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Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin β1, only some integrin β7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from severe COVID-19 patients every third to seventh day during hospitalization and every third month after recovery. We profiled the antigen-specific immune cell dynamics by combining single cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-Seq, B cell receptor (BCR)-Seq with oligo-tagged antigen baits. While the proportion of Spike Receptor Binding Domain-specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen specific cells, which was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to one year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen specific B cell responses in longitudinally sampled COVID-19 infected patients.

Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment in vitro. Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry. One hundred forty-three proteins were detected and their relative abundance was calculated using a reference sample generated from all PDSs. We performed heatmap analysis of all the detected proteins to display their expression patterns across different PDSs together with pathway enrichment analysis to reveal which processes that were connected to PDS protein composition. This protein dataset together with clinical information is useful to investigators studying the microenvironment of breast cancers. Further, after repopulating PDSs with either MCF7 or MDA-MB-231 cells, we quantified their gene expression profiles using RNA sequencing. These data were also compared to cells cultured in conventional 2D conditions, as well as to cells cultured as xenografts in immune-deficient mice. We investigated the overlap of genes regulated between these different culture conditions and performed pathway enrichment analysis of genes regulated by both PDS and xenograft cultures compared to 2D in both cell lines to describe common processes associated with both culture conditions. Apart from our described analyses of these systems, these data are useful when comparing different experimental model systems. Downstream data analyses and interpretations can be found in the research article "Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment" [1].

The atypical protein kinase C isoform ι (PKCι) is upregulated, which cooperates with mutated KRAS (mu-KRAS) to promote the development of pancreatic cancers. However, the exact role of PKCι in KRAS-mediated pancreatic tumorigenesis is not fully defined. In the present study, we demonstrate that mu-KRAS upregulates and activates PKCι, accompanied by dephosphorylation of large tumor suppressor (LATS), a key member of the growth-inhibiting Hippo signaling pathway. As a result, Yes-associated protein 1 (YAP1; a transcriptional coactivator) is dephosphorylated and translocates to the nucleus, which promotes transcription of downstream target genes to sustain the transformed growth of pancreatic cancer cells. In contrast, when PKCι is suppressed by the chemical inhibitor or small-hairpin RNA, the levels of phosphorylated LATS and YAP1 are elevated and YAP1 is excluded from the nucleus, which enhances the susceptibility of pancreatic cancer cells harboring mu-KRAS to apoptosis. These findings shed new light on the mechanisms underlying the pancreatic tumorigenesis initiated by mu-KRAS, and suggest that the PKCι-YAP1 signaling may potentially be therapeutically targeted for restricting the growth and inducing apoptosis in pancreatic tumors expressing mu-KRAS.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant disease with 5-year survival rate of less than 6%. Activating mutations of Kras (mu-Kras) are often detected in most of PDAC patients. Although it has been known that oncogenic Kras is the driver of pancreatic cancer initiation and development, the underlying mechanisms by which mu-Kras promotes PDAC remain poorly understood. Here, we identify that PKCι is one of the crucial factors for supporting the survival of pancreatic cancer cells expressing mu-Kras. Our study demonstrates that after the knockdown of PKCι, the expression of the transcriptional co-activator YAP1 is decreased, which hinders the expression of the downstream target gene Mcl-1, and subsequently sensitizes pancreatic cancer MiaPaCa and PANC-1 cells experssing mu-Kras to apoptosis. In comparison, the suppression of PKCι has little impact on the viability of non-neoplastic pancreatic HPDE6-C7 cells. Moreover, the transient overexpression of oncogenic Kras in HPDE6-C7 elevates the expression of PKCι and YAP1 concomitantly. The upregulated YAP1 in HPDE6-C7/ mu-Kras cells is abolished once PKCι is suppressed, suggesting the linear relationship among mu-Kras, PKCι and YAP1. This phenomenon is further proven by the co-upregulation of PKCι and YAP1 in HPDE6-C7 cells stably transfected with mu-Kras. Taken together, our findings suggest that PKCι acts through promoting YAP1 function to promote the survival of pancreatic cancer cells expressing mu-Kras. It appears that targeting PKCι-YAP1 signaling is a feasible strategy for developing new therapeutics for treating pancreatic cancer patients.

Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that are blocked in differentiation. Acute promyelocytic leukemia (APL) is a rare form of AML, which generally presents with a t(15;17) translocation causing expression of the fusion protein PML-RARA. Pharmacological doses of all-trans retinoic acid (ATRA) induce granulocytic differentiation of APL cells leading to cure rates of >80% if combined with conventional chemotherapy. Autophagy is a lysosomal degradation pathway for the removal of cytoplasmic content and recycling of macromolecules. ATRA induces autophagy in ATRA-sensitive AML and APL cells and autophagy inhibition attenuates ATRA-triggered differentiation. In this study, we aimed at identifying if the autophagy-linked FYVE-domain containing protein (ALFY/WDFY3) is involved in autophagic degradation of protein aggregates contributes to ATRA therapy-induced autophagy. We found that ALFY mRNA levels increase significantly during the course of ATRA-induced differentiation of APL and AML cell lines. Importantly ALFY depletion impairs ATRA-triggered granulocytic differentiation of these cells. In agreement with its function in aggrephagy, knockdown of ALFY results in reduced ATRA-induced proteolysis. Our data further suggest that PML-RARα is an autophagy substrate degraded with the help of ALFY. In summary, we present a crucial role for ALFY in retinoid triggered maturation of AML cells.

