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Article ; Online: Optimized immunofluorescence staining protocol for imaging germinal centers in secondary lymphoid tissues of vaccinated mice

Sigrid Fra-Bido / Simon A. Walker / Silvia Innocentin / Michelle A. Linterman

STAR Protocols, Vol 2, Iss 3, Pp 100499- (2021)

2021

Abstract: Summary: Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center ... ...

Abstract	Summary: Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues.For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).
Keywords	Antibody ; Cell Biology ; Immunology ; Microbiology ; Microscopy ; Model Organisms ; Science (General) ; Q1-390
Language	English
Publishing date	2021-09-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Heterochronic faecal transplantation boosts gut germinal centres in aged mice

Marisa Stebegg / Alyssa Silva-Cayetano / Silvia Innocentin / Timothy P. Jenkins / Cinzia Cantacessi / Colin Gilbert / Michelle A. Linterman

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 13

Abstract: Microbiota impacts all major aspects of physiology, but little is known about its effects on age-related changes in immune responses. Here the authors show that gut microbiota transfer between adult and old mice increases local but not systemic germinal ... ...

Abstract	Microbiota impacts all major aspects of physiology, but little is known about its effects on age-related changes in immune responses. Here the authors show that gut microbiota transfer between adult and old mice increases local but not systemic germinal centre responses regardless of age directionality.
Keywords	Science ; Q
Language	English
Publishing date	2019-06-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Heterochronic faecal transplantation boosts gut germinal centres in aged mice

Marisa Stebegg / Alyssa Silva-Cayetano / Silvia Innocentin / Timothy P. Jenkins / Cinzia Cantacessi / Colin Gilbert / Michelle A. Linterman

Nature Communications, Vol 10, Iss 1, Pp 1-

2019 Volume 13

Abstract	Microbiota impacts all major aspects of physiology, but little is known about its effects on age-related changes in immune responses. Here the authors show that gut microbiota transfer between adult and old mice increases local but not systemic germinal centre responses regardless of age directionality.
Keywords	Science ; Q
Language	English
Publishing date	2019-06-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: GIMAP6 is required for T cell maintenance and efficient autophagy in mice.

John C Pascall / Louise M C Webb / Eeva-Liisa Eskelinen / Silvia Innocentin / Noudjoud Attaf-Bouabdallah / Geoffrey W Butcher

PLoS ONE, Vol 13, Iss 5, p e

2018 Volume 0196504

Abstract: The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family ... ...

Abstract	The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2018-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Follicular Regulatory T Cells Can Access the Germinal Center Independently of CXCR5

Ine Vanderleyden / Sigrid C. Fra-Bido / Silvia Innocentin / Marisa Stebegg / Hanneke Okkenhaug / Nicola Evans-Bailey / Wim Pierson / Alice E. Denton / Michelle A. Linterman

Cell Reports, Vol 30, Iss 3, Pp 611-619.e

2020 Volume 4

Abstract: Summary: The germinal center (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunization. Control of the GC response is thought to require follicular regulatory T (Tfr) ...

Abstract	Summary: The germinal center (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunization. Control of the GC response is thought to require follicular regulatory T (Tfr) cells, a subset of suppressive Foxp3+ regulatory T cells located within GCs. Relatively little is known about the exact role of Tfr cells within the GC and how they exert their suppressive function. A unique feature of Tfr cells is their reported CXCR5-dependent localization to the GC. Here, we show that the lack of CXCR5 on Foxp3+ regulatory T cells results in a reduced frequency, but not an absence, of GC-localized Tfr cells. This reduction in Tfr cells is not sufficient to alter the magnitude or output of the GC response. This demonstrates that additional, CXCR5-independent mechanisms facilitate Treg cell homing to the GC. : Vanderleyden et al. show that CXCR5-deficient Treg cells can migrate into the B cell follicle. In the absence of CXCR5, fewer Tfr cells participate in the germinal center reaction, but the reduction in Tfr cell number does not affect the magnitude of the germinal center response. Keywords: follicular regulatory T cells, germinal center response, CXCR5
Keywords	Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2020-01-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans

Danika L Hill / Carly E Whyte / Silvia Innocentin / Jia Le Lee / James Dooley / Jiong Wang / Eddie A James / James C Lee / William W Kwok / Martin S Zand / Adrian Liston / Edward J Carr / Michelle A Linterman

eLife, Vol

2021 Volume 10

Abstract: Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us ... ...

