Kawakami Lab – Live imaging of autoimmunity

One of the key pathological features of Multiple Sclerosis, a human autoimmune disease, is mononuclear cell infiltration within the central nervous system (CNS). Among those cells, autoantigen-specific CD4+ T helper cells play a central role in initiation of CNS inflammation. The aim of our work is to visualize and to understand the cellular and molecular mechanisms of T cell infiltration into the CNS beyond the blood-brain barrier and induction of CNS inflammation.

We use intravital two-photon microscopy to visualize genetically labelled autoreactive T cells in Experimental Autoimmune Encephalomyelitis (EAE), an animal model for Multiple Sclerosis. For T cell labelling, conventional Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) are used. Additionally, we perform functional imaging by using calcium sensing proteins.. Flow cytometry, immune fluorescence confocal imaging and quantitative PCR are used to support findings from intravital imaging.

The encephalitogenicity of T cells depends on TCR specificity and host. We have shown previously that the encephalitogenic potential is also related to T cell activation in the CNS. Highly encephalitogenic T cells were activated in the CNS, whereas weakly encephalitogenic T cells were not. However, it is largely unknown what factors contribute to different degrees of T cell activation. We visualized and compared the behaviour of highly and weakly encephalitogenic T cells in the animal. We are especially interested in differences in antigen presentation within the CNS.

The encephalitogenic T cells migrate into the CNS through the Blood-Brain barrier, a tight membrane which separates CNS and periphery. Although integrina4, which is the target of Natalizumab, one of the most effective treatments for MS patients, is known to be involved, the detailed molecular mechanism for this step is still largely unknown. We are exploring the molecule which can be used as therapeutic target.

Varying calcium concentrations within migrating T cells were detected using a FRET-based calcium biosensor. Images were acquired by intravital two-photon microscopy. Encephalitogenic T cells are activated in the CNS; however, the percentage of activated T cells goes only up to 40%. By conventional immunological analysis, it is hard to answer where and how T cells are activated within the target organ. In contrast, by using a recently developed calcium sensing protein, we can visualize T cell activation in vivo at single cell level. To this end, we express Fluorescence Resonance Energy Transfer (FRET) based calcium sensing protein in T cells and use them for intravital two-photon imaging.

Encephalitogenic T cells exist in healthy individuals although they do not induce inflammation in the majority of cases. Recent findings suggest that gut microbiota trigger the first step of inflammation by stimulating T cells to initiate CNS infiltration. By using a transgenic mouse line with high numbers of encephalitogenic T cells in combination with activation sensors and intravital imaging, we try to visualize the stimulation in the gut-associated lymphoid tissues and unveil the mechanism of the process. In addition, we study the phenotypical changes of those encephalitogenic T cells following stimulation.

2018

2017

2016

Kawakami N. In vivo imaging in autoimmune diseases in the central nervous system. Allergol Int. 2016 Jul;65(3):235-42.

2014

2013

Pohl M, Kawakami N, Kitic M, Bauer J, Martins R, Fischer MT, Machado-Santos J, Mader S, Ellwart JW, Misu T, Fujihara K, Wekerle H, Reindl M, Lassmann H, Bradl M. T cell-activation in neuromyelitis optica lesions plays a role in their formation. Acta Neuropathol Commun. 2013 Dec 24;1:85.

Mues M, Bartholomäus I, Thestrup T, Griesbeck O, Wekerle H, Kawakami N*, Krishnamoorthy G. Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator. Nat Med. 2013 Jun;19(6):778-83. (* co-senior author)

Pesic M, Bartholomäus I, Kyratsous NI, Heissmeyer V, Wekerle H, Kawakami N. 2-photon imaging of phagocyte-mediated T cell activation in the CNS. J Clin Invest. 2013 Mar;123(3):1192-201.

Alumni

Isabel Bauer

Cuilien Du

Clara Seizer

Mary Claire Tuohy

Ping Fang, post doctoral fellow, UCLA, USA

Max Frank

Mariam Wörner

Nikolaos Kyratsous

Marija Pesic

Elham Saghaei

Ingo Barholomäus

I have been working on the function of encephalitogenic T cells during CNS inflammation for almost 20 years. I would like to understand how those encephalitogenic T cells migrate into the CNS, become activated and cause local inflammation. To answer these questions, I have establihsed and still use fluorescent protein based activation sensors and intravital imaging. Key research achievements include the first imaging of encephalitogenic T cells in a living animal (Nature 2009) and the detection of encephalitogenic T cell activation by using NFAT-GFP fusion proteins (JCI, 2013) and calcium sensing proteins (PNAS 2017).

Training

1992 – 1996 Bachelor Pharmaceutical Sciences, Osaka University, Japan

1996 – 1998 Master Pharmaceutical Sciences, Osaka University, Japan

1998 – 2001 PhD Pharmaceutical Sciences, Osaka University, Japan

2001 – 2002 Postdoctoral fellow at Dept. of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, Germany

Academic positions & appointments

2003 – 2011 Group leader at Dept. of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, Germany

Since 2011 Group leader at Institute of Clinical Neuroimmunology, LMU Munich, Germany

2013 Habilitation in Neuroimmunology, LMU Munich, Germany

2015 – 2020 Heisenberg fellowship

since 2015 Member and research group leader at the Biomedical Center, LMU Munich, Germany

Collaborative Research

2008 – 2011 Member and PI of the Collaborative Research Center 571 “Autoimmune reactions: From manifestations and mechanisms to therapy”

since 2016 Member and PI of the Collaborative Research Center 128 “Initiating/effector versus regulatory mechanisms in Multiple Sclerosis – progress towards tackling the disease”

Awards & honors

2001 – 2002 Research fellowship from Alexander von Humboldt Foundation

2006 Helmut Bauer prize for a Young Scientist in Multiple Sclerosis Research

2015 – 2020 Heisenberg fellowship

5 key papers

Mues M, Bartholomäus I, Thestrup T, Griesbeck O, Wekerle H, Kawakami N*, Krishnamoorthy G. Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator. Nat Med. 2013 Jun;19(6):778-83. (* co-senior author)

Pesic M, Bartholomäus I, Kyratsous NI, Heissmeyer V, Wekerle H, Kawakami N. 2-photon imaging of phagocyte-mediated T cell activation in the CNS. J Clin Invest. 2013 Mar;123(3):1192-201.

Bartholomäus I, Kawakami N, Odoardi F, Schläger C, Miljkovic D, Ellwart JW, Klinkert WE, Flügel-Koch C, Issekutz TB, Wekerle H, Flügel A. Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions. Nature. 2009 Nov 5;462(7269):94-8.