IMAGING IMMUNITY – from Nanoscale to Macroscale | Insights from Biophysics
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Talks from Abstract Submissions

Session chair: Pavle Andjus (Belgrade, Serbia)
 
Date: Thursday, 16 January, 2020, 9:30 AM - 10:15 AM

Contents

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9:30 AM -01

Diversity of innate immune cell subsets across spatial and temporal scales in an EAE mouse model (#13)

Celine Caravagna1, 2, Alexandre Jaouen1, 2, Sophie Desplat-Jego4, Keith Fenrich1, Elise Bergot4, Hervé Luche3, Pierre Grenot3, Geneviève Rougon1, 2, Marie Malissen4, 3, Franck Debarbieux1, 2

1 Aix Marseille University - CNRS, Institut de Neurosciences Timone, Marseille, France
2 Aix Marseille University - CNRS, CERIMED, Marseille, France
3 Aix Marseille University - INSERM, CIPHE, Marseille, France
4 Aix Marseille University - CNRS-INSERM, CIML, Marseille, France

Introduction

In both multiple sclerosis and its model experimental autoimmune encephalomyelitis (EAE), the extent of resident microglia activation and infiltration of monocyte-derived cells to the CNS is positively correlated to tissue damage.

Methods

To address the phenotype characterization of different cell subsets, their spatio-temporal distributions and contributions to disease development we induced EAE in Thy1-CFP//LysM-EGFP//CD11c-EYFP reporter mice. We combined high content flow cytometry, immunofluorescence and two-photon imaging in live mice

Results/Discussion

We have identified a stepwise program of inflammatory cells accumulation. First on day 10 after induction, EGFP+ neutrophils and monocytes invade the spinal cord parenchyma through the meninges rather than by extravasion. This event occurs just before axonal losses in the white matter. Once in the parenchyma, monocytes mature into EGFP+/EYFP+ monocyte-derived dendritic cells (moDCs) whose density is maximal on day 17 when the axonal degradation and clinical signs stabilize. Meanwhile, microglia is progressively activated in the grey matter and subsequently recruited to plaques to phagocyte axon debris.

Conclusions

LysM-EGFP//CD11c-EYFP mice appear as a powerful tool to differentiate moDCs from macrophages and to study the dynamics of immune cell maturation and phenotypic evolution in EAE.

References

  1. Jaouen A., Caravagna C., Desplat-Jego S., Fenrich K., Bergot E., Luche H., Grenot P., Rougon G., Malissen M., Debarbieux F. (2018) Diversity of innate immune cell subsets across spatial and temporal scales in a EAE mouse model. Scientific Reports 23;8(1):5146.
  2. Fenrich K.K., Weber P., Rougon G, Debarbieux F. (2013) Long and short term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury J Physiol. 591: 4895-4902

Acknowledgement

We thank Marion Compagnone and Florence Pelletier for mouse colony management. Drs Carole Colin and Jacques Durand for statitical analyses. This work was supported by Agence Nationale Recherche ANR15-CE16-0009-01 (to F.D., M.M.),  and Association de Recherche sur la Sclérose en Plaques ARSEP grants 2015 (to G.R., M.M.), a CIFRE fellowship (to A.J.), a Marie Skłodowska-Curie Actions fellowship from European Commission program H2020 (to C.C.), and core support from AMU, CNRS and INSERM

Characterization of the fluorescently labeled immune cells in Thy1-CFP//LysM-EGFP//CD11c-EYFP mice
A) LysM-EGFP and CD11c-EYFP expression in microglia and the various infiltrating immune cell  from EAE brain at day 17 B) Distribution histograms for microglia cells C)  Bar graph showing the expression of MHC class II, CD11c and CD11c-EYFP in microglia cells D) Quantification and distribution of fluorescent cells in the EAE spinal cord for control (PBS or CFA.PTX) or induced (MOG.CFA.PTX) mice at day 8, 13 or 17. The pie chart sizes are proportional to the total number of cells. The numbers indicate the percentage of the corresponding population
In vivo visualization of immune cell dynamics during EAE progression.
EGFP+ cells (green), EYFP+ cells (yellow), EGFP+/EYFP+ cells (pink, manually highlighted), CFP+ neurons (cyan), blood vessels (red). D = post-induction day. A) Dynamics of cell distribution during EAE at different post-induction days. B) Evolution of immune cell densities relative to pre-induction values  C)  Initial meningeal accumulation of EGFP+ cells and subsequent infiltration into the tissue. D)  Evolution of the number of CFP+ axons in determined regions of interest at different stages of EAE progression E) Evolution of identified individual axons F) Axonal damages
Keywords: MODALITY: Intravital 2P-fluorescence microscopy, DOMAIN: Neuroinflammation, preclinical imaging
9:50 AM -02

