Tony Kiat Hon Lim

Mapping the human DC lineage through the integration of high-dimensional techniques

Tracing development of the dendritic cell lineage Dendritic cells (DCs) are important components of the immune system that form from the bone marrow into two major cell lineages: plasmacytoid DCs and conventional DCs. See et al. applied single-cell RNA sequencing and cytometry by time-of-flight to characterize the developmental pathways of these cells. They identified blood DC precursors that shared surface markers with plasmacytoid DCs but that were functionally distinct. This unsuspected level of complexity in pre-DC populations reveals additional cell types and refines understanding of known cell types. Science, this issue p. eaag3009 In human blood, the immunological dendritic cell lineage contains many predendritic cell populations. INTRODUCTION Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises multiple subsets, including plasmacytoid DC (pDC) and two functionally specialized lineages of conventional DC (cDC1 and cDC2), whose origins and differentiation pathways remain incompletely defined. RATIONALE As DC are essential regulators of the immune response in health and disease, potential intervention strategies aiming at manipulation of these cells will require in-depth insights of their origins, the mechanisms that govern their homeostasis, and their functional properties. Here, we employed two unbiased high-dimensional technologies to characterize the human DC lineage from bone marrow to blood. RESULTS We isolated the DC-containing population (Lineage−HLA−DR+CD135+ cells) from human blood and defined the transcriptomes of 710 individual cells using massively parallel single-cell mRNA sequencing. By combining complementary bioinformatic approaches, we identified a small cluster of cells within this population as putative DC precursors (pre-DC). We then confirmed this finding using cytometry by time-of-flight (CyTOF) to simultaneously measure the expression of a panel of 38 different proteins at the single-cell level on Lineage−HLA−DR+ cells and found that pre-DC possessed a CD123+CD33+CD45RA+ phenotype. We confirmed the precursor potential of pre-DC by establishing their potential to differentiate in vitro into cDC1 and cDC2, but not pDC, in the known proportions found in vivo. Interestingly, pre-DC also express classical pDC markers, including CD123, CD303, and CD304. Thus, any previous studies using these markers to identify or isolate pDC will have inadvertently included CD123+CD33+ pre-DC. We provide here new markers that can be used to identify unambiguously pre-DC from pDC, including CD33, CX3CR1, CD2, CD5, and CD327. When CD123+CD33+ pre-DC and CD123+CD33− pDC were isolated separately, we observed that pre-DC have unique functional properties that were previously attributed to pDC. Although pDC remain bona fide interferon-α–producing cells, their reported interleukin-12 (IL-12) production and CD4 T cell allostimulatory capacity can likely be attributed to “contaminating” pre-DC. We then asked whether the pre-DC population contained both uncommitted and committed pre-cDC1 and pre-cDC2 precursors, as recently shown in mice. Using microfluidic single-cell mRNA sequencing (scmRNAseq), we showed that the human pre-DC population contains cells exhibiting transcriptomic priming toward cDC1 and cDC2 lineages. Flow cytometry and in vitro DC differentiation experiments further identified CD123+CADM1−CD1c− putative uncommitted pre-DC, alongside CADM1+CD1c− pre-cDC1 and CADM1−CD1c+ pre-cDC2. Finally, we found that pre-DC subsets expressed T cell costimulatory molecules and induced comparable proliferation and polarization of naïve CD4 T cells as adult DC. However, exposure to the Toll-like receptor 9 (TLR9) ligand CpG triggered IL-12p40 and tumor necrosis factor–α production by early pre-DC, pre-cDC1, and pre-cDC2, in contrast to differentiated cDC1 and cDC2, which do not express TLR9. CONCLUSION Using unsupervised scmRNAseq and CyTOF analyses, we have unraveled the complexity of the human DC lineage at the single-cell level, revealing a continuous process of differentiation that starts in the bone marrow (BM) with common DC progenitors (CDP), diverges at the point of emergence of pre-DC and pDC potential, and culminates in maturation of both lineages in the blood and spleen. The pre-DC compartment contains functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset and two CD45RA+CD123lo lineage-committed subsets. The discovery of multiple committed pre-DC populations with unique capabilities opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting. Human DC emerge from BM CDP, diverge at the point of emergence of pre-DC and pDC potential, and culminate in maturation of both lineages in the blood. The pre-DC compartment further differentiates into functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset (early pre-DC), which give rise to both cDC1 and cDC2 through corresponding CD45RA+CD123lo pre-cDC1 and pre-cDC2 lineage-committed subsets, respectively. Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies—single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)—to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based Heterogeneity in Phenotype and Frequency

