Single-cell Immune Profiling Research Articles

Single-cell profiling reveals pathogenic role and differentiation trajectory of granzyme K+CD8+ T cells in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune inflammatory disease mainly defined by T cell-dominated destruction of exocrine glands. Currently, CD8+ T cells are thought to be involved in the pathogenesis of pSS. However, the single-cell immune profiling of pSS and molecular signatures of pathogenic CD8+ T cells have not been well elucidated. Our multiomics investigation showed that both T cells and B cells, especially CD8+ T cells, were undergoing significant clonal expansion in pSS patients. TCR clonality analysis revealed that peripheral blood granzyme K+ (GZMK+) CXCR6+CD8+ T cells had higher a proportion of clones shared with CD69+CD103-CD8+ tissue-resident memory T (Trm) cells in labial glands in pSS. CD69+CD103-CD8+ Trm cells featured by high expression of GZMK were more active and cytotoxic in pSS compared with their CD103+ counterparts. Peripheral blood GZMK+CXCR6+CD8+ T cells with higher CD122 expression were increased and harbored a gene signature similar to Trm cells in pSS. Consistently, IL-15 was significantly elevated in pSS plasma and showed the capacity to promote differentiation of CD8+ T cells into GZMK+CXCR6+CD8+ T cells in a STAT5-dependent manner. In summary, we depicted the immune profile of pSS and further conducted comprehensive bioinformatics analysis and in vitro experimental investigations to characterize the pathogenic role and differentiation trajectory of CD8+ Trm cells in pSS.

Single-cell immune profiling reveals immune responses in oral lichen planus.

Oral lichen planus (OLP) is a common chronic inflammatory disorder of the oral mucosa with an unclear etiology. Several types of immune cells are involved in the pathogenesis of OLP. We used single-cell RNA sequencing and immune repertoire sequencing to characterize the mucosal immune microenvironment of OLP. The presence of tissue-resident memory CD8+ T cells are validated by multiplex immunofluorescence. We generated a transcriptome atlas from four OLP biopsy samples and their paired peripheral blood mononuclear cells (PBMCs), and compared them with two healthy tissues and three healthy PBMCs samples. Our analysis revealed activated tissue-resident memory CD8+ T cells in OLP tissues. T cell receptor repertoires displayed apperant clonal expansion and preferrential gene pairing in OLP patients. Additionally, obvious BCR clonal expansion was observed in OLP lesions. Plasmacytoid dendritic cells, a subtype that can promote dendritic cell maturation and enhance lymphocyte cytotoxicity, were identified in OLP. Conventional dendritic cells and macrophages are also found to exhibit pro-inflammatory activity in OLP. Cell-cell communication analysis reveals that fibroblasts might promote the recruitment and extravasation of immune cells into connective tissue. Our study provides insights into the immune ecosystem of OLP, serving as a valuable resource for precision diagnosis and therapy of OLP.

Abstract 5142: 50-parameter flow cytometry by CyTOF empowers comprehensive single-cell immune profiling of pulmonary immunosenescence in aged mice

