goodyear, C. S., Gori, A., Grogan, J. L., Grummitt, D., Grutzkau, A., Haftmann, C., Hahn, J., Hammad, H., Hammerling, G., Hansmann, L., Hansson, G., Harpur, C. M., Hartmann, S., Hauser, A., Hauser, A. E., Haviland, D. L., Hedley, D., Hernandez, D. C., Herrera, G., Herrmann, M., Hess, C., Hofer, T., Hoffmann, P., Hogquist, K., Holland, T., Hollt, T., Holmdahl, R., Hombrink, P., Houston, J. P., Hoyer, B. F., Huang, B., Huang, F. -P., Huber, J. E., Huehn, J., Hundemer, M., Hunter, C. A., Hwang, W. Y. K., Iannone, A., Ingelfinger, F., Ivison, S. M., Jack, H. -M., Jani, P. K., Javega, B., Jonjic, S., Kaiser, T., Kalina, T., Kamradt, T., Kaufmann, S. H. E., Keller, B., Ketelaars, S. L. C., Khalilnezhad, A., Khan, S., Kisielow, J., Klenerman, P., Knopf, J., Koay, H. -F., Kobow, K., Kolls, J. K., Kong, W. T., Kopf, M., Korn, T., Kriegsmann, K., Kristyanto, H., Kroneis, T., Krueger, A., Kuhne, J., Kukat, C., Kunkel, D., Kunze-Schumacher, H., Kurosaki, T., Kurts, C., Kvistborg, P., Kwok, I., Landry, J., Lantz, O., Lanuti, P., Larosa, F., Lehuen, A., LeibundGut-Landmann, S., Leipold, M. D., Leung, L. Y. T., Levings, M. K., Lino, A. C., Liotta, F., Litwin, V., Liu, Y., Ljunggren, H. -G., Lohoff, M., Lombardi, G., Lopez, L., Lopez-Botet, M., Lovett-Racke, A. E., Lubberts, E., Luche, H., Ludewig, B., Lugli, E., Lunemann, S., Maecker, H. T., Maggi, L., Maguire, O., Mair, F., Mair, K. H., Mantovani, A., Manz, R. A., Marshall, A. J., Martinez-Romero, A., Martrus, G., Marventano, I., Maslinski, W., Matarese, G., Mattioli, A. V., Maueroder, C., Mazzoni, A., Mccluskey, J., Mcgrath, M., Mcguire, H. M., Mcinnes, I. B., Mei, H. E., Melchers, F., Melzer, S., Mielenz, D., Miller, S. D., Mills, K. H. G., Minderman, H., Mjosberg, J., Moore, J., Moran, B., Moretta, L., Mosmann, T. R., Muller, S., Multhoff, G., Munoz, L. E., Munz, C., Nakayama, T., Nasi, M., Neumann, K., Ng, L. G., Niedobitek, A., Nourshargh, S., Nunez, G., O'Connor, J. -E., Ochel, A., Oja, A., Ordonez, D., Orfao, A., Orlowski-Oliver, E., Ouyang, W., Oxenius, A., Palankar, R., Panse, I., Pattanapanyasat, K., Paulsen, M., Pavlinic, D., Penter, L., Peterson, P., Peth, C., Petriz, J., Piancone, F., Pickl, W. F., Piconese, S., Pinti, M., Pockley, A. G., Podolska, M. J., Poon, Z., Pracht, K., Prinz, I., Pucillo, C. E. M., Quataert, S. A., Quatrini, L., Quinn, K. M., Radbruch, H., Radstake, T. R. D. J., Rahmig, S., Rahn, H. -P., Rajwa, B., Ravichandran, G., Raz, Y., Rebhahn, J. A., Recktenwald, D., Reimer, D., Reis e Sousa, C., Remmerswaal, E. B. M., Richter, L., Rico, L. G., Riddell, A., Rieger, A. M., Robinson, J. P., Romagnani, C., Rubartelli, A., Ruland, J., Saalmuller, A., Saeys, Y., Saito, T., Sakaguchi, S., Sala-de-Oyanguren, F., Samstag, Y., Sanderson, S., Sandrock, I., Santoni, A., Sanz, R. B., Saresella, M., Sautes-Fridman, C., Sawitzki, B., Schadt, L., Scheffold, A., Scherer, H. U., Schiemann, M., Schildberg, F. A., Schimisky, E., Schlitzer, A., Schlosser, J., Schmid, S., Schmitt, S., Schober, K., Schraivogel, D., Schuh, W., Schuler, T., Schulte, R., Schulz, A. R., Schulz, S. R., Scotta, C., Scott-Algara, D., Sester, D. P., Shankey, T. V., Silva-Santos, B., Simon, A. K., Sitnik, K. M., Sozzani, S., Speiser, D. E., Spidlen, J., Stahlberg, A., Stall, A. M., Stanley, N., Stark, R., Stehle, C., Steinmetz, T., Stockinger, H., Takahama, Y., Takeda, K., Tan, L., Tarnok, A., Tiegs, G., Toldi, G., Tornack, J., Traggiai, E., Trebak, M., Tree, T. I. M., Trotter, J., Trowsdale, J., Tsoumakidou, M., Ulrich, H., Urbanczyk, S., van de Veen, W., van den Broek, M., van der Pol, E., Van Gassen, S., Van Isterdael, G., van Lier, R. A. W., Veldhoen, M., Vento-Asturias, S., Vieira, P., Voehringer, D., Volk, H. -D., von Borstel, A., von Volkmann, K., Waisman, A., Walker, R. V., Wallace, P. K., Wang, S. A., Wang, X. M., Ward, M. D., Ward-Hartstonge, K. A., Warnatz, K., Warnes, G., Warth, S., Waskow, C., Watson, J. V., Watzl, C., Wegener, L., Weisenburger, T., Wiedemann, A., Wienands, J., Wilharm, A., Wilkinson, R. J., Willimsky, G., Wing, J. B., Winkelmann, R., Winkler, T. H., Wirz, O. F., Wong, A., Wurst, P., Yang, J. H. M., Yang, J., Yazdanbakhsh, M., Yu, L., Yue, A., Zhang, H., Zhao, Y., Ziegler, S. M., Zielinski, C., Zimmermann, J., Zychlinsky, A., UCL - SSS/DDUV - Institut de Duve, UCL - SSS/DDUV/GECE - Génétique cellulaire, Netherlands Organization for Scientific Research, German Research Foundation, European Commission, European Research Council, Repositório da Universidade de Lisboa, CCA - Imaging and biomarkers, Experimental Immunology, AII - Infectious diseases, AII - Inflammatory diseases, Biomedical Engineering and Physics, ACS - Atherosclerosis & ischemic syndromes, and Landsteiner Laboratory

