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Background: Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion., Methods: We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion., Results: During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1, encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes., Conclusions: Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP., Competing Interests: C.J.E.W. has received honoraria for lectures from Organox Ltd. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Organ shortage is a major challenge in kidney transplantation but the use of older donors, often with co-morbidities, is hampered by inconsistent outcomes. Methods of accurately stratifying marginal donor organs by clinical and histological assessment are lacking. To better understand organ variability, we profiled the transcriptomes of 271 kidneys from deceased donors at retrieval. Following correction for biopsy composition, we assessed molecular pathways that associated with delayed, and sub-optimal one-year graft function. Analysis of cortical biopsies identified an adaptive immune gene-rich module that significantly associated with increasing age and worse outcomes. Cellular deconvolution using human kidney reference single cell transcriptomes confirmed an increase in kidney-specific B and T cell signatures, as well as kidney macrophage, myofibroblast and fibroblast gene sets in this module. Surprisingly, innate immune pathway and neutrophil gene signature enrichment was associated with better outcomes. Thus, our work uncovers cellular molecular features of pathological organ ageing, identifiable at kidney retrieval, with translational potential., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

There is increasing interest in how immune cells in the meninges-the membranes that surround the brain and spinal cord-contribute to homeostasis and disease in the central nervous system 1,2 . The outer layer of the meninges, the dura mater, has recently been described to contain both innate and adaptive immune cells, and functions as a site for B cell development 3-6 . Here we identify organized lymphoid structures that protect fenestrated vasculature in the dura mater. The most elaborate of these dural-associated lymphoid tissues (DALT) surrounded the rostral-rhinal confluence of the sinuses and included lymphatic vessels. We termed this structure, which interfaces with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. Immune aggregates were present in DALT during homeostasis and expanded with age or after challenge with systemic or nasal antigens. DALT contain germinal centre B cells and support the generation of somatically mutated, antibody-producing cells in response to a nasal pathogen challenge. Inhibition of lymphocyte entry into the rostral-rhinal hub at the time of nasal viral challenge abrogated the generation of germinal centre B cells and class-switched plasma cells, as did perturbation of B-T cell interactions. These data demonstrate a lymphoid structure around vasculature in the dura mater that can sample antigens and rapidly support humoral immune responses after local pathogen challenge., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

B cells play a central role in humoral immunity but also have antibody-independent functions. Studies to date have focused on B cells in blood and secondary lymphoid organs but whether B cells reside in non-lymphoid organs (NLO) in homeostasis is unknown. Here we identify, using intravenous labeling and parabiosis, a bona-fide tissue-resident B cell population in lung, liver, kidney and urinary bladder, a substantial proportion of which are B-1a cells. Tissue-resident B cells are present in neonatal tissues and also in germ-free mice NLOs, albeit in lower numbers than in specific pathogen-free mice and following co-housing with 'pet-store' mice. They spatially co-localise with macrophages and regulate their polarization and function, promoting an anti-inflammatory phenotype, in-part via interleukin-10 production, with effects on bacterial clearance during urinary tract infection. Thus, our data reveal a critical role for tissue-resident B cells in determining the homeostatic 'inflammatory set-point' of myeloid cells, with important consequences for tissue immunity., (© 2023. The Author(s).)

