Cellular Indexing Of Transcriptomes Research Articles

Integrated spatial and multimodal single-cell transcriptomics reveal patient-dependent cell heterogeneity in splenic marginal zone lymphoma.

Biological hallmarks of splenic marginal zone lymphoma (SMZL) remain poorly described. Herein, we performed in-depth SMZL characterization through multimodal single-cell analyses of paired blood/spleen samples. The 3'-single-cell RNA-sequencing, Cellular Indexing of Transcriptomes and Epitopes by sequencing, and 5'-V(D)J single-cell RNA-sequencing datasets were integrated to characterize SMZL transcriptome profiles, including B-cell receptor and T-cell receptor repertoires. Hyperexpanded B-cell clones in the spleen were at a memory-like stage, whereas recirculating tumor B-cells in blood encompassed multiple differentiation stages, indicating an unexpected desynchronization of the B-cell maturation program in SMZL cells. Spatial transcriptomics showed the enrichment of T-effector and T-follicular helper (TFH) signatures in the nodular subtype of SMZL. This latter also exhibited gene-based cell-cell interactions suggestive of dynamic crosstalk between TFH and cancer cells in transcriptomics, further substantiated by using imaging mass cytometry. Our findings provide a comprehensive high-resolution description of SMZL biological hallmarks and characterize, for the first time in situ, inter- and intra-patient heterogeneity at both transcriptomic and protein levels. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

#2704 Effects of high dietary salt on immunome and microbiome composition – results from a randomized clinical trial

Abstract Background and Aims Increased dietary salt intake ranks among the most prominent nutritional risk factors worldwide and has been tied to arterial hypertension and all-cause mortality. The mechanisms responsible are incompletely understood and have recently been extended to include inflammatory and microbiome-associated mechanisms. Building on our preliminary data, the present study investigates whether a moderate increase in salt intake in healthy subjects primes host physiology to a more transiently unstable state, potentially leading to changes in the microbiome-immune axis. Method We conducted a prospective, randomized, double-blinded trial to evaluate the effect of high dietary salt in healthy participants (NCT03024567), where the intervention group (n = 19) was given 6 g of salt (NaCl capsules) in addition to the habitual salt intake, resulting in an average doubling of the salt intake recommended by expert societies. A placebo group (n = 19) received gelatin capsules for 14 days. Clinical parameters, fecal samples and PBMC were collected at baseline and day 14. Shotgun metagenomic sequencing, metabolomics (stool and serum), and single-cell sequencing (Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq)) of whole PBMCs and CD4+ sorted T cells (∼150 k single cells) was performed. Results Microbiome analysis confirms previous findings showing an increased dissimilarity of microbiome composition (Jensen-Shannon Divergence) under a high-salt. Differential abundance of the microbiome's functional space, as quantified by functional gut-specific modules, picked up a salt-specific module (M00494, NatK-NatR (sodium extrusion) two-component regulatory system) in the salt intervention group. CITEseq revealed a significant reduction of circulating NK cells and an increase in classical monocytes as well as changes to several T cell subsets. Furthermore, within several immune cell subsets, we detected differently expressed genes (DEGs) solely in the salt group. We found 602 DEG in naïve conventional T cells (Tconv) and 202 DEGs in non-naïve Tconv, e.g. belonging to NF-kB and T cell receptor signaling pathways. Conclusion Our placebo-controlled study is the first to utilize different omics techniques to investigate and identify high-salt-induced changes in the microbiome and immunome in healthy individuals that may be relevant for health-to-disease transition.

The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

CITEViz: interactively classify cell populations in CITE-Seq via a flow cytometry-like gating workflow using R-Shiny