Subjects
0301 basic medicine, Consensus, Immunology, Consensu, Cell Separation, Biology, Article, Flow cytometry, 03 medical and health sciences, 0302 clinical medicine, Guidelines, Allergy and Immunology, medicine, Cell separation, Immunology and Allergy, Humans, guidelines, flow cytometry, immunology, medicine.diagnostic_test, BIOMEDICINE AND HEALTHCARE. Basic Medical Sciences, Cell sorting, Flow Cytometry, Cell selection, Data science, 3. Good health, 030104 developmental biology, Phenotype, [SDV.IMM]Life Sciences [q-bio]/Immunology, BIOMEDICINA I ZDRAVSTVO. Temeljne medicinske znanosti, 030215 immunology, Human
Abstract

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

Published
2019

30. Vitamin D regulates microbiome-dependent cancer immunity.

Author

Giampazolias E, Pereira da Costa M, Lam KC, Lim KHJ, Cardoso A, Piot C, Chakravarty P, Blasche S, Patel S, Biram A, Castro-Dopico T, Buck MD, Rodrigues RR, Poulsen GJ, Palma-Duran SA, Rogers NC, Koufaki MA, Minutti CM, Wang P, Vdovin A, Frederico B, Childs E, Lee S, Simpson B, Iseppon A, Omenetti S, Kelly G, Goldstone R, Nye E, Suárez-Bonnet A, Priestnall SL, MacRae JI, Zelenay S, Patil KR, Litchfield K, Lee JC, Jess T, Goldszmid RS, and Reis E Sousa C

Subjects
Animals, Female, Humans, Male, Mice, Immunotherapy, Intestinal Mucosa immunology, Intestinal Mucosa microbiology, Intestinal Mucosa metabolism, Mice, Inbred C57BL, Diet, Cell Line, Tumor, Calcifediol administration & dosage, Calcifediol metabolism, Vitamin D-Binding Protein genetics, Vitamin D-Binding Protein metabolism, Bacteroides fragilis metabolism, Gastrointestinal Microbiome drug effects, Immune Checkpoint Inhibitors therapeutic use, Immune Checkpoint Inhibitors pharmacology, Neoplasms immunology, Neoplasms microbiology, Neoplasms therapy, Vitamin D administration & dosage, Vitamin D metabolism
Abstract

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis , which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Published
2024
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31. Myeloid C-type lectin receptors in innate immune recognition.