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response., Competing Interests: Declaration of interests R.K.G. has received honoraria for consulting and educational activities from Gilead, GSK, Janssen, and Moderna., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Classic Hodgkin lymphoma (cHL) has a rich immune infiltrate, which is an intrinsic component of the neoplastic process. Malignant Hodgkin Reed-Sternberg cells (HRSCs) create an immunosuppressive microenvironment by the expression of regulatory molecules, preventing T-cell activation. It has also been demonstrated that mononuclear phagocytes (MNPs) in the vicinity of HRSCs express similar regulatory mechanisms in parallel, and their presence in tissue is associated with inferior patient outcomes. MNPs in cHL have hitherto been identified by a small number of canonical markers and are usually described as tumor-associated macrophages. The organization of MNP networks and interactions with HRSCs remains unexplored at high resolution. Here, we defined the global immune-cell composition of cHL and nonlymphoma lymph nodes, integrating data across single-cell RNA sequencing, spatial transcriptomics, and multiplexed immunofluorescence. We observed that MNPs comprise multiple subsets of monocytes, macrophages, and dendritic cells (DCs). Classical monocytes, macrophages and conventional DC2s were enriched in the vicinity of HRSCs, but plasmacytoid DCs and activated DCs were excluded. Unexpectedly, cDCs and monocytes expressed immunoregulatory checkpoints PD-L1, TIM-3, and the tryptophan-catabolizing protein IDO, at the same level as macrophages. Expression of these molecules increased with age. We also found that classical monocytes are important signaling hubs, potentially controlling the retention of cDC2 and ThExh via CCR1-, CCR4-, CCR5-, and CXCR3-dependent signaling. Enrichment of the cDC2-monocyte-macrophage network in diagnostic biopsies is associated with early treatment failure. These results reveal unanticipated complexity and spatial polarization within the MNP compartment, further demonstrating their potential roles in immune evasion by cHL., (© 2023 by The American Society of Hematology.)

Rationale: Obesity affects 40% of U.S. adults, is associated with a proinflammatory state, and presents a significant risk factor for the development of severe coronavirus disease (COVID-19). To date, there is limited information on how obesity might affect immune cell responses in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: To determine the impact of obesity on respiratory tract immunity in COVID-19 across the human lifespan. Methods: We analyzed single-cell transcriptomes from BAL in three ventilated adult cohorts with ( n  = 24) or without ( n  = 9) COVID-19 from nasal immune cells in children with ( n  = 14) or without ( n  = 19) COVID-19, and from peripheral blood mononuclear cells in an independent adult COVID-19 cohort ( n  = 42), comparing obese and nonobese subjects. Measurements and Main Results: Surprisingly, we found that obese adult subjects had attenuated lung immune or inflammatory responses in SARS-CoV-2 infection, with decreased expression of IFN-α, IFN-γ, and TNF-α (tumor necrosis factor α) response gene signatures in almost all lung epithelial and immune cell subsets, and lower expression of IFNG and TNF in specific lung immune cells. Peripheral blood immune cells in an independent adult cohort showed a similar but less marked reduction in type-I IFN and IFNγ response genes, as well as decreased serum IFNα, in obese patients with SARS-CoV-2. Nasal immune cells from obese children with COVID-19 also showed reduced enrichment of IFN-α and IFN-γ response genes. Conclusions: These findings show blunted tissue immune responses in obese patients with COVID-19, with implications for treatment stratification, supporting the specific application of inhaled recombinant type-I IFNs in this vulnerable subset.

Tumor behavior is intricately dependent on the oncogenic properties of cancer cells and their multi-cellular interactions. To understand these dependencies within the wider microenvironment, we studied over 270,000 single-cell transcriptomes and 100 microdissected whole exomes from 12 patients with kidney tumors, prior to validation using spatial transcriptomics. Tissues were sampled from multiple regions of the tumor core, the tumor-normal interface, normal surrounding tissues, and peripheral blood. We find that the tissue-type location of CD8 + T cell clonotypes largely defines their exhaustion state with intra-tumoral spatial heterogeneity that is not well explained by somatic heterogeneity. De novo mutation calling from single-cell RNA-sequencing data allows us to broadly infer the clonality of stromal cells and lineage-trace myeloid cell development. We report six conserved meta-programs that distinguish tumor cell function, and find an epithelial-mesenchymal transition meta-program highly enriched at the tumor-normal interface that co-localizes with IL1B-expressing macrophages, offering a potential therapeutic target., Competing Interests: Declaration of interests In the past 3 years, S.A.T. has consulted for Roche and Genentech and is a Scientific Advisory Board member of Qiagen, Foresite labs, Biogen, and GSK, as well as a consultant and equity holder as co-founder of Transition Bio., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lupus nephritis is a serious complication of systemic lupus erythematosus, mediated by IgG immune complex (IC) deposition in kidneys, with limited treatment options. Kidney macrophages are critical tissue sentinels that express IgG-binding Fcγ receptors (FcγRs), with previous studies identifying prenatally seeded resident macrophages as major IC responders. Using single-cell transcriptomic and spatial analyses in murine and human lupus nephritis, we sought to understand macrophage heterogeneity and subset-specific contributions in disease. In lupus nephritis, the cell fate trajectories of tissue-resident (TrMac) and monocyte-derived (MoMac) kidney macrophages were perturbed, with disease-associated transcriptional states indicating distinct pathogenic roles for TrMac and MoMac subsets. Lupus nephritis-associated MoMac subsets showed marked induction of FcγR response genes, avidly internalized circulating ICs, and presented IC-opsonized antigen. In contrast, lupus nephritis-associated TrMac subsets demonstrated limited IC uptake, but expressed monocyte chemoattractants, and their depletion attenuated monocyte recruitment to the kidney. TrMacs also produced B cell tissue niche factors, suggesting a role in supporting autoantibody-producing lymphoid aggregates. Extensive similarities were observed with human kidney macrophages, revealing cross-species transcriptional disruption in lupus nephritis. Overall, our study suggests a division of labor in the kidney macrophage response in lupus nephritis, with treatment implications - TrMacs orchestrate leukocyte recruitment while MoMacs take up and present IC antigen.