BackgroundThe rapid advancement of new genomic sequencing technology has enabled the development of multi-omic single-cell sequencing assays. These assays profile multiple modalities in the same cell and can often yield new insights not revealed with a single modality. For example, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) simultaneously profiles the RNA transcriptome and the surface protein expression. The surface protein markers in CITE-Seq can be used to identify cell populations similar to the iterative filtration process in flow cytometry, also called “gating”, and is an essential step for downstream analyses and data interpretation. While several packages allow users to interactively gate cells, they often do not process multi-omic sequencing datasets and may require writing redundant code to specify gate boundaries. To streamline the gating process, we developed CITEViz which allows users to interactively gate cells in Seurat-processed CITE-Seq data. CITEViz can also visualize basic quality control (QC) metrics allowing for a rapid and holistic evaluation of CITE-Seq data.ResultsWe applied CITEViz to a peripheral blood mononuclear cell CITE-Seq dataset and gated for several major blood cell populations (CD14 monocytes, CD4 T cells, CD8 T cells, NK cells, B cells, and platelets) using canonical surface protein markers. The visualization features of CITEViz were used to investigate cellular heterogeneity in CD14 and CD16-expressing monocytes and to detect differential numbers of detected antibodies per patient donor. These results highlight the utility of CITEViz to enable the robust classification of single cell populations.ConclusionsCITEViz is an R-Shiny app that standardizes the gating workflow in CITE-Seq data for efficient classification of cell populations. Its secondary function is to generate basic feature plots and QC figures specific to multi-omic data. The user interface and internal workflow of CITEViz uniquely work together to produce an organized workflow and sensible data structures for easy data retrieval. This package leverages the strengths of biologists and computational scientists to assess and analyze multi-omic single-cell datasets. In conclusion, CITEViz streamlines the flow cytometry gating workflow in CITE-Seq data to help facilitate novel hypothesis generation.

NFĸB signaling drives myocardial injury via CCR2+ macrophages in a preclinical model of arrhythmogenic cardiomyopathy.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.

501 Systemic Immunological Suppression in Glioblastoma Is Targeted by Concurrent VEGF and PD-1 Inhibition in a Dose-Dependent Manner: Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) From a Randomized Controlled Trial

INTRODUCTION: Anti-PD1 blockade has had poor efficacy in recurrent glioblastoma (rGBM) across three RCTs. VEGF, a proangiogenic factor that is upregulated in rGBM, contributes to tumor-associated immunosuppression and is widely targeted through bevacizumab. Preclinical data indicate a potential dose-dependent effect of anti-VEGF therapy on immunomodulation. Hence, a combination of anti-PD-1 and anti-VEGF agents is a promising approach for rGBM, for which an RCT was conducted. METHODS: Patients with rGBM were administered nivolumab (240mg IV q2weeks) plus either standard dose bevacizumab (10mg/kg) or low dose bevacizumab (3mg/kg) IV q2weeks, with 1:1 randomization. Stratification factors were age, performance status, extent of resection, and MGMT methylation status. Primary endpoint was overall survival (OS) at 12 months. CITE-Seq was performed on 16 patients across trial arms, both pre- and on-therapy, yielding total 32 immunological profiles. For control, CITE-seq was done on three patients receiving anti-VEGF therapy alone. RESULTS: 90 patients were enrolled with median age of 60.6 years and 45 patients per arm. Both arms had similar OS at 12 months (P = 0.14). Significantly higher OS was found in posthoc analysis for patients aged = 60 years with standard dose. Distinct immune cell populations were identified and differential changes in myeloid-derived suppressor cells (MDSCs) found across trial arms on-therapy. Network medicine approach identified VEGF as target to reduce MDSCs, with VEGF primarily expressed by responders’ MDSCs. Differential gene expression analysis identified increased pro-inflammatory signatures in standard dose arm. CONCLUSIONS: Standard dose bevacizumab was associated with increased inflammatory response and reduction of immunosuppressive MDSC. These mechanistic insights potentially explain survival benefit seen with concurrent VEGF and PD-1 inhibition in elderly rGBM patients.

