Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Anti-Phospholipid Antibodies and Coronavirus Disease 2019: Vaccination Does Not Trigger Early Autoantibody Production in Healthcare Workers.

A molecular mimicry between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins supports the possibility that autoimmunity takes place during coronavirus disease 2019 (COVID-19) contributing to tissue damage. For example, anti-phospholipid antibodies (aPL) have been reported in COVID-19 as a result of such mimicry and thought to contribute to the immunothrombosis characteristic of the disease. Consistently, active immunization with the virus spike protein may elicit the production of cross-reactive autoantibodies, including aPL. We prospectively looked at the aPL production in healthcare workers vaccinated with RNA- (BNT162b2, n. 100) or adenovirus-based vaccines (ChAdOx1, n. 50). Anti-cardiolipin, anti-beta2 glycoprotein I, anti-phosphatidylserine/prothrombin immunoglobulin G (IgG), IgA, and IgM before and after vaccination were investigated. Anti-platelet factor 4 immunoglobulins were also investigated as autoantibodies associated with COVID-19 vaccination. Additional organ (anti-thyroid) and non-organ (anti-nuclear) autoantibodies and IgG against human proteome were tested as further post-vaccination autoimmunity markers. The antibodies were tested one or three months after the first injection of ChAdOx1 and BNT162b2, respectively; a 12-month clinical follow-up was also performed. Vaccination occasionally induced low titers of aPL and other autoantibodies but did not affect the titer of pre-existing autoantibodies. No significant reactivities against a microarray of approximately 20,000 human proteins were found in a subgroup of ChAdOx1-vaccinees. Consistently, we did not record any clinical manifestation theoretically associated with an underlying autoimmune disorder. The data obtained after the vaccination (two doses for the RNA-based and one dose for the adenovirus-based vaccines), and the clinical follow-up are not supporting the occurrence of an early autoimmune response in this cohort of healthcare workers.

The USR domain of USF1 mediates NF-Y interactions and cooperative DNA binding.

The trimeric CCAAT-binding NF-Y is a "pioneer" Transcription Factor -TF- known to cooperate with neighboring TFs to regulate gene expression. Genome-wide analyses detected a precise stereo-alignment -10/12 bp- of CCAAT with E-box elements and corresponding colocalization of NF-Y with basic-Helix-Loop-Helix (bHLH) TFs. We dissected here NF-Y interactions with USF1 and MAX. USF1, but not MAX, cooperates in DNA binding with NF-Y. NF-Y and USF1 synergize to activate target promoters. Reconstruction of complexes by structural means shows independent DNA binding of MAX, whereas USF1 has extended contacts with NF-Y, involving the USR, a USF-specific amino acid sequence stretch required for trans-activation. The USR is an intrinsically disordered domain and adopts different conformations based on E-box-CCAAT distances. Deletion of the USR abolishes cooperative DNA binding with NF-Y. Our data indicate that the functionality of certain unstructured domains involves adapting to small variation in stereo-alignments of the multimeric TFs sites.

Integrated longitudinal immunophenotypic, transcriptional and repertoire analyses delineate immune responses in COVID-19 patients.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.

Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest. EMSA with recombinant Ala and Glu mutants confirm a role of Ser320, but not Ser326, in stabilization of DNA binding. Transactivation assays of the CCAAT-dependent MDR1 and RHOB promoters show loss in transcription function for Ser320Glu and Ser320Ala NF-YA mutants. Phylogenetic analysis of NF-YA proteins indicates that Ser320 is indeed evolutionarily conserved. We conclude that phosphorylation of this residue belongs to the core mechanisms of DNA-binding control, possibly driven by the necessity to unfasten binding of or to evict NF-Y from CCAAT sites under specific conditions of growth regulation.-Bernardini, A., Lorenzo, M., Nardini, M., Mantovani, R., Gnesutta, N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.

NF-Y activates genes of metabolic pathways altered in cancer cells.

The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells.

Interactions and CCAAT-binding of Arabidopsis thaliana NF-Y subunits.

BACKGROUND: NF-Y is a transcription factor that recognizes with high specificity and affinity the widespread CCAAT box promoter element. It is formed by three subunits: NF-YA and the NF-YB/NF-YC- heterodimer containing histone fold domains (HFDs). We previously identified a large NF-Y gene family in Arabidopsis thaliana, composed of 29 members, and characterized their expression patterns in various plant tissues. METHODS: We used yeast Two-hybrids assays (Y2H), pull-down and Electrophoretic Mobility Shift Assay (EMSA) in vitro experiments with recombinant proteins to dissect AtNF-YB/AtNF-YC interactions and DNA-binding with different AtNF-YAs. RESULTS: Consistent with robust conservation within HFDs, we show that heterodimerization is possible among all histone-like subunits, including the divergent and related LEC1/AtNF-YB9 and L1L/AtNF-YB6 required for embryo development. DNA-binding to a consensus CCAAT box was investigated with specific AtNF-YB/AtNF-YC combinations and observed with some, but not all AtNF-YA subunits. CONCLUSIONS: Our results highlight (i) the conserved heterodimerization capacity of AtNF-Y histone-like subunits, and (ii) the different affinities of AtNF-YAs for the CCAAT sequence. Because of the general expansion of NF-Y genes in plants, these results most likely apply to other species.