Established Tumors Developing in the Anterior Chamber Induce Depletion of Effector CD8+ CTL in Lymphoid Tissues

Abstract

Purpose: : Certain immunogenic tumors grow progressively in the anterior chamber (a.c) of the eye despite the induction of tumor-specific CD8+ cytolytic T lymphocyte (CTL) responses which suggests that CTL responses are somehow inhibited within the ocular tumor microenvironment. The purpose of these studies was to follow the fate of transferred tumor-specific CTL effectors in mice with established ocular tumors.

Methods: : The EL-4 thymoma transduced to express OVA as a surrogate tumor antigen was utilized and OT-I T cell receptor (TCR) transgenic mice, in which the majority of CD8+ T cells express a TCR which recognizes OVA 257-264 (SIINFEKL) peptide complexed with MHC Class I K^b were used as a source of naïve OVA-specific CTL precursors. Effector CTL were generated by in vitro stimulation of OT-I lymphocytes with SIINFEKL peptide for three days and then 3.0 X 10⁶ CTL were intravenously (i.v.) transferred into mice before or seven days after 10⁴ - 10⁵ E.G7-OVA cells were injected into the a.c. of Thy 1.1 congenic B6.PL mice. E.G7-OVA tumors (CD45+, Thy 1.2+, CD8 negative), transferred CTL (CD45+,Thy 1.2+, CD8+), and tumor associated CD11b+ myeloid cells were monitored in vivo by flow cytometric analysis of secondary lymphoid organs, blood, and collagenase digested tumor bearing eyes.

Results: : i.v. transfer of OVA-specific CTL prior to a.c. injection of 10⁵ E.G7-OVA reduced ocular tumor numbers by 70% four days after challenge in comparison to control mice given PBS i.v. Elimination of tumors in the a.c. was associated with ocular infiltration of transferred CD8+ CTL and a 5-fold increase in CD11b+, F4/80+, GR-1 negative myeloid cells in comparison to control mice. Transferred CTL were also observed in the draining lymph nodes (0.39+/- 0.21 %), spleen (1.8 +/- 0.58%), and blood (3.3 +/- 0.74 %). In contrast, four days after CTL transfer of mice with established day 7 eye tumors, the frequency of transferred CTL were markedly reduced in the draining lymph nodes (0.07 +/- 0.04 %) and spleen (0.23 +/-0.18%) and CTL were not observed within ocular tumors suggesting that transferred CTL were deleted or sequestered to other sites as a result of the established ocular tumor microenvironment.

Conclusions: : Tumors developing within the a.c. may inhibit tumor-specific CTL responses by limiting the number of tumor-specific CD8+ CTL within the ocular tumor microenvironment by systemic deletion or sequestration of CTL away from the eye.

Keywords: immunomodulation/immunoregulation • immune tolerance/privilege • tumors