Unbiased Clustering Research Articles

Single cell mapping identifies expansion of Cytotoxic CD4 T cells in the atherosclerotic plaque of subjects with Type 2 Diabetes

Abstract Patients with Type 2 diabetes (T2D) are at greater risk of atherosclerotic cardiovascular disease (ASCVD) and fail to fully respond to standard lipid lowering drugs. While the role of macrophages in T2D plaque pathology has been investigated, no studies have systematically investigated the heterogeneity of CD4 T cells in human plaques and how they respond to cholesterol reduction. To identify the specific T cell alterations in plaques from patients with T2D, we performed scRNAseq of human carotid plaques from 22 subjects (54.5% T2D) were being treated with cholesterol lowering drugs (average LDL cholesterol of 1.2 mmol/L) undergoing carotid endarterectomy. Data were processed and integrated using Seurat and Slingshot algorithms. We identified a total of 24 immune cell clusters by unbiased clustering of CD45+ cells, including CD4 T cells that presented similar frequencies between T2D (~11%) and non-T2D patients (~10%). CD4 T cells comprised of 13 sub clusters: naïve, central memory (CM), effector memory (EM), T regulatory (Tregs), cytotoxic (CTL), and gamma delta (γδ) T cells. One cluster of CD4 CTLs (cluster 5) expressing TRDC and TRGC2 surface markers along with cytotoxicity markers GZMB, GNLY, and PRF1 was significantly expanded in T2D plaques (p=0.032). This CD4CTL expressed high levels of transcription factors (TFs) involved in T cell differentiation such as RUNX3 and ZEB2 and contained sets of genes involved in cytotoxicity, chemotaxis and IL-1 signaling. Trajectory analysis using Slingshot inferred that CD4 CTLs clusters in the plaques differentiate from CD4 EM T cells. Plaques from patients with T2D are highly enriched in CTL/γδ T cells and suggest that their cytotoxic potential contributes to the increased cardiovascular risk. This Work is supported by AHA SFRN 20SFRN35210252 and the AHA-SFRN Training Program.

Abstract 2496: The landscape of the pre-leukemic bone marrow niche in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a heterogenous disease that develops due to genetic mutations occurring within hematopoietic stem/progenitor cells (HSPCs), giving rise to clonal proliferation. Mutated leukemic stem cells (LSCs) can remain dormant within the bone marrow (BM) for decades without disease, suggesting the possibility that LSCs could alter the normal crosstalk between HSCs and cells that comprise the BM niche. Therefore, remodeling of the BM niche, in addition to the acquisition of genetic mutations in HSPCs, may be required for full-blown leukemic transformation. Uncovering novel mechanisms by which LSCs remodel their microenvironment may be critical in the hopes of developing novel targeted therapies to completely eradicate the disease. Our lab has previously developed a murine model of AML (MllPTD/WT; Flt3ITD/WT (PTD; ITD)) that recapitulates important hallmarks of the human disease. This model develops AML sporadically with a preleukemic phase in which they display normal white blood counts (WBCs) and absence of leukemic blasts in the bone marrow. Recently, we observed a persistent decline in red blood cells (RBCs) in these mice that preceded leukemia development without any increases in BM cellularity. Thus, this unique model provides us with an opportunity to evaluate changes in the BM niche that could correlate with LSC emergence. Using this model we performed single cell RNA-sequencing on preleukemic BM niche cells compared to wild-type (WT) controls. Upon performing unbiased clustering, we found 17 stromal cell clusters, with 5 clusters of Lepr+ mesenchymal stromal cells, 3 clusters of endothelial cells, 4 fibroblast clusters, 3 osteo-lineage cell clusters 1 pericyte cluster, and 1 chondrocytes cluster. There was an increase in pericytes, with a shift away from the normal niche regulatory populations including LepR+ mesenchymal cells and endothelial cells in the preleukemic mice. Interestingly, in contrast to other studies examining the BM niche after full blown AML, we found an increase in fibroblasts, specifically CD55+ fibroblasts, that was confirmed by immunohistochemical microscopy. This increase in CD55+ fibroblasts was found to be a result of increased proliferation of this subpopulation. Differential gene expression analysis in revealed down-regulation of several collagen genes including: Col1a1, Col1a2, Col3a1, Col4a1, and Col6a1. Alterations of ECM proteins in preleukemic BM were confirmed by immunohistochemical analysis, suggesting remodeling of the ECM is an important mechanism by which LSCs lead to alterations in the BM niche and contributes to disease progression. Overall, we have confirmed that LSCs remodel their BM niche leading to an increase in CD55+ fibroblasts expressing decreased levels of critical ECM proteins. These changes might support the maintenance/generation of preleukemic clones at early stages that could ultimately evolve into leukemic transformation. Citation Format: Chinmayee Goda, Rohan Kulkarni, Alexander Rudich, Malith Karunasiri, Girish Rajgolikar, Bethany Mundy-Bosse, Ramiro Garzon, Elaine Mardis, Katherine Miller, Adrienne M. Dorrance. The landscape of the pre-leukemic bone marrow niche in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2496.