Polymorphic variants of the FTO (fat mass and obesity) gene associate with body mass index in humans, but the underlying molecular mechanisms have not been firmly determined. FTO is linked to energy homeostasis via amino acid sensing and is thought to activate the mammalian target of rapamycin complex 1, a negative regulator of autophagy. FTO localises both to the nucleus and the cytoplasm, and in this study we identify a functional nuclear localisation signal (NLS) in the N-terminus of FTO, as well as nuclear localization information in its very C-terminus. Inhibition of FTO nuclear transport has no effect on autophagy and in contrast to a previously proposed role of FTO in autophagy, we find no difference in starvation-induced autophagy in control cells compared to a panel of cell types depleted of FTO. Future studies that further characterise the cellular functions of FTO will be important to understand why variants in FTO are associated with body weight.

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients.

Autophagy, a highly conserved lysosomal degradation pathway, was initially characterized as a bulk degradation system induced in response to starvation. In recent years, autophagy has emerged also as a highly selective pathway, targeting various cargoes such as aggregated proteins and damaged organelles for degradation. The key factors involved in selective autophagy are autophagy receptors and adaptor proteins, which connect the cargo to the core autophagy machinery. In this review, we discuss the current knowledge about the only mammalian adaptor protein identified thus far, autophagy-linked FYVE protein (ALFY). ALFY is a large, scaffolding, multidomain protein implicated in the selective degradation of ubiquitinated protein aggregates by autophagy. We also comment on the possible role of ALFY in the context of disease.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid and/or arsenic trioxide represents a paradigm in targeted cancer therapy because these drugs cause clinical remission by affecting the stability of the fusion oncoprotein promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARA). The authors of previous studies have implicated the ubiquitin-proteasome pathway as the main mechanism involved in therapy-induced PML/RARA degradation. Here we have investigated a role of autophagy, a protein degradation pathway that involves proteolysis of intracellular material within lysosomes. We found that both all-trans retinoic acid and arsenic trioxide induce autophagy via the mammalian target of rapamycin pathway in APL cells and that autophagic degradation contributes significantly both to the basal turnover as well as the therapy-induced proteolysis of PML/RARA. In addition, we observed a correlation between autophagy and therapy-induced differentiation of APL cells. Given the central role of the PML/RARA oncoprotein in APL pathogenesis, this study highlights an important role of autophagy in the development and treatment of this disease.

There is growing evidence that macroautophagic cargo is not limited to bulk cytosol in response to starvation and can occur selectively for substrates, including aggregated proteins. It remains unclear, however, whether starvation-induced and selective macroautophagy share identical adaptor molecules to capture their cargo. Here, we report that Alfy, a phosphatidylinositol 3-phosphate-binding protein, is central to the selective elimination of aggregated proteins. We report that the loss of Alfy inhibits the clearance of inclusions, with little to no effect on the starvation response. Alfy is recruited to intracellular inclusions and scaffolds a complex between p62(SQSTM1)-positive proteins and the autophagic effectors Atg5, Atg12, Atg16L, and LC3. Alfy overexpression leads to elimination of aggregates in an Atg5-dependent manner and, likewise, to protection in a neuronal and Drosophila model of polyglutamine toxicity. We propose that Alfy plays a key role in selective macroautophagy by bridging cargo to the molecular machinery that builds autophagosomes.

Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. Here we demonstrate that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity.

Upon completion of cytokinesis, the midbody ring is transported asymmetrically into one of the two daughter cells where it becomes a midbody ring derivative that is degraded by autophagy. In this study we showed that the ubiquitin-binding autophagy receptor SQSTM1/p62 and the interacting adaptor protein WDFY3/ALFY form a complex with the ubiquitin E3 ligase TRAF6 and that these proteins, as well as NBR1, are important for efficient clearance of midbody ring derivatives by autophagy. The number of ubiquitinated midbody ring derivatives decreases in TRAF6-depleted cells and we showed that TRAF6 mediates ubiquitination of the midbody ring localized protein KIF23/MKLP1. We conclude that TRAF6-mediated ubiquitination of the midbody ring is important for its subsequent recognition by ubiquitin-binding autophagy receptors and degradation by selective autophagy.

Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells. © 2011 Landes Bioscience.

Accumulation of ubiquitinated proteins in cytoplasmic and/or nuclear inclusions is a hallmark of several diseases associated with premature cell death. SQSTM1/p62 is known to bind ubiquitinated substrates and aid their aggregation and degradation by macroautophagy. We show here that p62 is required to recruit the large phosphoinositide-binding protein ALFY to cytoplasmic p62 bodies generated upon amino acid starvation or puromycin-treatment. ALFY, as well as p62, is required for formation and autophagic degradation of cytoplasmic ubiquitin-positive inclusions. Moreover, both p62 and ALFY localize to nuclear promyleocytic leukemia (PML) bodies. The Drosophila p62 homologue Ref(2) P accumulates in ubiquitinated inclusions in the brain of flies carrying mutations in the ALFY homologue Blue cheese, demonstrating that ALFY is required for autophagic degradation of p62-associated ubiquitinated proteins in vivo. We conclude that p62 and ALFY interact to organize misfolded, ubiquitinated proteins into protein bodies that become degraded by autophagy.