Development and function of inflammatory dendritic cells

Lead Research Organisation: King's College London
Department Name: Immunology Infection and Inflam Diseases

Abstract

The vaccination method relies on the activation and expansion of cells called T lymphocytes that can recognise the antigens of pathogens. After vaccination, these T cells react quicker and better to pathogen encounter and can mediate immune protection.

The activation of T lymphocytes is controlled by a family of cells called dendritic cells that capture pathogens and present pathogen derived antigens to T cells on presenter molecules called MHC (major histocompatibility complex).

Several populations of dendritic cells have been described. They harbour distinct immune function important for the establishment of protective immunity. However, some members of the dendritic cell family are ill described and their specific contribution to the protective T cell responses not yet defined. In particular, during the small inflammation caused by vaccination (or infection), a new type of cells is produced in large quantities by the immune system called inflammatory DCs (iDCs). How iDCs are produced and to what extent they contribute to the development of protective immune responses has still to be understood.

This proposal intends to :
- better characterise these cells,
- understand how the immune system produces them and
- determine how they impact the development of adaptive immunity induced by vaccination

This research is important for human and animal health because it will enable to produce better vaccines. Indeed understanding iDCs biology is key to tailor vaccine adjuvants that will maximise the induction of protective immune responses required for the success of vaccination.

Technical Summary

Inflammatory dendritic cells (iDCs) are monocyte-derived antigen presenting cells arising during inflammation.

Ly6ChiCD115+ inflammatory monocytes give rise to inflammatory DCs (iDCs) and iNOS-producing microbicidal phagocytes (iMPs) through CCR2-dependent mobilization. This proposal addresses a) the mechanisms underlying the generation of iDCs from Ly6ChiCD115+, b) the contribution of iDCs to T cell responses. We aim at:

1- Characterising monocytes subsets acting as progenitors for iDCs (pre-iDCs).

The applicant laboratory has recently evidenced that Ly6Chigh blood monocytes are heterogeneous and contain CCR2-dependent progenitors (Ly6ChighFlt3+CD11c-MHCII+ and Ly6ChighFlt3+CD11c+MHCII+) poised to iDCs differentiation and poorly able to generate iMPs. This proposal intends to fully assess the pre-iDCs progeny in various inflammatory settings.

2- Identifying transcriptional networks controlling iDCs development.

We have acquired preliminary evidence that IRF4, CIITA and MHCII expression is heterogeneous within naive Ly6Chigh monocytes subsets. We hypothesise the existence of regulatory network integrating positive master regulators (SFPI1) or negative master regulators (NFIL3) regulators of monocyte fate and iDCs lineage commitment. These hypotheses will be tested in vitro and in vivo by genetic experiments.

3- Producing a cre recombinase knock in mouse model targeting specifically iDCs.

To this end, we will use the CD209a gene that is highly expressed in iDC-committed monocytes subsets.

4- Visualising iDCs lineage by fate-mapping.

The CD209acre line will be crossed to ROSAloxSTOPloxTomato reporter to assess the dynamic of iDCs populations.

5- Assessing iDCs contribution to T cell responses.

We will perform inducible, conditional iDC ablation (CD209acre x Csf1rloxSTOPloxDTR) and conditional gene inactivation in iDCs (CD209acre x Ablox/lox) to define their contribution to CD4+ T cell responses in models of vaccination.

Planned Impact

Industrial development
This research may generate commercially-exploitable research tools and possibly intellectual property as well. Indeed, understanding the generation of inflammatory cells should improve the ability to screen for the most effective adjuvant relevant for vaccine design.
Also, in-depth analysis of inflammatory dendritic cells (an their transcriptional underpinnings) might lead to the identification of pharmacologically targetable pathways susceptible to provide a level of manipulation of inflammatory immune responses. This might serve the rational design of new anti-inflammatory therapies.
We will take advantage of industrial outreach structures dedicated to foster industrialization projects at King's College London. We will try also to develop industrial interactions that may lead to the application for a CASE studentship attached to this project.

Outreach, education, training and capacity-building
To publicise our research findings, we will attend national and international conferences. All research findings funded by this grant will be published using open-access mechanisms (such as PMC) to facilitate access of the findings to a wide public.
In addition, insertion of the PI into the teaching faculty at King's College London might facilitate the diffusion of new findings to a wide public of new scientist and health professional in training.

Societal beneficiaries
The research in this proposal has both medical and societal impact since it is expected to improve health status through the rational design of vaccines by a better understanding of the cells mediating the immune responses triggered by vaccines.
Indirectly, this might have a broad impact on the health status of the human and animal population as well. Indeed, mechanisms unravelled by our research might benefit to the rationale design of new vaccines against old (malaria) or emerging (Ebola virus) pathogens.

