A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast

A.D.R. performed the majority of the bioinformatic analysis and interpretation of the data. S.P. contributed to the study design, sample processing, analysis and interpretation of the data. J.S. contributed to the sample processing. D.J.K. and P.H. contributed to the data processing, batch correction and cell cluster identification. A.S. contributed to the design of the sample batches and contributed to the analysis of the raw data. A.J.T. contributed to the analysis of the data and Figure design. L.J.P. performed the immune histochemistry validations. K.H. assisted A.D.R. with the inferCNV analysis and interpretation. P.H. assisted with the subclustering of immune cells and scVI integration analysis. A.Q.S. performed the immunofluorescence quantification. K.K. performed all the scRNA-seq library preparation and sequencing. R.B.M., I.G., J.J.G., V.S. and J.L.J. provided the human tissues and the metadata from the 55 donors. A.D.R., S.P., J.C.M. and W.T.K. wrote the paper. J.C.M. and W.T.K. conceptualized and supervised the study.Peer reviewe

Loss of the interleukin-6 receptor causes immunodeficiency, atopy, and abnormal inflammatory responses

Abstract: IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and this is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R. We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3 and ZNF341, genes encoding different components of the IL-6 signalling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R.J.E.D.T. is supported by the MRC (RG95376 and MR/L006197/1). KB is supported by the European Research Council (ERC StG 310857) and the Austrian Science Fund (P29951-B30). This work is supported, in part, by the intramural research program of the NIAID, NIH. A.J.T. is supported by the Wellcome Trust (104807/Z/14/Z) and the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. KGCS is supported by the Medical Research Council (program grant MR/L019027) and is a Wellcome Investigator. M.G. and S.T. are supported in part by Cancer Research UK. RCA and MT are supported by a DOC fellowship of the Austrian Academy of Sciences. This research was made possible through access to the data and findings generated by two pilot studies for the 100,000 Genomes Project. The enrolment for one pilot study was coordinated by the NIHR BioResource (preprint from doi: https://doi.org/10.1101/507244) and the other by Genomics England Limited (GEL), a wholly owned company of the Department of Health in the UK. Over 90% of participants in the pilot studies have been enrolled in the NIHR BioResource. These pilot studies were mainly funded by grants from the National Institute for Health Research (NIHR) in England to the University of Cambridge and GEL, respectively. Additional funding was provided by the BHF, MRC, NHS England, the Wellcome Trust, amongst many other funders. The pilot studies use data provided by patients and their close relatives and collected by the NHS and other healthcare providers as part of their care and support. We thank all volunteers for their participation, and also gratefully acknowledge NIHR Biomedical Research Centres, NIHR BioResource Centres, NHS Trust Hospitals, NHS Blood and Transplant and staff for their contribution. ST is on the scientific advisory board for Ipsen, and is a consultant for Kallyope Inc. The authors declare no competing financial interests

Single-cell multi-omics analysis of the immune response in COVID-19

Peer reviewedPublisher PD

Single-cell multi-omics analysis of the immune response in COVID-19

Funder: Lister Institute of Preventive Medicine; doi: https://doi.org/10.13039/501100001255Funder: University College London, Birkbeck MRC Doctoral Training ProgrammeFunder: The Jikei University School of MedicineFunder: Action Medical Research (GN2779)Funder: NIHR Clinical Lectureship (CL-2017-01-004)Funder: NIHR (ACF-2018-01-004) and the BMA FoundationFunder: Chan Zuckerberg Initiative (grant 2017-174169) and from Wellcome (WT211276/Z/18/Z and Sanger core grant WT206194)Funder: UKRI Innovation/Rutherford Fund Fellowship allocated by the MRC and the UK Regenerative Medicine Platform (MR/5005579/1 to M.Z.N.). M.Z.N. and K.B.M. have been funded by the Rosetrees Trust (M944)Funder: Barbour FoundationFunder: ERC Consolidator and EU MRG-Grammar awardsFunder: Versus Arthritis Cure Challenge Research Grant (21777), and an NIHR Research Professorship (RP-2017-08-ST2-002)Funder: European Molecular Biology Laboratory (EMBL)Abstract: Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy

