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Dendritic cells (DC) are the most potent inducers and regulators of immune responses, responsible for communication within immune system. The ability of DC to act both as the inducers of immune responses and as regulatory/suppressive cells led to the interest in their immunotherapeutic use in different disease types, ranging from cancer to autoimmunity, and as a tool to prevent the rejection of transplanted tissues and organs. Over the last years, several groups including ours have demonstrated the feasibility of obtaining monocyte-derived DC with different functions, by modulating the conditions and the duration of DC maturation. The current chapter provides a detailed protocol of generating type-1-, type-2-, and type-17-polarized DC for testing the cytokine-producing abilities of these cells and their effectiveness in inducing Th1, Th2, and Th17 responses of CD4+ T cells and CTL responses of naïve and memory CD8+ T cells.
Dendritic cells (DC) are the most potent inducers and regulators of immune responses, responsible for intercellular communication between other immune cells. They act as sentinel cells in the peripheral tissues, being key to the development of effective immune responses to the pathogens residing in different cellular compartments and susceptible to different immune mechanisms (1–8). In line with their central role in pathogen control, DC dysfunction has been implicated in the pathogenesis and progression of a wide range of disease conditions, ranging from autoimmunity to chronic infections and cancer, with multiple pathogens developing ways to interfere with DC functions as a mean to avoid eradication by the immune system (3, 9–15).
Both the efficiency of DC as an effective element of immune system and their susceptibility to pathogen-induced dysfunction result from an enormous plasticity of the DC system (1, 7, 8). Distinct DC subsets or DC developing or maturing in different conditions show striking functional differences (1, 2, 5–8, 16–19). One aspect of DC function that is a subject to strict regulation is their ability to induce such effector immune cells as Th1-, Th2-, or Th17-type CD4+ Th cells or cytotoxic CD8+ T cells (CTLs) (1, 7, 8) as opposed to regulatory T(reg) cells (20–25).
In contrast to the inhibitory Tregs, all the above effector T-cell types have been shown to provide essential elements of protection against different classes of pathogens and have been implicated in different forms of autoimmunity. Th1-type CD4+ T cells (key producers of IFN-γ and lymphotoxin) and CD8+ CTLs (main type of antigen-specific killer cells) are generally considered as the effector cells key to our ability to effectively fight intracellular bacteria and viruses, as well as to eliminate tumor cells. In addition, Th1 cells provide support for the production of several immunoglobulin classes by B cells. Th2 cells, producing mainly IL-4 and IL-5, are an essential component of our defenses against intestinal parasites and contribute to the majority of antibody production. The more recently discovered IL-17-producing Th cells (Th17 cells) are required for the protection against certain bacteria. Moreover, Th17 cells have been implicated to play a role in the development and/or maintenance of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and colitis (26–40).
The ability of DC to act both as the inducers of immune responses and as regulatory/suppressive cells led to the interest in their immunotherapeutic use in different disease types, ranging from cancer to autoimmunity, and as a tool to prevent the rejection of transplanted tissues and organs (24, 41–50). Taking into account the plasticity of DC and their ability to adopt different functions, it is important to match the desired type of the DC to the type of their clinical or laboratory application.
Over the last years, we and multiple other groups demonstrated the feasibility of obtaining monocyte-derived DC with different functions, by modulating the conditions of their early development (51, 52), the conditions of their maturation (53–60), or the length of DC maturation period (54, 61). The current chapter provides a detailed protocol of generating type-1-, type-2-, and type-17-polarized DC, the protocols used to test the cytokine-producing capacity of these cells, and their ability to induce Th1, Th2, and Th17 responses of CD4+ Th cells as well as the CTL responses of naïve and memory CD8+ T cells.
Our serum-supplemented conditions of DC culture involve FCS, rather than human serum, since DC obtained in the presence of human serum do not express CD1a and show a relative resistance to maturation. FCS/IMDM-based media allow the generation of type-1-polarized DC (DC1), using a combination of TNF-α and IFN-γ (or LPS and IFN-γ). In contrast, the generation of fully mature DC1 in serum-free media (such as AIM-V or CellGenix) requires the addition of IFN-α and poly-I:C (αDC1, Ref. (60).
The SEB-based model of naïve Th cell priming was first described in Ref. (51). It is based on the ability of SEB to activate a substantial proportion of naïve T cells (66, 67). This allows to use it as a substitute of the TCR-transgenic models that are not available in the human system. In contrast, the traditional allogeneic MLR model does not allow to induce any detectable amounts of IL-12 within the first 3 days of DC–Th cell interaction, most likely due to 100–1,000-fold lower frequency of responsive T cells. The possible applications and the typical results obtained with use of the described protocols can be found in our previous publications (51, 53, 54, 57–59, 62, 64, 68–72).
Based on the past experiences on introducing the described protocol in other labs, we would like to draw your attention to the following issues critical for its outcome.
This work was supported by the NCI grants CA95128, CA101944, and CA114931.