Talking T Cells

A collaborative research team led by Laura Downie, Holly Chinnery, and Scott Mueller from the University of Melbourne and Melbourne’s Doherty Institute, has made a breakthrough discovery about the immune cell landscape of the cornea. Previously classified as dendritic cells, a new imaging technique has discovered that a large number of cells at the surface of the healthy cornea are T cells.

Here, the research team tells us about the inspiration for this project, the challenges it presented, and what the next steps might be.

What inspired you to explore the immune cell composition of the cornea?
 

Holly Chinnery: The three of us were all independently running preclinical or clinical studies on corneal immune cells: Laura was capturing confocal images in patients, I was assessing changes to corneal immune cells during ocular surface inflammation and injury, and Scott was characterizing the T cell recruitment in the corneas of Herpes-infected mice. We converged our ideas about what we thought was happening in this unique tissue, as in each setting, we could see overlapping morphological features of corneal immune cells.

The most striking observation was the similarities between the highly motile T cells in Scott’s models with the motile immune cells that we were observing in the clinic. This Eureka moment inspired us to pursue the idea of using our mice to learn about the behaviors of immune cell subsets in the cornea, and translate this across to the behaviors of immune cells in human corneas.

Can you outline your research and the key methods you used to obtain results?
 

Laura Downie: By integrating a range of preclinical and clinical research methods, our research has re-defined understanding of the immune cell types present in healthy human corneas. The most exciting method is a new approach called functional-In Vivo Confocal Microscopy (Fun-IVCM). Fun-IVCM enables corneal immune cells to be dynamically tracked in living humans, over the depth of the tissue. To perform the technique, we capture high-resolution en face images of the cornea over time, using a clinical confocal microscope device. We then piece these images together using fixed anatomical features to generate time-lapse videos. With that we see how different types of corneal immune cells behave, how they interact with the corneal nerves, and also how they interact with each other.

Fun-IVCM thus provides a direct window to an intact neuro-immune system in living humans. It’s amazing! Using this method, we were able to separate subtypes of corneal immune cells, based on their shape and dynamics, and show that many of the immune cells resident in the healthy human cornea are T cells, and not another type of immune cell (dendritic cell), as has always been thought.

To complement these in vivo cell analyses, we also performed multi-parameter flow cytometry and immunohistochemistry on human donor tissue, and used specific cell markers to phenotype the cells, providing further evidence for the presence of T cells in healthy human corneas.

Were there any unexpected or surprising results during the course of this research?
 

Scott Mueller: Our imaging of immune cell dynamics before and after contact lens wear revealed a surprisingly rapid change in immune cell behavior. This suggests that small changes in the environment at the ocular surface can be sensed by the local immune cells.

How might the presence of T cells in the cornea contribute to the overall health and function of the eye?
 

Scott Mueller: The presence of T cells in the healthy cornea raises several questions as to the potential roles of these cells in maintaining healthy tissue and protecting against disease. We speculate that T cells in the cornea play similar roles to those in the body’s other tissues, such as the skin and mucosal tissues, where many T cells enter and form long-lived populations of memory cells that can protect against infections. Our prior work in mice showed that this is likely to be the case (1).  Yet, T cells can also contribute to autoimmune disease and other conditions and we will need to learn more about what subtypes of T cells are found in the cornea and whether these are always protective or pathogenic.

Did you encounter any challenges or limitations while conducting the study?
 

Holly Chinnery: A major challenge was gaining access to healthy human corneal tissue, for obvious reasons (they are highly valuable for transplantation). This made it challenging to generate evidence that the immune cells were likely T cells, and not dendritic cells, as we previously thought. We addressed this by collaborating with our colleagues at the University of New South Wales and Westmead Institute, as they were performing flow cytometry of human corneal tissue. We asked them if they would share some of their control tissue data with us – and sure enough, there were T cells in their epithelial preparations.

Another challenge was the development of the time-lapse Fun-IVCM movies, which was critical to identify the cell subsets (based on their dynamic characteristics), as well as evaluate interactions between cells, and between cells and sensory nerves. This time-lapse imaging approach requires repeated imaging of a small area of the cornea (400 x 400 microns) every few minutes over a period of 20-30 minutes. This is not a trivial exercise, not least because of the minor movements of the participants' eyes, and in trying to capture the same area in the corneal stroma where the nerve landmarks are absent. Once the images were captured, we applied our skills in analyzing immune cells using mouse intravital imaging to track the movement and morphologies of the cells. But once we worked out how to capture the Fun-IVCM images, we were on our way!

Are there any follow up studies to further explore the presence of T cells in the cornea?
 

We are very excited about the study findings, which we consider have important implications for understanding both normal human corneal immunology, as well as the pathobiology of corneal disease, and the effect of different ocular surface therapeutic interventions. For example, in the current study we were able to show that corneal immune cell behaviors were altered in response to just a few hours of contact lens wear (i.e., an acute inflammatory stimulus), as well as in people with seasonal allergy (being a representative chronic inflammatory stimulus). We also found that people taking immunomodulatory therapies to treat their allergy had a degree of normalization of the corneal immune cell features. Together, these findings suggest Fun-IVCM imaging could be clinically useful for identifying patients with different profiles of eye inflammation, and also detecting treatment responses.

We now appreciate that there is much still to learn about the corneal immune compartment, including the role of T cells in tissue homeostasis and in different disease states. At our FrontTear Research Centre at the University of Melbourne, we have a program of studies planned to investigate these questions, across a range of eye and systemic conditions.