&NA; Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra‐epithelial (ie)ILC1‐like cells that represent a broader category of NK cells in mucosal and non‐mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals and tissues. It also provides a global, comprehensive, and detailed description of ILC heterogeneity in humans across patients and tissues. Graphical Abstract Figure. No caption available. HighlightsComprehensive profiling of human ILCs across tissuesDetailed description of previously defined ILC subsets except helper‐type ILC1ieILC1‐like cells are present in several tissues and functionally similar to NK cellsIdentification of markers expressed on ILCs, including functional IL‐18R &NA; Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. Simoni et al. (2016) profile human ILCs using mass cytometry across tissues. The results provide a global, comprehensive, and detailed description of ILC populations and their heterogeneity across individuals and tissues.

Microfluidic enrichment for the single cell analysis of circulating tumor cells

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

KRAS and BRAF mutations are rare and related to DNA mismatch repair deficiency in gastric cancer from the East and the West: results from a large international multicentre study.

Background:Inhibitors of the epidermal growth factor (EGFR) signaling pathway have a major role in the treatment of KRAS wild-type colorectal cancer patients. The EGFR pathway has been shown to be activated in gastric cancer (GC). However, published data on KRAS and BRAF mutation status is limited in GC and has not been compared between GC from different geographic regions.Methods:The prevalence of KRAS and BRAF mutations was established in 712 GC: 278 GC from the United Kingdom, 230 GC from Japan and 204 GC from Singapore. The relationship between KRAS/BRAF mutation status, DNA mismatch repair (MMR) status, clinicopathological variables and overall survival was analysed.Results:Overall, 30 (4.2%) GC carried a KRAS mutation. In total, 5.8% of the UK GC, 4% of Japan GC and 1.5% of Singapore GC were KRAS mutant. KRAS mutant GC had fewer lymph node metastases in the UK cohort (P=0.005) and were more frequent in elderly patients in the Japan cohort (P=0.034). KRAS mutations were more frequent in MMR-deficient GC in the UK and the Japanese cohort (P<0.05). A BRAF mutation was only detected in a single Japanese GC.Conclusions:This large multicentre study demonstrated that KRAS mutations and DNA MMR deficiency have a role in a small subgroup of GC irrespective of country of origin, suggesting that this subgroup of GC may have developed along a common pathway. Further studies need to establish whether concomitant mutations or amplifications of other EGFR signalling pathway genes may contribute to the activation of this pathway in GC.

Platelet-rich plasma has no effect on increasing free fat graft survival in the nude mouse.

BACKGROUND Free fat grafts have an unpredictable survival rate, which may be dependent on host bed vascularity. Therefore, the authors hypothesized that the presence of growth factors in platelet-rich plasma (PRP), may enhance free fat graft survival. METHODS Free fat grafts and autologous PRP were harvested from a healthy female and processed using the Coleman technique and the Medtronic Magellan system respectively. The experiment comprised two groups of 12 nude mice each with injection of free fat grafts into the scalp. The experimental group comprised the combination of 0.8 ml of free fat graft and 0.2 ml of PRP. The control group comprised the combination of 0.8 ml of free fat graft and 0.2 ml of normal saline. The mice were euthanized after 16 weeks and the fat grafts explanted and measured for weight and volume. Histology was performed with Oil Red O stain. Statistical analysis of the weight and volume in between groups was performed using the independent samples T-test (SPSS v11). The Mann-Whitney test was used to compare the ranking of six histological parameters between the two groups. RESULTS The mean weight and volume for the experimental arm were 0.503 g and 0.545 ml respectively. The mean weight and volume for the control arm were 0.500 g and 0.541 ml respectively. The weight, volume and histological parameters between the two groups were not statistically significant. A mouse from each group died of unknown causes. CONCLUSION PRP did not enhance free fat graft survival in the nude mouse.