Abstract 3882: tumor adaptations to PI3K inhibition increases and its reversibility decreases as a function of time in drug

Abstract The PI3K pathway is a key regulator of metabolism, cell proliferation and migration and some of its components (e.g. PIK3CA, HER2 and PTEN) are frequently altered in cancer by genetic events that deregulate its output. However, inhibitors of components of the pathway have only modest antitumor effects and, in some instances, have resulted in “tumor flares” in patients upon cessation of therapy. Low efficacy has been attributed to toxicity and to adaptive resistance due to relief of feedback. Inhibition of the PI3K pathway has been associated with increased receptor expression and decreased expression of the PTEN phosphatase, a major negative regulator of the pathway. Now we show that changes in expressions of RTKs, PTEN, anti-apoptosis proteins like Mcl1, and transcription factors known to drive oncogenic and survival programs in tumors like c-Myc, c-Jun, FOXOs are induced upon continuous long-term PI3K inhibition (2 weeks and longer). These changes increase as a function of duration of inhibition and become less reversible with time in drug. Unbiased clustering of RNA seq data of cells treated with PI3K inhibitors revealed clusters of gene expression changes based upon short or long-term inhibition. Correspondingly the mRNA of the RTKs and transcription factors change with similar kinetics as that of the proteins and become less reversible with duration of inhibition. Moreover, the persister cells treated with PI3K inhibitors for 2 weeks or longer grow faster than the treatment naive cells upon drug removal both in vitro and in vivo and the degree of reversibility of the increased proliferation rate is inversely proportional to the duration of inhibition. Persister cells treated with PI3K inhibitors for more than two weeks have higher levels of expression of a oncogenic transcription factors, RTKs, Mcl1, and of pAKT after one week of drug wash-out and are less sensitive to re-inhibition of PI3K. However, combined and staggered inhibition of PI3K with Mcl1/Bcl2 inhibitors lead to greater reduction of cell survival and increased cell death selectively in the models that upregulate Mcl1 in response to PI3K inhibitors. The data suggests that induction of adaptive resistance is in part responsible for the continued persistence of tumor masses after the initial response to targeted therapy but also selectively sensitizes them towards Mcl1/Bcl2 inhibitors due to the upregulation of an anti-apoptotic program. Therefore, pulsatile targeted inhibition of PI3K in combination with anti-apoptotic BH3 domain protein inhibitors in a subset of breast cancer patients may provide a wide therapeutic window and can circumvent chronic adaptation induced resistance and drive potent cell death thereby improving therapeutic outcomes. Citation Format: Radha Mukherjee, Kiran Gireesan Vanaja, Malvika Sharma, Yangjingyi Ruan Ruan, Hilla Solomon, Elisa deStanchina, Sarat Chandarlapaty, Neal Rosen. tumor adaptations to PI3K inhibition increases and its reversibility decreases as a function of time in drug. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3882.

