Human memory B cells show plasticity and adopt multiple fates upon recall response to SARS-CoV-2.

The B cell response to different pathogens uses tailored effector mechanisms and results in functionally specialized memory B (Bm) cell subsets, including CD21+ resting, CD21-CD27+ activated and CD21-CD27- Bm cells. The interrelatedness between these Bm cell subsets remains unknown. Here we showed that single severe acute respiratory syndrome coronavirus 2-specific Bm cell clones showed plasticity upon antigen rechallenge in previously exposed individuals. CD21- Bm cells were the predominant subsets during acute infection and early after severe acute respiratory syndrome coronavirus 2-specific immunization. At months 6 and 12 post-infection, CD21+ resting Bm cells were the major Bm cell subset in the circulation and were also detected in peripheral lymphoid organs, where they carried tissue residency markers. Tracking of individual B cell clones by B cell receptor sequencing revealed that previously fated Bm cell clones could redifferentiate upon antigen rechallenge into other Bm cell subsets, including CD21-CD27- Bm cells, demonstrating that single Bm cell clones can adopt functionally different trajectories.

Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer.

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. In vitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic T cells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.

Distinct immunological signatures discriminate severe COVID-19 from non-SARS-CoV-2-driven critical pneumonia.

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19.

Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

Signature of long-lived memory CD8+ T cells in acute SARS-CoV-2 infection.

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.

SLiM-binding pockets: an attractive target for broad-spectrum antivirals.

Short linear motif (SLiM)-mediated interactions offer a unique strategy for viral intervention due to their compact interfaces, ease of convergent evolution, and key functional roles. Consequently, many viruses extensively mimic host SLiMs to hijack or deregulate cellular pathways and the same motif-binding pocket is often targeted by numerous unrelated viruses. A toolkit of therapeutics targeting commonly mimicked SLiMs could provide prophylactic and therapeutic broad-spectrum antivirals and vastly improve our ability to treat ongoing and future viral outbreaks. In this opinion article, we discuss the therapeutic relevance of SLiMs, advocating their suitability as targets for broad-spectrum antiviral inhibitors.

A Consensus Binding Motif for the PP4 Protein Phosphatase.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.