Abstract
Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.
Keywords: Antigen presentation, Bone marrow derived dendritic cells, CD4 T cells, Self-antigens, Bacterial antigens, Apoptotic cells
Background
T cell lymphocytes express on their surface the T cell receptor (TCR), which allows the recognition of cellular (self) or microbial (non-self) antigens that are processed and presented as peptides bound to the major histocompatibility complex (MHC) molecules by antigen presenting cells (APC). APC are able to process antigens and to present them to T cells, and MHC-TCR interactions are critical steps for T cell activation during both infectious and autoimmune responses.Previous works have described a mechanism of regulation of antigen presentation based on the stimulation of Pattern Recognition Receptors (PRRs), such as toll-like receptors (TLRs) (Blander and Medzhitov, 2004 and 2006). Indeed, TLR signals specifically from phagosomes containing microbial pathogens favor the presentation of non-self-antigens within MHC-II molecules. On the other hand, self-antigens generated after phagocytosis of apoptotic cells are directed to lysosomal degradation because of the absence of TLR stimuli. However, the segregation of self and non-self-antigens does not occur when both derive from infected apoptotic cells and are simultaneously carried by the same phagosome, which is optimally tailored by TLR signals for antigen presentation. Such mechanism of phagosome maturation and antigen presentation upon TLR triggering has been demonstrated in vitro using bone marrow derived dendritic cells (BMDC) and apoptotic murine B cells–either primary or A20 B cell line–previously incubated with the TLR4 ligand lipopolysaccharide (LPS), which is internalized by B cells and mimics bacterial infection (Blander and Medzhitov, 2004 and 2006; Campisi et al., 2016). Despite its elegance, this experimental system fails to reproduce bacterial invasion of the eukaryotic target cell. Furthermore, no T cell traceable antigens are present in the apoptotic cargo that internalized LPS. I developed an in vitro alternative protocol where A20 cells are directly infected by the cell invasive bacteria Listeria monocytogenes expressing a recombinant antigen, allowing to assess the capacity of BMDC to present self and non-self-antigens derived from the same infected apoptotic cargo (Campisi et al., 2016).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The following conditions should be prepared (at least in duplicate):
Notes
Recipes
Acknowledgments
L.C. was supported by an Arthritis Foundation Research Fellowship Award. The protocol described herein was based on the paper Campisi et al. (2016). L.C. thanks Ivan Marazzi for advise and support. 1H3.1 TCR transgenic mice used in the paper Campisi et al. (2016) were a kind gift from Adrian Morelli to J. Blander’s laboratory under material transfer agreement (MTA). The author declares no conflict of interest.
References
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