Isopentenyl pyrophosphate secreted from Zoledronate-stimulated myeloma cells, activates the chemotaxis of γδT cells

https://doi.org/10.1016/j.bbrc.2015.05.118Get rights and content

Highlights

  • The conditioned medium from RPMI8226 myeloma (MM) cells treated with zoledronic acid (ZOL) activates the γδT cell migration.

  • RPMI8226 cells secrete isopentenyl pyrophosphate (IPP), a metabolite of mevalonate pathway.

  • A micro total analysis system-based chamber analysis clearly shows the γδT cell chemotaxis by IPP.

  • IPP activates the chemotaxis of γδT cells toward target myeloma cells treated with ZOL.

Abstract

γδT cell receptor (TCR)-positive T cells, which control the innate immune system, display anti-tumor immunity as well as other non-immune-mediated anti-cancer effects. γδT cells expanded ex vivo by nitrogen-containing bisphosphonate (N-BP) treatment can kill tumor cells. N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for γδT cells. We have previously observed that as they get closer, migrating γδT cells increase in speed toward target multiple myeloma (MM) cells. In the present study, we investigated the γδT cell chemotactic factors involving using a micro total analysis system-based microfluidic cellular analysis device. The addition of supernatant from RPMI8226 MM cells treated with the N-BP zoledronic acid (ZOL) or the addition of IPP to the device induced chemotaxis of γδT cells and increased the speed of migration compared to controls. Analysis of the ZOL-treated RPMI8226 cell supernatant revealed that it contained IPP secreted in a ZOL-dose-dependent manner. These observations indicate that IPP activates the chemotaxis of γδT cells toward target MM cells treated with ZOL.

Introduction

γδT cell receptor (TCR)-positive T cells, which control the innate immune system, display anti-tumor immunity as well as other non-immune-mediated anti-cancer effects. We have previously demonstrated that nitrogen-containing bisphosphonate (N-BP) treatment expands γδT cells ex vivo and that these expanded cells can kill tumor cells in a major histocompatibility complex (MHC)-unrestricted manner [1], [2], [3]. N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP) [4], which is a stimulatory antigen for γδT cells. γδT cells stimulated by the N-BP zoledronic acid (ZOL) recognize tumor cells via several molecules including IPP, MHC class I-related chain gene A (MICA), ICAM-1, CD166, and CD266 [2], [5], [6], [7], [8].

Following the Encyclopedia of DNA Elements Projects, the analyses of proteomes as well as cellomes have attracted considerable attention across the life science. Research in these fields has recently focused on the elucidating vital mechanisms for the development of novel therapeutic strategies against various diseases. Essential in parallel is the development of new devices for clarifying the mechanisms of cellular functions, including chemotaxis and secretion of hormones. A transwell chamber system has traditionally been used to analyze cell migration toward stimulators. However, this device is often inadequate in providing a reproducible, controllable, and stable linear gradient. Furthermore, a transwell chamber system is not compatible with live cell imaging [9], [10]. To overcome these problems, the lab-on-chip or micro total analysis system (μTAS) has been recently applied. A μTAS controls the microenvironment of cells, enabling their biological behaviors, including chemotaxis, differentiation, and cell–cell interactions, to be analyzed [10], [11], [12], [13]. The microvalve is a particularly useful tool for investigating the fast-response of cells [10]. We have developed a micro total analysis system (μTAS)-based microfluidic cellular analysis device, which possesses a minute-volume (240 nL) chamber integrated with a microsample injector controlled by a microvalve that permits the injection of a small amount (several nL) of a solute [14], [15]. We have previously demonstrated real-time monitoring of antibody secretion from B cells using this microchip [14]. Moreover, because of the minute size of this chamber, we can analyze the behavior of individual cells in response to stimulators, including drugs and chemokines. Importantly, fluid convection or stirring has no influence on a concentration gradient and the solute can consequently spread in a diffusion-dependent manner (Supplementary Fig. S1). In the present study, we investigated γδT cell chemotaxis using a μTAS-based microchamber device. Our results show that ZOL-treated MM cells secrete the metabolite of mevalonate pathway IPP and that IPP activates the γδT cell migration.

Section snippets

Reagents

The human RPMI8226 MM cell line was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and was cultured in RPMI1640 (Gibco, Tokyo, Japan) containing 10% heat-inactivated FCS (Gibco), l-glutamine (Gibco), and 1% penicillin-streptomycin (Gibco). The synthetic pyrophosphomonoester compound 2-methyl-3-butenyl-1-pyrophosphate was kindly provided by Dr. Yoshimasa Tanaka (Kyoto University, Kyoto, Japan). Recombinant human interleukin-2 (rhIL-2) was

Migration of γδT cells was activated by RPMI8226 CM

To investigate whether RPMI8226 MM cells treated with ZOL secreted chemotactic factors for γδT cells, the CM from RPMI8226 cells treated by ZOL was applied into a microchamber containing γδT cells (5 × 105/mL) via a microinjector. After the injection of the CM, the γδT cell migration was investigated under a microscope and the continuous time-lapse recording was carried out. While γδT cells treated by the 10% FCS-containing culture medium (control) moved randomly (Supplementary Movie S1), the

Discussion

By N-BP treatment, IPP was accumulated on the surface of the tumor cells and IPP activates γδT cells via γδTCRs [1], [2], [3], [4], [8], [18], [19]. In a previous study, we have observed that migrating γδT cells increase their speed toward MM as well as solid cancer target cells as they move closer to them [1], [2], and from this we hypothesized that cancer cells secreted the chemotactic factors for γδT cells into the supernatant. We focused on the ZOL-stimulated metabolites in the γδT cells,

Conflict of interest statements

Conflict-of-interest disclosure: E. Ashihara, S. Kimura, Y. Nakagawa, H. Hirai, T. Miida, S. Yamato, S. Shoji, and Taira Maekawa disclose no financial conflict of interest. T. Munaka, M. Kanai, and H. Abe. are employees of Shimadzu Corporation.

Acknowledgments

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan (23591404, 26461436 to EA, 21591201 to SK, 24790175 to SN, 25461615, 11068019 to HH, and 23112507, 24390244, 25112706, 11068019 to TM). This work was also supported by Mitsubishi Pharma Medical Research Foundation (The 26th Grants for Basic Research of Hematology to EA), the Kobayashi Foundation for Cancer Research (The 6th Grants for Clinical Research of Oncology to

References (23)

  • O. Toutirais et al.

    DNAX accessory molecule-1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vgamma9Vdelta2 T cells

    Eur. J. Immunol.

    (2009)
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