Abstract 608: Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma

Abstract The frequency of CD8+ tumor infiltrating lymphocytes (TILs) has been positively associated with overall survival in several cancers. The interaction of T cell receptors (TCRs) on T cells with Major Histocompatibility Complex (MHC)-peptide complexes on cancer cells elicits the cytotoxic activity of CD8+ T cells. Here we set out to understand how the strength of this interaction, or T cell avidity, shapes the phenotype of CD8+ TILs and how that dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We then used this method to isolate from tumors of Meth A tumor-bearing mice (28 days after tumor challenge), Low (K off < 15 min), Medium (75 min > K off > 45 min), and High (K off > 105 min) avidity CD8+ TILs responding to the tumor rejection neoepitope, PDPRMUT of this fibrosarcoma (Ebrahimi-Nik et al. JCI Insight 2019). Unbiased clustering of single cell RNA sequencing, TCR sequencing, and CITE-seq of these PDPRMUT -specific CD8+ TILs revealed five clusters: Stem-Like (LY108+Tcf7+), Effector-Exhausted (Eff-Exh PD-1Low,CX3CR1+) Terminally-Exhausted-1 (Term-Exh1 PD-1+TIM3High), Terminally-Exhausted-2 (Term-Exh2 PD-1HighToxHigh), and NK-Like (PD-1Low, KlrHigh). The low avidity CD8+ TILs have more than twice the proportion of Eff-Exh (60.0%) when compared to high avidity CD8+ TILs (28.39%). The high avidity CD8+ TILs have more Stem-Like (12.37% vs 6.31%), Term-Exh1 (27.38% vs 21.88%), and Term-Exh2 (24.03% vs 5.33%) cells than low avidity CD8+ TILs. Furthermore, trajectory inference of these clusters, by scVelo, predicts that Stem-Like cells differentiate into Eff-Exh cells that can differentiate into either Term-Exh1 or Term-Exh2. Our data demonstrates that low avidity Eff-Exh cells preferentially differentiate into Term-Exh1, while high avidity Eff-Exh cells differentiate into both Term-Exh1 and Term-Exh2. To validate these results, we isolated CD8+ TILs responding to PDPRMUT with low or high avidity from tumors of 21 Meth A bearing mice 28 days after tumor challenge and analyzed their exhaustion phenotype by flow cytometry. There were significantly more TIM3+PD-1+ cells (Paired t-test; P < 0.0001) and TOX+PD-1+ cells (Paired t-test; P < 0.05) in the high avidity CD8+ TILs than the low avidity CD8+ TILs. We conclude that CD8+ TILs with high avidity are more exhausted and less effector like than low avidity CD8+ TILs. This finding has major implications for T cell-based cancer immunotherapy. Citation Format: Summit Singhaviranon, Joseph P. Dempsey, Adam T. Hagymasi, Pramod K. Srivastava. Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 608.

177 International Authorship Trends in Academic Neurosurgery and Effect of Double-Blind Review

INTRODUCTION: There is a lack of data on international publication rates and whether reviewer biases exist that may affect diversity of authorship. METHODS: Articles published within Neurosurgery, Journal of Neurosurgery (JNS), and World Neurosurgery (WNS) were collected from 2000-2019. Country affiliation data was assigned and analyzed with respect to journal, year of publication, neurosurgical subspeciality, and relative impact. RESULTS: Across 23,325 articles, lead authorship consisted of United States (42.9%, N = 10,005), Japan (9.9%, N = 2,305), China (9.3%, N = 2,167), Germany (5.1%, N = 1,187), South Korea (3.3%, N = 773), Italy (3.3%, N = 773), Canada (3.3%, N = 769), and other nations (22.9%, N = 5,346). Rates of publications did not differ across United States, Asian countries, South/Latin American countries, or African countries pre- and post-initiation of double-blind review in Neurosurgery (p > 0.05). Annual percentage of publications showed highest relative decrease for the United States (-0.48% per year, r2 = 0.194) and increase for China (0.87% per year, r2 = 0.761). WNS published fewer articles from the United States compared to Neurosurgery or JNS (Mean difference [MD] -20.6%, p < 0.001), and more articles from Asian (MD +23.5%, p < 0.001), Latin/South American (MD +1.4%, p < 0.001), and African countries (MD +0.8%, p < 0.001). Unbiased co-authorship clustering by country demonstrated that certain countries were more likely to publish together based on geopolitical characteristics. CONCLUSIONS: There is a higher relative contribution to neurosurgery publications from authors in the United States compared to other countries. This effect is not due to potential biases in single-blind review, as evidenced by a natural experiment comparing authorship rates at Neurosurgery pre- and post-initiation of double-blind review in 2011. Additional research is needed to determine whether other biases contribute to differences in publication rates across countries.

Analysis of human lung mast cells by single cell RNA sequencing.

Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MCT and MCTC, where MCT express the mast cell protease tryptase and MCTC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MCT subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG. However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak.

