Hervé Luche
Abstract:
Massive changes in Phenomics: Functional phenotyping made in CyTOF
GREGORI E., NARDINI J., IVANOV R, MARTINEZ M., MALISSEN M., LUCHE H.*
Introduction
The identification of the major cellular players involved in the progression of a type of cancer is a key step for the success of new immunotherapies for effective personalized medicine. Complex relationships exist between tumor cells and immune system cells, and each component of innate immunity or adaptive immunity, such as T lymphocytes, macrophages or neutrophils, may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment, it is necessary to use an experimental approach capable of characterizing the heterogeneity of the cell types present, the evolution of their relative proportion and their fine functional specificity. This approach should lead to the identification of new cellular biomarkers characteristic of the different stages of cancer progression in order to improve its diagnosis and to identify new targets for therapeutic treatments on specific time windows.
The possibility of "modifying" the murine genome is currently the most reliable approach for linking the expression of a gene to a given physiological function. To characterize the impact of a gene involved in the anti-tumor response, cellular phenotyping of leukocytes infiltrating a tumor but also those present in peripheral organs is necessary. Essentially based on extracellular labeling, this primary screen aims to quantify the different cell populations present in a MC38 tumor model. This first approach describing cellular heterogeneity does not evaluate the effector functions of immune cells. Mass cytometry is the technique of choice in that respect. Mass cytometry is an emerging technique for the analysis of single cells (1). The principle of mass cytometry is similar to flow cytometry, but the detection unit differs when antibody / metal conjugates are used. The surface markers (1), but also the functional markers (2) and the RNA (3) can now be studied simultaneously by mass cytometry.
The effector / function of immune cells are traditionally analyzed by extensive characterization of cytokine production, detection of regulatory factors for transcription and more recently intracellular signaling. We study a set of these markers at the level of the single cell in mice grafted with MC38 tumors treated or not by an immuno-oncological agent. Phospho-epitopes are also traced to investigate TILs activation status. The simultaneous analysis by mass cytometry of a set of molecules with several stages of tumor progression on the main cell lines of immune cells should increase the chances of identifying the mode of action of an immuno-oncological treatment.
References
- Bendall SC et al. 2012. A deep profiler's guide to cytometry. Trends in Immunology 33(7): 323-32.
- Bondemiller B et al. 2012 Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators. Nature Biotechnology 30(9):858-67
- Frei AP et al. 2016. Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nature Methods, 13(3):269-75.
*Centre d’Immunophénomique, CIPHE, PHENOMIN, INSERM US012, CNRS UMS3367, UM2 Aix-Marseille Université, 13288 Marseille. herve.luche@inserm.fr
Biography:
I studied developmental immunology in the lab of Hans-Joerg FEHLING during my PhD and in the lab of Bernard and Marie MALISSEN for my post-doctoral studies. I engineered two knock-in mouse strains to address lineage commitment issues, among which a Cre activable RFP line. I combined detection of fluorescent proteins with multi-parametric flow cytometry panels to trace the lineage of reporter positive cells. I applied high resolution flow cytometry schemes with mouse mutants of the TCR signaling pathway and transcriptomic to understand the pattern of genes that were involved in lineage decisions at the molecular level.
In 2012, I joined the Immuno-phenotyping module at CIPHE, the Centre for ImmunoPHEnomics - (INSERM/US012, AMU, CNRS/UMS3367) in Marseille-Luminy. With its cutting-edge expertise in mouse genetics and immunology, CIPHE aims to develop and analyze, in a massively parallel and standardized mode, mouse KO/KI models allowing understanding the function of the mouse immune system under normal and infectious conditions. CIPHE is a fantastic place to attempt deciphering the immune-phenome of a given gene in the context of an inflammatory response or pathogen challenge. In charge of the R&D of our phenotyping service, I am developing new high content (>15 parameters) flow cytometry panels of immune cells at basal, inflammatory or infectious conditions. I contribute to national and international phenotyping efforts (International Mouse Phenotyping Consortium (IMPC)). Finally, I am I am leading the mass cytometry effort at CIPHE towards functional phenotyping.
Scientific interests:
Understand how cells integrate signals and communicate with their environment to actually change their transcriptional programs, fate or acquire a new function. Knock-in mouse strains generation to address lineage commitment issues. Blend phenomic datasets, functional assays and transcriptomic datasets to help deciphering the function of a gene on the immune system.
Favorite cytometry applications:
Multi-parametric flow and mass cytometry and more recently spectral cytometry
Multi-Fluorescent reporter study using lineage tracing mice
Phospho-Flow
Integrative Data Analysis
Featured publications:
- Luche H, Ardouin L, Chelbi R, Carpentier S, Shawket A, Montanana Sanchis F, Santa Maria C, Grenot P, Alexandre Y, Grégoire C, Fries A, Vu Manh TP, Tamoutounour S, Crozat K, Tomasello E, Jorquera A, Fossum E, Bogen B, Azukizawa H, Bajenoff M, Henri S, Dalod M, Malissen B.Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery. Immunity. 2016 Aug 16;45(2):305-18. doi: 10.1016/j.immuni.2016.07.019
- Tricot S, Meyrand M, Sammicheli C, Elhmouzi-Younes J, Corneau A, Bertholet S, Malissen M, Le Grand R, Nuti S, Luche H, Cosma A. “Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments.”, Cytometry A. 2015 Apr;87(4):357-68. doi: 10.1002/cyto.a.22648. Epub 2015 Feb 20
- Luche H, Nageswara Rao T, Kumar S, Tasdogan A, Beckel F, Blum C, Martins VC, Rodewald HR, Fehling HJ. In vivo fate mapping identifies pre-TCRα expression as an intra- and extrathymic, but not prethymic, marker of T lymphopoiesis. J Exp Med. 2013 Apr 8;210(4):699-714. doi: 0.1084/jem.20122609
- Luche H, Ardouin L, Teo P, See P, Henri S, Merad M, Ginhoux F, Malissen B. The earliest intrathymic precursors of CD8α(+) thymic dendritic cells correspond to myeloid-type double-negative 1c cells. Eur J Immunol. 2011 Aug;41(8):2165-75. doi: 10.1002/eji.201141728
- Irion S, Luche H, Gadue P, Fehling HJ, Kennedy M, Keller G. Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat Biotechnol. 2007 Dec;25(12):1477-82. PubMed PMID: 18037879
- Luche H, Weber O, Nageswara Rao T, Blum C, Fehling HJ. Faithful activation of an extra-bright red fluorescent protein in "knock-in" Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol. 2007 Jan;37(1):43-53. PubMed PMID: 17171761