Abstract High-parameter flow cytometry is essential for human and mouse studies to discover novel immunological mechanisms of cancer, infections, and immunosenescence. It plays an increasingly important role in cancer research to ensure clinical therapeutic success but is limited by the large amount of cell samples needed for staining controls. CyTOF® technology has transformed flow cytometry by enabling 50-plus-marker analysis per tube of sample, with easy panel design and no need for single-stained or autofluorescence controls. Flow cytometry by CyTOF provides an efficient and unbiased approach to discovering novel subsets and unique functional states of immune cells, maximizing insights from precious samples. Immunosenescence perturbs lung cancer onset and development, yet the mechanisms remain largely unknown. To study the pulmonary immune populations in aged (75 weeks old) and young adult (6-8 weeks old) mice, we built a 50-parameter panel (2 for single live-cell identification, 6 for live-cell sample barcoding, and 42 for immune profiling). A core panel of 32 antibodies was selected from Standard BioTools™ catalogs to detect key cell lineage and functional surface markers. A complementary panel with 10 Maxpar® antibodies was added to further study functional cell states. Maxpar Pathsetter™ software was used to create an automated analytical model for high-dimensional analysis. The 50-parameter panel successfully identified over 30 lymphoid and myeloid cell subsets including but not limited to T cells, B cells, NK cells, alveolar macrophages (AMs), dendritic cells, and neutrophils. The panel enabled high-fidelity detection of over 15 functional markers mediating proliferation, activation, inhibition, migration, tissue residence, and cellular metabolism. Automated in-depth analysis by Maxpar Pathsetter efficiently identified many aging-associated alterations in cell frequencies and functional states such as the enrichment of PD-1+ T cells and CD27- γδ T cells that could potentially perturb anti-tumor immunity. Moreover, CyTOF technology is uniquely advanced in characterizing autofluorescent cells such as AMs. A pro-inflammatory state (higher expression of MHC-II, CD80, and PD-L1) was specifically defined for AMs in aged lungs. This study demonstrates comprehensive single-cell immune profiling of mouse tissues with the products and solutions provided by Standard BioTools. Both the 50-parameter panel and Maxpar Pathsetter analytical model can be customized for deep characterization of specific immune populations according to the requirements of various cancer studies in mice. By utilizing end-to-end solutions offered by Standard BioTools, flow cytometry by CyTOF can significantly facilitate the mechanistic studies of mouse models to expand the understanding of human cancers and accelerate therapeutic development. Citation Format: Wenxi Xu, Stephen Li, Alexandre Bouzekri, Lauren Tracey, Christina Loh. 50-parameter flow cytometry by CyTOF empowers comprehensive single-cell immune profiling of pulmonary immunosenescence in aged mice. [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 5142.

Single-cell immune profiling reveals thymus-seeding populations, T cell commitment, and multilineage development in the human thymus.

T cell development in the mouse thymus has been studied extensively, but less is known regarding T cell development in the human thymus. We used a combination of single-cell techniques and functional assays to perform deep immune profiling of human T cell development, focusing on the initial stages of prelineage commitment. We identified three thymus-seeding progenitor populations that also have counterparts in the bone marrow. In addition, we found that the human thymus physiologically supports the development of monocytes, dendritic cells, and NK cells, as well as limited development of B cells. These results are an important step toward monitoring and guiding regenerative therapies in patients after hematopoietic stem cell transplantation.

CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA). Methods We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel. Results We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson's diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion. Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 - Figure 1 shows an example patient). To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease. To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001). Conclusions Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Molecular Mechanisms Governing CAR-T Cell Response in MM Patients Using Single Cell Transcriptomics