Abstract	Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us to better understand the cellular and molecular mechanisms that underpin successful vaccination in order to improve vaccine design for at-risk groups. Here, by studying the breadth of anti-haemagglutinin (HA) IgG, serum cytokines, and B and T cell responses by flow cytometry before and after influenza vaccination, we show that formation of circulating T follicular helper (cTfh) cells was associated with high-titre antibody responses. Using Major Histocompatability Complex (MHC) class II tetramers, we demonstrate that HA-specific cTfh cells can derive from pre-existing memory CD4+ T cells and have a diverse T cell receptor (TCR) repertoire. In older people, the differentiation of HA-specific cells into cTfh cells was impaired. This age-dependent defect in cTfh cell formation was not due to a contraction of the TCR repertoire, but rather was linked with an increased inflammatory gene signature in cTfh cells. Together, this suggests that strategies that temporarily dampen inflammation at the time of vaccination may be a viable strategy to boost optimal antibody generation upon immunisation of older people.
Keywords	vaccines ; ageing ; influenza ; germinal centre ; antibodies ; t follicular helper cells ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2021-11-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Cellular Stress in the Context of an Inflammatory Environment Supports TGF-β-Independent T Helper-17 Differentiation

Verena Brucklacher-Waldert / Cristina Ferreira / Marisa Stebegg / Olivier Fesneau / Silvia Innocentin / Julien C. Marie / Marc Veldhoen

Cell Reports, Vol 19, Iss 11, Pp 2357-

2017 Volume 2370

Abstract: T helper-17 (Th17) cells are associated with inflammatory disorders and cancer. We report that environmental conditions resulting in cellular stress, such as low oxygen, glucose, and isotonic stress, particularly enhance the generation of Th17 cells. ... ...

Abstract	T helper-17 (Th17) cells are associated with inflammatory disorders and cancer. We report that environmental conditions resulting in cellular stress, such as low oxygen, glucose, and isotonic stress, particularly enhance the generation of Th17 cells. Pharmacological inhibition of cell stress reduces Th17 cell differentiation while stress inducers enhance the development of Th17 cells. The cellular stress response results in Th17 cell development via sustained cytoplasmic calcium levels and, in part, XBP1 activity. Furthermore, in an inflammatory environment, conditions resulting in cell stress can bring about de novo Th17 cell differentiation, even in the absence of transforming growth factor β (TGF-β) signaling. In vivo, cell stress inhibition enhances resistance to Th17-mediated autoimmunity while stress-exposed T cells enhance disease severity. Adverse metabolic environments during inflammation provide a link between adaptive immunity and inflammation and may represent a risk factor for the development of chronic inflammatory conditions by facilitating Th17 cell differentiation.
Keywords	cell stress ; Th17 cells ; T cell differentiation ; autoimmunity ; interleukin-17 ; metabolic stress ; T cell activation ; T helper cells ; Biology (General) ; QH301-705.5
Language	English
Publishing date	2017-06-01T00:00:00Z
Publisher	Elsevier
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Rejuvenating conventional dendritic cells and T follicular helper cell formation after vaccination

Marisa Stebegg / Alexandre Bignon / Danika Lea Hill / Alyssa Silva-Cayetano / Christel Krueger / Ine Vanderleyden / Silvia Innocentin / Louis Boon / Jiong Wang / Martin S Zand / James Dooley / Jonathan Clark / Adrian Liston / Edward Carr / Michelle A Linterman

eLife, Vol

2020 Volume 9

Abstract: Germinal centres (GCs) are T follicular helper cell (Tfh)-dependent structures that form in response to vaccination, producing long-lived antibody secreting plasma cells and memory B cells that protect against subsequent infection. With advancing age the ...

