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 glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galα(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galα(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

When ␣(1,2)fucosyltransferase cDNA is expressed in cells that normally express large amounts of the terminal carbohydrate Gal␣(1,3)Gal, and therefore the ␣(1,3)galactosyltransferase (GT), the Gal␣(1,3)Gal almost disappears, indicating that the presence of the ␣(1,2)fucosyltransferase (HT) gene/enzyme alters the synthesis of Gal␣(1,3)Gal. A possible mechanism to account for these findings is enzyme location within the Golgi apparatus. We examined the effect of Golgi localization by exchanging the cytoplasmic tails of HT and GT; if Golgi targeting signals are contained within the cytoplasmic tail sequences of these enzymes then a "tail switch" would permit GT first access to the substrate and thereby reverse the observed dominance of HT. Two chimeric glycosyltransferase proteins were constructed and compared with the normal glycosyltransferases after transfection into COS cells. The chimeric enzymes showed K m values and cell surface carbohydrate expression comparable with normal glycosyltransferases. Coexpression of the two chimeric glycosyltransferases resulted in cell surface expression of Gal␣(1,3)Gal, and virtually no HT product was expressed. Thus the cytoplasmic tail of HT determines the temporal order of action, and therefore dominance, of these two enzymes.Glycosylation is a complex form of posttranslational modification of proteins where glycosyltransferases, located in the endoplasmic reticulum and Golgi apparatus, catalyze the sequential transfer of monosaccharides from nucleotide sugars to saccharide acceptors resulting in mature oligosaccharides (1). Thus the large number of both N-and O-linked carbohydrate side chains, consisting of structures of three or 4 simple monosaccharides, can have the different glycosylation patterns generated by the sequential and coordinated action of more than 100 different glycosyltransferases (2). In this process, the product of one transferase serves as the substrate for the next transferase (3), with the differences in the glycosylation pattern being due to variation in the topology and function and the level of expression of the glycosyltransferase within the Golgi complex (3).Evidence to date based on immunoelectron microscopy and cell fractionation studies suggests that the glycosyltransferases are sequentially distributed within the Golgi compartments in approximately the observed order of glycosylation (4). A number of studies using hybrid molecules have examined mechanisms for the sorting and retention of glycosyltransferases within the Golgi and have identified the transmembrane domains of ␣(2,6)sialyltransferase, ␤(1,4)galactosyltransferase, and N-acetylglucosaminyltransferase I as playing a central role in their Golgi localization (5); however, cytoplasmic tail and stem region sequences in ␣(2,6)sialyltransferase and Nacetylglucosaminyltransferase I also contain important structural elements within their cytoplasmic domains that augment the efficiency of Golgi localization of these proteins (5). Thus the sequences that localize glycosyltransferases within di...