Abstract 25: Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features

Abstract Introduction: Autologous TIL cell therapies such as lifileucel have demonstrated durable responses in patients (pts) with various solid tumors including melanoma and lung cancer. The TIL drug product (DP) phenotype was previously assessed and extensively characterized based on the bulk population. The current research further characterizes the TIL DP at a single-cell level using high-dimension multimodal sequencing and explores features that have a potential to inform future development of TIL therapies. Methods: Manufactured lifileucel TIL DP from 20 pts (best overall response [BOR]: 6 complete response [CR], 4 partial response, 5 stable disease, 5 progressive disease [PD]) from the registrational melanoma C144-1 trial were analyzed using single-cell RNA and T-cell receptor (TCR) sequencing, resulting in 153K single-cell transcriptomes. To aid with cell-type annotation, 130 surface proteins on TIL DP samples from a subset of 4 pts (BOR: 2 CR, 2 PD) were detected via Cellular Indexing of Transcriptomes and Epitopes by sequencing. Multimodal weighted nearest-neighbor analysis combined with manual cluster annotation (via known marker genes and proteins), cell-type-specific gene signatures, and automated annotation were used to develop a single-cell TIL (scTIL) reference map, resulting in high-resolution subsets of cells. TCR clonotypes from lifileucel TIL DP were compared to those from respective pretreatment tumors previously identified by bulk TCR sequencing. Results: Mapping of the full dataset to the novel scTIL reference revealed a CD8+ TIL-dominant composition of responder TIL DP. TIL from pts with BOR PD exhibited reduced CD8+ effector memory (Tem)-like and resident memory (Trm)-like T cells. Differential expression analysis showed that responder CD8+ TIL had increased expression of TCR signaling genes while responder CD4+ TIL exhibited gene expression consistent with a more cytotoxic-like phenotype. Relative to responders, CD8+ TIL of pts with BOR PD had lower expression of the CD39−CD69− (stem-like) gene signature and higher expression of the CD39+CD69+ (terminal differentiation) gene signature. Pseudotime trajectory analysis found that both CD8+ and CD4+ TIL of pts with BOR PD were more terminally differentiated. Single-cell TCR sequencing revealed that CD8+ T-cell clusters had greater clonal overlap than CD4+ T-cell clusters. Expanded clonotypes in pretreatment tumor tissues were more prevalent in TIL TCR repertoires of responders and were more likely to be expressed by CD8+ (specifically CD8+ Tem- and Trm-like TIL) than CD4+ T cells. Conclusions: Collectively, these results shed additional light on the cellular and molecular characteristics of ex vivo expanded lifileucel TIL DP and signal additional features with a potential to guide future TIL product development. Citation Format: Joe Dean, Joe Yglesias, Hequn Yin, Rongsu Qi. Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 25.

An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

S100A8-enriched microglia populate the brain of tau-seeded and accelerated aging mice.

Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.

Multimodal and spatially resolved profiling identifies distinct patterns of T cell infiltration in nodal B cell lymphoma entities

The redirection of T cells has emerged as an attractive therapeutic principle in B cell non-Hodgkin lymphoma (B-NHL). However, a detailed characterization of lymphoma-infiltrating T cells across B-NHL entities is missing. Here we present an in-depth T cell reference map of nodal B-NHL, based on cellular indexing of transcriptomes and epitopes, T cell receptor sequencing, flow cytometry and multiplexed immunofluorescence applied to 101 lymph nodes from patients with diffuse large B cell, mantle cell, follicular or marginal zone lymphoma, and from healthy controls. This multimodal resource revealed quantitative and spatial aberrations of the T cell microenvironment across and within B-NHL entities. Quantitative differences in PD1+TCF7− cytotoxic T cells, T follicular helper cells or IKZF3+ regulatory T cells were linked to their clonal expansion. The abundance of PD1+TCF7− cytotoxic T cells was associated with poor survival. Our study portrays lymphoma-infiltrating T cells with unprecedented comprehensiveness and provides a unique resource for the investigation of lymphoma biology and prognosis.

Deciphering metabolic crosstalk in context: lessons from inflammatory diseases.

Metabolism plays a crucial role in regulating the function of immune cells in both health and disease, with altered metabolism contributing to the pathogenesis of cancer and many inflammatory diseases. The local microenvironment has a profound impact on the metabolism of immune cells. Therefore, immunological and metabolic heterogeneity as well as the spatial organization of cells in tissues should be taken into account when studying immunometabolism. Here, we highlight challenges of investigating metabolic communication. Additionally, we review the capabilities and limitations of current technologies for studying metabolism in inflamed microenvironments, including single-cell omics techniques, flow cytometry-based methods (Met-Flow, single-cell energetic metabolism by profiling translation inhibition (SCENITH)), cytometry by time of flight (CyTOF), cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), and mass spectrometry imaging. Considering the importance of metabolism in regulating immune cells in diseased states, we also discuss the applications of metabolomics in clinical research, as well as some hurdles to overcome to implement these techniques in standard clinical practice. Finally, we provide a flowchart to assist scientists in designing effective strategies to unravel immunometabolism in disease-relevant contexts.

The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective.

The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Associations Between Rheumatoid Arthritis Clinical Factors and Synovial Cell Types and States.

