To better understand primary and recall T cell responses during COVID-19, it is important to examine unmanipulated SARS-CoV-2-specific T cells. Using peptide-HLA tetramers for direct ex vivo analysis, we characterized CD8 + T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8 + T cells directed towards subdominant epitopes – B7/N 257 , A2/S 269 and A24/S 1208 – CD8 + T cells specific for the immunodominant B7/N 105 epitope were detected at high frequency in pre-pandemic samples, and at increased frequency during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8 + T cells in pre-pandemic samples from children, adults and elderly individuals predominantly displayed a naïve phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N 105 + CD8 + T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N 105 -specific CD8 + T cells, and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.

The intracellular parasite Toxoplasma scavenges sphingolipids from its host mammalian cell by establishing a close relationship with the host Golgi. It subverts the Golgi's structure, hijacks selected Rab Golgi-derived vesicles within its parasitophorous vacuole, and retrieves sphingolipids from these vesicles.

The highly reduced protozoan parasite Giardia lamblia has minimal machinery for cellular processes such as protein trafficking. Giardia trophozoites maintain diverse and regulated secretory pathways but lack an identifiable Golgi complex. During differentiation to cysts, however, they produce specialized compartments termed encystation-specific vesicles (ESVs). ESVs are hypothesized to be unique developmentally regulated Golgi-like organelles dedicated to maturation and export of pre-sorted cyst wall proteins. Here we present a functional analysis of this unusual compartment by direct interference with the functions of the small GTPases Sar1, Rab1 and Arf1. Conditional expression of dominant-negative variants revealed an essential role of Sar1 in early events of organelle neogenesis, whilst inhibition of Arf1 uncoupled morphological changes and cell cycle progression from extracellular matrix export. The latter led to development of `naked cysts', which lacked water resistance and thus infectivity. Time-lapse microscopy and photobleaching experiments showed that putative Golgi-like cisternae in Giardia develop into a network capable of exchanging soluble cargo at a high rate via dynamic, tubular connections, presumably to synchronize maturation. The minimized and naturally pulsed trafficking machinery for export of the cyst wall biopolymer in Giardia is a simple model for investigating basic principles of neogenesis and maturation of Golgi compartments.

Irreversible failure of pancreatic β-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.

Graphical abstractHighlights► Identification and characterisation of Toxoplasma sphingolipid synthase (TgSLS). ► Demonstration of TgSLS inositol phosphorylceramide synthase activity. ► Identification of inositol phosphorylceramide in Toxoplasma extracts. ► Delineation of role of host sphingolipid biosynthesis in Toxoplasma proliferation. ► Host biosynthesis non-essential for proliferation, de novo synthesis could be key.