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K.C. McKenna, J.A. Kapp; Simultaneous Enumeration of Ocular Tumor Cells and Tumor Infiltrating Lymphocytes by Flow Cytometry . Invest. Ophthalmol. Vis. Sci. 2003;44(13):762.
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Purpose: Injection of E.G7-OVA tumor cells into the anterior chamber (a.c.) of the eye of mice primes tumor-specific CTL responses. However, CTL fail to eliminate the ocular tumor. The mechanism of immune evasion in this ocular tumor model is not fully understood. The purpose of this study was to develop a method for tracking tumor cells and tumor infiltrating lymphocytes in the eye by flow cytometry. Methods: E.G7-OVA tumor cells (5.0 X 103), the EL-4 (Thy 1.2+) thymoma expressing chicken ovalbumin (OVA) as a surrogate tumor antigen, were injected into the a.c. of B6. PL (Thy 1.1+) mice. At various times, mice were sacrificed, whole body perfusion was performed, and then ipsilateral and contralateral eyes were removed and digested with collagenase. Single cell suspensions of eyes were stained with combinations of anti-CD45, anti Thy 1.2, anti-CD4, anti-CD8, anti αß TCR, and anti CD11b antibodies and then flow cytometric analysis was performed. Results: E.G7-OVA cells were confined to ipsilateral eyes, detectable one day after injection, and increased in frequency 570-fold by day 8. Recipient CD45+ cells expressing CD4, CD8, αß TCR and CD11b were also detected during this time. Conclusions: The E.G7-OVA ocular tumor model in B6 PL mice is ideal for enumerating both tumor cells and immune cells migrating to the anterior chamber. . This model will be used to determine whether the inability of the immune response to reject a.c. tumors is the result of (1) a kinetic difference between tumor growth and the induction of a CTL response, (2) failure of CTL to migrate to the eye, (3) inactivation of CTL in the eye, or (4) antigenic variation of tumor cells in the eye.
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