Under normal conditions, cells of the immune system are largely excluded from the eye. However, under inflammatory conditions, autoreactive or pathogen-specific T cells can gain entry to the retina, which can be detrimental. Thus, drug development for several autoimmune conditions has focused on the prevention of T cell trafficking to immune-privileged tissues. However, one unforeseen complication of such treatment is the inability to control infections, including ocular toxoplasmosis, the leading cause of infectious posterior uveitis worldwide. An ongoing T cell-mediated immune response is necessary to limit Toxoplasma gondii replication and destruction of the retina. The factors that support the immune response by allowing T cell entry into and/or influencing migratory behavior within the eye following infection remain poorly understood. Chemokines are crucial for T cell migration to sites of inflammation and our preliminary analysis demonstrates increased levels of CXCL10 transcript in the retina and that the majority of T cells that migrated into this site express CXCR3 (the receptor for CXCL10). In addition, administration of CXCL10-specific blocking antibodies leads to a decrease in T cell number in the eye and a corresponding increase in ocular pathology. Finally, preliminary data generated in this laboratory using multi-photon microscopy shows that T cells in the eyes of mice chronically with infected T. gondii exhibit a range of migratory behaviors. This proposal will focus specifically on the role of the CXCL10 on the CD8+ T cell response, which will be tested in the following specific aims:
SPECIFIC AIM I. Do CXCL10 and CXCR3 affect the trafficking of CD8+ T cells during ocular toxoplasmosis? In order to examine the role of CXCL10 in T cell trafficking to the eye, a T cell transfer system will be developed, where activated T cells will be transferred to chronically infected mice and tracked using flow cytometry and immunohistochemistry (IHC). To test the hypothesis that CXCR3 and CXCL10 are required for T cell trafficking to the eye, anti-CXCL10 antibodies and CXCR3-/- T cells will be used. The effect of cell transfer on parasite burden in the presence or absence of chemokine will be examined using real-time PCR and IHC.
SPECIFIC AIM II. Do CXCL10 and CXCR3 influence T cell migratory behavior in the retina during ocular toxoplasmosis? As chemokines influence various aspects of T cell movement and function (20), multi-photon intravital imagining of the retina is proposed, using fluorescently-labeled antigen-specific T cells and transgenic parasites to assess how the absence of CXCR3 or CXCL10 influence the behavior of T cells. Several parameters of migration will be assessed, including, velocity, meandering index, and motility coefficient. The studies outlined in this proposal will define the role of CXCL10 and CXCR3 in T cell-mediated control of T. gondii in the retina. Moreover, we envisage that our results will provide proof of principle for the utility of this model for examining the role of additional cytokines, chemokines, and immunotherapeutics.

Public Health Relevance

Toxoplasma gondii infection of the eye is a cause of blindness. Many people are infected with this parasite, but do not develop eye disease due to a strong response by the immune system. This proposal will identify how the immune system prevents the parasite from causing destruction of the eye.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21EY021314-02
Application #
8258720
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Mckie, George Ann
Project Start
2011-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
2
Fiscal Year
2012
Total Cost
$240,000
Indirect Cost
$90,000
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104