Publications

10 25 50
 
Description Since our last submission, we have also focused our attention to the human counterpart of inflammatory DCs. We were able to identify a recently described subset of DCs (DC3) as an immediate precursor for human inflammatory DCs differentiated in response of GM-CSF. In addition, we studied the DC3 ontogeny, and we provided evidence showing that they originate from a population of progenitors with granulocyte, monocyte and dendritic cell potential (GMDP) through a developmental pathway independent from the common dendritic cell progenitor (CDP) and the monocyte progenitor (cMoP). Finally, we also identify a specific ability of DC3s to induce the differentiation of naive T cells into CD8+CD103+ effector cells.
Exploitation Route A better understanding of human inflammatory phagocytes origin and function has the potential to enable their targeting for immunotherapy purposes in human inflammatory diseases.
Sectors Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description A website dedicated to monocytes in inflammation targeted for a non-specialist is under construction
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title CRISPR-Cas9 
Description Generation of cancer cell lines using CRISPR-Cas9 to knockout key cytokines (ie. GM-CSF) that may influence the development on mononuclear phagocytes in the context of inflammation and cancer. 
Type Of Material Cell line 
Provided To Others? No  
Impact Using this method we are able to evaluate the impact of key cytokines in the development of mononuclear phagocytes. 
 
Title GENERATION OF FEEDER CELL LINES EXPRESSING HUMAN CYTOKINES 
Description One of the tested protocols for the generation of human hematopoietic progenitors cells (HSPC) from pluripotent stem cells provides the use of mouse stromal cell lines (i.e. MS5, OP9). Human ES or iPS are co-cultured for 10-15 days with mouse stromal cells supporting hematopoietic differentiation and expansion of the human cells. In order to improve this system as well as identify key factors indispensable for HSPC and dendritic cells (DC) differentiation, human cytokines involved in HSC maintenance in the bone marrow niche (i.e. CXCL12, SCF, Flt3L and TPO) have been cloned into retroviral expression vectors. These vectors have been subsequently used to transduce MS5 and OP9 feeders and the validation of the generated cell lines is ongoing at the moment. 
Type Of Material Cell line 
Provided To Others? No  
Impact With the generation of feeder cell lines expressing the key human cytokines responsible for HSC maintenance and expansion we are in the process of establishing an in vitro platform to improve the differentiation of human pluripotent cells into hematopoietic progenitor cells. This tool will allow us to obtain larger numbers of in vitro generated HSPC that we will further differentiated into human DCs subsets, applying the already established protocol previously validated on cord blood-derived HSPC. 
 
Title Immunodeficient mouse model to study human tumor-infiltrating immune cells 
Description Using the previously described mouse melanoma cell lines (B16) expressing human Flt3L and human GM-CSF we established an in vivo model to study the infiltration of human lymphoid and myeloid cells in a in vivo model of lung metastasis. To this end, B16 cells expressing human cytokines were injected i.v. in immunodeficient mice (i.e. NSG), followed by the injection of human peripheral blood mononuclear cells (PBMCs) intra-venously. Subsequently lungs were collected and analysed by flow cytometry and the presence of infiltrating human immune cells was detected. More importantly, the effect of the human cytokines Flt3L and GM-CSF on the composition of tumor-infiltrating cells can be study in these experimental settings. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact This method allows us to study the contribution of the human cytokines GM-CSF and Flt3L in shaping the composition of tumor-infiltrating human myeloid and lymphoid cells in in vivo settings. 
 
Title Mouse melanoma cell lines expressing human cytokines 
Description We generated two new cell lines of mouse melanoma (B16) expressing the human cytokines Flt3L and GM-CSF. The genomic sequence of human Flt3L and GM-CSF were cloned into a retroviral vector (pMX) carrying an internal ribosome entry site (IRES) upstream the coding sequence for GFP. Mouse melanoma cells B16 were then transduced using retriviral particles and sorted according to their GFP expression, in order to generated two melanoma cells line constitutively expressing the human factors Flt3L and GM-CSF. The cell lines were validated by measuring the expression of human cytokines in the cells' supernatants by ELISA. 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact These lines can be used in in vivo setting (e.g. immunodeficient mice) to study the effect of Flt3L and GM-CSF in the composition of human tumor-infiltrating immune cells. 
 
