Live imaging of neuroimmunological processes

Research group „Live imaging of neuroimmunological processes“ (Francesca Odoardi)

Field of research

Immune processes require strictly coordinated regulations of their cellular and molecular components in terms of time and space. Targeted therapeutic manipulations of misled immune reactions therefore require an exact knowledge of their time course and dynamics in vivo. The introduction of two-photon laser scanning microscopy for the first time made it possible to visualize immune responses in their natural environment, i.e. in real time within the living organism. This group aims to apply and optimize intravital imaging technologies to analyze the autoimmune attack of the CNS in models of experimental autoimmune encephalitis (EAE).

Why two-photon laser scanning microscopy?

Traditional optical microscopic techniques, including confocal microscopy, which use linear (one-photon) absorption for contrast generation, are hindered in the generation of high quality pictures deep in tissues due to strong and multiple light scattering. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy, has overcome this limitation by providing large depth penetration with reduced phototoxicity/photobleaching (Fig.1). We established this technique in our lab to visualize effector T cell motility in living animals during EAE. The group currently exploits this methodological approach to investigate the following items: how autoaggressive immune T cells invade the nervous system (aim 1) and how they coordinate the autoimmune attack within their target organ (aim 2). Furthermore, the group will use intravital imaging combined with molecular techniques to get deeper insight about the mechanisms of established and newly developed therapies of MS (aim 3).

Principles of two-photon technique.

Principles of two-photon technique.

The two-photon excitation phenomenon is based on the quasi-simultaneous absorption of energy from two photons, each of which contributes one half of the energy required to induce fluorescence. Since the energy of a photon is inversely proportional to its wavelength, the two absorbed photons must have a wavelength about twice that required for one-photon excitation (a); the resulting fluorescence emission varies with the square of the excitation intensity: consequently almost no fluorescence from outside the focal plane is generated and optical sectioning is obtained by the two photon effect, rather than by the use of a pinhole as in the confocal laser scanning microscope (1-photon) (b) The probability of a two-photon absorption event occurring within a fluorophore is extremely low: this problem is overcome by using a femtosecond-pulsed laser which concentrates the output into brief bursts (100 fs) improving the two-photon rate 100,000-fold, compared to a continuous-wave laser operating at the same average power level (c). The Titanium:Sapphire crystal-based laser we are currently using allows wavelength tunability from ~700 – 1000nm providing two-photon excitation of fluorophores in the ultraviolet to green region of the light spectrum (~350 – 500 nm).

 
1.) Effector T cells on their way to the target organ
Encephalitogenic T cells after transfer into recipient animals do not enter directly into the CNS, but rather follow a precise migratory pattern: in the first 24 h, they localize mainly in the lung and in parathymic lymph nodes; afterwards they enter into the blood and spleen. Finally after 3 days, they massively infiltrate meninges and spinal cord and this coincides with the onset of the clinical disease (Fig.2). Live imaging of effector T cells in the preclinical phase in the peripheral immune organs revealed that most of the T cells – independently from the antigen specificity – move at high speed in an apparently random way, establishing transient contact with local stromal cells. The addiction of the specific antigen induces a rapid and dramatic motility change: in ~10 minutes the cells slow down and become tethered to local sessile cells; a part of them form clusters (Fig.3).
On their way to the CNS the effector T cells undergo a profound modification of their expression profile acquiring a specific migratory phenotype, an essential requirement for organ invasion. Factors regulating the migratory properties of an effector T cell population are currently being evaluated.

In vivo imaging of TMBP-GFP infiltration in the leptomeninges during EAE

In vivo imaging of TMBP-GFP infiltration in the leptomeninges during EAE

Panoramic view of the leptomeningeal vessel tree of the dorsal spinal cord obtained by assembling 18 individual 2-photon microscopy scans. The newly incoming effector T MBP-GFP cells (green dots) 2 days after transfer are located mainly inside the vessels labeled by texas red conjugated dextran (red) (a). The T cell number progressively increases in the following hours. In parallel, a part of the cells starts to extravasate (b). Three days post transfer most of the T cells accumulate in the perivascular environment strictly located close to the abluminal surface of the vessel (c). Twelve hours later the cells are diffusely distributed within the pia mater (d). Scale bar 200 µm. Collagen fibers (blue) are visualized by second harmonic generation.

In vivo imaging of effector TMBP-GFP cells in the pre-clinical phase

In vivo imaging of effector TMBP-GFP cells in the pre-clinical phase

Intravital imaging of effector T cell motility in the spleen day 3 post transfer. The vast majority of TMBP-GFP cells (>80%, green dots) is in permanent motion (yellow dot lines), with only a few scattered T cells attached to anchoring points (orange dots) (a, b). The motile TMBP-GFP cells apparently move at random as indicated by their trajectories (c), following a stop and go mode. After i.v. administration of a high dose of specific antigen, the cells completely change their motility pattern by stopping (c,d,e) and forming clusters around potential antigen presenting cells (texas red conjugated dextran-positive cells, red dots) (f).