Recent studies have uncovered diverse cell types and states in the rheumatoid arthritis (RA) synovium; however, limited data exist correlating these findings with patient-level clinical information. Using the largest cohort to date with clinical and multicell data, we determined associations between RA clinical factors with cell types and states in the RA synovium. The Accelerated Medicines Partnership Rheumatoid Arthritis study recruited patients with active RA who were not receiving disease-modifying antirheumatic drugs (DMARDs) or who had an inadequate response to methotrexate (MTX) or tumor necrosis factor inhibitors. RA clinical factors were systematically collected. Biopsies were performed on an inflamed joint, and tissue were disaggregated and processed with a cellular indexing of transcriptomes and epitopes sequencing pipeline from which the following cell type percentages and cell type abundance phenotypes (CTAPs) were derived: endothelial, fibroblast, and myeloid (EFM); fibroblasts; myeloid; T and B cells; T cells and fibroblasts (TF); and T and myeloid cells. Correlations were measured between RA clinical factors, cell type percentage, and CTAPs. We studied 72 patients (mean age 57 years, 75% women, 83% seropositive, mean RA duration 6.6 years, mean Disease Activity Score-28 C-reactive Protein 3 [DAS28-CRP3] score 4.8). Higher DAS28-CRP3 correlated with a higher T cell percentage (P < 0.01). Those receiving MTX and not a biologic DMARD (bDMARD) had a higher percentage of B cells versus those receiving no DMARDs (P < 0.01). Most of those receiving bDMARDs were categorized as EFM (57%), whereas none were TF. No significant difference was observed across CTAPs for age, sex, RA disease duration, or DAS28-CRP3. In this comprehensive screen of clinical factors, we observed differential associations between DMARDs and cell phenotypes, suggesting that RA therapies, more than other clinical factors, may impact cell type/state in the synovium and ultimately influence response to subsequent therapies.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in human beings, challenging the concept of TRAIL as a potent anticancer agent. Herein, we aimed to define mechanisms by which TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). Multiple immunocompetent syngeneic, orthotopic models of CCA were used. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of CD45+ cells in murine tumors from the different CCA models was conducted. In multiple immunocompetent murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/- mice resulted in a significant reduction in tumor volumes compared with wild-type mice. Tumor-bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs owing to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent nuclear factor-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of immune cells from murine tumors showed enrichment of a nuclear factor-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis owing to enhanced expression of cellular FLICE inhibitory protein, an inhibitor of proapoptotic TRAIL signaling. Accordingly, cellular FLICE inhibitory protein knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. Our findings highlight the therapeutic potential of targeting TRAIL+ cancer cells for treatment of a poorly immunogenic cancer.

Elucidating granulocytic myeloid-derived suppressor cell heterogeneity during Staphylococcus aureus biofilm infection.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated immature myeloid cells with immunosuppressive activity that expand during chronic inflammation, such as cancer and prosthetic joint infection (PJI). Myeloid-derived suppressor cells can be broadly separated into 2 populations based on surface marker expression and function: monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic myeloid-derived suppressor cells (G-MDSCs). Granulocytic myeloid-derived suppressor cells are the most abundant leukocyte infiltrate during PJI; however, how this population is maintained in vivo and cellular heterogeneity is currently unknown. In this study, we identified a previously unknown population of Ly6G+Ly6C+F4/80+MHCII+ MDSCs during PJI that displayed immunosuppressive properties ex vivo. We leveraged F4/80 and MHCII expression by these cells for further characterization using cellular indexing of transcriptomes and epitopes by sequencing, which revealed a distinct transcriptomic signature of this population. F4/80+MHCII+ MDSCs displayed gene signatures resembling G-MDSCs, neutrophils, and monocytes but had significantly increased expression of pathways involved in cytokine response/production, inflammatory cell death, and mononuclear cell differentiation. To determine whether F4/80+MHCII+ MDSCs represented an alternate phenotypic state of G-MDSCs, Ly6G+Ly6C+F4/80-MHCII- G-MDSCs from CD45.1 mice were adoptively transferred into CD45.2 recipients using a mouse model of PJI. A small percentage of transferred G-MDSCs acquired F4/80 and MHCII expression in vivo, suggesting some degree of plasticity in this population. Collectively, these results demonstrate a previously unappreciated phenotype of F4/80+MHCII+ MDSCs during PJI, revealing that a granulocytic-to-monocytic transition can occur during biofilm infection.

Transcriptomic and Proteomic Characterization of the Immune Response to Elective Spinal Reconstructive Surgery: Impact of Aging and Comparison with Traumatic Injury Response.