Bladder infection affects a hundred million people annually, but our understanding of bladder immunity is incomplete. We found type 17 immune response genes among the most up-regulated networks in mouse bladder following uropathogenic Escherichia coli (UPEC) challenge. Intravital imaging revealed submucosal Rorc + cells responsive to UPEC challenge, and we found increased Il17 and IL22 transcripts in wild-type and Rag2 -/- mice, implicating group 3 innate lymphoid cells (ILC3s) as a source of these cytokines. NCR-positive and negative ILC3 subsets were identified in murine and human bladders, with local proliferation increasing IL17-producing ILC3s post infection. ILC3s made a more limited contribution to bladder IL22, with prominent early induction of IL22 evident in Th17 cells. Single-cell RNA sequencing revealed bladder NCR-negative ILC3s as the source of IL17 and identified putative ILC3-myeloid cell interactions, including via lymphotoxin-β-LTBR. Altogether, our data provide important insights into the orchestration and execution of type 17 immunity in bladder defense., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Improving the efficacy of immune checkpoint therapies will require a better understanding of how immune cells are recruited and sustained in tumors. Here, we used the photoconversion of the tumor immune cell compartment to identify newly entering lymphocytes, determine how they change over time, and investigate their egress from the tumor. Combining single-cell transcriptomics and flow cytometry, we found that while a diverse mix of CD8 T cell subsets enter the tumor, all CD8 T cells retained within this environment for more than 72 h developed an exhausted phenotype, revealing the rapid establishment of this program. Rather than forming tumor-resident populations, non-effector subsets, which express TCF-1 and include memory and stem-like cells, were continuously recruited into the tumor, but this recruitment was balanced by concurrent egress to the tumor-draining lymph node. Thus, the TCF-1+ CD8 T cell niche in tumors is highly dynamic, with the circulation of cells between the tumor and peripheral lymphoid tissue to bridge systemic and intratumoral responses., (© 2022 Li et al.)

IgG antibodies form immune complexes (IC) that propagate inflammation and tissue damage in autoimmune diseases such as systemic lupus erythematosus. IgG IC engage Fcγ receptors (FcγR) on mononuclear phagocytes (MNP), leading to widespread changes in gene expression that mediate antibody effector function. Bromodomain and extra-terminal domain (BET) proteins are involved in governing gene transcription. We investigated the capacity of BET protein inhibitors (iBET) to alter IgG FcγR-mediated MNP activation. We found that iBET dampened IgG IC-induced pro-inflammatory gene expression and decreased activating FcγR expression on MNPs, reducing their ability to respond to IgG IC. Despite FcγR downregulation, iBET-treated macrophages demonstrated increased phagocytosis of protein antigen, IgG IC, and apoptotic cells. iBET also altered cell morphology, generating more amoeboid MNPs with reduced adhesion. iBET treatment impaired chemotaxis towards a CCL19 gradient in IC-stimulated dendritic cells (DC) in vitro , and inhibited IC-induced DC migration to draining lymph nodes in vivo , in a DC-intrinsic manner. Altogether, our data show that iBET modulates FcγR-mediated MNP activation and migration, revealing the therapeutic potential of BET protein inhibition in antibody-mediated diseases., Competing Interests: Author NS and RP are employees of and shareholders in GlaxoSmithKline (GSK). This study received funding from GSK. GSK had the following involvement with the study: funded RNA sequencing. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Banham, Lee, Ferdinand, Matthews, Jing, Smithers, Prinjha and Clatworthy.)

Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (B EM ) and find that many B EM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, B EM cells may exit the lymph node to enter distant tissues, while some B EM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of B EM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific B EM cells and transport of antigen back to GC may support affinity maturation to antigenic drift., (© 2022. The Author(s).)

Background: Transplantation is an effective treatment for end-stage lung disease, but the donor organ shortage is a major problem. Ex-vivo lung perfusion (EVLP) of extended criteria organs enables functional assessment to facilitate clinical decision-making around utilization, but the molecular processes occurring during EVLP, and how they differ between more or less viable lungs, remain to be determined., Methods: We used RNA sequencing of lung tissue to delineate changes in gene expression occurring in 10 donor lungs undergoing EVLP and compare lungs that were deemed non-transplantable (n = 4) to those deemed transplantable (n = 6) following perfusion., Results: We found that lungs deemed unsuitable for transplantation had increased induction of innate immune pathways and lower expression of oxidative phosphorylation related genes. Furthermore, the expression of SCGB1A1, a gene encoding an anti-inflammatory secretoglobin CC10, and other club cell genes was significantly decreased in non-transplantable lungs, while CHIT-1 was increased. Using a larger validation cohort (n = 17), we confirmed that the ratio of CHIT1 and SCGB1A1 protein levels in lung perfusate have potential utility to distinguish transplantable from non-transplantable lungs (AUC .81)., Conclusions: Together, our data identify novel biomarkers that may assist with pre-transplant lung assessment, as well as pathways that may be amenable to therapeutic intervention during EVLPAQ6., (© 2021 The Authors. Clinical Transplantation published by John Wiley & Sons Ltd.)

Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide-based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir-24-3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co-localization with proteins involved in the RNA-induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir-24-3p action increased expression of genes controlled by this microRNA, including heme oxygenase-1 and sphingosine-1-phosphate receptor 1. The expression of genes not under the control of mir-24-3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation., (© 2022 The American Society of Transplantation and the American Society of Transplant Surgeons.)

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories., (© 2022. The Author(s).)

We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods., (© 2022. The Author(s).)

