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textabstractBackground: Synthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel‐Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOECs. Objective: To generate a VWF‐deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells. Methods: We used CRISPR/CRISPR‐associated protein 9 editing in single‐donor cord blood–derived BOECs (cbBOECs) to generate clonal VWF−/− cbBOECs. Clones were selected using high‐throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized. Results: Two VWF−/− BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin‐3 and the cargo proteins angiopoietin (Ang)‐2, interleukin (IL)‐6, and IL‐8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang‐2 was relocated to the cell periphery and colocalized with Tie‐2. Conclusions: CRISPR editing of VWF provides a robust method to create VWF‐ deficient BOECs that can be directly compared to their wild‐type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang‐2/Tie‐2 interaction contributes to angiogenic abnormalities in VWD patients.

Background: A large global effort is ongoing to develop vaccines against SARS-CoV-2, the causative agent of COVID-19. While there is accumulating information on the antibody response against SARS-CoV-2, less is known about the SARS-CoV-2 antigens that are targeted by CD8 T cells. Such knowledge will be of high value to gain fundamental insights into the antigenic landscape of SARS-CoV-2 recognized by CD8 T cells, to develop tool allowing focused analysis of the SARS-CoV-2 specific T cell responses directly ex vivo, and to understand whether current vaccine designs are covering the CD8 T cell recognized antigens. Methods: To address this issue, we have analyzed samples from 18 COVID-19 patients for CD8 T cell recognition of 500 predicted SARS-CoV-2-derived epitopes restricted to 10 common HLA-A and HLA-B alleles. For each HLA allele, the top 50 epitopes were selected based on predicted binding affinity and likelihood of successful proteasomal processing. To probe for CD8 T cell recognition of the selected epitope-HLA complexes, we made use of our in-house technology based on multiplexing of peptide HLA (pHLA) multimers conjugated to fluorescent dyes. Results: In addition to previous studies showing CD8 T cell reactivity towards epitopes derived from the spike protein of SARS-CoV-2, we have identified several CD8 T cell recognized epitopes derived from the ORF1ab, including one epitope displaying clear immunodominant properties across patients positive for HLA-A*01:01. Investigation of the functional status of part of the identified responses (including 4 responses specific for the immunodominant epitope) revealed that the T cell responses were highly dysfunctional. In addition the SARS-CoV-2 specific CD8 T cell responses displayed an increased expression of NKG2A in comparison with bulk CD8 T cells, which may explain their dysfunctional state. Conclusions: Our data suggest that part of the ORF1ab encodes multiple CD8 T cell antigens including one immunodominant epitope. Noteworthy these epitopes were derived from a part of the viral genome that is not included in the majority of vaccine candidates in development, and this may potentially influence their clinical activity and safety profile. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.