Specific Cell Subsets Research Articles

Abstract A029: Re-purposing non-oncology agent itraconazole to target the dynamic cellular ecosystem of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival of only 10% and persists as the third most common cause of cancer-related death in Western societies. Precision medicine is a likely future for all cancer treatment, but may have its greatest impact in less common, high-mortality and molecularly heterogeneous cancers, including pancreatic cancer. Fortunately, in the era of Big Data, bioinformatic analyses of genomic datasets have helped us gain new insights of how novel molecular-guided therapies can modulate signalling within tumors and their ecosystem (Vennin et al. 2017; Waddell, N, Pajic, M, Patch, AM. et al. 2015; Chou A. et al. 2018; Lau, MT. et al 2020). In this study, we delineate, at single-cell resolution, the complex stromal mechanisms by which a clinically-used, orally available anti-fungal agent itraconazole improves the overall anti-tumor response in PDAC. Single-cell RNAseq analysis of the genetically-engineered KPC model (LSL- KrasG12D;LSL-Trp53 R172H/+) revealed significant downregulation of the pro-tumorigenic CD105+ cancer-associated fibroblast (CAF) signature post-itraconazole therapy in vivo. Moreover, single-cell trajectory inference of CAFs subsets revealed a marked effect of treatment on myofibroblasts (myCAFs), which produce the fibrotic PDAC desmoplasia. In support of these findings, immunofluorescence analyses further demonstrated decreased deposition of collagen and altered matrix remodelling within itraconazole-treated tumors. Within the antigen-presenting apCAF and inflammatory iCAF populations, itraconazole treatment led to decreased pro-tumorigenic Igf1-Itga6:Itgb4 signalling to specific tumor cell subsets, which was functionally associated with reduced metastatic colonisation in vivo. apCAFs also showed decreased expression of H2-Aa and CD74, major antigens expressed by apCAFs which act as decoys for corresponding receptors on CD4+/CD8+ T cells, thus leading to their impaired activation (Lakins et al, 2018). These potentially promising effects on the immune environment of PDAC were supported by significant positive changes within macrophage populations, including enrichment of M1-like pro-inflammatory macrophage signatures (confirmed via immunofluorescence), and further associated with positive signalling via Cxcl9-Cxcr3 and Tnf1rs-1a/1b ligand-receptors. scRNAseq revealed dampening of regulatory T cell homing and functional potency signals within the pancreatic tumor microenvironment was associated with increased CD8+ T-cell infiltration post-itraconazole treatment, and importantly, significantly improved response to immune checkpoint blockade in an advanced in vivo model of pancreatic cancer. In summary, these integrated bioinformatic and molecular studies provide scientific rationale for the development of itraconazole and immunotherapy combination in pancreatic cancer. Citation Format: Diego Chacon-Fajardo, Sean Porazinski, Jennifer Man, Howard Yim, Emad El-Omar, Anthony Joshua, Marina Pajic. Re-purposing non-oncology agent itraconazole to target the dynamic cellular ecosystem of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A029.

Presence of onco-fetal neighborhoods in hepatocellular carcinoma is associated with relapse and response to immunotherapy.

Onco-fetal reprogramming of the tumor ecosystem induces fetal developmental signatures in the tumor microenvironment, leading to immunosuppressive features. Here, we employed single-cell RNA sequencing, spatial transcriptomics and bulk RNA sequencing to delineate specific cell subsets involved in hepatocellular carcinoma (HCC) relapse and response to immunotherapy. We identified POSTN+ extracellular matrix cancer-associated fibroblasts (EM CAFs) as a prominent onco-fetal interacting hub, promoting tumor progression. Cell-cell communication and spatial transcriptomics analysis revealed crosstalk and co-localization of onco-fetal cells, including POSTN+ CAFs, FOLR2+ macrophages and PLVAP+ endothelial cells. Further analyses suggest an association between onco-fetal reprogramming and epithelial-mesenchymal transition (EMT), tumor cell proliferation and recruitment of Treg cells, ultimately influencing early relapse and response to immunotherapy. In summary, our study identifies POSTN+ CAFs as part of the HCC onco-fetal niche and highlights its potential influence in EMT, relapse and immunotherapy response, paving the way for the use of onco-fetal signatures for therapeutic stratification.