Author

Reis E Sousa C, Yamasaki S, and Brown GD

Subjects
Humans, Immunity, Innate, Myeloid Cells metabolism, Signal Transduction, Receptors, Pattern Recognition metabolism, Lectins, C-Type metabolism, Neoplasms metabolism
Abstract

C-type lectin receptors (CLRs) expressed by myeloid cells constitute a versatile family of receptors that play a key role in innate immune recognition. Myeloid CLRs exhibit a remarkable ability to recognize an extensive array of ligands, from carbohydrates and beyond, and encompass pattern-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and markers of altered self. These receptors, classified into distinct subgroups, play pivotal roles in immune recognition and modulation of immune responses. Their intricate signaling pathways orchestrate a spectrum of cellular responses, influencing processes such as phagocytosis, cytokine production, and antigen presentation. Beyond their contributions to host defense in viral, bacterial, fungal, and parasitic infections, myeloid CLRs have been implicated in non-infectious diseases such as cancer, allergies, and autoimmunity. A nuanced understanding of myeloid CLR interactions with endogenous and microbial triggers is starting to uncover the context-dependent nature of their roles in innate immunity, with implications for therapeutic intervention., Competing Interests: Declaration of interests C.R.e.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, and Bicycle Therapeutics, all unrelated to this work. C.R.e.S. also has an additional appointment as Visiting Professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London. G.D.B. holds an honorary professorship at the University of Cape Town., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2024
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32. Distinct ontogenetic lineages dictate cDC2 heterogeneity.

Author

Minutti CM, Piot C, Pereira da Costa M, Chakravarty P, Rogers N, Huerga Encabo H, Cardoso A, Loong J, Bessou G, Mionnet C, Langhorne J, Bonnet D, Dalod M, Tomasello E, and Reis E Sousa C

Subjects
Animals, Mice, Cell Differentiation, Dendritic Cells, Sialic Acid Binding Immunoglobulin-like Lectins
Abstract

Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet + cDC2As and T-bet - cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H + pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H - CD8α + pre-cDC2As in tissues, which differentiated into T-bet + cDC2As. In contrast, a Siglec-H - fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet - cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment., (© 2024. The Author(s).)

Published
2024
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33. SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells.

Author

Henry CM, Castellanos CA, Buck MD, Giampazolias E, Frederico B, Cardoso A, Rogers NC, Schulz O, Lee S, Canton J, Faull P, Snijders AP, Mohapatra B, Band H, and Reis E Sousa C

Subjects
CD8-Positive T-Lymphocytes, Proto-Oncogene Proteins c-cbl, Ubiquitination, Dendritic Cells, Syk Kinase, Cross-Priming, Ubiquitin-Protein Ligases
Abstract

Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8 + T cells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8 + T cells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity., Competing Interests: Declaration of interests C.R.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, and Bicycle Therapeutics, all unrelated to this work. C.R.S. also has an additional appointment as Visiting Professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London. H.B. received a research grant from Nimbus Therapeutics for unrelated work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2023
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34. Interplay between CXCR4 and CCR2 regulates bone marrow exit of dendritic cell progenitors.

Author

Pereira da Costa M, Minutti CM, Piot C, Giampazolias E, Cardoso A, Cabeza-Cabrerizo M, Rogers NC, Lebrusant-Fernandez M, Iliakis CS, Wack A, and Reis E Sousa C

Subjects
Bone Marrow Cells metabolism, Inflammation metabolism, Signal Transduction, Animals, Bone Marrow metabolism, Dendritic Cells metabolism, Receptors, CCR2 metabolism, Receptors, CXCR4 metabolism
Abstract

Conventional dendritic cells (cDCs) are found in most tissues and play a key role in initiation of immunity. cDCs require constant replenishment from progenitors called pre-cDCs that develop in the bone marrow (BM) and enter the blood circulation to seed all tissues. This process can be markedly accelerated in response to inflammation (emergency cDCpoiesis). Here, we identify two populations of BM pre-cDC marked by differential expression of CXCR4. We show that CXCR4 lo cells constitute the migratory pool of BM pre-cDCs, which exits the BM and can be rapidly mobilized during challenge. We further show that exit of CXCR4 lo pre-cDCs from BM at steady state is partially dependent on CCR2 and that CCR2 upregulation in response to type I IFN receptor signaling markedly increases efflux during infection with influenza A virus. Our results highlight a fine balance between retention and efflux chemokine cues that regulates steady-state and emergency cDCpoiesis., Competing Interests: Declaration of interests C.R.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, and Bicycle Therapeutics, all unrelated to this work. C.R.S. also has an additional appointment as a visiting professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2023
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35. DNGR-1-mediated cross-presentation of dead cell-associated antigens.