Evaluation of the Fukuoka Consensus Guidelines for intraductal papillary mucinous neoplasms of the pancreas: Results from a systematic review of 1,382 surgically resected patients

BACKGROUND International consensus guidelines to guide management of intraductal papillary mucinous neoplasms (IPMN) were revised in Fukuoka and published in 2012. However, despite widespread acceptance of the Fukuoka Consensus Guidelines (FCG), the utility of these guidelines have not been well-validated. This systematic review was performed to evaluate the clinical utility of the FCG. DESIGN A computerized search of the PubMed and Scopus databases was performed to identify all studies evaluating the utility of the FCG in surgically resected IPMN. IPMN were stratified according to the FCG as high risk (HR), worrisome risk (WR), and low risk (LR). HR and WR IPMN were termed FCG+ve and LR IPMN were termed FCG-ve. RESULTS Seven studies analyzing 1,382 patients were included. There were 402 malignant neoplasms (29%), including 242 invasive IPMNs. There were 1,000 IPMN classified as FCG+ve. The FCG+ve group had a positive predictive value (PPV) ranging from 27 to 62% and the FCG-ve group had negative predictive value ranging from 82 to 100%. Pooled analysis demonstrated that there was 362 of 1,000 (36%) malignant FCG+ve IPMN and 342 of 382 (90%) benign FCG-ve IPMN. PPV of the HR group and the WR groups alone were 104 of 158 (66%) and 75 of 261 (29%), respectively. Forty of 382 (11%), including 22 (6%) invasive FCG-ve IPMN, were malignant. Twenty-six malignant including 18 invasive FCG-ve IPMN were reported from a single study. When the results from this study were excluded, there were only 14 of 241 malignant neoplasms (6%), including 4 of 241 (2%) invasive FCG-ve IPMN in the remaining 6 studies. CONCLUSION The FCG+ve criteria had a similarly low PPV compared with the 2006 consensus criteria. Stratification of IPMN into HR and WR groups resulted in a higher PPV in the HR group. Some malignant and even invasive IPMN may be missed by the FCG criteria.

Human fetal dendritic cells promote prenatal T-cell immune suppression through arginase-2

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Loss of tumor suppressor KDM6A amplifies PRC2-regulated transcriptional repression in bladder cancer and can be targeted through inhibition of EZH2