Structure-function relationship of phase-separated liposomes containing diacylglycerol analogues.

The composition and morphology of lipid-based nanoparticles can influence their overall in vivo behavior. Previously, we demonstrated that phase separation in liposomes composed of DSPC and a diacylglycerol lipid analogue (DOaG) drives the in vivo biodistribution towards a specific subset of endothelial cells in zebrafish embryos. In the absence of traditional targeting functionalities (e.g., antibodies, ligands), this selectivity is mediated solely by the unique liposome morphology and composition, characterized by a DOaG-rich lipid droplet within the DSPC-rich phospholipid bilayer. The phase separation is induced due to the geometry of DOaG lipid and its ability to create non-bilayer phases in lipid membranes. To investigate the underlying principles of phase separation and to optimize the liposome colloidal stability, we performed a structure-function relationship study by synthesizing a library of DOaG analogues with varying molecular properties, such as the number, length and sn-position of the acyl chains, as well as the degree of saturation or carbonyl substituents. We assessed the ability of these lipid analogues to assemble into phase-separated liposomes and studied their morphology, colloidal stability, and in vivo biodistribution in zebrafish embryos. We found that analogues containing unsaturated, medium length (C16-C18) fatty acids were required to obtain colloidally stable, phase-separated liposomes with cell-specific biodistribution patterns. Moreover, we observed that using the pure DOaG isomer, with acyl chains at the sn-1,3 positions, leads to more colloidally stable liposomes than when a mixture of sn-1,2 and sn-1,3 isomers is used. Similarly, we observed that incorporating a DOaG analogue with fatty tails shorter than DSPC, as well as PEGylation, endows liposomes with long term stability while retaining cell-selective biodistribution. Diacylglycerols are known to promote fusion, lipid polymorphism, signaling and protein recruitment on lipid membranes. In this study, we showed that diacylglycerol derivatives can induce phase separation in liposomes, unlocking the potential for cell-specific targeting in vivo. We believe that these findings can be the foundation for future use of diacylglycerols in lipid-based nanomedicines and could lead to the development of novel targeted delivery strategies.

CRISPR Knock-Ins in Organoids to Track Tumor Cell Subpopulations.

The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.

Current Concepts of Leukemic Stem Cells: Origin, Characteristics, and its Clinical Implications in Acute Myeloid Leukemia

Despite significant advancements in therapy, patients diagnosed with acute myeloid leukemia (AML) continue to face poor outcomes, often experiencing relapses even after achieving initial complete remission. The occurrence of relapse is attributed to the inability of conventional treatment to eliminate a specific subset of cells within the bone marrow known as leukemic stem cells (LSCs). These specialized cells exhibit self-renewal capacity and have the ability to proliferate and differentiate into leukemic blasts. The accumulation of multiple genetic mutations in LSCs makes them resistant to standard chemotherapy. Several studies have been conducted to identify the phenotypic characteristics and genetic signatures of LSCs, with the aim of differentiating them from normal hematopoietic stem cells (HSCs). Understanding the role of LSCs in AML treatment resistance has paved the way for the development of targeted and more precise treatments, especially for relapsed AML patients, without affecting the healthy HSCs. This review elaborates on the origin, phenotypic and genotypic characteristics of LSCs, and their role in the biology of AML, with a brief note on therapies targeting LSCs.

Advances in Single-Cell Techniques for Linking Phenotypes to Genotypes.