Major surgery triggers trauma-like stress responses linked to age, surgery duration, and blood loss, resembling polytrauma. This similarity suggests elective surgery as a surrogate model for studying polytrauma immune responses. We investigated stress responses across age groups and compared them with those of polytrauma patients. Patients undergoing major spinal reconstruction surgery were divided into older (age >65 years, n = 5) and young (age 18 to 39 years, n = 6) groups. A comparison group consisted of matched trauma patients (n = 8). Blood samples were collected before, during, and after surgery. Bone marrow mononuclear cells and peripheral blood mononuclear cells were analyzed using cellular indexing of transcriptomes and epitopes sequencing or single-cell RNA sequencing. Plasma was subjected to dual-platform proteomic analysis (SomaLogic and O-link). Response to polytrauma was highest within 4 hours. By comparison, the response to surgery was highest at 24 hours. Both insults triggered significant changes in cluster of differentiation 14 monocytes, with increased inflammation and lower major histocompatibility complex-class 2 expression. Older patient's cluster of differentiation 14 monocytes displayed higher inflammation and less major histocompatibility complex-class 2 suppression; a trend was also seen in bone marrow mononuclear cells. Although natural killer cells were markedly activated after polytrauma, they were suppressed after surgery, especially in older patients. In plasma, innate immunity proteins dominated at 24 hours, shifting to adaptive immunity proteins by 6 weeks with heightened inflammation in older patients. Senescence-associated secretory phenotype proteins were higher in older patients at baseline and further elevated during and after surgery. Although both major surgery and polytrauma initiate immune and stress responses, substantial differences exist in timing and cellular profiles, suggesting major elective surgery is not a suitable surrogate for the polytrauma response. Nonetheless, distinct responses in young vs older patients highlight the utility of elective spinal in studying patient-specific factors affecting outcomes after major elective surgery.

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.

Precision Lineage Deconvolution in Mixed Phenotype Acute Leukemia Using Cite-Seq Derived Hematopoietic Stages Identifies Lineage Dynamics Associated with Treatment Response

Introduction: Mixed phenotype Acute Leukemia (MPAL) is challenging due to ambiguous biology and lack of formal therapeutic guidelines. Although MPAL itself is rare, mixed phenotype lineage expression in secondary AML (sAML-MP) occurs with greater frequency and is associated with poor clinical outcomes. Multimodal single cell sequencing with cellular indexing of transcriptomes and epitopes (CITE-seq) offers more precise lineage characterization and assessment of lineage dynamics. Here we demonstrate a novel, unbiased approach to quantitative multistage hematopoietic lineage assessment in MPAL and sAML-MP using CITE-seq data. In addition, we demonstrate that this approach can be translated from single cell to bulk analysis. Methods: Sample preparation: Four MPAL and 6 sAML-MP samples displaying T-cell and myeloid subsets were identified and flow sorted into T and myeloid subgroups. RNA-seq was performed on each subset and analyzed using DESeq2. ComBat was used for batch correction when integrating with disparate data sources. Single cell lineage deconvolution: CITE-seq samples were processed using Seurat. A single cell deconvolution library was constructed using previously published hematopoietic stage clustering and labeling, and applied to CITE-seq data from five MPAL samples in the same dataset. Deconvolution algorithm parameters were optimized through a bootstrapping approach on in-silico sample mixtures. Bulk and translated lineage deconvolution: Bulk lineage deconvolution was performed using a published library of 13 stages of hematopoiesis derived from healthy donors.This was applied to bulk RNA-seq data from a cohort of published MPAL samples as well as the 6 sAML-MP and 4 MPAL samples described above. A pseudobulk was created for each CITE-seq lineage stage. Bulk lineage deconvolution was then re-run on the MPAL and sAML-MP samples using this library. Clinical Outcomes: A Wilcoxon signed-rank test was used to analyze differences among deconvolution-derived patient clusters, including response to induction therapy, specific gene mutations, and likelihood of transplant. Results: Lineage evolution with treatment: Deconvolution was applied on 17,848 MPAL cells and 35,038 control PBMC/BMMC cells. Antibody Derived Tag (ADT) features were consistent with lineage deconvolution stage assignments. CD38 was negatively correlated with HSC (-0.34, p &amp;lt; 0.001) and LMPP (-0.18, p &amp;lt; 0.001) while CD34 was positively correlated with both HSC (0.11, p &amp;lt; 0.001) and LMPP (0.27, p &amp;lt; 0.001) stages. MPAL 5 in particular was assessed at diagnosis and at two later time points. Unsupervised hierarchical clustering of all timepoints resulted in 10 clusters ( Figure A). The diagnostic time point was largely contained in clusters 4 (LMPP character, N = 1545 cells, 37.13%) and 9 (HSC character, N = 756 cells, 18.16%). The majority of both relapse timepoints were within cluster 4, with 76.77% (N=357 cells) of T1 and 73.92% (N=1100 cells) of T2 located in it. Bulk Unsupervised Analysis: Unsupervised analysis of sorted MPAL (N = 8), sorted AML-MP (N = 12), and unsorted MPAL (N = 24) bulk samples resulted in six distinct clusters, each with a distinct lineage signature ( Figure B). Some were enriched for more differentiated stages such as monocyte or GMP (clusters 6, 5), while others were enriched earlier stages such as LMPP (cluster 2). Secondary AML-MP samples were notably enriched in cluster 4. We found significant associations between lineage clusters and mutations in RUNX1 (p &amp;lt; 0.01), FLT3 (p = 0.004). Clinical Outcomes: Bulk RNA deconvolution cluster was significantly associated with complete remission (p = 0.0009). On average, individuals with an incomplete/no response to induction chemotherapy had a higher NK signature (p = 0.001), while individuals assigned to transplant had decreased LMPP character (p = 0.0495). Among the single-cell stages, decreased CLP 2 levels were associated with a poor response (p = 0.0224) while CMP / LMPP (p = 0.040) and CD4 N2 (p = 0.022) levels were decreased in patients who were able to undergo HSCT. Conclusions: Precise identification of lineage signatures in mixed phenotype leukemias shows promise in identifying clinically meaningful biological subsets of these diseases. Prospective analysis of lineage-derived biomarkers should be performed to undertake identification of formal risk stratification and treatment schemas.

