1. 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

2. 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

3. Corneal, unlike limbal, APC are universally
MHC class II-negative and immature in the epithelium and stroma
Limbus
Red = CD (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:
Hamrah et al., The Corneal Stroma is Endowed with Significant Numbers of Resident Dendritic Cells.
Invest Ophthalmol Vis Sci, 2003; 44:

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

5. 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

6. 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.

7. CD11c-eYFP

8. MHC class II-eGFP
Day 5 Inflammation

9. MHC class II-eGFP
Day 5 inflammation

10. Normal
Inflamed

11. 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

12. 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.