Single-cell analysis has become an essential tool in modern biological research, providing unprecedented insights into cellular behavior and heterogeneity. By examining individual cells, this approach surpasses conventional population-based methods, revealing critical variations in cellular states, responses to environmental cues, and molecular signatures. In the context of cancer, with its diverse cell populations, single-cell analysis is critical for investigating tumor evolution, metastasis, and therapy resistance. Understanding the phenotype-genotype relationship at the single-cell level is crucial for deciphering the molecular mechanisms driving tumor development and progression. This review highlights innovative strategies for selective cell isolation based on desired phenotypes, including robotic aspiration, laser detachment, microraft arrays, optical traps, and droplet-based microfluidic systems. These advanced tools facilitate high-throughput single-cell phenotypic analysis and sorting, enabling the identification and characterization of specific cell subsets, thereby advancing therapeutic innovations in cancer and other diseases.

DVsc: An Automated Framework for Efficiently Detecting Viral Infection from Single-cell Transcriptomics Data.

Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable tool for studying cellular heterogeneity in various fields, particularly in virological research. By studying the viral and cellular transcriptomes, the dynamics of viral infection can be investigated at a single-cell resolution. However, limited studies have been conducted to investigate whether RNA transcripts from clinical samples contain substantial amounts of viral RNAs, and a specific computational framework for efficiently detecting viral reads based on scRNA-seq data has not been developed. Hence, we introduce DVsc, an open-source framework for precise quantitative analysis of viral infection from single-cell transcriptomics data. When applied to approximately 200 diverse clinical samples that were infected by more than 10 different viruses, DVsc demonstrated high accuracy in systematically detecting viral infection across a wide array of cell types. This innovative bioinformatics pipeline could be crucial for addressing the potential effects of surreptitiously invading viruses on certain illnesses, as well as for designing novel medicines to target viruses in specific host cell subsets and evaluating the efficacy of treatment. DVsc supports the FASTQ format as an input and is compatible with multiple single-cell sequencing platforms. Moreover, it could also be applied to sequences from bulk RNA sequencing data. DVsc is available at http://62.234.32.33:5000/DVsc.

Immune profiling and tracking of two-dimensional transition metal dichalcogenides in cells and tissues

Two-dimensional (2D) transition metal dichalcogenides (TMDs), such as molybdenum disulfide (MoS2) and tungsten disulfide (WS2), have attracted considerable interest in biomedicine due to their unique combination of physicochemical properties. The effect of nanomaterials on immune cells and their biodistribution are critical aspects of their clinical translation. However, understanding the interactions of these emerging 2D nanomaterials with the complex pool of peripheral blood mononuclear cells (PBMCs) at the single-cell level and their in situ localization in the main organs still needs to be discovered, preventing their translation in medical settings. Here, we report in-depth immune profiling of water-based and defect-free 2D formulations of MoS2 and WS2 through the simultaneous label-free tracking of their immune cell interactions, both ex vivo in human PBMCs and in vivo in mice by high-dimensional analytical approaches, as well as their biodistribution. For comparison, we studied graphene, the hitherto most explored 2D material for biomedical applications. First, we assessed the impact at the protein and gene level by multiplex protein arrays and RNA sequencing, demonstrating a very modest effect of MoS2 and WS2 on immune cell functionality compared to graphene. Then, a single-cell view of the effects of MoS2 and WS2 on 16 primary human immune cell types in terms of viability and functionality was obtained by single-cell mass cytometry by time-of-flight (CyTOF). We explored over 30 markers looking at multiple cell parameters. Finally, we present evidence that MoS2 and WS2 are visible, without the need for labeling, at the single-cell and tissue level by CyTOF, imaging mass cytometry, and multiplexed ion beam imaging by time-of-flight (MIBI-TOF). In particular, MoS2 and WS2 could be detected in the molybdenum (95Mo) and tungsten (180–186W) channels, respectively, which are not used for commercial mass cytometry tags, allowing for the simultaneous interrogation of a wide variety of biological parameters ex vivo and in vivo following intravenous administration of the TMDs. Indeed, we demonstrated the accumulation of TMDs in the main organs by MIBI-TOF and they could also be identified in specific immune cell subsets by CyTOF. Among the two TMDs studied, WS2 exhibited the highest brightness and signal intensity in all the cell subpopulations and tissues analyzed. In conclusion, we identified TMDs as immune-compatible nanoplatforms, traceable at the single-cell and tissue (sub-organ) levels, thus opening up new perspectives for their exploration in biomedicine.

