Human Peripheral Blood Mononuclear Cells
Human peripheral blood mononuclear cells (PBMCs) include a mixture of cells composed of lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells obtained from human blood or buffy coats. In humans, the frequencies of these populations vary across individuals, but lymphocytes are the most abundant, constituting in the range of 70–90 %. PBMCs are typically employed in studies where immune-regulatory effects of food bioactives are to be scrutinized. Main read-out systems include proliferation measurements, evaluation of surface activation markers by flow cytometry and quantification of the cytokine profile produced after adding the food bioactive to the cell culture. Chapter 15 fully explains the principal features and isolation procedures of PBMCs from human blood. Moreover, protocols to perform proliferation assays and evaluation of the anti-inflammatory properties with compounds from food origin are also described.
T Lymphocytes or T-Cells
As mentioned earlier, PBMCs are an important source of lymphocytes. Of special interest are T lymphocytes or T-cells (45–70 % of PBMCs in human peripheral blood), which are produced by stem cells in the bone marrow as progenitors and then migrate to the thymus where they mature into T cells. After completing their maturation, T-cells enter the bloodstream and recirculate between blood and secondary lymphoid organs until they encounter their cognate antigen. After antigen presentation by DCs, along with other appropriate stimuli, the cells may proliferate and differentiate into different subsets of effector cells (Santana and EsquivelGuadarrama 2006). Originally, two main types of effector T cells, called T-helper 1 (Th1) and 2 (Th2) cells, were distinguished by their cytokine secretion patterns. Th1 cells secrete mainly IL-2, IFNγ and TNFα, and Th2 cells secrete IL-4, IL-13 and IL-5 (Romagnani 2000). Recently, a new lineage of T cells characterized by their ability to secrete a proinflammatory cytokine, IL-17, and thus designated Th17 cells has been discovered. This new T cell type has been related to autoimmune diseases (Jing and Dong 2013). Another subset, named regulatory T cells (Treg), acts by inhibiting, between others T cell responses by the production of cytokines, such as IL-10 and TGF-β and/or via cell–cell interactions (Jutel and Akdis 2011). T cell cultures are a valuable tool in food research, especially to perform studies within the food allergy field. To study effects of food bioactives on T cells, it is necessary to activate the T cells by a polyclonal activator, either a mitogen like phytohaemagglutinin or monoclonal antibodies against CD3 and CD28. In food allergy, their main applications include analysis of immunological responses towards food protein antigens to gain further insights into the mechanisms responsible for the development of oral tolerance or for the triggering of food allergies. Chapter 16 describes the main applications in food allergy research, isolation techniques, and culture conditions for PBMC-derived T cells. Furthermore, critical parameters of the model, together with the experimental read outs are discussed.