 
2.) Effector T cells invade the CNS scanning the tissue on at least three levels

The CNS parenchyma is effectively shielded from the blood circulation by specialized vessels, nevertheless the brain is susceptible to autoimmune attacks. By using two-photon laser scanning microscopy, we were able to visualize the behavior of effector T cells during the incipient autoimmune process. We observed that the incoming cells remained in close association with pial blood vessels, crawling on surfaces within the outline of the vessels (Fig.4). This behaviour was specific to the CNS: in peripheral organs, for example in peripheral nerves, muscle or subcutaneous tissue, TMBP–GFP cells mainly rolled along the inner surface of the vessels (Fig 5). After diapedesis the effector T cells continued their scan on the abluminal surface and the underlying leptomeningeal membrane (Fig 4). Based on these observations we are currently investigating the driving forces of T cell motility within the target tissue. In particular we are interested in dissecting the role of chemokines and metalloproteinases a) in the early EAE in the 3 different phases of the meningeal infiltration; and b) in the fully established disease in the CNS parenchyma.
Once inside the leptomeningeal area and in the parenchyma, the effector T cells are reactivated and triggered to release inflammatory cytokines. Two-photon live imaging in the target organs revealed that the invading cells present two different motility patterns: a major proportion of the autoimmune T cells meanders rapidly through the CNS tissue, whereas a minor, though significant, part of the cells seem to become attached to a fixed point. By combining in vivo imaging with confocal microscopy on fixed slides we learned that the stationary cells exhibit polarization of the TCR receptor/LFA1, resembling the immunological synapse, in the contact zone with MHC class II-expressing cells and so are presumably in the process of antigen recognition. Prompted from these observations we are currently trying to functionally characterize these potential antigen-presenting cells (APCs). Moreover, by using a genetically encoded calcium biosensor, we are interested in visualizing the interaction between APCs and the effector T cells in different CNS compartments and in different phases of the disease correlating the calcium signaling pattern with their locomotive behavior.

Effector T cells scan the leptomeningeal area in three different phases

Effector T cells scan the leptomeningeal area in three different phases

Migratory tracks (green lines) of TMBP-GFP cells during the EAE course imaged by in vivo two-photon laser scanning microscopy at the indicated time points (10 minutes time recording). In the early phase the encephalitogenic T cells crawl on the intraluminal surface of the leptomeningeal vessels continuously monitoring the surface (day 2); just after diapedesis, the T cells maintain their scanning behavior on the abluminal surface of the vessels (day 2.5); finally the T cells diffusely distribute over the surface of the neuropil extensively scrutinizing it (day 3).

Crawling behavior is specific to T cells in CNS vessels.

Crawling behavior is specific to T cells in CNS vessels.

Intravital two-photon recording of ear and saphenous vessels. Differently from that observed in the letomeninges, the TMBP-GFP cells mainly roll in the peripheral vessels (labeled with texas red conjugated dextran) appearing in time projection imaging as several dots.

 
3.) How do therapies of autoimmune reactions really work?

The possibility of visualizing effector T cells in their natural environment opens the possibility of better understanding the functional mechanisms behind therapeutic approaches.
Natalizumab is a recombinant humanized IgG4 monoclonal antibody that binds to the Α-4 subunit of the Α4-β1 integrin and inhibits the Α-4-mediated adhesions of leukocytes to their counterreceptors. It has been introduced for the treatment of autoimmune diseases such as multiple sclerosis and Crohn disease. We used our in vivo set-up to explore the mechanism of this well-established treatment. Within a few hours the VLA-4 antibody completely stopped the crawling and transmigration of the TMBP-GFP cells into the CNS parenchyma (Fig. 6). Accordingly the development of neurological deficits was strongly reduced.
Infusion of autoantigenic proteins targeting a specific T cell population has been proposed as a highly efficient therapy to suppress the unfolding of organ-specific autoimmune diseases. We reinvestigated the effect of administration of a high dose of soluble antigen in different phases of EAE. In vivo imaging revealed that administration of the antigen in the preclinical phase completely prevented the disease: antigen specific T cells were paralyzed in the peripheral organs and driven to activation-induced cell death. The same treatment in the clinical phase induced an equivalent change of effector T cells motility pattern in the EAE lesions with a drastic increase of stationary cells (Fig. 3 and Fig. 7). The disease outcome, however, was completely different from the one observed in the preclinical treatment. There was an equally strong aggravation of the clinical symptoms due to a burst of cytokine release within the target organ (Fig. 7). These data support our current working hypothesis that the level of re-activation of effector T cells in the CNS tissues determines the clinical outcome, instigating us to search for therapeutic targets in order to dampen effector T cell activation in the CNS tissues.

VLA-4 antibody treatment completely abolishes intracellular crawling

VLA-4 antibody treatment completely abolishes intracellular crawling

Intravital two-photon recording of effector T cells 2.5 days after transfer: the cells in this phase are mainly located inside the vessels (a,b). The intravenous infusion of VLA-4 antibody during the recording induced an immediate TMBP-GFP detachment from the intraluminal surface, without interfering with the motility of the extravasated cells (c,d). Green: TMBP-GFP cells; red: Texas red conjugated dextran. Time of recording in relation with the antibody treatment is shown. Scale bar 50 µm.

