No abstract

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in Spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of Spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

SUMMARY CD4+Foxp3+ regulatory T cells (Tregs) are known to control the progression of autoimmune diabetes, but when, where and how they exert their influence in this context are questions still under vigorous debate. Exploiting a transgene encoding the human diphtheria toxin receptor, we punctually and specifically ablated Foxp3+ cells in the BCD2.5/NOD mouse model of autoimmune diabetes. Strikingly, overt diabetes developed within three days. The earliest detectable event was the activation of natural killer cells (NK) directly within the insulitic lesion, notably induction of gene expression already by seven hours. Increased expression of IL-12p40 by neighboring dendritic cells followed shortly thereafter. Interferon (IFN)-γ impacted extensively on the gene-expression program of the local CD4+ effector cell population, unleashing it to aggressively attack the islets, and very crucial for the development of diabetes. Thus, Tregs rein in pancreatic autoimmunity in situ through control of a central innate immune system player, NK cells.

Interference with inhibitory immunological checkpoints controlling T cell activation provides new opportunities to augment cancer immunotherapies. Whereas cytotoxic T lymphocyte -associated antigen-4 blockade has shown promising preclinical and clinical results, therapeutic CD4 + CD25 + T reg cell depletion has failed to consistently enhance immunebased therapies. Using B16/BL6, a transplantable murine melanoma model, we show a dichotomy between the effects of T reg cell depletion on tumor rejection dependent on whether depletion occurs before (prophylactic) or after (therapeutic) tumor engraftment. Failure to promote rejection with therapeutic depletion is not related to lack of T reg cell depletion, to elimination of CD25 + effector T cells, or to a failure to enhance systemic antitumor T cell responses, but correlates with failure of effector cells to infi ltrate the tumor and increase the intratumor ratio of effector T cell/T reg cell. Finally, systemic antitumor responses generated upon therapeutic T reg cell depletion are signifi cantly stronger than those generated in the presence of T reg cells, and are capable of eliciting rejection of established tumors after transfer into immunoablated recipients receiving combination immunotherapy. The data demonstrate a dissociation between measurable systemic responses and tumor rejection during CD25-directed T reg cell depletion, and suggest an alternative, clinically applicable strategy for the treatment of established tumors.

Passage through the β-selection developmental checkpoint requires productive rearrangement of Tcrb gene segments and formation of a pre-T cell receptor (pre-TCR) on the surface of CD4–CD8– thymocytes. How other receptors influence β-selection is less well understood. Here, we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associates with the pre-TCR and influences β-selection by regulating steady-state localization of immature thymocytes within thymic sub-regions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. These data designate CXCR4 as a co-stimulator of the pre-TCR during β-selection.

Most antigenic peptides presented on MHC class I molecules are generated by proteasomes during protein breakdown. It is unknown whether these peptides are protected from destruction by cytosolic peptidases. In cytosolic extracts, most antigenic peptides are degraded by the metalloendopeptidase, thimet oligopeptidase (TOP). We therefore examined whether TOP destroys antigenic peptides in vivo. When TOP was overexpressed in cells, class I presentation of antigenic peptides was reduced. In contrast, TOP overexpression didn't reduce presentation of peptides generated in the endoplasmic reticulum or endosomes. Conversely, preventing TOP expression with siRNA enhanced presentation of antigenic peptides. TOP therefore plays an important role in vivo in degrading peptides released by proteasomes and is a significant factor limiting the extent of antigen presentation.

Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep.

The origin and intermediate host for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be determined. Coronaviruses found to be closely related to SARS-CoV-2 include RaTG13 derived from bat and two clusters (PCoV-GD and PCoV-GX) of coronaviruses identified in pangolin. Here, we studied the infectivity and antigenicity patterns of SARS-CoV-2 and the three related coronaviruses. Compared with the other three viruses, RaTG13 showed almost no infectivity to a variety of cell lines. The two pangolin coronaviruses and SARS-CoV-2 showed similar infectious activity. However, in SARS-CoV-2-susceptible cell lines, the pangolin coronaviruses presented even higher infectivity. The striking difference between the SARS-CoV-2 and pangolin coronaviruses is that the latter can infect porcine cells, which could be partially attributed to an amino acid difference at the position of 498 of the spike protein. The infection by SARS-CoV-2 was mainly mediated by Furin and TMPRSS2, while PCoV-GD and PCoV-GX mainly depend on Cathepsin L. Extensive cross-neutralization was found between SARS-CoV-2 and PCoV-GD. However, almost no cross-neutralization was observed between PCoV-GX and SARS-CoV-2 or PCoV-GD. More attention should be paid to pangolin coronaviruses and to investigate the possibility of these coronaviruses spreading across species to become zoonoses among pigs or humans.