Single Cell Analysis Identifies Expression of PD-L2 on Human Bone Marrow Hematopoietic Stem Cells

The identification of specific and reliable markers for human hematopoietic stem cells (HSCs) is crucial to enable their prospective isolation allowing the investigation of unique physiological HSC functions within the blood cell hierarchy. Despite significant advancements in identification of surface markers for human bone marrow HSC isolation, the enriched populations remain heterogeneous. Here we applied single cell CITE-Seq (cellular indexing of transcriptomes and epitopes) of FACS-enriched hematopoietic stem and progenitor cells (HSPCs) to decipher differentiation hierarchies and to identify markers of most immature populations. We FACS-isolated over 60.000 high-quality human bone marrow HSPCs by sorting CD34 + and CD34 +CD38 - cells from 15 different healthy donors. These were subjected to scCITE-Seq using the BD Rhapsody system with a targeted gene panel comprising 600 most relevant genes of early HSC differentiation and 50 antibodies. To validate differentially expressed surface marker genes, we performed extensive in vitro and in vivo studies of FACS-purified subpopulations of human bone marrow and mobilized peripheral blood samples (mPB) from healthy donors. Our multi-omics based single HSPC profiling indicated Programmed death ligand 2 (PD-L2 / CD273) as a surface marker expressed on the most immature HSCs within the hematopoietic compartment defined as CD34 +CD38 -CD45RA -CD90 +. Prospective FACS-based isolation allowed a deeper understanding of transcriptomic differences depending on PD-L2 positivity. A transcriptomic principal component analysis of CD273 hi and CD273 low expressing HSCs isolated pairwise from four healthy donors proved distinct clustering accompanied by discriminative molecular signatures. Stem cell genes as HOPX, MLLT3 and MPL were enriched in CD273 hi HSCs, and CDK6 was downregulated, further validating our single-cell analysis. The differential expression of stem cell genes was validated at protein level. Single cell tracking using video-microscopy confirmed a delayed entry into cell cycle in CD273 hi HSCs, suggesting deeper quiescence of these cells. Furthermore, their mitochondrial potential was reduced. In vivo transplantations of both populations in immunocompromised NSGs are ongoing and will reveal the long-term repopulation potential of CD273 hi HSCs. Multiple in vitro differentiation assays demonstrated CD273 positivity to be associated with an increased stem cell phenotype with higher expression of EPCR (CD201) and CD90 in liquid culture, a delayed differentiation and high capability of generating multipotent colonies. Interestingly, CD273 surface expression was strongly upregulated in HSCs upon in vitro culture. Since PD-L2 is known as a ligand for the Programmed death receptor 1 (PD1), we investigated the immunomodulating function of PD-L2 expressing HSPCs by establishing an allogeneic lymphocyte reaction assay. In addition to their stem cell-like characteristics, PD-L2 expressing HSPCs mediated diminished T cell proliferation in allogeneic co-culture settings. Furthermore, cytokine profiling of co-culture supernatants indicated increased secretion of anti-inflammatory cytokines (IL-10 and TGF-β1), accompanied by reduced secretion of pro-inflammatory cytokines (IL-17A) and a diminished expression of the pro-inflammatory receptor CD69 on CD8 + T cells. These findings showed that CD34 + cells isolated from mobilized peripheral blood of healthy donors possess immunomodulating capabilities, and that PD-L2 expression indicates an immunocompromising HSC phenotype. In conclusion, this study demonstrates differential surface expression of PD-L2 within the most immature human HSCs. PD-L2 expression is associated with HSC quiescence and delayed in vitro differentiation. Furthermore, these results provide valuable insights into the immunomodulating features of HSCs expressing PD-L2.

Single-Cell-Multiomics Demonstrates Molecular Efficacy of a Clinical Lentiviral Vector for Gene Therapy of RPS19-Deficient Diamond-Blackfan Anemia

It has previously been demonstrated that lentiviral vector-mediated gene transfer of human codon-optimized RPS19 to hematopoietic stem cells by the gene therapy vector (CLIN-LV-EFS-coRPS19-PRE*) corrects the anemic phenotype of RPS19-deficient mice, supporting development of this vector for clinical gene therapy of RPS19-deficient Diamond-Blackfan Anemia (DBA). In this study, we evaluate the molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* in CD34+ cells from RPS19-deficient DBA patients. CD34+ cells from two healthy donors and three DBA patients with confirmed heterozygous mutations in RPS19 were transduced using GMP-like reagents according to a protocol developed for clinical use. To evaluate the therapeutic effect of RPS19-gene transfer, transduced (GT) and untransduced (Mock) CD34+ cells were cultured under conditions supporting erythroid and myeloid progenitor proliferation and differentiation. In order to obtain a comprehensive molecular characterization of the therapeutic mechanisms, cells from day 9 of culture were subjected to CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) analysis, a single-cell-multiomics method combining the advantages of FACS and scRNA-Seq for simultaneously analyzing transcriptomes alongside cell surface protein abundance at the single cell level. In GT cells, transduced (coRPS19-positive) cells were identified based on transgene expression. At day 9, 84-91% of erythroid progenitors in the GT-DBA samples expressed the coRPS19-transgene, while the frequency in myeloid progenitors was 29-56%. To reveal the molecular therapeutic effect of GT, we compared coRPS19-positive cells to Mock-treated cells from the same individuals. Expression of coRPS19 in DBA erythroid progenitor cells led to a significant induction of genes associated with terminal erythropoiesis (HEMGN, HBB, AHSP, EPB42 and GYPA) and down-regulation of genes associated with apoptosis and p53 activation (BAX, MDM2, ZMAT3 and MIR34AHG). GT also induced up-regulation of the large non-coding RNA LINC01133 and down-regulation of XACT. Interestingly, the two most-significantly changed genes in coRPS19-positive erythroid cells in all DBA samples were RPL22L1 and CD70. RPL22L1 is an RNA-binding component of the 60S ribosomal subunit that regulates pre-mRNA splicing but is not required for global cap-dependent translation. CD70 mRNA and protein exclusively expressed in erythroid progenitors in Mock-treated DBA samples and down-regulated in coRPS19-positive cells. CD70 is thus a new potential marker of DBA erythroid progenitor cells with a possible role in DBA pathogenesis. Myeloid progenitor DBA cells primarily responded to GT by up-regulation of ribosomal protein genes, suggesting RPS19-deficiency in myeloid progenitors leads to reduced ribosome biogenesis without the nucleolar stress observed in erythroid cells. To summarize, GT-induced changes in gene and protein expression agree with restoration of healthy ribosome biogenesis and elimination of nucleolar stress-induced p53 activation in erythroid progenitor cells responsible for the DBA phenotype, demonstrating molecular efficacy of CLIN-LV-EFS-coRPS19-PRE* supporting development for clinical gene therapy. In addition, the GT-induced reversal of RPS19-deficiency reveals several genes with potential relevance in DBA diagnostics and pathogenesis, such as RPL22L1 and CD70 for further investigation.