Oleic acid availability impacts thymocyte preprogramming and subsequent peripheral Treg cell differentiation.

The nature of activation signals is essential in determining T cell subset differentiation; however, the features that determine T cell subset preference acquired during intrathymic development remain elusive. Here we show that naive CD4+ T cells generated in the mouse thymic microenvironment lacking Scd1, encoding the enzyme catalyzing oleic acid (OA) production, exhibit enhanced regulatory T (Treg) cell differentiation and attenuated development of experimental autoimmune encephalomyelitis. Scd1 deletion in K14+ thymic epithelia recapitulated the enhanced Treg cell differentiation phenotype of Scd1-deficient mice. The dearth of OA permitted DOT1L to increase H3K79me2 levels at the Atp2a2 locus of thymocytes at the DN2-DN3 transition stage. Such epigenetic modification persisted in naive CD4+ T cells and facilitated Atp2a2 expression. Upon T cell receptor activation, ATP2A2 enhanced the activity of the calcium-NFAT1-Foxp3 axis to promote naive CD4+ T cells to differentiate into Treg cells. Therefore, OA availability is critical for preprogramming thymocytes with Treg cell differentiation propensities in the periphery.

Role of peripheral inflammation in driving central nervous system inflammatory signature in pathogenesis of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) impacts an estimated 6.5 million people in the United States, however, it cannot yet be prevented, slowed or cured. Although the sporadic AD form comprises around 95% of cases, animal models of familial AD are the ones used for basic and translational research. Most of these models are based on human mutations that cause amyloid‐β (Aβ) pathological accumulation. However, the failure of the majority of Aβ‐related clinical trials, has begged the necessity of an alternative approach to address AD. Interestingly, most of the genetic and non‐genetic risk factors for developing sporadic AD are associated with inflammation. Thus, it has been suggested that microglia, the immune cells in the central nervous system, play a key role in the pathogenesis of AD, and that people with systemic inflammatory diseases have a higher incidence of developing AD. In this context, we hypothesize that a specific subset of cells in peripheral blood contributes to AD development through its communication with microglial cells in the brain parenchyma.MethodTo address this hypothesis, we analyzed peripheral blood mononuclear cells (PBMCs) from sex‐ and age‐matched healthy and AD subjects using mass cytometry (CyTOF) and cellular indexing of transcriptomes and epitopes by sequencing (CITE‐seq) technologies. Moreover, we used in vitro and histological approaches to study interactions between PBMCs and human microglial cells.ResultOur preliminary data show a different proportion of peripheral blood immune cell types between healthy controls and AD patients. Remarkably, a specific inflammatory subset of immune cells characterized by the upregulation of pro‐inflammatory signaling pathways and cytokines was detected in blood of AD patients. In addition, T cells in contact with microglia were detected in the parenchyma of AD brains by histological analysis, suggesting adrive of microglial cells towards an inflammatory phenotype that could contribute to AD pathology. We corroborated the pathogenic component of AD blood as healthy PBMCs developed an inflammatory phenotype after stimulation with AD plasma.ConclusionTogether, our data demonstrate the importance of peripheral immune cells in AD disease and reinforce that studying AD as a multifactorial disease could be key to its prevention and treatment.

Intrinsic factors and CD1d1 but not CD1d2 expression levels control invariant natural killer T cell subset differentiation

Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood.

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture-derived EVs are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 h post-intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig-tailed macaques. Using this ex vivo system, we found that labelled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions.

An increase in regulatory T cells in peripheral blood correlates with an adverse prognosis for malignant melanoma patients – A study of T cells and natural killer cells

Malignant melanoma is a highly immunogenic tumour, and the immune profile significantly influences cancer development and response to immunotherapy. The peripheral immune profile may identify high risk patients. The current study showed reduced levels of CD4+ T cells and increased levels of CD8+ T cells in peripheral blood from malignant melanoma patients compared with controls. Percentages of peripheral CD56dimCD16+ NK cells were reduced and CD56brightCD16−KIR3+ NK cells were increased in malignant melanoma patients. Late stage malignant melanoma was correlated with low levels of CD4+ T cells and high levels of CD56brightCD16−KIR3+ NK cells. Finally, high levels of Tregs in peripheral blood were correlated with poor overall survival and disease-free survival. The results indicate that changes in specific immune cell subsets in peripheral blood samples from patients at the time of diagnosis may be potential biomarkers for prognosis and survival. Further studies will enable clarification of independent roles in tumour pathogenesis.

Spatiotemporal Impact of Sars-Cov-2 Infection on the Transcriptome of Bone Marrow Megakaryocytes

Megakaryocytes, integral to platelet production, predominantly reside in the bone marrow, undergoing regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections have been shown to influence megakaryocyte transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptome. In this study, we hypothesized that SARS-CoV-2 infection could impact the transcriptome of bone marrow megakaryocytes, particularly those in direct proximity to the blood. Utilizing spatial transcriptomic analysis and machine learning techniques, we observed that the transcriptome of healthy mouse bone marrow megakaryocytes exhibited minimal alteration based on proximity to sinusoid vessels. Similar findings were observed during peak SARS-CoV-2 viremia, when the disease primarily affected the lungs. Conversely, a significant divergence in the transcriptome of megakaryocytes was observed during systemic inflammation, even when SARS-CoV-2 was no longer detectable in the lungs or bone marrow. Under these conditions, the transcriptional landscape was enriched in genes associated with structural and translational activities, platelet degranulation, netosis, and auto-immunity. Of the approximately ~19,000 genes identified in megakaryocytes during systemic inflammation, machine learning pinpointed 30 genes significantly altered in cells closely adjacent to sinusoid vessels, with gene ontology attributing these changes primarily to protein deSUMOylation. Intriguingly, the type-I interferon signature and calprotectin (S100A8/A9) were not induced under any condition. Inflammatory cytokines were elevated in the blood of COVID-19 mice, but not in bone marrow plasma, suggesting a preferential impact of inflammation on this specific subset of cells. Collectively, our data indicate that distinct subpopulations of bone marrow megakaryocytes may emerge based on spatial localization and the stage of COVID-19 pathogenesis.

Intradermal Vaccination with Vididencel in MRD+ AML-Patients Leads to Increase in Antigen Presenting Cells and T-Cells to the Injection Site, Visualized Using Imaging Mass Cytometry, Showing Local Immune Cell Interactions Leading to Systemic Immune Responses

Background. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine being developed as maintenance treatment in AML patients. Intradermal vaccination using vididencel is known to induce a local skin reaction, with local antigen processing by skin dendritic cells and direct presentation to T cells as well as dendritic cells, crawling out of the skin and presentation of the antigen in the draining lymph nodes for a full-blown immune response. It was previously shown that vididencel is able to induce an influx of immune cells in the skin using standard immunohistochemistry (van de Loosdrecht, 2018). In a phase 2 study vididencel was investigated as AML maintenance treatment after induction of CR1 with chemotherapy (ADVANCE-II, Clintrials.gov: NCT03697707). Using imaging mass cytometry up to 43 markers can be assessed in the same tissue section, to allow in depth analysis of the immune cells on a single cell level infiltrating in the dermis at the injection site. Methods. AML patients, in first complete remission with measurable residual disease (MRD +) and not planned for HSCT at inclusion, received 4 biweekly doses of vididencel followed by 2 booster doses at week 14 and 18. Skin biopsies of the injection site were taken 2 days after the 1 st, 4 th and 6 th vaccination. Control samples were collected from unvaccinated/unaffected skin in two patients and from healthy controls. Samples were fixed and embedded in paraffin, and stained by standard immunohistochemistry for immune cell markers like CD4, CD8 and CD68 in the central lab. For patients treated at Haukeland University Hospital (N=6), tissue sections were prepared, mounted and stained with the panel of 29 antibodies labelled with heavy metal isotopes for use in the Hyperion mass cytometry imaging. For each sample a region of interest (ROI) of 2.8mm 2 was scanned based on the localization of the immune infiltrates, guided by standard H&E staining from neighboring slides. Results In 88.5% of patients injection site reactions were reported in this phase 2 study, characterized by redness, swelling and warmth at the site of intradermal injection, which were mild (maximum grade 1 or 2), and were of short duration (generally resolved within 12 days). Skin biopsies of a subset of 6 patients were analyzed using Hyperion. In standard single stain immunohistochemistry a large influx of CD4, CD8 and CD68 cells was observed in contrast to normal skin. Imaging mass cytometry confirmed these dense immune infiltrations. Using additional markers to characterize monocytes, dendritic cells and T-cells revealed that these cells are in close proximity and suggestive for cell-to-cell interaction (Figure 1). At sites of dense immune infiltration, influx of GranzymeB expressing CD8 T-cells and CD38 positive CD4 T-cells were observed as well as CD1c+ or CD141+ dendritic cells with high HLA-DR expression. These T-cells and dendritic cells are in close contact suggestive for patients' dendritic cells migrating from blood to skin, inducing a local immune response. Conclusion/discussion. Vaccination with vididencel triggered an inflammatory skin reaction in the majority of patients, with redness, warmth and swelling, of short duration, indicative for an active immune response at the site of injection. Skin biopsies using standard immunohistochemistry showed influx of activated CD4 and CD8 T cells as well as antigen presenting cell, including dendritic cells.Extended analysis with multiparameter imaging mass cytometry showed specific immune cell subsets infiltrating in these site of injection sites. The observed co-localization of the different immune subsets following vaccination supports the hypothesis of local antigen capturing by blood derived and local antigen presenting cells (Zuo et al, 2021). The local immune response may ultimately leads to a systemic immune response and disease control.

Label-Free High Dimensional Single Cell Morphological Profiling of Different Hematological Malignancies By Ghost Cytometry

Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern 'omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6) (Figure 1). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information (Figure 2). For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified unique cell populations across blood cell populations that were distinct from the healthy BM-MNC controls. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Keywords: flow cytometry, acute leukemia, multiple myeloma, label-free cell analysis, biomarker, liquid biopsy, ghost cytometry, artificial intelligence, machine learning Figure 1. Research design overview. Bone marrow samples from patients and non-diseased controls will be analyzed by Ghost Cytometry to assess disease by conventional markers and identify novel morphometric subpopulations. Morphological information is acquired as label-free GMI signals without any labeling. The label-free GMI information is transformed into two-dimensional space by UMAP dimensionality reduction method to identify subpopulations of interest. Both samples are stained with known cell surface markers for validation of unsupervised classification. Figure 2. Identification of disease-specific cell subpopulations by unsupervised approach. Using an unsupervised machine learning technique called UMAP, label-free morphologically distinct cellular subpopulations can be identified in B-ALL, AML, MM when compared to healthy donor controls (left panels, inside the dotted circles). For validation of the subpopulations, known cell surface markers (CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM) and cell trace marker (CellTracker) were overlaid. The intensity increases monotonically through the colormaps. The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population.

CCR5 promoter region polymorphisms in systemic lupus erythematosus.

This study investigated the impacts of CCR5 promoter region polymorphisms on the development of systemic lupus erythematosus (SLE) by comparing CCR5 genotypes and haplotypes from SLE patients with ethnically matched controls. A total of 382 SLE patients (289 European-derived and 93 African-derived) and 375 controls (243 European-derived and 132 African-derived) were genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms through polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Previous data from CCR5Δ32 analysis was included in the study to infer the CCR5 haplotypes and as a possible confounding factor in the binary logistic regression. European-derived patients showed a higher frequency of CCR5 wild-type genotype (conversely, a reduced frequency of Δ32 allele) and a reduced frequency of the HHG*2 haplotype compared to controls; both factors significantly affecting disease risk [p=.003 (OR 3.5, 95%CI 1.6-7.5) and 2.0% vs. 7.2% (residual p=2.9E-5), respectively]. Additionally, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between African-derived patients and controls [10% vs. 20.5% (residual p=.003), 29.4% vs. 17.4% (residual p=.003) and 3.9% vs. 0.8% (residual p=.023), respectively]. Considering the clinical manifestations of the disease, the CCR5Δ32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when all patients were grouped for comparison [pcorrected =.012 (OR 3.0; 95%CI 3.0-333.3) and pcorrected =.0006 (OR 6.8; 95%CI 1.9-24.8), respectively]. In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces the CCR5Δ32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also described a reduced frequency of HHA/HHB and an increased frequency of HHC and HHG*2 haplotypes in African-derived patients, which could modify the CCR5 protein expression in specific cell subsets.

17-β Estradiol (E2) distinctly regulates the expression of IL-4 and IL-13 in Th2 cells via modulating the interplay between GATA3 and PU.1

17-β Estradiol (E2) has long standing known functions in regulating human physiology as well as immune system. E2 is known to elicit a protective role against experimental autoimmune encephalomyelitis (EAE) and has been used as a drug for treatment against multiple sclerosis. Moreover, E2 regulates the adaptive immune system by directly affecting the T helper cell subsets differentiation and antibody secretion mediated by B cells. Reports have shown that E2 promotes Th1 and Treg cell differentiation; whereas it attenuated the Th17 and Tfh cell differentiation. Albeit multiple and contrasting studies, the mechanisms of behind E2 action on Th2 cells remained understudied. Hence, we sought to dissect the impact of E2 in Th2 cell differentiation. In this study, we elucidated the molecular mechanisms behind E2-mediated regulation of the differentiation of Th2 cells. We observed that E2 significantly attenuated the IL-4-secreting Th2 population in an ERα-dependent manner. We validated these findings using ICI 182, 780, an antagonist to ERα, not ERβ and ectopically overexpressing ERα in Th2 cells. We further determined that ERα alters the recruitment of GATA3 and PU.1 to Il4 gene by directly interacting with them. This altered recruitment was observed to be stronger at Il4 than Il13 locus. Interestingly, we detected a distinct recruitment of GATA3 and PU.1 at Il13 gene; however, there was no E2-mediated broad alteration in the recruitment of histone-modifiers at Il13 locus. These findings suggest that E2 regulates Il4 in a distinctly separate mechanism as opposed to Il13 locus in Th2 cells.

Single cell transcriptomics shows that malaria promotes unique regulatory responses across multiple immune cell subsets

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.

Combatting ageing in dermal papilla cells and promoting hair follicle regeneration using exosomes from human hair follicle dermal sheath cup cells.

Dermal papilla cells (DPCs) undergo premature ageing in androgenetic alopecia and senescent alopecia. As critical components of hair follicle reconstruction, DPCs are also prone to senescence invitro, resulting in a diminished hair follicle inductivity capacity. Dermal sheath cup cells (DSCCs), a specific subset of hair follicle mesenchymal stem cells, intimately linked to the function of DPCs. The primary objective of this research is to investigate the anti-ageing effect of exosomes derived from DSCCs (ExoDSCCs ) on DPCs. Exosomes were utilized to treat H2 O2 -induced DPCs or long-generation DPCs(P10). Our findings demonstrate that ExoDSCCs(P3) promote the proliferation, viability and migration of senescent DPCs while inhibiting cell apoptosis. The expression of senescence marker SA-β-Gal were significantly downregulated in senescent DPCs. When treated with ExoDSCCs(P3) , expression of inducibility related markers alkaline phosphatase and Versican were significantly upregulated. Additionally, ExoDSCCs(P3) activated the Wnt/β-catenin signalling invitro. In patch assay, ExoDSCCs(P3) significantly promoted hair follicle reconstruction in senescent DPCs. In summary, our work highlights that ExoDSCCs(P3) may restore the biological functions and improve the hair follicle induction ability of senescent DPCs. Therefore, ExoDSCCs(P3) may represent a new strategy for intervening in the ageing process of DPCs, contributing to the prevention of senile alopecia.