Modulation of tumor sensitivity to effector mechanisms of cytotoxic lymphocytes

Abstract

Today, ample evidence demonstrates a clear role for the immune system in the battle against cancer. However, the relatively high rate of mutation and proliferation of tumor cells, in combination with the selective pressure exerted by the immune system, can potentially lead to the generation of genetically altered tumor cells, which are able to evade recognition by the immune system and continue to grow and form tumors. Increased knowledge of the mechanisms allowing tumors to escape from the immune system is of great importance in facilitating the design of effective immunotherapeutic regimens against cancer. The work described in this thesis was aimed at identifying new mechanisms of tumor escape as well as possible ways to counteract them.

We have identified TNF-alpha as a potent modulator of MHC class I antigen presentation in tumors. TNF-alpha-treatment led to enhanced expression of several molecules in the MHC class I antigen processing and presentation pathway, including the IFN-inducible subunits of the proteasome, LMP2, LMP7 and MECL-1, the transporters associated with antigen presentation (TAP) and MHC class I heavy chain. These changes resulted in increased stability of surface MHC class I complexes, presumably due to an increased supply of peptides suitable for binding to MHC class I molecules, and enhanced susceptibility of TNF-alpha-treated tumors to antigen-specific lysis by cytotoxic T-lymphocytes (CTLs). Our results suggest a role for TNF-alpha as a potent immunomodulator in IFN-gamma unresponsive tumors.

Investigating the possible effects of cytokines on the sensitivity of tumor cells to different CTL effector mechanisms, we found that IFN-gamma protects uveal melanoma cells from CTL-mediated lysis. We also demonstrated that despite potent upregulation of antigen presentation in uveal melanoma cells, IFN-gamma-treated tumor cells were less sensitive to lysis by CTL. Granzyme B is an apoptosis-inducing effector molecule released by CTLs upon triggering of the T-cell receptor. IFNgamma-treated uveal melanoma cells bound less granzyme B than their untreated, or TNF-alpha-treated, counterparts. Cleavage of the granzyme B substrate Bid was reduced in uveal melanoma cells following treatment with IFN-gamma.

This correlated with a reduced expression of the cationindependent mannose-6-phosphate receptor (CI-MPR), a receptor for granzyme B, and decreased CTL-lysis of IFN-treated uveal melanoma cells. In another study, we examined the regulatory role of IFN-gamma on the sensitivity of uveal melanoma cells to the lytic activity of perforin, another major constituent of cytolytic granules. We demonstrated that IFN-gamma induces resistance of uveal melanoma cells to plasma membrane lysis by perforin.

This was not a result of proteolytic inactivation of perform by either cathepsin B, known to protect CTL from perforin-mediated suicide, or other proteases. Protection from perforin lysis correlated with IFN-gamma-induced growth arrest in the G1-phase of the cell cycle, and reduced binding of perform to IFN-gamma-treated OCM1 cells. In light of the current data, we propose a mechanism were IFN-gamma-induced growth arrest leading to structural changes in the plasma membrane results in decreased perforin binding capacity of the tumor cell and protection from perforin. Our results demonstrate that, in response to IFN-gamma, tumors can escape the immune system through the active acquisition of a CTL-resistant phenotype, characterized by impaired sensitivity to granule-mediated killing.

The second major effector mechanism employed by CTL is the engagement of death receptors expressed on target cells. The production of soluble Fas ligand (sFasL) completely protected uveal melanoma cells from killing via Fas. Inhibition of metalloproteases on the surface of tumor cells prevented shedding of Fast, and rendered uveal melanoma cells sensitive to Fasmeditated lysis by CTL. The protective effect of Fast, was not due to tumor counter-attack or reduced lytic potential of CTL, but transfer of sFasL-containing culture supernatant protected normally Fas-sensitive cells from killing induced both by FasLexpressing lymphocytes and a agonistic antibody to Fas. We speculated that soluble Fast, bind to Fas receptors expressed on tumor cells, thereby preventing their activation by Fas-inducing effector molecules. Our findings demonstrate the existence of a novel mechanism of tumor escape from death receptor-mediated killing by cytotoxic lymphocytes, and point to a new rationale for the use of metalloprotease inhibitors as cancer therapeutic agents.