Characterization of Liver CD8 T Cell Subsets that are Associated with Protection Against Pre-erythrocytic Plasmodium Parasites
Murine models of malaria, such as Plasmodium berghei (Pb) and Plasmodium yoelii (Py), have been used for decades to identify correlates of protection associated with immunization using radiation-attenuated sporozoites (RAS). To date, RAS is the only known immunization regimen to consistently deliver 100 % sterilizing immunity and is considered the “gold standard” of protection against malaria. The ability to isolate lymphocytes directly from the liver of immune mice has facilitated the identification of correlates of protection at the site of infection. Liver CD8 T cells have been identified as a key factor in mediating protection against challenge with infectious Plasmodium sporozoites. Liver CD3 + CD8 T cells can further be divided into subsets based on the expression of specific surface molecules and the increase of CD8 effector memory (TEM) cells (identified by the phenotype CD44+CD62L−) has been shown to mediate protection by releasing of IFN-γ while CD8 central memory (TCM) cells (CD44+CD62L+) are important for maintaining long-term protection.
Identification of multiple CD8 T cell subsets present in the liver relies on the ability to detect multiple surface markers simultaneously. Polychromatic flow cytometry affords the user with the ability to distinguish multiple lymphocyte populations as well as subsets defined within each population. In this chapter we present a basic 9-color surface staining panel that can be used to identify CD8 TEM, CD8 TCM, short-lived effector cells (SLECs), and memory precursor cells (MPECs) as well as identify those cells which have recently undergone degranulation (surface expression of CD107a). This panel has been designed to allow for the addition of intracellular staining for IFN-γ on other available channels (such as PE) as is discussed in another chapter for analysis of functional CD8 T cell responses.
Key wordsMalaria Flow cytometry CD8 T cells CD8 T cell subsets Liver
The research described in this chapter was supported by the US Army Medical Research and Materiel Command. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense. Described procedures must be conducted under an IACUC-approved protocol in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NRC, 2011).
The authors would like to thank the current Krzych lab members for their assistance with the performance of the assays.
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