Abnormalities in intron retention characterize patients with systemic lupus erythematosus

Regulation of intron retention (IR), a form of alternative splicing, is a newly recognized checkpoint in gene expression. Since there are numerous abnormalities in gene expression in the prototypic autoimmune disease systemic lupus erythematosus (SLE), we sought to determine whether IR was intact in patients with this disease. We, therefore, studied global gene expression and IR patterns of lymphocytes in SLE patients. We analyzed RNA-seq data from peripheral blood T cell samples from 14 patients suffering from systemic lupus erythematosus (SLE) and 4 healthy controls and a second, independent data set of RNA-seq data from B cells from16 SLE patients and 4 healthy controls. We identified intron retention levels from 26,372 well annotated genes as well as differential gene expression and tested for differences between cases and controls using unbiased hierarchical clustering and principal component analysis. We followed with gene-disease enrichment analysis and gene-ontology enrichment analysis. Finally, we then tested for significant differences in intron retention between cases and controls both globally and with respect to specific genes. Overall decreased IR was found in T cells from one cohort and B cells from another cohort of patients with SLE and was associated with increased expression of numerous genes, including those encoding spliceosome components. Different introns within the same gene displayed both up- and down-regulated retention profiles indicating a complex regulatory mechanism. These results indicate that decreased IR in immune cells is characteristic of patients with active SLE and may contribute to the abnormal expression of specific genes in this autoimmune disease.

DNA Damage Response-Related Proteins Are Prognostic for Outcome in Both Adult and Pediatric Acute Myelogenous Leukemia Patients: Samples from Adults and from Children Enrolled in a Children's Oncology Group Study.

The survival of malignant leukemic cells is dependent on DNA damage repair (DDR) signaling. Reverse Phase Protein Array (RPPA) data sets were assembled using diagnostic samples from 810 adult and 500 pediatric acute myelogenous leukemia (AML) patients and were probed with 412 and 296 strictly validated antibodies, respectively, including those detecting the expression of proteins directly involved in DDR. Unbiased hierarchical clustering identified strong recurrent DDR protein expression patterns in both adult and pediatric AML. Globally, DDR expression was associated with gene mutational statuses and was prognostic for outcomes including overall survival (OS), relapse rate, and remission duration (RD). In adult patients, seven DDR proteins were individually prognostic for either RD or OS. When DDR proteins were analyzed together with DDR-related proteins operating in diverse cellular signaling pathways, these expanded groupings were also highly prognostic for OS. Analysis of patients treated with either conventional chemotherapy or venetoclax combined with a hypomethylating agent revealed protein clusters that differentially predicted favorable from unfavorable prognoses within each therapy cohort. Collectively, this investigation provides insight into variable DDR pathway activation in AML and may help direct future individualized DDR-targeted therapies in AML patients.

Single-cell RNA sequencing reveals tumor heterogeneity, microenvironment, and drug-resistance mechanisms of recurrent glioblastoma.

Glioblastomas are highly heterogeneous brain tumors. Despite the availability of standard treatment for glioblastoma multiforme (GBM), i.e., Stupp protocol, which involves surgical resection followed by radiotherapy and chemotherapy, glioblastoma remains refractory to treatment and recurrence is inevitable. Moreover, the biology of recurrent glioblastoma remains unclear. Increasing evidence has shown that intratumoral heterogeneity and the tumor microenvironment contribute to therapeutic resistance. However, the interaction between intracellular heterogeneity and drug resistance in recurrent GBMs remains controversial. The aim of this study was to map the transcriptome landscape of cancer cells and the tumor heterogeneity and tumor microenvironment in recurrent and drug-resistant GBMs at a single-cell resolution and further explore the mechanism of drug resistance of GBMs. We analyzed six tumor tissue samples from three patients with primary GBM and three patients with recurrent GBM in which recurrence and drug resistance developed after treatment with the standard Stupp protocol using single-cell RNA sequencing. Using unbiased clustering, nine major cell clusters were identified. Upregulation of the expression of stemness-related and cell-cycle-related genes was observed in recurrent GBM cells. Compared with the initial GBM tissues, recurrent GBM tissues showed a decreased proportion of microglia, consistent with previous reports. Finally, vascular endothelial growth factor A expression and the blood-brain barrier permeability were high, and the O6 -methylguanine DNA methyltransferase-related signaling pathway was activated in recurrent GBM. Our results delineate the single-cell map of recurrent glioblastoma, tumor heterogeneity, tumor microenvironment, and drug-resistance mechanisms, providing new insights into treatment strategies for recurrent glioblastomas.

