THEME 4 IN VIVO IMAGING

“Skin resident memory T cells proliferate in response to virus infection.” Multi-colour imaging the skin using dual laser two-photon microscopy to identify tissue-resident T cells responding to virus infection. Image shows a single frame of a movie identifying virus-infected cells (light blue), skin collagen fibres (dark blue), nerve fibres (green), hairs (white) and tissue-resident memory T cells (red).

 “Achieving visualisation of multiple cell types and structures by intra-vital imaging provides new insight into biology.”

In this theme, we are developing microscopes with increased capacity to image at high speed deep within living animal tissues. This work is being combined with development of advanced surgical and engineering methods to enable imaging of live tissues without disturbing their function. We are developing labelling techniques and novel transgenic systems to visualise multiple cell populations and structural features in living animals or their tissues. These objectives provide highly visual, novel understanding of the biology of immune cells as they migrate, interact and function in their natural environment.

PROF. WILLIAM (BILL) HEATH

AT A GLANCE

In 2018, Imaging CoE scientists successfully used intravital two-photon microscopy to monitor the function and fate of tissue-resident memory T cells and other immune cells in skin, after viral infections or exposure to tumour cells, and in the liver during development of immunity and beyond. They also visualised B cells in lymph nodes and the spleen during the development of antibody responses.

These studies are beginning to reveal important roles for immune cells in control of infections and cancer and to outline critical factors that influenced generation and maintenance of immune populations in animal models. Our work sets the scene to enable manipulation of immunity in favour of protection from invading pathogens such as Plasmodium berghei, which causes malaria. It’s our use of two-photon microscopy and novel transgenic mouse models that has achieved sophisticated imaging to enable visualisation of immune cells in complex tissue environments.

The immune system is comprised of a variety of cell types that must interact with each other during initiation of immunity and then later during the battle with invading microbes. Interactions that initiate immunity occur deep within lymphoid tissues, i.e. the spleen and lymph nodes, which are challenging organs to visualise. To address this goal, Imaging CoE scientists are creating tools to simultaneously monitor multiple populations of cells, improving microscopes for deeper penetration of vital organs, and developing methodologies for labelling cells and structures within these tissues.

In this field, our scientists are now working to solve major questions about immune cell interactions with invading pathogens, how immune responses to infections begin and how cells co-operate to fight microbes and cancer cells. We anticipate that by mapping the behaviour of various immune cell populations during the various phases of immunity, we will be able to build insight into the regulation of this complex but vital system.

 

ACTIVITY PLAN

  1. Develop clearing techniques for spleen, liver and lymph nodes to enable whole organ imaging leading to advances in our understanding immune cell functions during immune responses.
  2. Identify multiple cell types within tissues using an immunohistocytometry approach to identify and map the location of multiple different cell types simultaneously in static images.
  3. Improve depth of intra-vital imaging by developing adaptive optics, rapid axial scanning and real-time image processing for 3D imaging deep in living tissues, e.g. spleen.
  4. Mathematically model immune surveillance of the liver, lymph nodes and spleen by identifying and measuring immune cells subset movement and function within these tissues, mapping tissue architecture and then modelling behaviour.

HIGHLIGHT

Tissue resident memory t cells proliferate locally

Memory T cells develop from naïve T cells after exposure to new infections or vaccines. These cells are crucial for protecting against future infections by the same organism, the basis of vaccination. There are three main types of memory T cells, central memory T cells, effector memory T cells and tissue-resident memory T cells (TRM cells). While the former two populations recirculate throughout the body, TRM cells remain permanently within any tissue they seed during the initial infection or vaccination. Their tissue localisation places them in the front-line for preventing future infections. While it has been clear that TRM cells can survive long-term, perhaps indefinitely, in tissues such as the skin and brain in the absence of any further infections, it was unknown how they responded to re-infection or how unrelated infections affected their survival.

Imaging CoE scientists used advance two-photon imaging and flow cytometry approaches to show that upon re-infection with the same virus, skin TRM cells engaged virus-infected cells, proliferated in situ in response to local antigen encounter
and remained in the skin, where they exclusively reside.

As a consequence of proliferation, new secondary TRM cells formed from pre-existing primary TRM cells. In addition to this source, new primary TRM cells could develop, recruited from precursors in the circulating memory T cell pool.

By examining responses to multiple different antigens in the one animal, we showed that new antigens could recruit new waves of TRM cells and that these new waves did not displace cells of the pre-existing TRM cell pool. Our studies revealedthat multiple TRM cell specificities could be stably maintained within the skin.

Our results reveal the complexity of TRM cell maintenance(in the skin) and provide insight into how we can maintain immunity to multiple infective agents without diminishing individual responses.

 

Specific TRM cells (green) attacking virus-infected skin cells (light blue), while non-specific TRM cells (red) ignore infection

ACHIEVEMENT

Developing live imaging of the liver and its multiple immune cell types

In 2018, work in this Imaging CoE research theme has focused on improving liver imaging. We have developed imaging surgery and engineering to reduce image artefacts and improve tissue stability. This has led to unprecedented quality of live-cell multi-photon imaging of this tissue
(see image below).

Our investigations have revealed that during most immune responses, activated killer T lymphocytes enter the liver and convert into a liver-resident population that are stably maintained in this organ. These cells are important for fighting intracellular infections of the liver, such as pre-erythrocytic malaria, and can be visualised migrating around the liver in the small blood vessels called sinusoid (see accompanying image).

By studying multiple waves of T cells entering the liver, Imaging CoE investigators revealed that individual populations decayed in number over time, but that new populations did not displace older populations, enabling large numbers of T cells with multiple specificities to inhabit the liver over time.

This is likely important to maintain immunity to the variety of parasites, viruses and bacteria that may infect the liver over a life-time under natural conditions.

This research theme has also enabled visualisation of another population of T lymphocytes within the liver, i.e. the helper T lymphocytes. Like killer T lymphocytes, they can be seen migrating within the sinusoids in an ameboid-like patrolling manner. We have revealed that upon infection with malaria parasites, these cells form clusters throughout the liver, presumably to attack the invading pathogen. We are currently developing tools and reagents for imaging multiple components of this response.

In collaboration with associate investigator Professor Steve Lee (ANU), we have obtained images of the spleen and other tissues with the newly-developed multi-photon system, revealing detailed cellular images at unprecedented speed.

 

Migration of killer T lymphocyte (green) through liver sinusoid (red).