Author

Henry CM, Castellanos CA, and Reis e Sousa C

Subjects
Humans, CD8-Positive T-Lymphocytes, Receptors, Immunologic, Antigens metabolism, Dendritic Cells, Cross-Priming, Neoplasms metabolism
Abstract

Conventional dendritic cells type 1 (cDC1) are critical for inducing protective CD8 + T cell responses to tumour and viral antigens. In many instances, cDC1 access those antigens in the form of material internalised from dying tumour or virally-infected cells. How cDC1 extract dead cell-associated antigens and cross-present them in the form of peptides bound to MHC class I molecules to CD8 + T cells remains unclear. Here we review the biology of dendritic cell natural killer group receptor-1 (DNGR-1; also known as CLEC9A), a C-type lectin receptor highly expressed on cDC1 that plays a key role in this process. We highlight recent advances that support a function for DNGR-1 signalling in promoting inducible rupture of phagocytic or endocytic compartments containing dead cell debris, thereby making dead cell-associated antigens accessible to the endogenous MHC class I processing and presentation machinery of cDC1. We further review how DNGR-1 detects dead cells, as well as the functions of the receptor in anti-viral and anti-tumour immunity. Finally, we highlight how the study of DNGR-1 has opened new perspectives into cross-presentation, some of which may have applications in immunotherapy of cancer and vaccination against viral diseases., Competing Interests: Declaration of Competing Interest C.R.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, Bicycle Therapeutics and Sosei Heptares. C.R.S. has an additional appointment as a Visiting Professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Published
2023
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36. Loss of secreted gelsolin enhances response to anticancer therapies.

Author

Lim KHJ, Giampazolias E, Schulz O, Rogers NC, Wilkins A, Sahai E, Strid J, and Reis e Sousa C

Subjects
Actins metabolism, Animals, Antigens, Neoplasm metabolism, CD8-Positive T-Lymphocytes, Doxorubicin metabolism, Homeodomain Proteins, Lectins, C-Type, Mice, Ovalbumin, Proto-Oncogene Proteins B-raf metabolism, Receptors, Immunologic metabolism, Gelsolin genetics, Gelsolin metabolism, Melanoma, Experimental
Abstract

Type 1 conventional dendritic cells (cDC1) play a critical role in priming anticancer cytotoxic CD8 + T cells. DNGR-1 (a.k.a. CLEC9A) is a cDC1 receptor that binds to F-actin exposed on necrotic cancer and normal cells. DNGR-1 signaling enhances cross-presentation of dead-cell associated antigens, including tumor antigens. We have recently shown that secreted gelsolin (sGSN), a plasma protein, competes with DNGR-1 for binding to dead cell-exposed F-actin and dampens anticancer immunity. Here, we investigated the effects of loss of sGSN on various anticancer therapies that are thought to induce cell death and provoke an immune response to cancer. We compared WT (wildtype) with Rag1 -/- , Batf3 -/- , Clec9a gfp/gfp , sGsn -/- or sGsn -/- Clec9a gfp/gfp mice implanted with transplantable tumor cell lines, including MCA-205 fibrosarcoma, 5555 Braf V600E melanoma and B16-F10 LifeAct (LA)-ovalbumin (OVA)-mCherry melanoma. Tumor-bearing mice were treated with (1) doxorubicin (intratumoral) chemotherapy for MCA-205, (2) BRAF-inhibitor PLX4720 (oral gavage) targeted therapy for 5555 Braf V600E , and (3) X-ray radiotherapy for B16 LA-OVA-mCherry. We confirmed that efficient tumor control following each therapy requires an immunocompetent host as efficacy was markedly reduced in Rag1 -/- compared with WT mice. Notably, across all the therapeutic modalities, loss of sGSN significantly enhanced tumor control compared with treated WT controls. This was an on-target effect as mice deficient in both sGSN and DNGR-1 behaved no differently from WT mice following therapy. In sum, we find that mice deficient in sGsn display enhanced DNGR-1-dependent responsiveness to chemotherapy, targeted therapy and radiotherapy. Our findings are consistent with the notion some cancer therapies induce immunogenic cell death (ICD), which mobilizes anticancer T cells. Our results point to cDC1 and DNGR-1 as decoders of ICD and to sGSN as a negative regulator of such decoding, highlighting sGSN as a possible target in cancer treatment. Further prospective studies are warranted to identify patients who may benefit most from inhibition of sGSN function., Competing Interests: Competing interests: KHJL, EG and CReS are named as contributors/inventors on a patent application on the use of sGSN for immunotherapies. CReS owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, Bicycle Therapeutics and Sosei Heptares. CReS holds a visiting professorship at Imperial College London and honorary professorships at University College London and King’s College London., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Published
2022
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37. DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential.