Abstract	Germinal centres (GCs) are T follicular helper cell (Tfh)-dependent structures that form in response to vaccination, producing long-lived antibody secreting plasma cells and memory B cells that protect against subsequent infection. With advancing age the GC and Tfh cell response declines, resulting in impaired humoral immunity. We sought to discover what underpins the poor Tfh cell response in ageing and whether it is possible to correct it. Here, we demonstrate that older people and aged mice have impaired Tfh cell differentiation upon vaccination. This deficit is preceded by poor activation of conventional dendritic cells type 2 (cDC2) due to reduced type 1 interferon signalling. Importantly, the Tfh and cDC2 cell response can be boosted in aged mice by treatment with a TLR7 agonist. This demonstrates that age-associated defects in the cDC2 and Tfh cell response are not irreversible and can be enhanced to improve vaccine responses in older individuals.
Keywords	germinal centre response ; ageing ; t follicular helper cells ; dendritic cells ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	570
Language	English
Publishing date	2020-03-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Immune response elicited by DNA vaccination using Lactococcus lactis is modified by the production of surface exposed pathogenic protein.

Daniela Pontes / Marcela Azevedo / Silvia Innocentin / Sébastien Blugeon / François Lefévre / Vasco Azevedo / Anderson Miyoshi / Pascal Courtin / Marie-Pierre Chapot-Chartier / Philippe Langella / Jean-Marc Chatel

PLoS ONE, Vol 9, Iss 1, p e

2014 Volume 84509

Abstract: In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid ... ...

Abstract	In this study, we compared immune responses elicited by DNA immunization using Lactococcus lactis or L. lactis expressing the Staphylococcus aureus invasin Fibronectin Binding Protein A (FnBPA) at its surface. Both strains carried pValac:BLG, a plasmid containing the cDNA of Beta-Lactoglobulin (BLG), and were designated LL-BLG and LL-FnBPA+ BLG respectively. A TH2 immune response characterized by the secretion of IL-4 and IL-5 in medium of BLG reactivated splenocytes was detected after either oral or intranasal administration of LL-FnBPA+ BLG. In contrast, intranasal administration of LL-BLG elicited a TH1 immune response. After BLG sensitization, mice previously intranasally administered with LL-BLG showed a significantly lower concentration of BLG-specific IgE than the mice non-administered. Altenatively administration of LL-FnBPA+ BLG didn't modify the BLG-specific IgE concentration obtained after sensitization, thus confirming the TH2 orientation of the immune response. To determine if the TH2-skewed immune response obtained with LL-FnBpA+ BLG was FnBPA-specific or not, mice received another L. lactis strain producing a mutated form of the Listeria monocytogenes invasin Internalin A intranasally, allowing thus the binding to murine E-cadherin, and containing pValac:BLG (LL-mInlA+ BLG). As with LL-FnBPA+ BLG, LL-mInlA+ BLG was not able to elicit a TH1 immune response. Furthermore, we observed that these difference were not due to the peptidoglycan composition of the cell wall as LL-FnBPA+ BLG, LL-mInlA+ BLG and LL-BLG strains shared a similar composition. DNA vaccination using LL-BLG elicited a pro-inflammatory TH1 immune response while using LL-FnBPA+ BLG or LL-mInlA+ BLG elicited an anti-inflammatory TH2 immune response.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2014-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

Daniela Pontes / Silvia Innocentin / Silvina Del Carmen / Juliana Franco Almeida / Jean-Guy Leblanc / Alejandra de Moreno de Leblanc / Sébastien Blugeon / Claire Cherbuy / François Lefèvre / Vasco Azevedo / Anderson Miyoshi / Philippe Langella / Jean-Marc Chatel

PLoS ONE, Vol 7, Iss 9, p e

2012 Volume 44892

Abstract: Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus ... ...

Abstract	Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.
Keywords	Medicine ; R ; Science ; Q
Subject code	570
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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