Description Analysis of Tissue resident memory T cells in breast cancer infiltrate 
Organisation Science and Life Foundation
Country Chile 
Sector Charity/Non Profit 
PI Contribution In collaboration with Alvaro Lladser, we have analysed the populations of Trm infiltrating breast cancer and evidenced a correlation between the infiltration of CD1c+CD163+CD14+ type 3 dendritic cells and CD8+CD103+ infiltration. In addition, In vitro experiments show that CD1c+CD163+CD14+ type 3 dendritic cells ca prime CD8+CD103+ effectors from native CD8+ T cells. These findings have important implications for our understanding of adpative immune responses against cancer.
Collaborator Contribution Providing methods and expertise in the analysis of Trm into tissue samples.
Impact Manuscript submitted to publication, currently in evaluation at Immunity. Disentangling the Heterogeneity of Human CD1c+ Dendritic Cells Identifies CD163+ Type 3 DCs as a Distinct DC Lineage Priming CD8+CD103+ T Cells Pierre Bourdely , Kristine Vaivode , Giorgio Anselmi , Rodrigo Nalio Ramos , Yoann 3 3,4 3 3 3 Missolo-Koussou , Sofia Hidalgo , Jimena Tosselo , Nicolas Nuñez , Wilfrid Richer , Anne 56643 Vincent-Salomon , Alka Saxena , Kristie Wood , Alvaro Lladser , Eliane Piaggio , Julie 3,9 1,2,7, 9,10, Helft , Pierre Guermonprez *
Start Year 2018
 
Description Analysis of human dendritic cells by mass spectrometry (CYTOF) 
Organisation Agency for Science, Technology and Research (A*STAR)
Department Singapore Immunology Network (SIgN)
Country Singapore 
Sector Academic/University 
PI Contribution WIth Charles Antoine Dutertre and Florent Ginhoux, we have developped new methods to analyse the heterogeneity of human dendritic cells by high dimensional anlysis using mass spectrometry.
Collaborator Contribution Provided figures in ms in press at Nature Comm. Engineered niches support the development of human dendritic cells in humanized mice. Anselmi G., Vaivode K., Dutertre C.A., Bourdely P., Newell E., Hickman O., Wood K., Saxena A., Helft J., Ginhoux J., Guermonprez P. Nature Communications. 2020. In press. doi: https://doi.org/10.1101/835223.
Impact Engineered niches support the development of human dendritic cells in humanized mice. Anselmi G., Vaivode K., Dutertre C.A., Bourdely P., Newell E., Hickman O., Wood K., Saxena A., Helft J., Ginhoux J., Guermonprez P. Nature Communications. 2020. In press. doi: https://doi.org/10.1101/835223.
Start Year 2017
 
Description Analysis of human dendritic cells diversity at homeostasis and in breast cancer (including inflammatory dendritic cells) 
Organisation Curie Institute Paris (Institut Curie)
Department Immunity and Cancer INSERM (U932)
Country France 
Sector Academic/University 
PI Contribution Dendritic cells (DC) are antigen presenting cells controlling T cell activation. In human, the diversity, developmental biology and functional capabilities of DC subsets are not fully understood. Here, we identify circulating CD88-CD1c+CD163+ DC (termed as type 3 DC or DC3) as an immediate precursor of inflammatory CD88-CD14+CD1c+CD163+FceRI+ DC. DC3 develop) via a specific pathway that is independent from the cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors and are activated by GM-CSF. Like classical DC, but unlike monocytes, DC3 drive the activation of naïve T cells. In vitro, DC3 display a distinctive ability to prime CD8+ T cells expressing a tissue-homing signature and the epithelial homing alpha-E integrin (CD103) through TGF-ß signaling. In vivo, DC3 infiltrate luminal breast cancer primary tumors and DC3 infiltration correlates positively with CD8+CD103+CD69+ tissue-resident memory T cells. Altogether, these findings define DC3 as a unique lineage of inflammatory DC endowed with a strong potential to regulate tumor immunity.
Collaborator Contribution access to single cell genomics and RNAseq facility data analysis bioinformatics
Impact Manuscript submitted for publication
Start Year 2017
 
Description Dr. Emmanuel Gautier 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution Our group, in collaboration with the group of Dr. Emmanuel Gautier (INSERM U1166), are working together on the key transcription factors that are required for the development of induced dendritic cell populations in the context of inflammation and cancer.
Collaborator Contribution This leads to the exchange of numerous protocols, reagents and expertise for example in the field of mononuclear phagocyte ontogeny and function.
Impact We have collaborated to utilise various mouse models deficient in IRF4, a key transcription factors that may be critical for induction of dendritic cells derived from monocytes.
Start Year 2016
 
