Single-Cell Transcriptomic Analysis of SARS-CoV-2 Reactive CD4 + T Cells.

The contribution of CD4+ T cells to protective or pathogenic immune responses to SARS-CoV-2 infection remains unknown. Here, we present large-scale single-cell transcriptomic analysis of viral antigen-reactive CD4+ T cells from 32 COVID-19 patients. In patients with severe disease compared to mild disease, we found increased proportions of cytotoxic follicular helper (TFH) cells and cytotoxic T helper cells (CD4-CTLs) responding to SARS-CoV-2, and reduced proportion of SARS-CoV-2 reactive regulatory T cells. Importantly, the CD4-CTLs were highly enriched for the expression of transcripts encoding chemokines that are involved in the recruitment of myeloid cells and dendritic cells to the sites of viral infection. Polyfunctional T helper (TH)1 cells and TH17 cell subsets were underrepresented in the repertoire of SARS-CoV-2-reactive CD4+ T cells compared to influenza-reactive CD4+ T cells. Together, our analyses provide so far unprecedented insights into the gene expression patterns of SARS-CoV-2 reactive CD4+ T cells in distinct disease severities. Funding: This work was funded by NIH grants U19AI142742 (P.V., A.S., C.H.O), U19AI118626 (P.V., A.S., G.S.), R01HL114093 (P.V., F.A., G.S.,), R35-GM128938 (F.A), S10RR027366 (BD FACSAria-II), S10OD025052 (Illumina Novaseq6000), the William K. Bowes Jr Foundation (P.V.), and Whittaker foundation (P.V., C.H.O.). Supported by the Wessex Clinical Research Network and National Institute of Health Research UK. Conflict of Interest: The authors declare no competing financial interests. Ethical Approval: Ethical approval for this study from the Berkshire Research Ethics Committee 20/SC/0155 and the Ethics Committee of La Jolla Institute for Immunology (LJI) was in place. Written consent was obtained from all subjects

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9(+) monocytes associated with COVID-19 hospitalization

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy

Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches

Genetic Sharing with Cardiovascular Disease Risk Factors and Diabetes Reveals Novel Bone Mineral Density Loci.

Bone Mineral Density (BMD) is a highly heritable trait, but genome-wide association studies have identified few genetic risk factors. Epidemiological studies suggest associations between BMD and several traits and diseases, but the nature of the suggestive comorbidity is still unknown. We used a novel genetic pleiotropy-informed conditional False Discovery Rate (FDR) method to identify single nucleotide polymorphisms (SNPs) associated with BMD by leveraging cardiovascular disease (CVD) associated disorders and metabolic traits. By conditioning on SNPs associated with the CVD-related phenotypes, type 1 diabetes, type 2 diabetes, systolic blood pressure, diastolic blood pressure, high density lipoprotein, low density lipoprotein, triglycerides and waist hip ratio, we identified 65 novel independent BMD loci (26 with femoral neck BMD and 47 with lumbar spine BMD) at conditional FDR < 0.01. Many of the loci were confirmed in genetic expression studies. Genes validated at the mRNA levels were characteristic for the osteoblast/osteocyte lineage, Wnt signaling pathway and bone metabolism. The results provide new insight into genetic mechanisms of variability in BMD, and a better understanding of the genetic underpinnings of clinical comorbidity

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies