THEME 4 IN VIVO IMAGING

Killer T cells (red) and helper T cells (green – large) migrate within the brain as a consequence of inflammation caused by malaria parasites (green, small) that infect the blood and sequester in the brain blood vessels (blue).

“Deep imaging by two-photon microscopy reveals immune cells entering the inflamed brain as malaria parasites circulate in blood vessels.”

In this theme we are developing advanced surgical and engineering methods to enable imaging of live tissues, without disturbing their function, or of fixed tissues, where large volumes are imaged to gain tissue-wide views of immunity. This work is being combined with developing microscopes with increased capacity to image at high speeds deep within living animal tissues. We are also developing novel transgenic systems and cell labelling techniques to visualise multiple cell populations and structural features in living animals or animal tissues. Our work provides a 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

The environment is swarming with unseen microorganisms that would continually cause disease given the opportunity. To combat on-going attack, mammals have developed sophisticated immune systems that comprise of a variety of cell types that work together in a co-ordinated fashion to rapidly expel invaders, and successfully prevent lethal infections. Most immune responses start within immune hubs called lymph nodes or in the spleen, which are organs located deep within the body, sites that are challenging to visualise. To better understand immunity, we aim to observe the coordinated responses of immune cells deep within tissues by creating tools to simultaneously monitor multiple populations of cells, by improving microscopes for deeper penetration of vital organs, and by developing methodologies for labelling cells and structures within these tissues.

In 2019, Imaging CoE scientists successfully used intravital two-photon microscopy combined with tissue clearing to visualise B cell interactions with dendritic cells in lymph nodes and the spleen during the development of antibody responses. This work uncovered an important communication method between immune cells which is essential for initiating immune responses.

In other studies, using a thick spleen sectioning approach combined with intravital two-photon imaging and extensive confocal analysis, scientists deciphered how helper T cells exert fine control over the development and subsequent migration program of killer T cells in the spleen in response to infection. These studies are beginning to reveal important roles for immune cells in control of infections, and the regulation of immunity to outline critical factors that influence generation and maintenance of the complex immune network.

To allow high-speed imaging deep within tissues, CoE scientists have developed adaptive optics for video-rate aberration correction in two-photon microscopy. This work greatly advances image resolution together with speed of image capture. Our pioneering studies help set the scene for advanced understanding of how the immune system fights infection and cancer.

In this field, our scientists are working to solve major questions including, how immune cells interact with invading pathogens, how immune responses to infections begin and how cells co-operate to fight microbes and cancer cells. By mapping the behaviour of various immune cell populations during the various phases of immunity, we will build insights 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

B cells recognise antigens on dendritic cells

B cells are responsible for the production of antibodies, which are secreted proteins that bind to infectious agents or toxins and neutralise their activity. While B cells recognise a vast spectrum of antigens, their initial activation, required for the generation and secretion of the antibody, usually requires presentation of the target antigen on a surface membrane. This enables receptor cross-linking and signal transduction and is achieved by the help of other cell types, which capture the relevant antigens and present them to B cells.

The two main cell types implicated in native antigen presentation to B cells are follicular dendritic cells, which reside in B cell follicles, and CD169+ macrophages, which line the antigen-exposed surfaces of these follicles in both the lymph nodes and the spleen. There is mounting evidence, however, that conventional dendritic cells (cDC) can also participate in native antigen presentation to B cells. cDC can be divided into two major subsets, cDC1 and cDC2, and this role has been largely attributed to cDC2.

Studies by CoE scientists have now revealed that cDC1 may also contribute to native antigen presentation to B cells for their initial activation. Using tissue clearing techniques, as well as live intravital imaging by two photon microscopy, cDC1 were shown to localise adjacent to regions occupied by B cells. By targeting antigens to these cDC1, interactions between these cDC and specific B cells were shown to be critical for initiation of antibody production.

Better understanding of how cDC1 participates in B cell priming is likely to improve our capacity to develop effective humoral vaccines.

Thick spleen section visualised by two-photon microscopy showing the location of cDC1 (green) in close proximity to B cell follicles (red) and T cell zones (blue).

ACHIEVEMENT

Imaging the migration patterns in a developing immune response

In 2019, work in this Imaging CoE research theme has focused on mapping immune cell migration and mathematically modelling this function. We have developed a mathematical model, for B cell migration within follicles, deciphering the interaction between B cells and bordering immune cells, movement of T cells in the liver, after modelling the migration within the sinusoids of this tissue.

In a challenging study, we have investigated the control helper T cells have over killer T cell migration, within the spleen during the onset of immunity to malaria. This study used thick spleen sectioning and two-photon microscopy to map the evolution of helper and killer T cell responses, showing that helper T cells are first to leave the initial site of immune cell interactions in the spleen, i.e. the white pulp, and move to the red pulp before exiting the spleen. This migration is mirrored by killer T cells which are slightly delayed in the migration, a process revealed to be coordinated by the helper T cells.

By using transgenic T cells specific for malaria and labelling techniques that enable clear distinction of helper and killer T cells and their surrounding environment, CoE scientists have discovered an unappreciated role for helper T cells in coordinating the movement of killer T cells. This understanding provides insight in how immune armies can be gathered in lymphoid tissues like the spleen and then directed to other areas of the body, such as the brain, to fight infection or cause immunopathology.

Scientists from the CoE have also played a role in understanding how the immune system controls cancer, visualising immune cells called tissue-resident memory T cells in close association with small pockets of cancer cells, kept in check but not fully eradicated by this interaction.

In other studies, CoE scientists have visualised immune cells as they undergo immune surveillance of tissues such as the liver and skin. These studies help understand how vaccines provide protection and how immune responses to infections ensure life-long immunity to reinfection.

Localisation of CD4 T cells (green) and CD8 T cells (red) in the spleen during initiation of immunity to malaria.