Single-cell profiling identifies T cell subsets associated with control of tuberculosis dissemination

To identify T cell subsets associated with control of tuberculosis, single-cell transcriptome and T cell receptor sequencing were performed on total T cells from patients with tuberculosis and healthy controls. Fourteen distinct subsets of T cells were identified by unbiased UMAP clustering. A GZMK-expressing CD8+ cytotoxic T cell cluster and a SOX4-expressing CD4+ central memory T cell cluster were depleted, while a MKI67-expressing proliferating CD3+ T cell cluster was expanded in patients with tuberculosis compared with healthy controls. The ratio of Granzyme K-expressing CD8+CD161−Ki-67− and CD8+Ki-67+ T cell subsets was significantly reduced and inversely correlated with the extent of TB lesions in patients with TB. In contrast, ratio of Granzyme B-expressing CD8+Ki-67+ and CD4+CD161+Ki-67− T cells and Granzyme A-expressing CD4+CD161+Ki-67− T cells were correlated with the extent of TB lesions. It is concluded that granzyme K-expressing CD8+ T cell subsets might contribute to protection against tuberculosis dissemination.

Preliminary Results of Single-cell Transcriptome Sequencing in Renal Arterial Lesions of Takayasu Arteritis

Objective To explore the preliminary application of single-cell RNA sequencing (scRNA-seq) in the renal arterial lesions in Takayasu arteritis (TA) patients. Methods This study included 2 TA patients with renal artery stenosis treated by bypass surgery in the Department of Vascular Surgery,Beijing Hospital.The obtained 2 renal artery samples were digested with two different protocols (GEXSCOPE kit and self-made digestion liquid) before scRNA-seq and bioinformatics analysis. Results A total of 2920 cells were obtained for further analysis.After unbiased cluster analysis,2 endothelial cell subsets,2 smooth muscle cell subsets,1 fibroblast subset,2 mononuclear macrophage subsets,1 T cell subset,and 1 undefined cell subset were identified.Among them,the two subsets of smooth muscle cells were contractile and secretory,respectively.The results of scRNA-seq indicated that enzymatic hydrolysis with GEXSCOPE kit produced a large number of endothelial cells (57.46%) and a small number of immune cells (13.21%).However,immune cells (34.64%) were dominant in the cells obtained by enzymatic hydrolysis with self-made digestive liquid. Conclusion scRNA-seq can be employed to explore the cellular heterogeneity of diseased vessels in TA patients.Different enzymatic digestion protocols may impact the proportion of different cells.

Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages.

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.

Dynamic activity in cis-regulatory elements of leukocytes identifies transcription factor activation and stratifies COVID-19 severity in ICU patients

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

Lineage-specific regulatory changes in hypertrophic cardiomyopathy unraveled by single-nucleus RNA-seq and spatial transcriptomics

Hypertrophic cardiomyopathy (HCM) is the most common cardiac genetic disorder characterized by cardiomyocyte hypertrophy and cardiac fibrosis. Pathological cardiac remodeling in the myocardium of HCM patients may progress to heart failure. An in-depth elucidation of the lineage-specific changes in pathological cardiac remodeling of HCM is pivotal for the development of therapies to mitigate the progression. Here, we performed single-nucleus RNA-seq of the cardiac tissues from HCM patients or healthy donors and conducted spatial transcriptomic assays on tissue sections from patients. Unbiased clustering of 55,122 nuclei from HCM and healthy conditions revealed 9 cell lineages and 28 clusters. Lineage-specific changes in gene expression, subpopulation composition, and intercellular communication in HCM were discovered through comparative analyses. According to the results of pseudotime ordering, differential expression analysis, and differential regulatory network analysis, potential key genes during the transition towards a failing state of cardiomyocytes such as FGF12, IL31RA, and CREB5 were identified. Transcriptomic dynamics underlying cardiac fibroblast activation were also uncovered, and potential key genes involved in cardiac fibrosis were obtained such as AEBP1, RUNX1, MEOX1, LEF1, and NRXN3. Using the spatial transcriptomic data, spatial activity patterns of the candidate genes, pathways, and subpopulations were confirmed on patient tissue sections. Moreover, we showed experimental evidence that in vitro knockdown of AEBP1 could promote the activation of human cardiac fibroblasts, and overexpression of AEBP1 could attenuate the TGFβ-induced activation. Our study provided a comprehensive analysis of the lineage-specific regulatory changes in HCM, which laid the foundation for targeted drug development in HCM.

