Elsevier

Immunology Letters

Volume 106, Issue 1, 15 July 2006, Pages 27-33
Immunology Letters

Functional characterization of OX40 expressed on human CD8+ T cells

https://doi.org/10.1016/j.imlet.2006.04.001Get rights and content

Abstract

In the present study, we investigated the expression of OX40 on human CD8+ T cells with regard to expression induction, costimulatory function and possible involvement in cytotoxicity. Human CD8+ T cells were purified from peripheral blood mononuclear cell (PBMC) of healthy donors and cocultured with allogeneic monocyte-derived dendritic cells. Flow cytometric analysis showed that expression of OX40 was induced on CD8+ T cells within 1 day and increased to the maximum levels on day 3. An addition of anti-OX40 ligand (OX40L) mAb suppressed CD25 expression, proliferation and IFN-γ production of CD8+ T cells, suggesting that OX40 functions as a costimulatory molecule not only for CD4+ T cells but also for CD8+ T cells. In parallel, coculture of pre-activated CD8+ T cells with OX40L-transfected murine epithelial cells (MMCE-OX40L) resulted in an increase in CD25 expression, proliferation and IFN-γ producing cells, compared with that with the mock control (MMCE-mock). Finally, non-specific cytotoxic activity of preactivated CD8+ T cells was examined using OKT3 hybridoma as target cells after coculture with these transfectants. Coculture with MMCE-OX40L induced slightly higher cytotoxicity of CD8+ T cells than that with MMCE-mock. These results indicate that OX40 is induced transiently on CD8+ T cells upon activation and its signals contribute to both clonal expansion and functional reinforcement.

Introduction

Effector functions of CD8+ T cells constitute an important part of cellular immunity, such as eradication of viruses, intracellular microbe-infected cells and tumor cells [1], [2]. Like CD4+ T cells, CD8+ T cells require both antigenic stimulation and costimulatory signals provided by antigen-presenting cells (APC) to proliferate and become competent effector CTL [3], [4], [5], [6], [7]. Recent evidence has indicated that, in addition to CD28, some members of the tumor-necrosis factor (TNF) receptor superfamily including 4-1BB [5], [8], CD27 [6], CD30 [9] and OX40 [10], [11], [12], [13], [14], [15], [16] mediate costimulatory signals for activation, proliferation and differentiation. However, immunological roles of OX40 in human CD8+ T cells remain undetermined while 4-1BB is known to be critically involved in amplification and maintenance of antigen-primed CD8+ T cells [5], [8], [17].

OX40 has been extensively studied in the context of costimulation of CD4+ T cells [18], [19], [20], [21], [22], [23], [24], [25], [26], [27]. Indeed, OX40 is preferentially expressed on CD4+ T cells transiently upon antigenic stimulation and the binding of OX40L expressed on APC to OX40 on T cells transmits strong signals into T cells leading to augmentation of cytokine secretion and cellular proliferation. Furthermore, OX40 has anti-apoptotic function during T cell activation by induction of the bcl-2 family molecules, which enables antigen-specific CD4+ T cells to survive and generates long-term memory [27]. On the other hand, the role of OX40 in CD8+ T cells is still controversial. Earlier studies with OX40-knockout mice or OX40L-deficient mice showed that these mice had apparently normal CD8+ T cells and unimpaired ability to mount CTL response after viral infection [11], [13], [28], suggesting that OX40 is dispensable in CD8+ T cells, whereas later reports using agonistic anti-OX40 mAb have suggested that OX40 signals augment CD8+ T cell response [12], [14], [29]. Moreover, cytotoxic activity of total splenocytes from OX40−/− mice was impaired [30]. In tumor immunity model, OX40 stimulation can enhance the expansion, survival and effector function of tumor-specific CD8+ T cell repertoire in tumor-burden mice when combined with GM-CSF-secreting whole cell vaccine [15]. In another report, both OX40L and OX40 were induced on CD8+ CTL clone upon prolonged stimulation with Epstein-Barr virus transformed autologous lymphoblastic cell lines, suggesting the importance of OX40/OX40L interaction in the recall response of CD8+ memory T cells [31].

It should be noted that above-mentioned studies on OX40 and CD8+ T cells were done in vivo either with agonistic anti-OX40 mAb or with gene-knockout mice. Considering that such mAb can interact with non-CD8+ T cells and that the gene-knockout can affect non-CD8+ T cells as well, it is difficult to conclude that OX40 signals directly alter CD8+ T cell functions. Moreover, the role of OX40 in human CD8+ T cells has scarcely been studied despite that these two species are immunologically different in many respects especially in cytokines and costimulatory molecules. As to OX40 and OX40L, expression of OX40L on endothelial cells in vitro and their costimulatory activity on CD4+ T cells via the OX40/OX40L system has been described solely in humans [32], [33], [34].

In the present study, we investigated the expression of OX40 on human CD8+ T cells with regard to expression induction, costimulatory function and possible involvement in cytotoxicity. We did simple in vitro experiments using human CD8+ T cells in order to settle some of the unsolved questions on OX40 and CD8+ T cells. Herewith we present evidence that OX40 signals directly augment activation, cytokine secretion, proliferation and possibly cytotoxicity of human CD8+ T cells.

Section snippets

Culture medium

RPMI-1640 (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 292 μg/ml l-glutamine (penicillin–streptomycin–glutamine; Life Technologies, Rockville, MD, USA) and 10% fetal calf serum was used as culture medium for culture of dendritic cells and CD8+ T cells. OKT3 hybridomas was cultured in the presence of 5.5 × 10−6 M 2-mercaptoethanol (Invitrogen, Carlsbad, CA, USA).

Human plasma, primary cells and cell lines

Heparinized peripheral blood from healthy volunteers was centrifuged and the plasma

Expression of OX40 on CD8+ T cells stimulated with allogeneic DCs

First we examined the mode of induction and the magnitude of OX40 expression on CD8+ T cells stimulated with allogeneic DCs in comparison with that of CD25. We chose allogeneic DCs as stimulants because they presented allo-antigens that were more physiological than artificial mitogens, such as anti-CD3 mAb, PHA and PMA. Five to seven percent of mature DCs expressed OX40L throughout the experiments. As shown in Fig. 1, fresh CD8+ T cells hardly expressed OX40, and after stimulation with

Discussion

It has been widely accepted that the OX40/OX40L system plays an essential role in CD4+ T cells but has minimal functions in CD8+ cells. Both OX40- and OX40L-knockout mice were shown to have apparently normal CD8+ T cells and could mount unimpaired CTL responses [28]. In the latter mice, cytotoxic activity of total splenocytes was significantly impaired compared with wild type mice [30], which might be ascribed to the suppressed help of CD4+ T cells. On the other hand, there is another line of

Acknowledgments

This work was partly supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan. We thank Novartis Pharma for providing recombinant human GM-CSF and Kyoto Red Cross Blood Center for buffy coats from normal healthy donors.

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    1

    These authors contributed equally to this work.

    2

    Present address: Wakayama Red-Cross Hospital, Wakayama, Japan.

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