Ocular Tumor Associated CD11b+ Myeloid Cells Inhibit CD8+ CTL Responses via Nitric Oxide Production

Abstract

Purpose:: We have previously demonstrated that the failure to reject tumor cells injected into the anterior chamber (AC) of the eye correlates with the accumulation of CD11b+ myeloid cells that inhibit CD8+ CTL responses in vitro. The purpose of these studies was to determine the mechanism of CD8+ CTL inhibition by ocular tumor associated CD11b+ cells.

Methods:: CD11b+ cells were isolated by flow cytometric cell sorting from collagenase-digested eyes of C57Bl/6 mice nine-ten days after E.G7-OVA tumor cells were injected in the AC. Ocular tumor associated CD11b+ cells were then added to OVA-specific (OT-I) CTL cultures stimulated with SIINFEKL peptide in the presence or absence of an inhibitory L-Arginine analog, L-NMMA, and lytic activity was measured. OT-I numbers and proliferation, evaluated by CFSE dilution, were also determined. mRNA expression for enzymes involved in L-Arginine metabolism in tumor bearing eyes was quantified relative to unchallenged eyes by RT-PCR. Tumor growth in the AC of wild type and inducible Nitric oxide synthase (iNOS/ NOS-2) deficient mice was measured.

Results:: The addition of ocular tumor associated CD11b+ cells significantly reduced the number of OT-I CTL in cocultures. The remaining OT-I had proliferated, as evidenced by CFSE dilution, suggesting that ocular tumor associated CD11b+ may induce apoptosis of CTL in vitro. Suppression of CTL responses by ocular tumor-associated CD11b+ cells was abrogated by the addition of L-NMMA, suggesting that L-arginine metabolism contributes to CTL suppression. L-arginine is metabolized by Nitric oxide synthases (NOS) and Arginases (ARG). Ocular NOS1, NOS2, and ARG1 but not NOS3 mRNA expression was increased as tumors developed and CD11b+ cells accumulated in the eye. Eleven days after tumor administration mRNAs for NOS1 increased 6-fold, NOS2 increased 100-fold, and ARG1 increased 19-fold in comparison to naïve eyes. Tumor growth in the AC of NOS2 deficient mice was reduced 2.05 ± 0.45 fold in three independent experiments suggesting that Nitric oxide production promotes tumor growth in the AC.

Conclusions:: Nitric oxide produced by ocular tumor associated CD11b+ cells may contribute to immune evasion by primary ocular tumors by inducing CD8+ CTL apoptosis within the tumor microenvironment.

Keywords: immune tolerance/privilege • immunomodulation/immunoregulation • nitric oxide