Author

Frederico B, Martins I, Chapela D, Gasparrini F, Chakravarty P, Ackels T, Piot C, Almeida B, Carvalho J, Ciccarelli A, Peddie CJ, Rogers N, Briscoe J, Guillemot F, Schaefer AT, Saúde L, and Reis e Sousa C

Subjects
Animals, Cell Differentiation, Ependyma, Mammals, Mice, Neuroglia, Spinal Cord, Neural Stem Cells
Abstract

Cells with latent stem ability can contribute to mammalian tissue regeneration after damage. Whether the central nervous system (CNS) harbors such cells remains controversial. Here, we report that DNGR-1 lineage tracing in mice identifies an ependymal cell subset, wherein resides latent regenerative potential. We demonstrate that DNGR-1-lineage-traced ependymal cells arise early in embryogenesis (E11.5) and subsequently spread across the lining of cerebrospinal fluid (CSF)-filled compartments to form a contiguous sheet from the brain to the end of the spinal cord. In the steady state, these DNGR-1-traced cells are quiescent, committed to their ependymal cell fate, and do not contribute to neuronal or glial lineages. However, trans-differentiation can be induced in adult mice by CNS injury or in vitro by culture with suitable factors. Our findings highlight previously unappreciated ependymal cell heterogeneity and identify across the entire CNS an ependymal cell subset wherein resides damage-responsive neural stem cell potential., Competing Interests: Declaration of interests C.R.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, Bicycle Therapeutics, Genor Biopharma, and Sosei Heptares, all unrelated to this work. C.R.S. also has an additional appointment as Visiting Professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2022
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38. RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency.

Author

Stok JE, Oosenbrug T, Ter Haar LR, Gravekamp D, Bromley CP, Zelenay S, Reis e Sousa C, and van der Veen AG

Subjects
Humans, RNA Editing, RNA, Double-Stranded, Autoimmune Diseases of the Nervous System genetics, Nervous System Malformations genetics, RNA Helicases metabolism
Abstract

RNA editing by the adenosine deaminase ADAR1 prevents innate immune responses to endogenous RNAs. In ADAR1-deficient cells, unedited self RNAs form base-paired structures that resemble viral RNAs and inadvertently activate the cytosolic RIG-I-like receptor (RLR) MDA5, leading to an antiviral type I interferon (IFN) response. Mutations in ADAR1 cause Aicardi-Goutières Syndrome (AGS), an autoinflammatory syndrome characterized by chronic type I IFN production. Conversely, ADAR1 loss and the consequent type I IFN production restricts tumor growth and potentiates the activity of some chemotherapeutics. Here, we show that another RIG-I-like receptor, LGP2, also has an essential role in the induction of a type I IFN response in ADAR1-deficient human cells. This requires the canonical function of LGP2 as an RNA sensor and facilitator of MDA5-dependent signaling. Furthermore, we show that the sensitivity of tumor cells to ADAR1 loss requires LGP2 expression. Finally, type I IFN induction in tumor cells depleted of ADAR1 and treated with some chemotherapeutics fully depends on LGP2 expression. These findings highlight a central role for LGP2 in self RNA sensing with important clinical implications., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Published
2022
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39. Epithelial colonization by gut dendritic cells promotes their functional diversification.

Author

Rivera CA, Randrian V, Richer W, Gerber-Ferder Y, Delgado MG, Chikina AS, Frede A, Sorini C, Maurin M, Kammoun-Chaari H, Parigi SM, Goudot C, Cabeza-Cabrerizo M, Baulande S, Lameiras S, Guermonprez P, Reis e Sousa C, Lecuit M, Moreau HD, Helft J, Vignjevic DM, Villablanca EJ, and Lennon-Duménil AM

Subjects
Actomyosin metabolism, Animals, Antigen Presentation, Antigens, CD metabolism, CD11b Antigen metabolism, Cell Differentiation, Cell Movement, Cells, Cultured, Immune Tolerance, Integrin alpha Chains metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Mucin-2 immunology, Tretinoin metabolism, Dendritic Cells immunology, Inflammation immunology, Intestinal Mucosa pathology, T-Lymphocytes immunology
Abstract

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103 + CD11b + cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification., Competing Interests: Declaration of interests C.R.S. has an additional appointment as professor in the Faculty of Medicine at Imperial College London. C.R.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, Oncurious NV, Bicycle Therapeutics, and Sosei Heptares, all unrelated to this work., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2022
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40. From mRNA sensing to vaccines.

Author

Fauci AS, Merad M, Swaminathan S, Hur S, Topol E, Fitzgerald K, Reis e Sousa C, Corbett KS, and Bauer S

Subjects
Animals, History, 21st Century, Humans, RNA, Messenger immunology, World Health Organization, COVID-19 immunology, COVID-19 Vaccines immunology, SARS-CoV-2 physiology, Vaccine Development history, mRNA Vaccines immunology
Abstract

The 2005 Immunity paper by Karikó et al. has been hailed as a cornerstone insight that directly led to the design and delivery of the mRNA vaccines against COVID-19. We asked experts in pathogen sensing, vaccine development, and public health to provide their perspective on the study and its implications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Published
2021
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41. Recruitment of dendritic cell progenitors to foci of influenza A virus infection sustains immunity.