Bladder cancer with loss of KDM6A expression is vulnerable to inhibition of EZH2. Cancer’s loss is targeted therapy’s gain A demethylating protein called KDM6A is a known tumor suppressor, and its function is often lost in bladder cancer as a result of inactivating mutations. There is no way to directly target the loss of the tumor suppressor, but Ler et al. found another strategy to effectively treat tumors with this mutation. The authors demonstrated that KDM6A-deficient cells are dependent on the function of another protein, called EZH2. Small-molecule inhibitors of EZH2 were effective against KDM6A-null bladder cancer in multiple mouse models, paving the way for further development of these drugs. Trithorax-like group complex containing KDM6A acts antagonistically to Polycomb-repressive complex 2 (PRC2) containing EZH2 in maintaining the dynamics of the repression and activation of gene expression through H3K27 methylation. In urothelial bladder carcinoma, KDM6A (a H3K27 demethylase) is frequently mutated, but its functional consequences and therapeutic targetability remain unknown. About 70% of KDM6A mutations resulted in a total loss of expression and a consequent loss of demethylase function in this cancer type. Further transcriptome analysis found multiple deregulated pathways, especially PRC2/EZH2, in KDM6A-mutated urothelial bladder carcinoma. Chromatin immunoprecipitation sequencing analysis revealed enrichment of H3K27me3 at specific loci in KDM6A-null cells, including PRC2/EZH2 and their downstream targets. Consequently, we targeted EZH2 (an H3K27 methylase) and demonstrated that KDM6A-null urothelial bladder carcinoma cell lines were sensitive to EZH2 inhibition. Loss- and gain-of-function assays confirmed that cells with loss of KDM6A are vulnerable to EZH2. IGFBP3, a direct KDM6A/EZH2/H3K27me3 target, was up-regulated by EZH2 inhibition and contributed to the observed EZH2-dependent growth suppression in KDM6A-null cell lines. EZH2 inhibition delayed tumor onset in KDM6A-null cells and caused regression of KDM6A-null bladder tumors in both patient-derived and cell line xenograft models. In summary, our study demonstrates that inactivating mutations of KDM6A, which are common in urothelial bladder carcinoma, are potentially targetable by inhibiting EZH2.

The spatial organization of intra-tumour heterogeneity and evolutionary trajectories of metastases in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has one of the poorest survival rates among cancers. Using multi-regional sampling of nine resected HCC with different aetiologies, here we construct phylogenetic relationships of these sectors, showing diverse levels of genetic sharing, spanning early to late diversification. Unlike the variegated pattern found in colorectal cancers, a large proportion of HCC display a clear isolation-by-distance pattern where spatially closer sectors are genetically more similar. Two resected intra-hepatic metastases showed genetic divergence occurring before and after primary tumour diversification, respectively. Metastatic tumours had much higher variability than their primary tumours, suggesting that intra-hepatic metastasis is accompanied by rapid diversification at the distant location. The presence of co-existing mutations offers the possibility of drug repositioning for HCC treatment. Taken together, these insights into intra-tumour heterogeneity allow for a comprehensive understanding of the evolutionary trajectories of HCC and suggest novel avenues for personalized therapy.

Mutational landscapes of tongue carcinoma reveal recurrent mutations in genes of therapeutic and prognostic relevance

BackgroundCarcinoma of the oral tongue (OTSCC) is the most common malignancy of the oral cavity, characterized by frequent recurrence and poor survival. The last three decades has witnessed a change in the OTSCC epidemiological profile, with increasing incidence in younger patients, females and never-smokers. Here, we sought to characterize the OTSCC genomic landscape and to determine factors that may delineate the genetic basis of this disease, inform prognosis and identify targets for therapeutic intervention.MethodsSeventy-eight cases were subjected to whole-exome (n = 18) and targeted deep sequencing (n = 60).ResultsWhile the most common mutation was in TP53, the OTSCC genetic landscape differed from previously described cohorts of patients with head and neck tumors: OTSCCs demonstrated frequent mutations in DST and RNF213, while alterations in CDKN2A and NOTCH1 were significantly less frequent. Despite a lack of previously reported NOTCH1 mutations, integrated analysis showed enrichments of alterations affecting Notch signaling in OTSCC. Importantly, these Notch pathway alterations were prognostic on multivariate analyses. A high proportion of OTSCCs also presented with alterations in drug targetable and chromatin remodeling genes. Patients harboring mutations in actionable pathways were more likely to succumb from recurrent disease compared with those who did not, suggesting that the former should be considered for treatment with targeted compounds in future trials.ConclusionsOur study defines the Asian OTSCC mutational landscape, highlighting the key role of Notch signaling in oral tongue tumorigenesis. We also observed somatic mutations in multiple therapeutically relevant genes, which may represent candidate drug targets in this highly lethal tumor type.