Unbiased single cell spatial analysis localises inflammatory clusters of immature neutrophils-CD8 T cells to alveolar progenitor cells in fatal COVID-19 lungs

Unbiased single cell spatial analysis localises inflammatory clusters of immature neutrophils-CD8 T cells to alveolar progenitor cells in fatal COVID-19 lungs

Investigating a CD8+ T cell age‐associated gene signature in Alzheimer's disease

AbstractBackgroundNeuroinflammation has been linked with the pathologic progression of Alzheimer’s disease (AD). Previous studies focused primarily on the innate immune system; however, there are few human studies evaluating the potential relationships between adaptive immune cells and the development of AD pathology. Alterations in the immune system occur with aging, likely contributing to increased infection, malignancy and inflammation in older adults. One of the most prominent immune changes with age includes the expansion of memory CD8+ T cells in peripheral blood. Notably, cytotoxic IL‐7 receptor alpha low (IL‐7Rα or CD127low) CD45RA+ effector memory (EM) CD8+ T cells (TEMRA) have been associated with dementia and mild cognitive impairment (MCI) due to AD. In addition, our lab recently discovered an age‐associated gene expression signature of IL‐7Rα low EM CD8+ T cells. Thus, we hypothesized that individuals with AD have altered levels of IL‐7Rα low EM CD8+ T cell aging gene expression.MethodsA total of 40 genes which include 9 IL‐7Rαlow aging genes and 31 genes previously reported to be associated with AD and/or memory were analyzed in peripheral blood of 82 participants with AD and 40 cognitively normal age‐balanced participants by quantitative polymerase chain reaction (qPCR). Gene expression data was analyzed utilizing one‐way ANOVA, principal component analysis (PCA), and unbiased hierarchical clustering analysis.ResultsEight genes were differentially expressed (P < 0.05) between cognitively normal participants and participants with MCI or dementia due to AD. These genes included 5 IL‐7Ralow aging genes (GZMH, NUAK1, TGFBR3, PRSS23, OSBPL5), 2 genes related to AD and/or memory (CD163, CYP4F3) and a gene related to both aging and AD (PADI4). The expression levels of IL‐7Ralow aging genes moderately correlated with those of AD and memory genes. PCA and clustering analysis suggest IL‐7Ralow aging genes may be associated with disease status in a subset of AD subjects.ConclusionsAn altered CD8+ T cell age‐associated gene signature is present in AD patients, warranting further studies investigating biological implications of CD8+ T cells, especially highly cytotoxic and inflammatory IL‐7Rαlow EM CD8+ T cells, in AD.

Neuroimmune proteins can differentiate between tauopathies

BackgroundTauopathies are a group of neurodegenerative diseases where there is pathologic accumulation of hyperphosphorylated tau protein (ptau). The most common tauopathy is Alzheimer’s disease (AD), but chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and argyrophilic grain disease (AGD) are significant health risks as well. Currently, it is unclear what specific molecular factors might drive each distinct disease and represent therapeutic targets. Additionally, there is a lack of biomarkers that can differentiate each disease in life. Recent work has suggested that neuroinflammatory changes might be specific among distinct diseases and offers a novel resource for mechanistic targets and biomarker candidates.MethodsTo better examine each tauopathy, a 71 immune-related protein multiplex ELISA panel was utilized to analyze anterior cingulate grey matter from 127 individuals neuropathologically diagnosed with AD, CTE, PSP, CBD, and AGD. A partial least square regression analysis was carried out to perform unbiased clustering and identify proteins that are distinctly correlated with each tauopathy correcting for age and gender. Receiver operator characteristic and binary logistic regression analyses were then used to examine the ability of each candidate protein to distinguish diseases. Validation in postmortem cerebrospinal fluid (CSF) from 15 AD and 14 CTE cases was performed to determine if candidate proteins could act as possible novel biomarkers.ResultsFive clusters of immune proteins were identified and compared to each tauopathy to determine if clusters were specific to distinct disease. Each cluster was found to correlate with either CTE, AD, PSP, CBD, or AGD. When examining which proteins were the strongest driver of each cluster, it was observed the most distinctive protein for CTE was CCL21, AD was FLT3L, and PSP was IL13. Individual proteins that were specific to CBD and AGD were not observed. CCL21 was observed to be elevated in CTE CSF compared to AD cases (p = 0.02), further validating the use as possible biomarkers. Sub-analyses for male only cases confirmed the results were not skewed by gender differences.ConclusionsOverall, these results highlight that different neuroinflammatory responses might underlie unique mechanisms in related neurodegenerative pathologies. Additionally, the use of distinct neuroinflammatory signatures could help differentiate between tauopathies and act as novel biomarker candidate to increase specificity for in-life diagnoses.

