Neuroblastoma express phosphatidlyserine: mechanism for immune evasion Free

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Introduction: We examined the effect of phosphatidylserine (PS) on tumor immunity. PS is a membrane phospholipid that is restricted to the inner surface of plasma membrane in living cells, but flips to the cell surface during apoptosis. PS may be also expressed on the surface of live tumor cells. PS was reported to promote tolerance, possibly by inhibiting antigen presentation and inflammation. The effect of tumor PS on the immune response against neuroblastoma (NB), a childhood malignancy, was examined in a murine model.

Methods: PS on NB cell surface was determined using Annexin-V (AnV) binding and flow cytometry. To block PS in vivo, NB cells were engineered to secrete AnV protein covalently fused to FLAG, which specifically binds and blocks PS. Western blot was used to determine AnV expression. A/J mice were injected with 104 NB cells subcutaneously and tumor growth was determined.

Results: NB cells were transfected with pre-pro-trypsin AnV-FLAG construct. Western blot analysis was used to quantify AnV production in supernatants and identify NB clones that secreted AnV (AnV-NB). Clones that proliferated in vitro at the same rate as wild type (wt) NB cells were selected. Supernatants from AnV-NB cells blocked PS on the surface of wt NB cells preventing staining with FITC-conjugated recombinant AnV. The findings suggest that secreted AnV blocked PS. The wt and AnV-NB cells were then injected subcutaneously into A/J mice. Tumor homogenates showed continuous AnV expression in vivo. In immunocompetent mice, wt tumors grew faster than AnV-NB cells. In mice depleted of T cells with anti-Thy1.2, no difference in growth rate between wt and AnV-NB tumors was noted. Mice were then immunized two times, 7 days apart, with AnV-NB or control cells. Seven days later mice were injected subcutaneously with 105 wt NB cells. Immunization with An-NB, but not control cells, protected mice from wt NB challenge.

Conclusions: The study demonstrated that mouse NB cells express PS on the cell surface. Blocking PS in vivo by engineering NB cells to secrete AnV slowed tumor growth in immunocompetent but not T cell depleted mice. Importantly, immunization with AnV-NB cells was protective. The findings suggest that PS inhibited anti-tumor T cell immunity in mice. The mechanism of PS action may be indirect inhibition of anti-tumor CTL responses, or activation of regulatory T cells. The studies support the hypothesis that PS expression is a potential mechanism for tumor immune evasion.