Novel Characterization of Resident Plasmacytoid Dendritic Cells in the Conjunctiva

Abstract

Purpose : Plasmacytoid dendritic cells (pDCs), a highly functional subtype of immune cells, bridge innate and adaptive immune responses. These cells, mainly located in bone marrow and secondary lymphoid organs, are typically not found in peripheral tissues under homeostatic conditions. This study aimed to evaluate the presence, distribution, and maturation level of pDCs in the limbus and bulbar conjunctiva during steady state.

Methods : Naïve 6-8 week old male wild-type C57BL/6 mice were euthanized; limbus and bulbar conjunctiva were excised, and underwent immunofluorescence (IF) staining with CD45 (pan-leukocyte marker), CD11c (conventional and plasmacytoid dendritic cell marker), and PDCA-1 (specific pDC marker) followed by confocal microscopy. Density of pDCs was measured via Imaris (Bitplane). Flow cytometry was performed on single cell suspensions of collagenase-digested limbus and bulbar conjunctiva labeled with CD45, Siglec-H, PDCA-1, B220/CD45R, CD40, CD80, CD86, I-A/I-E, or their respective isotypes. Intravital multi-photon microscopy (MPM) was carried out on 6-8 weeks old DPE-GFP×RAG1^-/- transgenic mice (pDCs tagged with GFP, called pDC-GFP) via an Ultima microscope with MaiTai Ti/Sapphire lasers set at 850nm and 900nm wavelengths. Fluorescent microcopy (Nikon Eclipse) was performed on pDC-GFP mice to study distribution of pDCs.

Results : Naïve limbus and bulbar conjunctiva host CD45⁺CD11^lowPDCA-1⁺ pDCs with a combined density of 472.5 ± 193.5 cells/mm². Flow cytometric analysis of the conjunctiva and limbal cells showed that resident pDCs express CD40 (63.2%), CD80 (69.1%), CD86 (31.5%), and MHC-II (65.8%). Intravital MPM confirmed presence of GFP-tagged pDCs in the limbus and bulbar conjunctiva of naïve pDC-GFP mice.In the limubs, pDCs accompanied limbal vessels. However, in the conjunctiva they were distributed evenly. Fluorescent microscopy demonstrated that pDCs are found at a lower density in the bulbar conjunctiva (151.7 ± 21.6 cells/mm²) versus limbus (335.9 ± 45.5, p=0.002).

Conclusions : The naïve limbus and bulbar conjunctiva are endowed with resident pDCs, which accompany limbal vessels. pDCs in these peripheral tissues are relatively mature as they express maturation and co-stimulatory markers. Currently, we are investigating the functions of these cells.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Intravital multiphoton micrograph of limbus and conjunctiva of pDC-GFP mouse, demonstrating GFP-tagged pDCs. Scale bar; 50µm