Traumatic brain injury is increasingly common in older individuals. Older age is one of the strongest predictors for poor prognosis after brain trauma, a phenomenon driven by the presence of extra-cranial comorbidities as well as pre-existent pathologies associated with cognitive impairment and brain volume loss (such as cerebrovascular disease or age-related neurodegeneration). Furthermore, ageing is associated with a dysregulated immune response, which includes attenuated responses to infection and vaccination, and a failure to resolve inflammation leading to chronic inflammatory states. In traumatic brain injury, where the immune response is imperative for the clearance of cellular debris and survey of the injured milieu, an appropriate self-limiting response is vital to promote recovery. Currently, our understanding of age-related factors that contribute to the outcome is limited; but a more complete understanding is essential for the development of tailored therapeutic strategies to mitigate the consequences of traumatic brain injury. Here we show greater functional deficits, white matter abnormalities and worse long-term outcomes in aged compared with young C57BL/6J mice after either moderate or severe traumatic brain injury. These effects are associated with altered systemic, meningeal and brain tissue immune response. Importantly, the impaired acute systemic immune response in the mice was similar to the findings observed in our clinical cohort. Traumatic brain-injured patient cohort over 70 years of age showed lower monocyte and lymphocyte counts compared with those under 45 years. In mice, traumatic brain injury was associated with alterations in peripheral immune subsets, which differed in aged compared with adult mice. There was a significant increase in transcription of immune and inflammatory genes in the meninges post-traumatic brain injury, including monocyte/leucocyte-recruiting chemokines. Immune cells were recruited to the region of the dural injury, with a significantly higher number of CD11b + myeloid cells in aged compared with the adult mice. In brain tissue, when compared with the young adult mice, we observed a more pronounced and widespread reactive astrogliosis 1 month after trauma in aged mice, sustained by an early and persistent induction of proinflammatory astrocytic state. These findings provide important insights regarding age-related exacerbation of neurological damage after brain trauma., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription 1-4 . This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins 5-7 . However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5 -/- ) and CSB (Csb m/m ; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5 -/- Csb m/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Langerhans cells (LC) are skin-resident antigen-presenting cells that regulate immune responses to epithelial microorganisms. Human papillomavirus (HPV) infection can promote malignant epithelial transformation. As LCs are considered important for controlling HPV infection, we compared the transcriptome of murine LCs from skin transformed by K14E7 oncoprotein and from healthy skin. We identified transcriptome heterogeneity at the single cell level amongst LCs in normal skin, associated with ontogeny, cell cycle, and maturation. We identified a balanced co-existence of immune-stimulatory and immune-inhibitory LC cell states in normal skin that was significantly disturbed in HPV16 E7-transformed skin. Hyperplastic skin was depleted of immune-stimulatory LCs and enriched for LCs with an immune-inhibitory gene signature, and LC-keratinocyte crosstalk was dysregulated. We identified reduced expression of interleukin (IL)-34, a critical molecule for LC homeostasis. Enrichment of an immune-inhibitory LC gene signature and reduced levels of epithelial IL-34 were also found in human HPV-associated cervical epithelial cancers., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Background: Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation. Oxidative damage to mitochondria is an early event in IR injury. Therefore, the uptake, safety, and efficacy of the mitochondria-targeted antioxidant MitoQ were investigated in models of transplant IR injury., Methods: MitoQ uptake by warm and cooled pairs of pig and declined human kidneys was measured when preserved in cold static storage or by hypothermic machine perfusion. Pairs of pigs' kidneys were exposed to defined periods of warm and cold ischaemia, flushed and stored at 4°C with or without MitoQ (50 nmol/l to 250 µmol/l), followed by reperfusion with oxygenated autologous blood in an ex vivo normothermic perfusion (EVNP). Pairs of declined human kidneys were flushed and stored with or without MitoQ (5-100 µmol/l) at 4°C for 6 h and underwent EVNP with ABO group-matched blood., Results: Stable and concentration-dependent uptake of MitoQ was demonstrated for up to 24 h in pig and human kidneys. Total blood flow and urine output were significantly greater in pig kidneys treated with 50 µmol/l MitoQ compared with controls (P = 0.006 and P = 0.007 respectively). In proof-of-concept experiments, blood flow after 1 h of EVNP was significantly greater in human kidneys treated with 50 µmol/l MitoQ than in controls (P ≤ 0.001). Total urine output was numerically higher in the 50-µmol/l MitoQ group compared with the control, but the difference did not reach statistical significance (P = 0.054)., Conclusion: Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation., (© The Author(s) 2021. Published by Oxford University Press on behalf of BJS Society Ltd. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Transplantation is the optimal treatment for most patients with end-stage kidney disease but organ shortage is a major challenge. Normothermic machine perfusion (NMP) has been used to recondition marginal organs; however, mechanisms by which NMP might benefit organs are not well understood. Using pairs of human kidneys obtained from the same donor, we compared the effect of NMP with that of cold storage on the global kidney transcriptome. We found that cold storage led to a global reduction in gene expression, including inflammatory pathway genes and those required for energy generation processes, such as oxidative phosphorylation (OXPHOS). In contrast, during NMP, there was marked upregulation OXPHOS genes, but also of a number of immune and inflammatory pathway genes. Using biopsies from kidneys undergoing NMP that were subsequently transplanted, we found that higher inflammatory gene expression occurred in organs with prolonged delayed graft function (DGF). Therefore, we used a hemoadsorber (HA) to remove pro-inflammatory cytokines. This attenuated inflammatory gene expression increased OXPHOS pathway genes and had potentially clinically important effects in reducing the expression of a DGF-associated gene signature. Together, our data suggest that adsorption of pro-inflammatory mediators from the perfusate represents a potential intervention which may improve organ viability., (© 2020 The Authors. American Journal of Transplantation published by Wiley Periodicals LLC on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background and Aims: Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown., Approach and Results: Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions., Conclusions: Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Thousands of kidneys from higher-risk donors are discarded annually because of the increased likelihood of complications posttransplant. Given the severe organ shortage, there is a critical need to improve utilization of these organs. To this end, normothermic machine perfusion (NMP) has emerged as a platform for ex vivo assessment and potential repair of marginal organs. In a recent study of 8 transplant-declined human kidneys on NMP, we discovered microvascular obstructions that impaired microvascular blood flow. However, the nature and physiologic impact of these lesions were unknown. Here, in a study of 39 human kidneys, we have identified that prolonged cold storage of human kidneys induces accumulation of fibrinogen within tubular epithelium. Restoration of normoxic conditions-either ex vivo during NMP or in vivo following transplant-triggered intravascular release of fibrinogen correlating with red blood cell aggregation and microvascular plugging. Combined delivery of plasminogen and tissue plasminogen activator during NMP lysed the plugs leading to a significant reduction in markers of renal injury, improvement in indicators of renal function, and improved delivery of vascular-targeted nanoparticles. Our study suggests a new mechanism of cold storage injury in marginal organs and provides a simple treatment with immediate translational potential., (© 2020 The American Society of Transplantation and the American Society of Transplant Surgeons.)