Relationship between therapeutic efficacy of doxorubicin-transferrin conjugate and expression of P-glycoprotein in chronic erythromyeloblastoid leukemia cells sensitive and resistant to doxorubicin

Background Conjugation of anti-neoplastic agents with human proteins is a strategy to diminish the toxic side effects of anthracycline antibiotics. We have developed a novel doxorubicin-transferrin (DOX-TRF) conjugate aimed to direct anticancer drugs against therapeutic targets that display altered levels of expression in malignant versus normal cells. Our previous work has shown that the cellular bio-distribution of the conjugate is dependent on a dynamic balance between influx and efflux processes. Here, we set out to investigate whether P-glycoprotein (P-gp) expression may affect DOXTRF conjugate-induced cellular drug accumulation and cytotoxicity. Results All experiments were carried out on human erythromyeloblastoid cells exhibiting P-gp over-expression (K562/DOX) and its drug sensitive parental line (K562). MTT cytotoxicity, flow cytometry, fluorescence microscopy and RT-PCR assessments revealed that the investigated conjugate (DOX-TRF) possesses a greater cytotoxic potential than free DOX. Conclusion Our data suggest that the newly developed DOXTRF conjugate is a less P-gp dependent substrate than free DOX and, consequently, may be used in a clinical setting to increase treatment efficacy in resistant human tumors.Grant no. 545/ 756 of the University of Lodz, Poland

The Toll-like Receptor 4 Polymorphism Asp299Gly Is Associated with an Increased Risk of Ovarian Cancer

Ovarian cancer (OC) is one of the most common cancers threatening women’s lives around the world. Epithelial ovarian tumors represent the most common ovarian neoplasms. Most OC patients are diagnosed at the advanced stage, and there is an urgent need to identify novel biomarkers of the disease. Single-nucleotide polymorphisms (SNPs) in TLR genes may serve as crucial markers of cancer susceptibility. We investigated the frequency of TLR polymorphisms in a group of 200 women, including 70 with OC. Four SNPs, two each in TLR4 (rs4986790 and rs4986791) and TLR9 (rs187084 and rs5743836), were analyzed using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). The digested fragments were separated and identified by multicapillary electrophoresis. The load quantification of human papillomavirus (HPV) types 16/18 was determined using a digital droplet PCR method. We found an increased frequency of heterozygous genotype and minor allele of the TLR4 rs4986790 SNP in women with OC compared with healthy controls, and this result remained highly significant after Bonferroni’s correction for multiple testing (p TLR SNPs. The findings suggest that the TLR4 Asp299Gly polymorphism could be a genetic risk factor for the development of OC

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Acknowledgements: We thank the staff at the Cambridge NIHR BRC Cell Phenotyping Hub and the Genomic and Histopathology Core at the CRUK Cambridge Institute for their constant support and assistance and S. Westermann for designing the schematic in Fig. 1. We acknowledge the role of the Breast Cancer Now Tissue Bank in collecting and making available the samples used in the generation of this publication and all the patients who donated. This study was primarily funded by an MRC project grant (MR/S036059/1) and supported by BBSRC project grant (BB/S006745/1), Breast Cancer Now project grant (2017MayPR907), CRUK career establishment award (17348) and CRUK programme foundation award (DCRPGF\100010) to W.T.K. and core funding from EMBL and CRUK (C9545/A29580) to J.C.M.Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.This study was primarily funded by an MRC project grant (MR/S036059/1), CRUK career establishment award (17348) and CRUK programme foundation award (DCRPGF\100010) to WTK and core funding from EMBL and CRUK (C9545/A29580) to JCM

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.This study was primarily funded by an MRC project grant (MR/S036059/1), CRUK career establishment award (17348) and CRUK programme foundation award (DCRPGF\100010) to WTK and core funding from EMBL and CRUK (C9545/A29580) to JCM