In vivo tracking of polyclonal human regulatory T cells reveals a role for innate immune cells in Treg transplant recruitment. (#18)

Jacinta Jacob2, Yasmin Mohseni1, 2, Alessia Volpe1, Qi Peng2, Suchita Nadkarni3, Rosalind Hannen3, Robert I. Lechler2, Federica Marelli-Berg3, Lesley Smyth4, Giovanna Lombardi2, Gilbert O. Fruhwirth1

1 King's College London, Imaging Chemistry & Biology, Biomedical Engineering and Imaging Sciences, London, United Kingdom
2 King's College London, MRC Centre for Transplantation, Peter Gorer Department of Immunobiology, School of Immunology and Microbial Science, London, United Kingdom
3 Queen Mary University London, William Harvey Research Institute, London, United Kingdom
4 University of East London, School of Health, Sport and Bioscience, London, United Kingdom

Introduction

In solid organ transplantation, organ demand outstrips supply with allograft rejection being another limitation. Adoptive transfer of regulatory T-cells (Tregs) protects from graft rejection1, and its safety has been shown in clinical trials. Antigen-specific Tregs are superior to polyclonal Tregs, with chimeric antigen receptors (CAR) emerging as an attractive option to produce them2. Important questions about in vivo fate, distribution and localization of Treg function remain unclear. Our goal was to identify a long-term in vivo tracking approach compatible with future clinical translation.

Methods

Human Tregs enriched via a GMP-compatible protocol were either directly labelled usgin 89Zr-oxine or lentivirally transduced with the reporter human sodium iodide symporter (NIS) fused to a fluorescent protein (FP) for preclinical evaluation3. Antigen-specific Tregs were produced using an expression platform for both a HLA-A2-specific CAR4 and the reporter. Tregs were characterized in vitro for phenotype, survival/expansion, suppressive capacity/activatability, and radiotracer (99mTcO4-) uptake if NIS-transduced. BALB/c Rag2‑/‑γc‑/‑ (BRG) mice were transplanted with A2+ human skin and ~6 weeks later traceable Tregs were i/v administered. Transplants were monitored by nanoSPECT/CT up to 40d for Treg recruitment/presence. Ex vivo tissue histology was employed to support in vivo data.

Results/Discussion

89Zr-labelling was unsuitable due to excessive cellular radiodamage affecting Treg survival/expansion at cellular label concentrations required for long-term in vivo Treg tracking (Fig.1). In contrast, reporter engineered Tregs fully retained phenotypes (CD4/CD25/FOXP3) and function (effector T-cell suppression, activatability/CD69) while showing stable reporter expression over weeks (Fig.2A-C). Antigen-specific CAR-Tregs showed significantly higher CD69 activation when challenged with A2+ B-cells compared to A2- B-cells (Fig.2D). Radiolabelling did not impact on Treg function (Fig.2E). NIS-FP+ Tregs were successfully tracked in vivo by SPECT/CT imaging (Fig.2F-G) and detectable from day 3 reaching a peak at day 8 and persisting in human skin grafts for at least 40 days. Importantly, we found early trafficking of Tregs to skin grafts to be markedly reduced by elimination of recipient innate Gr‑1+ immune cells (neutrophils and distinct monocyte subsets).

Conclusions

We showed first proof-of-principle of in vivo Treg tracking to transplants. This data suggests utility of radionuclide reporter gene imaging as a clinically compatible strategy for long-term in vivo Treg tracking in future clinical trials. Notably, with CAR-Treg therapy likely to lead the way, and thereby genetic engineering becoming a fundamental requirement, reporter gene technology does not add undue complications to this cell therapy.

References

1 Safinia N, et al. Cell Therapy in Organ Transplantation: Our Experience on the Clinical Translation of Regulatory T Cells. Frontiers in immunology 2018, 9:354.
2 Boardman DA et al. Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection. Am J Transplant 2017, 17(4):931.

3 Volpe A, Man F, Lim, L, Khoshnevisan A, Blower JE, Blower PJ, Fruhwirth GO. Radionuclide-fluorescence reporter gene imaging to track tumor progression in rodent tumor models. J Vis Exp 2018; 133:e57088. doi: 10.3791/57088.

Acknowledgement

We acknowledge funding from the Medical Research Council UK, the British Heart Foundation and Cancer Research UK. We declare no conflict of interest.

Figure 1
Figure 2
Keywords: Immunology, SPECT/CT, reporter gene, transplantation, regulatory T-cells