Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DR hi CD11c hi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR lo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

42 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a rapidly 43 unfolding pandemic, overwhelming health care systems worldwide 1 . Clinical manifestations of 44 Coronavirus-disease 2019 (COVID-19) vary broadly, ranging from asymptomatic infection to 45 acute respiratory failure and death 2 , yet the underlying mechanisms for this high variability are 46 still unknown. Similarly, the role of host immune responses in viral clearance of COVID-19 47 remains unresolved. For SARS-CoV (2002/03), however, it has been reported that CD4 + T cell 48responses correlated with positive outcomes 3,4 , whereas T cell immune responses to SARS-49CoV-2 have not yet been characterized. Here, we describe an assay that allows direct detection 50and characterization of SARS-CoV-2 spike glycoprotein (S)-reactive CD4 + T cells in peripheral 51blood. We demonstrate the presence of S-reactive CD4 + T cells in 83% of COVID-19 patients, 52as well as in 34% of SARS-CoV-2 seronegative healthy donors (HD), albeit at lower 53 frequencies. Strikingly, S-reactive CD4 + T cells in COVID-19 patients equally targeted N-54terminal and C-terminal epitopes of S whereas in HD S-reactive CD4 + T cells reacted almost 55exclusively to the C-terminal epitopes that are a) characterized by higher homology with spike 56 glycoprotein of human endemic "common cold" coronaviruses (hCoVs), and b) contains the S2 57 subunit of S with the cytoplasmic peptide (CP), the fusion peptide (FP), and the transmembrane 58 domain (TM) but not the receptor-binding domain (RBD). In contrast to S-reactive CD4 + T 59 cells in HD, S-reactive CD4 + T cells from COVID-19 patients co-expressed CD38 and HLA-60DR, indivative of their recent in vivo activation. Our study is the first to directly measure SARS-61CoV-2-reactive T cell responses providing critical tools for large scale testing and 62 characterization of potential cross-reactive cellular immunity to SARS-CoV-2. The presence of 63 pre-existing SARS-CoV-2-reactive T cells in a subset of SARS-CoV-2 naïve HD is of high 64interest but larger scale prospective cohort studies are needed to assess whether their presence 65 is a correlate of protection or pathology for COVID-19. Results of such studies will be key for 66 a mechanistic understanding of the SARS-CoV-2 pandemic, adaptation of containment 67 methods and to support vaccine development.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (T cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting T cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust T cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of T cell differentiation and a promising target for T cell-skewing vaccine adjuvants.