T cells are involved in the early identification and clearance of viral infections and also support the development of antibodies by B cells. This central role for T cells makes them a desirable target for assessing the immune response to SARS-CoV-2 infection. Here, we combined two high-throughput immune profiling methods to create a quantitative picture of the T-cell response to SARS-CoV-2. First, at the individual level, we deeply characterized 3 acutely infected and 58 recovered COVID-19 subjects by experimentally mapping their CD8 T-cell response through antigen stimulation to 545 Human Leukocyte Antigen (HLA) class I presented viral peptides (class II data in a forthcoming study). Then, at the population level, we performed T-cell repertoire sequencing on 1,015 samples (from 827 COVID-19 subjects) as well as 3,500 controls to identify shared "public" T-cell receptors (TCRs) associated with SARS-CoV-2 infection from both CD8 and CD4 T cells. Collectively, our data reveal that CD8 T-cell responses are often driven by a few immunodominant, HLA-restricted epitopes. As expected, the T-cell response to SARS-CoV-2 peaks about one to two weeks after infection and is detectable for several months after recovery. As an application of these data, we trained a classifier to diagnose SARS-CoV-2 infection based solely on TCR sequencing from blood samples, and observed, at 99.8% specificity, high early sensitivity soon after diagnosis (Day 3-7 = 83.8% [95% CI = 77.6-89.4]; Day 8-14 = 92.4% [87.6-96.6]) as well as lasting sensitivity after recovery (Day 29+/convalescent = 96.7% [93.0-99.2]). These results demonstrate an approach to reliably assess the adaptive immune response both soon after viral antigenic exposure (before antibodies are typically detectable) as well as at later time points. This blood-based molecular approach to characterizing the cellular immune response has applications in vaccine development as well as clinical diagnostics and monitoring.

Studies of mouse monoclonal CD4+ T cell repertoires have revealed several mechanisms of self-tolerance, however, which mechanisms operate in normal repertoires is unclear. Here, polyclonal CD4+ T cells specific for green fluorescent protein expressed in different organs were studied, allowing determination of the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas thymus-excluded peptides were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self-antigens. Thus, immune tolerance of polyclonal CD4+ T cells is maintained by distinct mechanisms, according to self-peptide expression patterns.

Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNFinduced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3 −/− mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the "two-faced" nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.

Triacylglycerol (TAG) serves as the major energy reserve in higher eukaryotic organisms ( 1 ). FFAs are mobilized from TAG through the hydrolytic action of lipases to provide substrates for oxidative metabolism in tissues, as well as substrates for synthesis of complex lipids and signaling molecules. Lipolysis, the breakdown of TAG to FFA and glycerol, occurs in an orderly and regulated manner, with different enzymes acting sequentially at each step ( 1-3 ). Hydrolysis of TAG to diacylglycerol is the fi rst step ( 4-6 ), and our laboratory, and others, recently identifi ed a novel TAG hydrolase in mice that we called desnutrin ( 5 ) [also known as TTS2.2, patatin-like phospholipase A domain containing 2, iPLA ( 4 ), or in humans, adipose triglyceride lipase (ATGL) ( 6 )]. Desnutrin is highly expressed in white and brown adipose tissue, where it is a major TAG lipase, but is also found at lower levels in most other tissues where it also plays an important role in TAG hydrolysis ( 5,7,8 ).Desnutrin contains an N-terminal patatin-like domain, spanning amino acids 8-180, that is characteristic of many plant lipid acyl hydrolases ( 5, 9 ). Enzymatic activity of desnutrin is predicted to be derived from an S47-D166 catalytic dyad that lies within an ␣ -␤ hydrolase fold in the patatin-like domain ( 5, 9 ). Mutation of the S47 residue to alanine has been shown to result in a complete loss of function in vitro, confi rming the predicted serine-esterase activity of this enzyme ( 10 ). However, the requirement of the D166 site has not yet been tested. Four individuals with point mutations in desnutrin/ATGL have been identifi ed, and all developed neutral lipid storage disease with myopathy (NLSDM) ( 11,12 ). In one, a duplication mutation within the N-terminal patatin-like domain caused a frameshift after L159 that is predicted to truncate the enzyme at amino acid position 178 as well as to cause a D166R mutaAbstract Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an ␣ -␤ hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V max than the full-length form without changes in K m , which may be explained by our fi nding of an interaction between the C-and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identifi ed two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich ...

Summary The vertebrate immune system is highly dependent on cell death for efficient responsiveness to microbial pathogens and oncogenically transformed cells. Cell death pathways are vital to the function of many immune cell types during innate, humoral and cellular immune responses. In addition, cell death regulation is imperative for proper adaptive immune self-tolerance and homeostasis. While apoptosis has been found to be involved in several of these roles in immunity, recent data demonstrate that alternative cell death pathways are required. Here, we describe the involvement of a programmed form of cellular necrosis called “necroptosis” in immunity. We consider the signaling pathways that promote necroptosis downstream of death receptors, type I transmembrane proteins of the tumor necrosis factor (TNF) receptor family. The involvement of necroptotic signaling through a “RIPoptosome” assembled in response to innate immune stimuli or genotoxic stress is described. We also characterize the induction of necroptosis following antigenic stimulation in T cells lacking caspase-8 or FADD function. While necroptotic signaling remains poorly understood, it is clear that this pathway is an essential component to effective vertebrate immunity.