Research paper
Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression

https://doi.org/10.1016/j.jim.2011.04.003Get rights and content

Abstract

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be ‘trapped’ on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system.

Introduction

CD8+ T cells are essential for the effective control and clearance of intracellular pathogens, and play a central role in tumor surveillance. In response to antigenic stimuli, CD8+ T cells can deploy an extensive array of effector functions, including perforin-mediated cytotoxicity, the induction of apoptosis via Fas and TNF-α (TNF from here on), and the production of numerous immunomodulatory cytokines and chemokines (Harty et al., 2000). Procedures to identify and isolate CD8+ T cells with these particular properties remain critically important for our understanding of CD8+ T cell function and the development of CD8+ T cell-based immunotherapeutic strategies.

Although basic analysis of CD8+ T cell function and phenotype is relatively straightforward through the use of flow cytometry, there are few procedures that permit the isolation of viable antigen-specific CD8+ T cells for further study. Staining with fluorescent MHC class I tetramers, or multimers with higher order valencies, remains the gold standard for the precise identification and isolation of antigen-specific CD8+ T cells (Altman et al., 1996, Wooldridge et al., 2009). However, this approach is limited to defined specificities, requires unique and costly reagents, and provides no assessment of functional capacity. Furthermore, it is clear that not all tetramer + CD8+ T cells are functional, at least as assessed by current techniques, and that not all functional cells can be identified with MHC class I tetramers due to threshold differences that distinguish activation from physical staining (Goepfert et al., 2000, Chattopadhyay et al., 2008). Currently, there are few available methods for the isolation of viable antigen-specific CD8+ T cells based on functional responses. As we have shown previously, exposure of CD107a on the CD8+ T cell surface after activation can be used to isolate viable antigen-specific CD8+ T cells capable of degranulation (Betts et al., 2003, Rubio et al., 2003). The only current techniques that enable the isolation of CD8+ T cells based on their ability to produce cytokines, however, are capture assays (Brosterhus et al., 1999, Douek et al., 2002b, Lichterfeld et al., 2004). These assays use a bi-specific antibody that recognizes the cytokine of interest, such as IFNγ, together with lineage specific markers, such as the pan-lymphocyte receptor CD45. Although useful, this procedure requires proprietary reagents and alternate procedural steps that can lead to specificity and sensitivity concerns.

Here, we sought to expand upon this repertoire of sorting methods by developing an assay to sort functional CD8+ T cells based on TNF production. TNF is an important cytokine produced by a variety of cell types, including CD8+ T cells, that has pleiotropic effects including apoptosis induction and complex immunoregulatory and systemic functions (Kull, 1988, Lazdins et al., 1997, Badovinac et al., 2000, Aggarwal, 2003). Compared to other cytokines, TNF production and release is somewhat unusual, in that TNF is produced initially as a membrane-bound protein that requires subsequent cleavage by the TNF-α converting enzyme (TACE) to be released (Bjornberg et al., 1994, Black et al., 1997). Both forms of TNF are biologically active, with potentially different functions. Thus, membrane-bound TNF binds preferentially to TNFRII to induce apoptosis, whereas soluble TNF binds primarily to TNFRI and exerts immunomodulatory properties (Chen and Goeddel, 2002, Wajant et al., 2003). Importantly, the TNF-α Processing Inhibitor 0 (TAPI-0) can directly prevent TNF release from the cell surface (Crowe et al., 1995, Pagan et al., 2003, Huse et al., 2006), effectively trapping TNF on the surface of the producing cell. Through the use of TAPI-0, we now demonstrate that human antigen-specific CD8+ T cells can be live-sorted based upon their ability to produce TNF. We show that this assay is highly specific and compatible with the isolation of mRNA from TNF-producing CD8+ T cells, as well as culture and in vitro expansion of antigen-specific CD8+ T cells.

Section snippets

Cells

Peripheral blood mononuclear cells (PBMC) were obtained from the University of Pennsylvania's Center for AIDS Research Human Immunology Core Facility in compliance with the guidelines set by the respective institutional internal review boards. PBMC were isolated by standard Hypaque–Ficoll separation and cryopreserved in fetal bovine serum (FBS; ICS Hyclone, Logan, Utah) containing 10% dimethyl sulfoxide (DMSO; Fisher Scientific, Pittsburgh, Pennsylvania). Individual peptide stimuli were

Prevention of TNF release from activated PBMC directly ex vivo

TNF is initially produced as a membrane bound protein that is subsequently cleaved by TACE to release soluble TNF (Black et al., 1997). To prevent this cleavage event, and thereby trap TNF on the producing cells, we used the TACE protease inhibitor TAPI-0. Initially, we used TNF ELISA assays to determine the optimal dosage of TAPI-0 necessary to reduce the cleavage of TNF (Fig. 1) and prevent its release from stimulated cells. We stimulated human PBMC directly ex vivo with SEB in the presence

Discussion

In this study, we have demonstrated a novel procedure by which to isolate activated TNF-producing lymphocytes. Through the use of a TACE inhibitor, we take advantage of the unique mechanism of TNF production in order to prevent TNF release from activated T cells, thereby allowing their direct identification and isolation. We find that this procedure is highly reproducible, yields similar results to intracellular cytokine staining, and is of sufficient sensitivity to identify rare populations of

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