Effect of soluble antigen treatment in the clinical phase of EAE

Effect of soluble antigen treatment in the clinical phase of EAE

Intravital two-photon imaging of TMBP-GFP day 3.5 post transfer in the EAE lesions before (a) and after (b) intravenous infusion of a high dose of soluble antigen. Ninety minutes after treatment the number of stationary cells (blue circles) significantly increased compared to the motile cells (yellow circles). The change of motility pattern was mirrored by an increased production of cytokines measured by real time PCR on sorted effector T cells at the indicated time points after treatment (c and d). Green: TMBP-GFP cells, red: Texas red conjugated dextran. Scale bar 10 µm.

 

Key technologies:

The lab is specialized in the use of intravital imaging by confocal/multi-photon microscopy, autoimmune disease models for MS (active/transfer EAE), construction of retroviral gene vectors including culture and transduction of different immune cells/hematogenic stem cells, gene expression profiling (FACS, real time PCR, microarray, proteomics).

Collaborations:

  • Department of Neuroimmunology, MPI for neurobiology (Prof. H. Wekerle)
  • Group of live imaging in CNS autoimmunity; MPI for Neuroimmunology (Dr. N. Kawakami).
  • Calcium Signaling Group in the University Medical Centre Hamburg-Eppendorf (Prof. A. Guse).
  • Laboratory of Medicinal Chemistry, University of Bath, (Prof. B.V.L. Potter).
  • Institute of Immunology; LMU-Munich (Prof. T. Brocker)
  • Department of Neuropathology, University of Göttingen (Prof. W. Brück).
  • Cellular dynamics lab; MPI for Neurobiology (Dr. O. Griesbeck).
  • Centre for brain research; Medical University of Vienna (Prof. H. Lassmann).
  • Division of Immunology, department of Pediatrics; Dalhousie University, (Dr. T.B. Issekutz)
  • Institute of Anatomy, University of Leipzig (Prof. I. Bechmann)

Relevant Publications:

Schläger C, Körner H, Krueger M, Vidoli S, Haberl M, Mielke D, Brylla E, Issekutz T, Cabañas C, Nelson PJ, Ziemssen T, Rohde V, Bechmann I, Lodygin D, Odoardi F, Flügel A.
Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.
Nature 530 (2016): 349-353. [Pubmed]

Odoardi F, Sie C, Streyl K, Ulaganathan VK, Schläger C, Lodygin D, Heckelsmiller K, Nietfeld W, Ellwart J, Klinkert WE, Lottaz C, Nosov M, Brinkmann V, Spang R, Lehrach H, Vingron M, Wekerle H, Flügel-Koch C, Flügel A. T cells become licensed in the lung to enter the central nervous system. Nature 488 (2012):675-9.

Cordiglieri C*, Odoardi F*, Zhang B, Nebel M, Kawakami N, Klinkert WE, Lodygin D, Lühder F, Breunig E, Schild D, Ulaganathan VK, Dornmair K, Dammermann W, Potter BV, Guse AH, Flügel A. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system. Brain. 2010 Jul;133(Pt 7):1930-43. Epub 2010 Jun 2. * equal contribution.

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

Müller N, van den Brandt J, Odoardi F, Tischner D, Herath J, Flügel A, Reichardt HM. A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats. J.Clin Invest. 2008 Apr; 118 (4):1405-16.

Odoardi F, Kawakami N, Klinkert WE, Wekerle H, Flügel A. Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease. Proc Natl Acad Sci U S A. 2007 Nov 20;104(47):18625-30.

Odoardi F, Kawakami N, Li Z, Cordiglieri C, Streyl K, Nosov M, Klinkert WE, Ellwart JW, Bauer J, Lassmann H, Wekerle H, Flügel A. Instant effect of soluble antigen on effector T cells in peripheral immune organs during immunotherapy of autoimmune encephalomyelitis. Proc Natl Acad Sci U S A. 2007 Jan 16;104(3):920-5.

Kawakami N, Odoardi F, Ziemssen T, Bradl M, Ritter T, Neuhaus O, Lassmann H, Wekerle H, Flügel A. Autoimmune CD4+ T cell memory: lifelong persistence of encephalitogenic T cell clones in healthy immune repertoires. J Immunol. 2005 Jul 1;175(1):69-81.

Kawakami N, Nägerl UV, Odoardi F, Bonhoeffer T, Wekerle H, Flügel A. Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion. J Exp Med. 2005 Jun 6;201(11):1805-14.

Kawakami N, Lassmann S, Li Z, Odoardi F, Ritter T, Ziemssen T, Klinkert WE, Ellwart JW, Bradl M, Krivacic K, Lassmann H, Ransohoff RM, Volk HD, Wekerle H, Linington C, Flügel A. The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis. J Exp Med. 2004 Jan 19;199(2):185-97.