RIPK1 mutations causing infantile-onset IBD with inflammatory and fistulizing features.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulator of necroptosis and inflammatory responses. We present the clinical features, genetic analysis and immune work-up of two patients with infantile-onset inflammatory bowel disease (IBD) resulting from RIPK1mutations. Whole exome and Sanger sequencing was performed in two IBD patients. Mass cytometry time of flight (CyTOF) was conducted for in-depth immunophenotyping on one of the patient's peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn's disease. The patients presented with severe colitis and perianal fistulas in the first months of life, without severe/atypical infections. Genetic studies identified pathogenic genetic variants in RIPK1 (Patient 1, A c.1934C>T missense mutation in Exon 11; Patient 2, c.580G>A missense mutation residing in Exon 4). Protein modeling demonstrated that the mutation in Patient 1 displaces a water molecule, potentially disrupting the local environment, and the mutation in Patient 2 may lead to disruption of the packing and conformation of the kinase domain. Immunofluorescence RIPK1 staining in rectal biopsies demonstrated no expression for Patient 1 and minimal expression for Patient 2, compared to controls and patients with active Crohn's disease. Using CyTOF unbiased clustering analysis, we identified peripheral immune dysregulation in one of these patients, characterized by an increase in IFNγ CD8+T cells along with a decrease in monocytes, dendritic cells and B cells. Moreover, RIPK1-deficient patient's immune cells exhibited decreased IL-6 production in response to lipopolysaccharide (LPS) across multiple cell types including T cells, B cells and innate immune cells. Mutations inRIPK1should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1's role in inflammasome activation, but also in epithelial cells, it is unclear whether IL1 blockade or allogeneic hematopoietic stem cell transplantation can suppress or cure the hyper-inflammatory response in these patients. Additional studies in humans are required to better define the role of RIPK1 in regulating intestinal immune responses, and how treatment can be optimized for patients with RIPK1 deficiency.

Cytokine/Chemokine assessment as a complementary diagnostic tool for inflammatory skin diseases.

Inflammatory skin conditions are the 4th leading cause of non-fatal health burden in the general population worldwide. The diagnosis of skin lesions due to systemic drug reactions, viral or bacterial exanthems, or in patients with psoriasis, atopic dermatitis or contact dermatitis is often difficult and relies heavily upon conventional histopathologic examination. Conversely, it is widely accepted that the cutaneous profile of inflammatory markers, or 'inflammatory signature', is differentially expressed in various skin conditions. In this pilot study, we investigated the possibility of inflammatory skin disease diagnosis from an immunological perspective in small punch biopsies. We collected lesional and perilesional punch biopsies from 139 patients suffering from a variety of inflammatory skin conditions and attending the Dermatology Department at the Princess Alexandra Hospital in Brisbane, Australia. Using bead-based immunoassays we were able to measure 13 out of 17 inflammatory markers from a pre-selected multi-analyte panel and to detect significant differences between lesional and perilesional biopsies from each individual patient. Hierarchical and unbiased clustering methods based on inflammatory signatures grouped psoriasis and atopic dermatitis lesions into individual clusters in contrast to other skin conditions, highlighting the potential of inflammatory signatures to be used as diagnostic differentiators and to inform alternative targets in anti-inflammatory treatment strategies.

Epigenetic Landscape Predicts Overall Survival and Remission Duration in Adult Acute Myeloid Leukemia