Background: CAR-T therapies have revolutionized cancer immunotherapy, especially for hematological malignancies, representing a promising advanced therapy for relapsed/refractory (R/R) multiple myeloma (MM) patients. Nevertheless, despite the high remission rates observed with BCMA CAR-T cells, not every patient achieves a complete response, and a significant number of patients still relapse. Application of single-cell technologies to study CAR-T cells has helped to shed light on important aspects of their expansion and evolution after administration. Methodology: CAR-T cells were FACS-isolated from infusion products (IP) as well as from bone marrow (BM) and peripheral blood (PB) from three MM patients (HCB08, IBS08 and HVA02), enrolled in the academic clinical trial CARTBCMA-HCB-01 (NCT04309981) assessing ARI0002h, an autologous CAR T-cell product targeting BCMA1, at different times after cell infusion (1 and 3 months). Single-cell analysis was performed using the Chromium Single-Cell Immune Profiling solution from 10X Genomics that allows simultaneous analysis of gene expression and paired T-cell receptors. Gene Regulatory Network (GRN) analysis was performed using SimiC, a novel computational method developed by our group, that infers regulatory dissimilarities2. Phenotypic analyses were performed by multidimensional flow cytometry. Results: scRNAseq analysis revealed that although CAR-T cells from IP presented similar phenotype, with CD4+ and CD8+ memory cells, and a high number of proliferative cells, CAR-T cells remaining after cell infusion were mainly resting CD8+ cells, with an effector/effector-memory phenotype. In addition, cell type distribution varied among patients, with a population of terminally differentiated effector cells in HCB08 patient, that presented a partial response and an early relapse, that increased from month 1 to 3 and presented a more exhausted phenotype. In contrast, IBS08 and HVA02 (complete responders) presented CD8+ cells in transition to central memory phenotype and a relatively constant pool of effector memory CD8+ CAR-T cells, respectively. Interestingly, CAR-T cells infiltrating BM presented increased expression of cytotoxic (GZMA, PRF1) and exhaustion (LAG3, HAVCR2, TIGIT) markers, compared to their PB counterparts. Functional characterization showed enrichment of T cell activation and cell-cell adhesion pathways in CAR-T cells from BM. GRN analysis at single-cell level with SimiC showed several regulons, such as IRF3 and PRDM1, presenting increased activity in BM. IRF3, a key transcriptional regulator of type I interferon response, was associated to DUSP2, IL32 and CCL5, genes associated to lymphocyte activation and inflammation. PRDM1, with crucial role in maintaining effector T cells homeostasis, was correlated with CST7, PRF1, MAP4K1 and CYTOR, genes associated with effector function and proliferation. All these data suggest that these regulons would be modulating the increased activation and inflammatory response of CAR-T cells observed in the BM. Further analysis using single-cell TCRseq allowed the identification of a hyperexpanded CAR-T cell clone in the BM of HCB08 patient (with an early relapse). Deeper characterization showed that this clone had higher expression of cytotoxicity (PRF1, GZMB) and activation markers (HLA-DRA, TNFRSF9). Strikingly, we also found increased expression of IL10, an immunosuppressor cytokine. Further analysis of this clone with SimiC showed increased activity of regulons from TFs related to exhausted CD8+ T cells (CREM, BHLHE40), meanwhile regulons from TFs associated to naïve and memory T cells (FOXP1, KLF2) had reduced activity. Interestingly, PRDM1, that is implicated in IL10 production in Treg, also presented increased activity in this clone. These data would suggest mechanistic insights regulating the phenotype of this hyperexpanded clone, that could represent a mechanism of therapeutic failure and/or early relapse. Conclusions: Overall, our data show that multiomic analysis are useful tools to characterize CAR-T cells and their dynamics after infusion, that presented different characteristics regarding their location (PB vs BM) and a heterogeneous behavior within the patients. Moreover, GRN analysis at single cell level offers an important tool to understand mechanisms modulating CAR-T cell response and to identify possible mechanisms of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

T cell receptor convergence is an indicator of antigen-specific T cell response in cancer immunotherapies.

T cells are potent at eliminating pathogens and playing a crucial role in the adaptive immune response. T cell receptor (TCR) convergence describes T cells that share identical TCRs with the same amino acid sequences but have different DNA sequences due to codon degeneracy. We conducted a systematic investigation of TCR convergence using single-cell immune profiling and bulk TCRβ-sequence (TCR-seq) data obtained from both mouse and human samples and uncovered a strong link between antigen-specificity and convergence. This association was stronger than T cell expansion, a putative indicator of antigen-specific T cells. By using flow-sorted tetramer+ single T cell data, we discovered that convergent T cells were enriched for a neoantigen-specific CD8+ effector phenotype in the tumor microenvironment. Moreover, TCR convergence demonstrated better prediction accuracy for immunotherapy response than the existing TCR repertoire indexes. In conclusion, convergent T cells are likely to be antigen-specific and might be a novel prognostic biomarker for anti-cancer immunotherapy.

Single Cell Immune Profiling Reveals Pro-Inflammatory Mechanisms Linked to Dysbiosis and Cardiovascular Disease in Children With CKD

Single Cell Immune Profiling Reveals Pro-Inflammatory Mechanisms Linked to Dysbiosis and Cardiovascular Disease in Children With CKD

Cellular basis of enhanced humoral immunity to SARS-CoV-2 upon homologous or heterologous booster vaccination analyzed by single-cell immune profiling

