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  1. Objective

    To obtain the comprehensive transcriptome profile of human citrulline‐specific B cells from patients with rheumatoid arthritis (RA).

    Methods

    Citrulline‐ and hemagglutinin‐specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood ofRApatients and healthy individuals. The transcriptome profile of the sorted cells was obtained byRNA‐sequencing, and expression of key protein molecules was evaluated by aptamer‐basedSOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays.

    Results

    Citrulline‐specific B cells, in comparison to citrulline‐negative B cells, from patients withRAdifferentially expressed the interleukin‐15 receptor α (IL‐15Rα) gene as well as genes related to protein citrullination and cyclicAMPsignaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositiveRApatients, the expression ofIL‐15Rα protein was enriched in citrulline‐specific B cells from the patients’ peripheral blood, and surprisingly, all B cells fromRApatients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production ofAREGdirectly led to increased migration and proliferation of fibroblast‐like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts.

    Conclusion

    To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing severalUSFood and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.

     
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  2. Graphical abstract [Formula: see text] 
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  3. null (Ed.)
    Abstract Background The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. Methods We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout ( CD8 and CD4 ) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. Results We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8 + T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8 + T-cell function. TCGA pan-cancer data confirmed that CD8 low Platelet high patients have a significantly lower survival probability compared to CD8 high Platelet low . Conclusions CD8 + T cells inhibit metastasis. When the balance between CD8 + T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8 + T-cell function. 
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  4. The protein p53 is a crucial tumor suppressor, often called “the guardian of the genome”; however, mutations transform p53 into a powerful cancer promoter. The oncogenic capacity of mutant p53 has been ascribed to enhanced propensity to fibrillize and recruit other cancer fighting proteins in the fibrils, yet the pathways of fibril nucleation and growth remain obscure. Here, we combine immunofluorescence three-dimensional confocal microscopy of human breast cancer cells with light scattering and transmission electron microscopy of solutions of the purified protein and molecular simulations to illuminate the mechanisms of phase transformations across multiple length scales, from cellular to molecular. We report that the p53 mutant R248Q (R, arginine; Q, glutamine) forms, both in cancer cells and in solutions, a condensate with unique properties, mesoscopic protein-rich clusters. The clusters dramatically diverge from other protein condensates. The cluster sizes are decoupled from the total cluster population volume and independent of the p53 concentration and the solution concentration at equilibrium with the clusters varies. We demonstrate that the clusters carry out a crucial biological function: they host and facilitate the nucleation of amyloid fibrils. We demonstrate that the p53 clusters are driven by structural destabilization of the core domain and not by interactions of its extensive unstructured region, in contradistinction to the dense liquids typical of disordered and partially disordered proteins. Two-step nucleation of mutant p53 amyloids suggests means to control fibrillization and the associated pathologies through modifying the cluster characteristics. Our findings exemplify interactions between distinct protein phases that activate complex physicochemical mechanisms operating in biological systems.

     
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  5. null (Ed.)
    Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. Methods To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. Results With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107a pos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. Conclusions Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit. 
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  6. null (Ed.)
  7. null (Ed.)
    Background Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer. Methods To identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in an unbiased manner, we performed genome-wide CRISPR immune screens and integrated our results with multi-omics clinical data to evaluate the role of tumor intrinsic factors in regulating two rate-limiting steps of cancer immunotherapy, namely, T cell tumor infiltration and T cell-mediated tumor killing. Results Our studies revealed two distinct types of immune resistance regulators and demonstrated their potential as therapeutic targets to improve the efficacy of immunotherapy. Among them, PRMT1 and RIPK1 were identified as a dual immune resistance regulator and a cytotoxicity resistance regulator, respectively. Although the magnitude varied between different types of immunotherapy, genetically targeting PRMT1 and RIPK1 sensitized tumors to T-cell killing and anti-PD-1/OX40 treatment. Interestingly, a RIPK1-specific inhibitor enhanced the antitumor activity of T cell-based and anti-OX40 therapy, despite limited impact on T cell tumor infiltration. Conclusions Collectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations. 
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