Author

Doran Spencer

Date

8-2011

Document Type

Dissertation

Degree Name

Ph.D.

Department

Department of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Background: In this dissertation, the role of regulatory T cells in influencing the course of uveitis in a murine model is described. Uveitis, inflammation of the intraocular environment, is a significant cause of visual impairment. Regulatory T cells are a naturally occurring, immunosuppressive population whose role in uveitis remains to be elucidated. We hypothesize that regulatory T cells act in the inflamed eye to contribute to the resolution of uveitis in a murine model. Methods: The main technique used to evaluate the uveitis is non-invasive, epifluorescent intravital microscopy of the iris, and the principal parameters evaluated are onset of arrival, overall numbers, and time of disappearance of effector and host cells. The uveitis model involves adoptive transfer of fluorescently-labeled, antigen-specific effector T cells (DO11.10) to a congenic recipient animal harboring differently fluorescent Foxp3[superscript +] regulatory T cells and/or CD11c[superscript +] dendritic cells. The effect of regulatory T cell absence on effector cell behavior, as observed via intravital microscopy, is achieved through use of Foxp3-DTR animals whose regulatory T cells can be depleted through administration of diphtheria toxin. Results: The principal findings of this study are that iris-infiltrating effector T cells and CD11c[superscript +] dendritic cells in our uveitis model are not observed to exhibit a statistically significant change in the observed parameters in the absence of systemic regulatory T cells. Other novel findings include: 1) regulatory T cells are not visible at baseline in the anterior segment of the naive murine eye, including iris, cornea, aqueous humor, ciliary body, except in the limbal vessel arcade of the peripheral cornea. The cells accumulate at the limbal vessel arcade, in contrast to the earskin, in an age-dependent manner, and first appear in both tissues at fourteen days of age. Regulatory T cells were observed in far greater numbers in the palpebral conjunctiva of mice than in the bulbar conjunctiva, which has not been described previously in rodents nor in humans. 2) Regulatory T cells and effector T cells were both observed in high numbers at early time points in the iris of the specific antigen-challenged eye, whereas few were present in the contralateral eye challenged with control protein. These two cells exhibited a consistent ratio of approximately 2-3:1 (effector: regulatory) during the first 48 hours after antigen challenge, before both cell populations decreased significantly. 3) CD11c[superscript +] dendritic cells were observed to exhibit very little day-to-day change in their distribution in the irides of previously unmanipulated animals. However, they would exhibit significant change in response to inflammatory stimulus, both in the experimental eye and, to a much lesser degree, in the untouched, contralateral (sympathizing) eye. 4) Iris-resident CD11c[superscript +] dendritic cells were not observed to take up fluorescently labeled antigen, as determined by double-labeling using intravital and confocal microscopy, in contrast to previous reports. Instead, iris-resident Lysozyme M[superscript +] phagocytic cells were observed to take up the labeled antigen, although their cells numbers did not change in response to antigenic challenge. Conclusions: Our results do not provide evidence of a role of regulatory T cells in contributing to the resolution of uveitis in our uveitis model, as determined by intravital microscopy of effector cell infiltration. This stands in contrast to other uveitis models, and may reflect model-specific differences. Notably, we use activated effector T cells in an adoptive transfer approach, which bypasses potential regulatory T cell control in the draining lymph node, where significant regulatory T cell activity has been noted in other uveitis models. This suggests that in self-limiting uveitis, the innately anti-inflammatory intraocular environment may be able to control inflammatory T cell activity without the contribution of regulatory T cells. Further investigations of the eye-intrinsic anti-inflammatory mechanisms of our model are warranted. The novel finding of regulatory T cells residing specifically in the limbus suggests a possible role of these cells in contributing to corneal immune privilege.

Identifier

doi:10.6083/M4GB2223

School

School of Medicine

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