Author

Cabeza-Cabrerizo M, Minutti CM, da Costa MP, Cardoso A, Jenkins RP, Kulikauskaite J, Buck MD, Piot C, Rogers N, Crotta S, Whittaker L, Encabo HH, McCauley JW, Allen JE, Pasparakis M, Wack A, Sahai E, and Reis e Sousa C

Subjects
Animals, Dendritic Cells immunology, Female, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Influenza A virus immunology, Orthomyxoviridae Infections immunology
Abstract

Protection from infection with respiratory viruses such as influenza A virus (IAV) requires T cell–mediated immune responses initiated by conventional dendritic cells (cDCs) that reside in the respiratory tract. Here, we show that effective induction of T cell responses against IAV in mice requires reinforcement of the resident lung cDC network by cDC progenitors. We found that CCR2-binding chemokines produced during IAV infection recruit pre-cDCs from blood and direct them to foci of infection, increasing the number of progeny cDCs next to sites of viral replication. Ablation of CCR2 in the cDC lineage prevented this increase and resulted in a deficit in IAV-specific T cell responses and diminished resistance to reinfection. These data suggest that the homeostatic network of cDCs in tissues is insufficient for immunity and reveal a chemokine-driven mechanism of expansion of lung cDC numbers that amplifies T cell responses against respiratory viruses.

Published
2021
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42. Maintenance and loss of endocytic organelle integrity: mechanisms and implications for antigen cross-presentation.

Author

Childs E, Henry CM, Canton J, and Reis e Sousa C

Subjects
Animals, Antigen Presentation, CD8-Positive T-Lymphocytes metabolism, Cross-Priming, Humans, Endosomes metabolism, Histocompatibility Antigens Class I metabolism, Phagocytes metabolism
Abstract

The membranes of endosomes, phagosomes and macropinosomes can become damaged by the physical properties of internalized cargo, by active pathogenic invasion or by cellular processes, including endocytic maturation. Loss of membrane integrity is often deleterious and is, therefore, prevented by mitigation and repair mechanisms. However, it can occasionally be beneficial and actively induced by cells. Here, we summarize the mechanisms by which cells, in particular phagocytes, try to prevent membrane damage and how, when this fails, they repair or destroy damaged endocytic organelles. We also detail how one type of phagocyte, the dendritic cell, can deliberately trigger localized damage to endocytic organelles to allow for major histocompatibility complex class I presentation of exogenous antigens and initiation of CD8 + T-cell responses to viruses and tumours. Our review highlights mechanisms for the regulation of endocytic organelle membrane integrity at the intersection of cell biology and immunology that could be co-opted for improving vaccination and intracellular drug delivery.

Published
2021
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43. Secreted gelsolin inhibits DNGR-1-dependent cross-presentation and cancer immunity.

Author

Giampazolias E, Schulz O, Lim KHJ, Rogers NC, Chakravarty P, Srinivasan N, Gordon O, Cardoso A, Buck MD, Poirier EZ, Canton J, Zelenay S, Sammicheli S, Moncaut N, Varsani-Brown S, Rosewell I, and Reis e Sousa C

Subjects
Actins metabolism, Amino Acid Sequence, Animals, Antigens, Neoplasm metabolism, CD8-Positive T-Lymphocytes drug effects, CD8-Positive T-Lymphocytes immunology, Cell Movement drug effects, Cell Proliferation drug effects, Cross-Priming drug effects, Cytoskeleton drug effects, Cytoskeleton metabolism, Dendritic Cells drug effects, Dendritic Cells immunology, Gelsolin chemistry, Gelsolin deficiency, Gene Expression Regulation, Neoplastic drug effects, Immune Checkpoint Inhibitors pharmacology, Immune Checkpoint Inhibitors therapeutic use, Mice, Inbred C57BL, Mutation genetics, Neoplasms drug therapy, Neoplasms genetics, Neoplasms pathology, Protein Binding drug effects, Survival Analysis, Mice, Cross-Priming immunology, Gelsolin metabolism, Immunity drug effects, Lectins, C-Type metabolism, Neoplasms immunology, Receptors, Immunologic metabolism, Receptors, Mitogen metabolism
Abstract

Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8 + T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8 + T cell responses., Competing Interests: Declaration of interests E.G., O.S., K.H.J.L., N.S., O.G., S.Z., S.S., P.C., and C.R.S. are named as inventors on a patent application on the use of sGSN for immunotherapies. C.R.S. owns stock options and/or is a paid consultant for Bicara Therapeutics, Montis Biosciences, Oncurious NV, Bicycle Therapeutics, and Sosei Heptares. C.R.S. holds a professorship at Imperial College London and honorary professorships at University College London and King’s College London. None of these activities are related to this work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2021
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44. An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

Author

Poirier EZ, Buck MD, Chakravarty P, Carvalho J, Frederico B, Cardoso A, Healy L, Ulferts R, Beale R, and Reis e Sousa C

Subjects
Alternative Splicing, Animals, Brain enzymology, Brain virology, Cell Line, DEAD-box RNA Helicases chemistry, Humans, Immunity, Innate, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Mice, Organoids enzymology, Organoids virology, RNA Virus Infections enzymology, RNA Virus Infections immunology, RNA Virus Infections virology, RNA Viruses genetics, RNA Viruses immunology, RNA, Double-Stranded metabolism, RNA, Small Interfering metabolism, Ribonuclease III chemistry, SARS-CoV-2 genetics, SARS-CoV-2 immunology, SARS-CoV-2 physiology, Virus Replication, Zika Virus genetics, Zika Virus immunology, Zika Virus physiology, Zika Virus Infection enzymology, Zika Virus Infection immunology, Zika Virus Infection virology, DEAD-box RNA Helicases genetics, DEAD-box RNA Helicases metabolism, RNA Interference, RNA Viruses physiology, RNA, Viral metabolism, Ribonuclease III genetics, Ribonuclease III metabolism, Stem Cells enzymology, Stem Cells virology
Abstract

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Published
2021
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45. SARS-CoV-2 detection by a clinical diagnostic RT-LAMP assay.

Author

Buck MD, Poirier EZ, Cardoso A, Frederico B, Canton J, Barrell S, Beale R, Byrne R, Caidan S, Crawford M, Cubitt L, Gandhi S, Goldstone R, Grant PR, Gulati K, Hindmarsh S, Howell M, Hubank M, Instrell R, Jiang M, Kassiotis G, Lu WT, MacRae JI, Martini I, Miller D, Moore D, Nastouli E, Nicod J, Nightingale L, Olsen J, Oomatia A, O'Reilly N, Rideg A, Song OR, Strange A, Swanton C, Turajlic S, Wu M, and Reis e Sousa C

Abstract

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive., Competing Interests: Competing interests: D. Miller and K. Gulati are employees of New England Biolabs, which provided the WarmStart Colorimetric LAMP 2X Master Mix used in this work. C. Swanton receives or has received grant support from Pfizer, AstraZeneca, Bristol-Myers Squibb (BMS), Roche-Ventana, Boehringer-Ingelheim, and Ono Pharmaceutical and has consulted for or received an honorarium from Pfizer, Novartis, GlaxoSmithKline, Merck Sharp & Dohme, BMS, Celgene, AstraZeneca, Illumina, Genentech, Roche-Venatana, GRAIL, Medicxi, and the Sarah Cannon Research Institute. C. Swanton also is a shareholder of Apogen Biotechnologies, Epic Bioscience, and GRAIL and has stock options in and is a cofounder of Achilles Therapeutics., (Copyright: © 2021 Buck MD et al.)

Published
2021
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46. Dendritic Cells Revisited.

Author

Cabeza-Cabrerizo M, Cardoso A, Minutti CM, Pereira da Costa M, and Reis e Sousa C

Subjects
Animals, Cell Lineage, Humans, Mice, Dendritic Cells, Immunity, Innate
Abstract

Dendritic cells (DCs) possess the ability to integrate information about their environment and communicate it to other leukocytes, shaping adaptive and innate immunity. Over the years, a variety of cell types have been called DCs on the basis of phenotypic and functional attributes. Here, we refocus attention on conventional DCs (cDCs), a discrete cell lineage by ontogenetic and gene expression criteria that best corresponds to the cells originally described in the 1970s. We summarize current knowledge of mouse and human cDC subsets and describe their hematopoietic development and their phenotypic and functional attributes. We hope that our effort to review the basic features of cDC biology and distinguish cDCs from related cell types brings to the fore the remarkable properties of this cell type while shedding some light on the seemingly inordinate complexity of the DC field.

Published
2021
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48. The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.