INTRODUCTION: Epigenetic dysregulation is a hallmark of Acute Myeloid Leukemia (AML). Although recurrent epigenetic-related mutations have been identified, the understanding of how they affect pathophysiology and leukemogenesis remains incomplete. We used proteomics to assess the epigenetic landscape in AML and identified new prognostic phenotypes and therapeutic targets. METHODS: Using Reverse Phase Protein Array (RPPA) 405 proteins (327 total; 78 post-translational modified) were measured in 818 samples from newly diagnosed adult AML patients and normalized to CD34+ cells. These included Histone H3 total and H3K4Me 1, 2, 3, K9Me2, K27Me3, K27Ac and K36Me3 Modification Marks (HMM) expression. Categorical clinical variables were compared with Fisher's Exact with simulated p-values (10000 replicates), and continuous ones with Wilcoxon or Kruskal-Wallis tests. Univariate (UV) and Multivariate (MV) models for Overall Survival (OS) and Remission Duration (RD) were built with Cox proportional hazards model (CoxPH). Differentially Expressed Proteins were determined using the Kruskal-Wallis test (p<0.05) and pairwise Dunn's tests with p-values adjusted with the False Discovery Rate method (p<0.05). RESULTS: Using unbiased hierarchical clustering, patients were clustered into 4 groups (Fig. 1, C1 to C4) based on the expression of 2 Repressive HMM (H3K27Me3 and H3K9Me2), and 5 Activating HMM (H3K27Ac, H3K36Me3, H3K4Me3, H3K4Me2, H3K4Me1). High repression and low activation HMM were prominent in C1, while C4 had the opposite pattern. Higher expression of all marks and Total H3 defined C2, and C3 had lower expression of all marks, and Total H3. Importantly, when normalized by Total H3, HMM expression of both C2 and C3 are close to average, representing a chromatin state between C1 and C4. Compared to the other groups, patients in C4 were older (66yo vs 62yo; p=0.006), and had more secondary AML (55% vs 43%; p=0.007) and unfavorable cytogenetic risk (55% vs 37%; p<0.001). Mutations weren't exclusively associated with any cluster: ASLX1 and NPM1 mutants were more frequent in C1 and C2 (27% vs 13%; p<0.001; 23% vs 15%; p=0.015), WT1 more in C1 and C3 (9% vs 3.7%, p=0.018), and EZH2, PTPN11, RAS and RUNX1 happened more in C3 and C4 (7.7% vs 1.7%, p<0.001; 13% vs 5.6%, p=0.002; 26% vs 14%, p<0.001; 22% vs 14%, p=0.015). The effect of HMM expression on OS (Fig. 2) showed that patients with high repression and low activation (C1) did significantly better (Median OS (MS) = 19mo, Median RD (MRD)>102mo), and those with the opposite pattern of low repression and high activation (C4) did significantly worse (MS=7mo, MRD=16mo). Patients with an intermediary chromatin state were close to the average OS (C2: MS=14mo, MRD=20mo; C3: MS=12mo, MRD=24mo). In the UV CoxPH model, C1, C3 and C4 predicted OS (HR=1, 1.3, 1.4, 2.3; p=ref., 0.007, 0.001), as did age, secondary AML, complex karyotype, and CEBPA, FLT3, KIT, JAK2, NPM1, PTPN11, SRSF2 and TP53 mutations. In the MV model C1, C3 and C4 were again predictive for OS (HR=1, 1.5, 2.4, 2.7; p=ref., 0.003, 0.006), together with age, secondary AML, and PTPN11, SRSF2 and TP53 mutations. For RD, in the UV model, all clusters predicted RD (HR=1, 1.9, 1.8, 2.7; p=ref., 0.002, 0.005, 0.001), as did age, secondary AML, complex karyotype, and FLT3, NPM1, RUNX1 and TP53 mutations. In the MV model of RD, all clusters were predictors (HR=1, 1.8, 1.8, 2.7; p=ref., 0.045, 0.038, 0.003), together with age, complex karyotype and RUNX1 mutation. Differentially Expressed Proteins, from the other 397 proteins, for C4 revealed high activation of VEGF, ErbB and FcεRI signaling pathways, unveiling potential new druggable targets. CONCLUSION: HMM expression found that opposing chromatin states, with a more closed (C1) or open (C4) chromatin, significantly affected prognosis. Moreover, the epigenetic landscape was useful to predict OS and RD: compact chromatin equals high OS and RD (C1), and a loose one means poor OS and RD (C4), whereas an intermediary state (C2/C3) leads to average OS and RD. The expression pattern of the worst OS group showed high activation of specific signaling pathways which could be targeted to improve OS and RD. It would be worthwhile to correlate the findings with ATAC-seq, ChIP-seq, RNA-seq and DNA methylation analysis, in order to determine the loci responsible for the observed phenotypes in each cluster, and also WGS to identify possible new drivers of chromatin aberrations in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal