Abstract
The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different subsets of PBMCs and immortalized B cells, in which SPPL2a is chemically inhibited and CD74-NTF levels are then assessed by flow cytometry or western blotting. The chemical inhibition of SPPL2a has been described elsewhere, but this is the first time that this inhibition has been reported as a protocol.
Keywords: SPPL2a, CD74-NTF, Inhibition, Chemical, L-685,458
Background
Signal peptide peptidase-like 2A (SPPL2a) is a transmembrane intracellular protease from the GxGD family (Voss et al., 2013). In both humans and mice, the absence of this enzyme results in the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF) in MHC class II-positive cells (Beisner et al., 2013; Bergmann et al., 2013; Schneppenheim et al., 2013; Kong et al., 2018). This accumulated CD74-NTF is selectively toxic to B cells in mice (Beisner et al., 2013; Bergmann et al., 2013; Schneppenheim et al., 2013) and conventional dendritic cells type 2 (cDC2) in both humans (CD1c+ DCs) and mice (CD11c+CD11c+ DCs). We identified the cells most sensitive to CD74-NTF accumulation and gained insight into the mechanism of CD74-NTF-mediated cytotoxicity, using a specific chemical inhibitor of SPPL2a (L-685,458) (Huttl et al., 2015) in peripheral blood mononuclear cells (PBMCs) and Epstein Barr virus-immortalized B cells (EBV-B) from healthy controls. We analyzed CD74-NTF accumulation by fluorescence-activated cell sorting (FACS) or western blotting (WB).
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Acknowledgments
We thank all the authors of our original research article (Kong et al., 2018). R.M.B. was funded by a European Molecular Biology Organization (EMBO) long-term fellowship. X.-F.K. was supported by the Jerome Lejeune Foundation, the Stony Wold-Herbert Fund, the Choh-Hao Li Memorial Fund Scholar Award, and the Shanghai Educational Development Foundation. The Laboratory of Human Genetics of Infectious Diseases was supported by grants from the St. Giles Foundation (J.-L.C.), The Rockefeller University Center for Clinical and Translational Science (grant UL1TR001866 from the National Center for Research Resources and the National Center for Advancing Sciences (NCATS) to R.M.-B. and X.-F.K.), the National Institutes of Health, the National Institute of Allergy and Infectious Diseases (5R01AI089970-02 and 5R37AI095983 to J.-L.C.), Institut National de la Santé et de la Recherche Médicale (INSERM), Paris Descartes University, and The Rockefeller University.
Competing interests
The authors have no competing interests to declare.
Ethics
This study was approved by and performed in accordance with the requirements of the institutional ethics committee of The Rockefeller University Hospital, New York, USA. Informed consent was obtained for all healthy control volunteers participating in the development of this protocol.
References
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