Description Dr. Federica Benvenuti 
Organisation International Centre for Genetic Engineering and Biotechnology
Country Italy 
Sector Academic/University 
PI Contribution Our group, in collaboration with the group of Dr. Federica Benvenuti (ICGEB), are working together on the mechanisms by which induced populations of dendritic cells modulate antigen presentation and T cell response against cancer.
Collaborator Contribution This leads to the exchange of numerous protocols and expertise for example in the field of antigen presentation, cancer immunology, CRISPR-Cas9 technology..
Impact We have generated lung adenocarcinoma cell lines with genetic deletion of key cytokines involved in inducing different dendritic cell populations and modulating immune suppression.
Start Year 2016
 
Description Dr. Loredana Saveanu. 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution Our group, in collaboration with the group of Dr. Loredana Saveanu (INSERM U1151/Bichat Hospital), are working together on the mechanisms by which Rab GTPases regulate corss presentation in DCs. This leads to the exchange of numerous protocols and expertise for example in the field of in vitro models of bone marrow derived DCs, lentiviral transduction, cellular imaging. In addition, we are working on the submission of parallel reviews about cross-presentation as a part of an special issue hosted in the journal frontiers in Immunology titled: "Unconventional antigen presentation: atypical cells and unusual pathways". An abstract of the review has already been accepted by the editors of the journal. Our contribution to their review will include bibliographic assessment, dessign and evaluation of the manuscript.
Collaborator Contribution This leads to the exchange of numerous protocols and expertise for example in the field of in vitro models of bone marrow derived DCs, lentiviral transduction, cellular imaging.
Impact The expected evaluation of the reviews for the publication in the journal will be in 01 Apr 2015.
Start Year 2006
 
Description Dr. Melanie Greter 
Organisation University of Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution Our group, in collaboration with the group of Dr. Melanie Greter (University of Zurich), are working together on the key transcription factors that are required for the development of induced dendritic cell populations in the context of inflammation and cancer.
Collaborator Contribution This leads to the exchange of numerous protocols, reagents and expertise for example in the field of mononuclear phagocyte ontogeny and function.
Impact We have collaborated to utilise various mouse models deficient in Ccfr2b, a key cytokine that may regulate the induction of suppressive mononuclear phagocytes.
Start Year 2016
 
Description Dr. Roxane Tussiwand 
Organisation University of Basel
Country Switzerland 
Sector Academic/University 
PI Contribution Our group, in collaboration with the group of Dr. Roxane Tussiwand (University of Basel), are working together on the key transcription factors that are required for the development of induced dendritic cell populations in the context of inflammation and cancer.
Collaborator Contribution This leads to the exchange of numerous protocols, reagents and expertise for example in the field of mononuclear phagocyte ontogeny and function.
Impact We have collaborated to utilise various mouse models deficient in IRF8, a transcription factors that may be critical for induction of dendritic cells derived from monocytes.
Start Year 2016
 
Description 2nd European workshop for young investigators in phagocytes 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Oral presentation of the data collected to postgraduate research students from 6 different universities scattered around Europe (UK, Switzerland and Germany). The talk was focused on the role of GM-CSF in the tumor microenvironment and its effect on the development of tumor-infiltrating myeloid cells both in mouse and human.
Year(s) Of Engagement Activity 2018
 
Description Barts and Queen's Mary University, CRUK Cancer centre, UK 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2019
 
Description CPTP, INSERM, Toulouse, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2018
 
Description Centre for Molecular Medicine, Newcastle University, British Society of Immunology, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2019
 
Description Dendritic cells in cancer immunotherapy Workshop, Champalimaud Fundation, Lisbon 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact talk on human dendritic cells
Year(s) Of Engagement Activity 2019
 
Description EMBO Workshop, Antigen Presentation and Processing, Paris 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2019
 
Description EMBO Workshop, Mesenchymal stromal cells and immunity, Athens, Greece 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2019
 
Description GSK Immunology Catalyst, Stevenage, UK Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk on dendritic cells
Year(s) Of Engagement Activity 2017
 
Description Institut Necker Enfants Malades, Paris, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2018
 
Description Institut Pasteur, Immunology Seminar, Paris, France 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2019
 
Description KCL-RTG summer school, London, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact talk on dendritic cells
Year(s) Of Engagement Activity 2019
 
Description KEYSTONE Meeting "Mononuclear Phagocytes", Austin, Texas, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk
Year(s) Of Engagement Activity 2017
 
Description KEYSTONE Meeting "Myeloid cells", Breckenridge, Colorado, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk on human dendritic cells
Year(s) Of Engagement Activity 2018
 
Description Mannheim/Heidelberg University/DKFZ, "Hallmarks of cancer" lecture, Mannheim 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact lecture
Year(s) Of Engagement Activity 2019