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Purpose: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model., Methods: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy., Results: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min., Conclusions: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is assumed to be a key location for the generation of adaptive immune mechanisms of the ocular surface, but functional studies of CALT are still lacking. The purpose of this study was to establish an animal model that enables functional analysis of immune mechanisms going on within CALT. In addition, the use of two-photon microscopy, a new optical method, was evaluated for examining complex immunologic interactions of CALT by volume (three-dimensional [3-D]) and time-dependence (four-dimensional [4-D]) in vivo., Methods: The conjunctiva of female BALB/c mice was repeatedly challenged with topical Chlamydia trachomatis serovar C or a solution of ovalbumin and cholera toxin B. Two-photon microscopy was conducted on explanted, unfixed, and unstained eyes with adjacent nictitating membranes., Results: After three to five stimulations, CALT was detected exclusively in the nictitating membrane of 73% (C. trachomatis) or 70% (ovalbumin/ cholera toxin) of the animals. CALT mainly consisted of CD45R/B220+ B cells and CD4+ and CD8+ T cells. Electron microscopy showed intraepithelial lymphocytes and follicles consisting of lymphocytes, dendritic cells, and macrophages. Two-photon microscopy based on tissue autofluorescence allowed all components of CALT to be detected three dimensionally. High-resolution images were generated in tissue depths of 65 microm below the mucosal surface., Conclusions: This study introduces a novel mouse model for functional investigations of CALT. Topical stimulation with C. trachomatis or ovalbumin/cholera toxin B reliably leads to CALT generation at the nictitating membrane. The use of two-photon microscopy enables groundbreaking 3-D and, in the future, intravital 4-D investigations of immunologic processes initiated in CALT.