Optimum culture conditions for specific and nonspecific activation of whole blood and PBMC for intracellular cytokine assessment by flow cytometry

Abstract

The assessment of cytokine production is an important component of studies of cell-mediated immune responses (CMI) to immunological challenges. In this study, we present a method to enhance the detection of cytokine-producing cells by allowing antigen-specific cells to expand in long-term culture. We investigated the influence of the degree of dilution of whole blood and the duration of the incubation period on whole blood as well as peripheral blood mononuclear cells (PBMCs), cultured in the absence or presence of mitogens, superantigens or specific antigens, for intracellular cytokine production (IFNγ, TNFα, IL-2, IL-4, IL-10 and IL-13) by CD4⁺ and CD8⁺ T lymphocytes using four-colour flow cytometry. Whole blood was diluted 1/1, 1/2, 1/5 and 1/10, and cultured for 6, 24, 48, 72 and 120 h in the presence of antibodies against the co-stimulatory molecules CD28 and CD49d, and, during the last 4 h of culture, in the presence of brefeldin A. Optimum conditions for detection of a high number of IFNγ-positive cells were observed after 72 h of culture in blood diluted 1/10. Median frequencies of IFNγ⁺ cells obtained after activation by PMA-ionomycin, PHA or SEA-B were 29.3%, 20.0% and 6.8% for CD4⁺ cells, and 67.8%, 20.6% and 6.8% for CD8⁺ cells. In blood samples diluted 1/5 or 1/10, and cultured in the presence of cytomegalovirus (CMV) or varicella-zoster virus (VZV), mean peak levels of 2.8% and 1.4% IFNγ⁺CD4⁺ cells were recorded at 120 h. The levels of cells producing cytokines other than IFNγ were generally much lower and, in the case of IL-4 and IL-13, difficult to distinguish from background levels recorded in cultures with medium only. Kinetic studies of cytokine production by PBMCs showed a pattern similar to that of whole blood with peak levels of IFNγ-producing cells recorded at 72 h. The increased levels of IFNγ production after culture for 72 h were due to an expansion of the numbers of cytokine-producing cells responsive to a specific stimulus. Antigen-specific cells are usually present only at low levels in peripheral blood and may not be detected following simple activation for a few hours. To reach a level of detection in such cases, culture of diluted blood for several days is recommended.

Introduction

Activation of specific T cells by antigen-presenting cells is an early and crucial step in the development of antigen-specific immune responses, resulting in a wide variety of biological events such as production and secretion of cytokines (Arai et al., 1990, Scott et al., 1990, Finklemann, 1995), cell-surface expression or up-regulation of cell adhesion molecules, ligands for molecules expressed on other cells, and cytokine receptors (Kaye and Janeway, 1984, Andersson et al., 1994, Jeannin et al., 1999), and eventually also the proliferation of antigen-specific clones (Vasseur et al., 1999). Cytokines regulate the initiation and maintenance of the immune response and are essential to determine the nature of the immune response and what effector mechanisms should be selected and propagated (Cher and Mosmann, 1987).

Numerous techniques, such as enzyme-linked immunosorbent assay (ELISA; Ledur et al., 1995), enzyme-linked immunospot (ELISpot; Klinman and Nutman, 1994), limiting dilution assay (LDA) (Hernandez-Fuentes et al., 2003), polymerase chain reaction (PCR; Reiner et al., 1993) and in situ hybridisation (ISH; Hoefakker et al., 1995), have been employed for the detection and measurement of cytokine responses, usually in bulk cultures of peripheral blood mononuclear cells (PBMCs) or cell lines. Andersson and co-workers introduced a system for fixation, permeabilisation and staining of cells for detection of intracellular cytokines by indirect immunofluorescence (Andersson et al., 1988). Furthermore, they evaluated the use of flow cytometry as a tool for the assessment of cytokine-producing cells, but reported that the low intracellular concentration of several cytokines, as well as the low frequency of antigen-specific responding cells, did not allow the use of flow cytometry for this purpose (Sander et al., 1991). Monensin and brefeldin A, which block cytoplasmic protein transport, thus causing accumulation of cytokines in the Golgi complex, were later shown to increase the intracellular concentration of cytokines (Jung et al., 1993), permitting the use of flow cytometry for assessment of intracellular cytokines (Prussin and Metcalfe, 1995, Openshaw et al., 1995, Picker et al., 1995, North et al., 1996, Waldrop et al., 1998).

In all of the studies mentioned above, cytokines or cytokine-producing cells were detected in conventional cultures of isolated PBMCs. Whole blood cultures had earlier been found to be valuable for measuring DNA synthesis and presented in a limited number of reports (Gratzner, 1982, Gonchoroff et al., 1985, Carayon and Bord, 1992). We developed a new method to study cell-mediated immune response (CMI) by assessment of proliferative responses and intracellular cytokine production, employing whole blood cultures and measuring results by flow cytometry (Gaines et al., 1996, Gaines and Biberfeld, 2000). Another improvement in flow cytometric analysis of cytokine expression in whole blood has recently been described (Suni et al., 1998), in which T cell responses to specific antigens are assessed after brief culture (6 h) in the presence of antibodies to co-stimulatory molecules, CD28/CD49d. Over recent years, this method has become predominant for the assessment of intracellular cytokines (Maino et al., 1995, Picker et al., 1995, Asanuma et al., 2000). Attempts to improve the sensitivity of this method by incubating whole blood cultures up to 24 h have been made, although without success (North et al., 1996, Sewell et al., 1997, Asanuma et al., 2000, Bueno et al., 2001). However, in these studies, whole blood was cultured undiluted or diluted only 1/2. We and others (Leroux et al., 1985, Bloemena et al., 1989, Gaines et al., 1996) have earlier reported that whole blood must be diluted more than 1/2 to permit culturing for longer periods. To the best of our knowledge, no study employing long-time culture of highly diluted whole blood for the detection of intracellular cytokines has been reported.

We investigated the influence of the degree of dilution of whole blood and the duration of the incubation period on intracellular cytokine production (IFNγ, TNFα, IL-2, IL-4, IL-10 and IL-13) by CD4⁺ and CD8⁺ T lymphocytes cultured in the absence or presence of mitogens, superantigens or specific antigens and assessed this by four-colour flow cytometry. We consistently recorded higher frequencies of responding T cells in cultures where highly diluted blood was incubated for several days. Kinetic studies of cytokine production by PBMCs were also performed showing a reactivity pattern similar to that of whole blood with peak levels of IFNγ-producing cells at 72 h. The increased levels of IFNγ production after culture of whole blood or PBMC for 72 h are due to an expansion of the numbers of cytokine-producing cells responsive to a specific stimulus (Gaines et al., 1996, Gaines and Biberfeld, 2000).