Author

Canton J, Blees H, Henry CM, Buck MD, Schulz O, Rogers NC, Childs E, Zelenay S, Rhys H, Domart MC, Collinson L, Alloatti A, Ellison CJ, Amigorena S, Papayannopoulos V, Thomas DC, Randow F, and Reis e Sousa C

Subjects
Animals, Cell Death, Coculture Techniques, Dendritic Cells immunology, HEK293 Cells, Histocompatibility Antigens Class I metabolism, Humans, Lectins, C-Type genetics, Ligands, Mice, NADPH Oxidases metabolism, Phagosomes genetics, Phagosomes immunology, Phosphorylation, RAW 264.7 Cells, Reactive Oxygen Species metabolism, Receptors, Immunologic genetics, Receptors, Mitogen genetics, Signal Transduction, Syk Kinase metabolism, T-Lymphocytes immunology, Antigen Presentation, Cross-Priming, Dendritic Cells metabolism, Lectins, C-Type metabolism, Phagosomes metabolism, Receptors, Immunologic metabolism, Receptors, Mitogen metabolism, T-Lymphocytes metabolism
Abstract

Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8 + T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.

Published
2021
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49. Antagonistic Inflammatory Phenotypes Dictate Tumor Fate and Response to Immune Checkpoint Blockade.

Author

Bonavita E, Bromley CP, Jonsson G, Pelly VS, Sahoo S, Walwyn-Brown K, Mensurado S, Moeini A, Flanagan E, Bell CR, Chiang SC, Chikkanna-Gowda CP, Rogers N, Silva-Santos B, Jaillon S, Mantovani A, Reis e Sousa C, Guerra N, Davis DM, and Zelenay S

Subjects
Animals, Dinoprostone metabolism, Humans, Immunotherapy, Inflammation genetics, Interferon-gamma metabolism, Killer Cells, Natural immunology, Mice, Neoplasms therapy, Phenotype, Prognosis, Prostaglandin-Endoperoxide Synthases genetics, Receptors, Prostaglandin E, EP2 Subtype metabolism, Receptors, Prostaglandin E, EP4 Subtype metabolism, T-Lymphocytes, Cytotoxic immunology, Tumor Microenvironment immunology, Immune Checkpoint Inhibitors therapeutic use, Inflammation immunology, Neoplasms immunology, Tumor Escape immunology
Abstract

Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes., Competing Interests: Declaration of Interests The COX-IS is the subject of a patent application (WO2019243567A1) in which E.B., C.P.B., and S.Z. are listed as inventors., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2020
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50. Cross-presentation of dead-cell-associated antigens by DNGR-1 + dendritic cells contributes to chronic allograft rejection in mice.

Author

Balam S, Kesselring R, Eggenhofer E, Blaimer S, Evert K, Evert M, Schlitt HJ, Geissler EK, van Blijswijk J, Lee S, Reis e Sousa C, Brunner SM, and Fichtner-Feigl S

Subjects
Animals, CD8-Positive T-Lymphocytes immunology, Female, Interferon-gamma immunology, Lymphocyte Activation immunology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Allografts immunology, Antigen Presentation immunology, Antigens, Surface immunology, Cross-Priming immunology, Dendritic Cells immunology, Graft Rejection immunology, Lectins, C-Type immunology, Receptors, Immunologic immunology
Abstract

The purpose of this study was to elucidate whether DC NK lectin group receptor-1 (DNGR-1)-dependent cross-presentation of dead-cell-associated antigens occurs after transplantation and contributes to CD8 + T cell responses, chronic allograft rejection (CAR), and fibrosis. BALB/c or C57BL/6 hearts were heterotopically transplanted into WT, Clec9a -/- , or Batf3 -/- recipient C57BL/6 mice. Allografts were analyzed for cell infiltration, CD8 + T cell activation, fibrogenesis, and CAR using immunohistochemistry, Western blot, qRT 2 -PCR, and flow cytometry. Allografts displayed infiltration by recipient DNGR-1 + DCs, signs of CAR, and fibrosis. Allografts in Clec9a -/- recipients showed reduced CAR (p < 0.0001), fibrosis (P = 0.0137), CD8 + cell infiltration (P < 0.0001), and effector cytokine levels compared to WT recipients. Batf3-deficiency greatly reduced DNGR-1 + DC-infiltration, CAR (P < 0.0001), and fibrosis (P = 0.0382). CD8 cells infiltrating allografts of cytochrome C treated recipients, showed reduced production of CD8 effector cytokines (P < 0.05). Further, alloreactive CD8 + T cell response in indirect pathway IFN-γ ELISPOT was reduced in Clec9a -/- recipient mice (P = 0.0283). Blockade of DNGR-1 by antibody, similar to genetic elimination of the receptor, reduced CAR (P = 0.0003), fibrosis (P = 0.0273), infiltration of CD8 + cells (p = 0.0006), and effector cytokine levels. DNGR-1-dependent alloantigen cross-presentation by DNGR-1 + DCs induces alloreactive CD8 + cells that induce CAR and fibrosis. Antibody against DNGR-1 can block this process and prevent CAR and fibrosis., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Published
2020
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