Abstract 3980: The immunomodulatory agent L-MTP-PE induces activation and expansion of human γδ T cells capable of lysing osteosarcoma cells.
Authors: Mellinghoff Sibylle , Altvater Bianca , Dirksen Uta , Juergens Heribert , Rossig Claudia , Ahlmann Martina 
Published: August 2014

The outcome of patients with metastatic osteosarcoma remains poor despite aggressive multimodality therapy. Liposomal Muramyl Tripeptide Phosphatidyl Ethanolamine (L-MTP-PE) is a non-specific immune modulator that activates monocytes and macrophages and has shown antitumor activity in osteosarcoma. The mechanism of action of L-MTP-PE remains unresolved. Here we investigated the effects of the agent on lymphocyte subpopulations.

Whole peripheral blood from 9 healthy donors were stimulated with L-MTP-PE for 24 hours. Compared to non-stimulated controls, a proliferative response of CD3+ lymphocytes was found (1.77±0.06 fold expansion, p=&lt;0.001). The strongest effect was observed on the γδ TCR+ CD3+ T cell subpopulation which expanded 5.0±1.0 fold. To further evaluate the effects of L-MTP-PE on γδ T cell proliferation, mononuclear cells were isolated from peripheral blood of 5 healthy donors and cultured in the presence of L-MTP-PE. On day 11, γδ T cells had expanded 4.6±0.6 fold compared to medium-controls. Addition of interleukin-2 (IL-2) to the cultures increased expansion of γδ T cells up to 9.8±1.6. In comparison, the known γδ T-cell stimulatory aminobisphosphonate agent zoledronic acid (ZA) combined with IL-2 induced 25.8±7.2-fold expansion. γδ T cell stimulation by ZA was previously shown to involve inhibition of the mevalonate pathway enzyme farnesyl diphosphate synthase and subsequent increase of isopentyl pyrophosphate levels. To explore the mechanism of γδ T cell expansion by L-MTP-PE, coincubation experiments were repeated in the presence of the HMG-CoA reductase inhibitor mevastatin. Addition of mevastatin resulted in a 5.1±1.8-fold reduction of L-MTP-PE induced γδ T cell expansion, confirming a critical role of the mevalonate pathway in γδ T cell expansion and thus a mechanism comparable to ZA. To explore the in vitro antitumor activity of L-MTP-PE activated γδ T cells, 24 hour coincubation experiments were performed with the osteosarcoma cell lines HOS and SAOS on day 6 of cultures. In a flow-cytometry based cytotoxicity assay, substantial cytolysis of osteosarcoma cells from both cell lines was observed after coincubation with both L-MTP-PE- and L-MTP-PE/IL-2-stimulated γδ T cells. At an effector-to-target-ratio of 1:1, L-MTP-PE-stimulated γδ T cells reduced viable cell counts of HOS and SAOS to 41.4±2.7% and 54.0±7.6%, respectively, and L-MTP-PE/IL-2-stimulated γδ T cells decreased cell counts to 33.7±2.0% and 23.0±0.9%. In comparison, ZA/IL-2-stimulated γδ T cells resulted in reduced viability to 46.2±1.0% (HOS) and 38.9±3.8% (SAOS).

We conclude that L-MTP-PE induces effective in vitro expansion of γδ T cells via a mechanisms involving the mevalonate pathway, in a manner similar to aminobisphosphonate agents. L-MTP-PE-activated γδ T cells have the potential to lyse osteosarcoma cells and may thus be involved in the antitumor effects of this agents.

Citation Format: Sibylle Mellinghoff, Bianca Altvater, Uta Dirksen, Heribert Juergens, Claudia Rossig, Martina Ahlmann. The immunomodulatory agent L-MTP-PE induces activation and expansion of human γδ T cells capable of lysing osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2013-3980