Use of Intravital Multi-Photon Microscopy to Study In Vivo Migratory Kinetics of Corneal Bone Marrow-Derived Cells Pedram Hamrah, M.D., Dimosthenis Mantopoulos, MD; Lixin Zheng, MD; Ulrich von Andrian, MD, PhD Massachusetts Eye & Ear Infirmary, Department of Ophthalmology & Immune Disease Institute, Harvard Medical School Financial Disclosure: The authors have no financial disclosure related to this project Support: NEI K12-EY016335, New England Corneal Transplant Research Fund, Falk Medical Research Trust

Antigen-presenting Cells Sentinels of the immune system Dendritic cells, macrophages and B cells Dendritic Cells and macrophages are the professional antigen-presenting cells (APC) of the cornea Implicated in corneal transplantation and allergic immunity, microbial keratitis, and dry eye disease

Corneal, unlike limbal, APC are universally MHC class II-negative and immature in the epithelium and stroma Limbus Red = CD45 (Bone marrow marker) Green = Class II Limbus Green = Class II (Maturation marker) Periphery Red = CD11c (DC marker) Green = CD80 (B7 costimulatory marker) Center Red = CD11c Green = CD80 Hamrah et al., Novel Characterization of MHC class II-negative Population of Resident Corneal Langerhans cell-type Dendritic Cells. Invest Ophthalmol Vis Sci, 2002; 43:639-646 Hamrah et al., The Corneal Stroma is Endowed with Significant Numbers of Resident Dendritic Cells. Invest Ophthalmol Vis Sci, 2003; 44:581-589

Hamrah and Dana, Immune homeostasis of the eye: Antigen Presenting Cells in the Eye and Ocular Surface. Encyclopedia of the Eye. Elsevier. In press

Advantages of Multi-Photon Microscopy Deeper imaging into scattering specimens Reduced out of plane photobleaching and photodamage in optically thick specimens Access to nonlinear signals other than fluorescence such as second harmonic scattering

Purpose and Methods The purpose of this study was to dissect migratory properties of antigen presenting cells by novel multi-photon intravital microscopy. Multi-photon intravital microscopy (MPM) of the cornea was applied to investigate localization and trafficking properties of corneal APCs in transgenic mice in steady state and inflammation in vivo.

CD11c-eYFP

MHC class II-eGFP Day 5 Inflammation

MHC class II-eGFP Day 5 inflammation

Normal Inflamed

Results Intravital MPM studies of the normal cornea demonstrated that APCs were sparsely distributed centrally and more dense in the periphery Epithelial and stromal APCs were distinguished by second harmonic generation that visualizes stromal collagen While APCs demonstrated continuous sampling motions in steady state, cells generally did not migrate laterally During inflammation, increased numbers of APCs were demonstrated, exhibiting extreme morphological changes An increase in lateral and vertical migration was shown particularly in stromal subpopulations

Conclusions Our studies are the first to demonstrate long-term migratory kinetics of corneal APCs in steady state and inflammation through high-resolution intravital multi-photon microscopy. Collectively, these models allow for dissecting molecular regulation of APC recruitment to, and migration in the cornea.