Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.
Published by Elsevier Inc.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19. Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.

How the microbiota affects health and disease is a crucial question. In mice, gut Clostridium bacteria are potent inducers of colonic interleukin (IL)-10-producing Foxp3 regulatory T cells (Treg), which play key roles in the prevention of colitis and in systemic immunity. In humans, although gut microbiota dysbiosis is associated with immune disorders, the underlying mechanism remains unknown. In contrast with mice, the contribution of Foxp3 Treg in colitis prevention has been questioned, suggesting that other compensatory regulatory cells or mechanisms may exist. Here we addressed the regulatory role of the CD4CD8 T cells whose presence had been reported in the intestinal mucosa and blood. Using colonic lamina propria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) from healthy individuals, and those with colon cancer and irritable bowel disease (IBD), we demonstrated that CD4CD8αα (DP8α) T lymphocytes expressed most of the regulatory markers and functions of Foxp3 Treg and secreted IL-10. Strikingly, DP8α LPL and PBL exhibited a highly skewed repertoire toward the recognition of Faecalibacterium prausnitzii, a major Clostridium species of the human gut microbiota, which is decreased in patients with IBD. Furthermore, the frequencies of DP8α PBL and colonic LPL were lower in patients with IBD than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. Moreover, PBL and LPL from most patients with active IBD failed to respond to F. prausnitzii in contrast to PBL and LPL from patients in remission and/or healthy donors. These data (i) uncover a Clostridium-specific IL-10-secreting Treg subset present in the human colonic LP and blood, (ii) identify F. prausnitzii as a major inducer of these Treg, (iii) argue that these cells contribute to the control or prevention of colitis, opening new diagnostic and therapeutic strategies for IBD, and (iv) provide new tools to address the systemic impact of both these Treg and the intestinal microbiota on the human immune homeostasis.

Dendritic cells (DCs) are the most potent antigen-presenting cells involved in initiating the immune response, presenting antigens to T cells and leading to T cell proliferation. In an immature state, DCs lack accessory signals required for T cell stimulation but are highly specialised to capture antigens. Full DC maturation changes the cell surface phenotype and facilitates stimulation of T cell proliferative responses. To examine the degree of DC maturity associated with vernal keratoconjunctivitis (VKC), the authors examined the phenotype and antigen-presentation capability of blood derived DCs from VKC patients and from normal controls.
Flow cytometry was used to identify the cell surface expression of markers of DC maturity (CD83, CD86, major histocompatibility complex class II) and mixed leucocyte reactions to assess DC induction of T cell proliferation.
DCs derived from VKC patients were of a more mature phenotype than those from normal controls. However, these VKC DCs had reduced capability for induction of T cell proliferation compared with DCs from controls.
The increased maturity of DCs in VKC patients correlates with the heightened immune responsiveness associated with this disorder. A number of mechanisms may underlie the impaired ability of DCs in atopy to stimulate T cell proliferation. This impairment of DC induction of T cell activation is likely to be one factor which contributes to the modified inflammatory response seen in VKC patients and the recognised susceptibility of these patients to viral infection.