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The functional relevance of pre-existing cross-immunity to SARS-CoV-2 is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)-reactive and SARS-CoV-2-cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that pre-existing spike- and S816-830-reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti-SARS-CoV-2-S1-IgG antibodies. Spike-cross-reactive T cells were also activated after primary BNT162b2 COVID-19 mRNA vaccination displaying kinetics similar to secondary immune responses. Our results highlight the functional contribution of pre-existing spike-cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity following primary SARS-CoV-2 immunization and the high rate of asymptomatic/mild COVID-19 disease courses.

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.

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRAS G12V in mouse intestinal organoids, while transgenic BRAF V600E activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with β-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.

BackgroundSARS-CoV-2 mRNA vaccination of healthy individuals is highly immunogenic and protective against severe COVID-19. However, there are limited data on how disease-modifying therapies (DMTs) alter SARS-CoV-2 mRNA vaccine immunogenicity in patients with autoimmune diseases.MethodsAs part of a prospective cohort study, we investigated the induction, stability and boosting of vaccine-specific antibodies, B cells and T cells in patients with multiple sclerosis (MS) on different DMTs after homologous primary, secondary and booster SARS-CoV-2 mRNA vaccinations. Of 126 patients with MS analysed, 105 received either anti-CD20-based B cell depletion (aCD20-BCD), fingolimod, interferon-β, dimethyl fumarate, glatiramer acetate, teriflunomide or natalizumab, and 21 were untreated MS patients for comparison.ResultsIn contrast to all other MS patients, and even after booster, most aCD20-BCD- and fingolimod-treated patients showed no to markedly reduced anti-S1 IgG, serum neutralising activity and a lack of receptor binding domain-specific and S2-specific B cells. Patients receiving fingolimod additionally lacked spike-reactive CD4+ T cell responses. The duration of fingolimod treatment, rather than peripheral blood B and T cell counts prior to vaccination, determined whether a humoral immune response was elicited.ConclusionsThe lack of immunogenicity under long-term fingolimod treatment demonstrates that functional immune responses require not only immune cells themselves, but also access of these cells to the site of inoculation and their unimpeded movement. The absence of humoral and T cell responses suggests that fingolimod-treated patients with MS are at risk for severe SARS-CoV-2 infections despite booster vaccinations, which is highly relevant for clinical decision-making and adapted protective measures, particularly considering additional recently approved sphingosine-1-phosphate receptor antagonists for MS treatment.

While evidence for pre-existing SARS-CoV-2-cross-reactive CD4+ T cells in unexposed individuals is increasing, their functional significance remains unclear. Here, we comprehensively determined SARS-CoV-2-cross-reactivity and human coronavirus-reactivity in unexposed individuals. SARS-CoV-2-cross-reactive CD4+ T cells were ubiquitous, but their presence decreased with age. Within the spike glycoprotein fusion domain, we identified a universal immunodominant coronavirus-specific peptide epitope (iCope). Pre-existing spike- and iCope-reactive memory T cells were efficiently recruited into mild SARS-CoV-2 infections and their abundance correlated with higher IgG titers. Importantly, the cells were also reactivated after primary BNT162b2 COVID-19 mRNA vaccination in which their kinetics resembled that of secondary immune responses. Our results highlight the functional importance of pre-existing spike-cross-reactive T cells in SARS-CoV-2 infection and vaccination. Abundant spike-specific cross-immunity may be responsible for the unexpectedly high efficacy of current vaccines even with single doses and the high rate of asymptomatic/mild infection courses.

COVID-19, coronavirus disease 2019; d28, day 28 after first vaccination; d49, day 49 after first vaccination; ∼d28, around day 28 after COVID-19 symptom onset; ∼d49, around day 49 after COVID-19 symptom onset; ∼d94, around day 94 after COVID-19 symptom onset; RT-PCR, real-time PCR; S1, SARS-CoV-2 spike glycoprotein subunit 1; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; S-I, peptide mix representing the SARS-CoV-2 spike glycoprotein N-terminal part; sIgA, secretory IgA; S-II, peptide mix representing the SARS-CoV-2 spike glycoprotein C-terminal part mix.