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

Macrophages play a central role in intestinal immunity, but inappropriate macrophage activation is associated with inflammatory bowel disease (IBD). Here, we identify granulocyte-macrophage colony stimulating factor (GM-CSF) as a critical regulator of intestinal macrophage activation in patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis. We find that GM-CSF drives the maturation and polarization of inflammatory intestinal macrophages, promoting anti-microbial functions while suppressing wound-healing transcriptional programs. Group 3 innate lymphoid cells (ILC3s) are a major source of GM-CSF in intestinal inflammation, with a strong positive correlation observed between ILC or CSF2 transcripts and M1 macrophage signatures in IBD mucosal biopsies. Furthermore, GM-CSF-dependent macrophage polarization results in a positive feedback loop that augmented ILC3 activation and type 17 immunity. Together, our data reveal an important role for GM-CSF-mediated ILC-macrophage crosstalk in calibrating intestinal macrophage phenotype to enhance anti-bacterial responses, while inhibiting pro-repair functions associated with fibrosis and stricturing, with important clinical implications., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1β, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1β and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species.

The thymus provides a nurturing environment for the differentiation and selection of T cells, a process orchestrated by their interaction with multiple thymic cell types. We used single-cell RNA sequencing to create a cell census of the human thymus across the life span and to reconstruct T cell differentiation trajectories and T cell receptor (TCR) recombination kinetics. Using this approach, we identified and located in situ CD8αα + T cell populations, thymic fibroblast subtypes, and activated dendritic cell states. In addition, we reveal a bias in TCR recombination and selection, which is attributed to genomic position and the kinetics of lineage commitment. Taken together, our data provide a comprehensive atlas of the human thymus across the life span with new insights into human T cell development., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Advances in single-cell technologies are transforming our understanding of cellular identity. For instance, the application of single-cell RNA sequencing and mass cytometry technologies to the study of immune cell populations in blood, secondary lymphoid organs and the renal tract is helping researchers to map the complex immune landscape within the kidney, define cell ontogeny and understand the relationship of kidney-resident immune cells with their circulating counterparts. These studies also provide insights into the interactions of immune cell populations with neighbouring epithelial and endothelial cells in health, and across a range of kidney diseases and cancer. These data have translational potential and will aid the identification of drug targets and enable better prediction of off-target effects. The application of single-cell technologies to clinical renal biopsy samples, or even cells within urine, will improve diagnostic accuracy and assist with personalized prognostication for patients with various kidney diseases. A comparison of immune cell types in peripheral blood and secondary lymphoid organs in healthy individuals and in patients with systemic autoimmune diseases that affect the kidney will also help to unravel the mechanisms that underpin the breakdown in self-tolerance and propagation of autoimmune responses. Together, these immune cell atlases have the potential to transform nephrology.

Background: The Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design., Results: This study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types. We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific., Conclusions: In conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.