SARS-CoV-2 vaccine booster dose can induce a robust humoral immune response, however, its cellular mechanisms remain elusive. Here, we investigated the durability of antibody responses and single-cell immune profiles following booster dose immunization, longitudinally over 6 months, in recipients of a homologous BBIBP-CorV/BBIBP-CorV or a heterologous BBIBP-CorV/ZF2001 regimen. The production of neutralizing antibodies was dramatically enhanced by both booster regimens, and the antibodies could last at least six months. The heterologous booster induced a faster and more robust plasmablast response, characterized by activation of plasma cells than the homologous booster. The response was attributed to recall of memory B cells and the de novo activation of B cells. Expanded B cell clones upon booster dose vaccination could persist for months, and their B cell receptors displayed accumulated mutations. The production of antibody was positively correlated with antigen presentation by conventional dendritic cells (cDCs), which provides support for B cell maturation through activation and development of follicular helper T (Tfh) cells. The proper activation of cDC/Tfh/B cells was likely fueled by active energy metabolism, and glutaminolysis might also play a general role in promoting humoral immunity. Our study unveils the cellular mechanisms of booster-induced memory/adaptive humoral immunity and suggests potential strategies to optimize vaccine efficacy and durability in future iterations.

425.3: Single-Cell Immune Profiling of Human Intestinal Allografts Reveals Differential Phenotypes of Alloreactive T-Cell Clones in Quiescent vs Chronically Rejecting Allografts

Introduction: The success of intestinal transplantation (ITx) is limited by high rejection rates. We have demonstrated that host-vs-graft (HvG) alloreactive T-cell clones are enriched in intestinal allografts during early rejection and persist despite rejection resolution. Recipient T cells in the mucosa eventually take on resident memory T cell (TRM) features, possibly posing a constant threat of late rejection. Methods: We integrated clonotype, alloreactivity and gene expression profiles of FACS-sorted allograft recipient T cells to assess the functional phenotype of donor-reactive mucosal T cells in association with graft outcomes using the 10x single cell RNA sequencing platform and our method for identifying donor-reactive TCRs from pre- and post-Tx mixed lymphocyte reactions (MLRs) (Fig.1A). Results: Recipient mucosal T cells from 6 quiescent and 5 chronically rejecting allograft specimens and a non-transplant control intestine shared at least five transcriptionally-defined clusters (Fig.1B-C): multifunctional (IL17A+, IL22+, TNF+) TRMs (CD69+, ITGAE+, CXCR6+); cytotoxic γδ and CD8 αβ T cells with mixed effector T cell (Teff) and TRM features (CD69dim, RUNX3+, TBX21+, GZMB+); follicular helper T cells (Tfh: CXCR5+, PDCD1+, Bcl6+); nonTRMs (CCR7+, KLF2+, S1PR1+); and regulatory T cells (FOXP3+, CTLA4+). Pre-Tx MLR-defined HvG clones were mainly detected in TRM, Teff/TRM and Tfh clusters in both quiescent and chronically rejecting allografts. Hyporesponsiveness of circulating recipient T cells to donor vs third-party antigens in post-Tx MLR indicated partial tolerance of circulating recipient T cells to donor antigens post-Tx (Fig.1D). There was a significantly decreased likelihood of detecting HvG clones defined by post- compared to pre-Tx MLR in late quiescent (but not chronically rejecting) allografts (Fig.1E). Quiescent allografts contained a significantly greater percentage of pre-Tx HvG clones that became tolerant in post-Tx MLR (tolerant HvG) compared to chronically rejecting allografts (Fig.2A-C). Tolerant HvG among quiescent allografts were dominated by TRM profiles. The fraction of tolerant HvG cells in TRM clusters was significantly higher in quiescent compared to rejecting allografts (Fig. 2D). Patients with quiescent allografts tended to show a higher level and longer persistence of tolerant HvG clones in ileum compared to PBMCs (Fig.2E). Cells classified as missing HvG (HvG in pre-Tx MLR and not detected in post-Tx MLR or circulation) and de novo H’vG (unmappable in pre-Tx MLR and unstimulated cells but HvG in post-Tx MLR) in chronically rejecting allografts showed significantly higher expression of genes related to cytotoxic Teff functions compared to those in quiescent allografts (Fig.2F). Conclusion: Single cell immune profiling reveals distinct contributions of pre-existing HvG-reactive T cells that potentially became tolerant in allografts with TRM transcriptional profiles in quiescent vs chronically rejecting allografts.

783TiP Phase I study of HFB301001, a novel OX40 agonist monoclonal antibody, in patients with solid tumors selected via Drug Intelligence Science (DIS)

OX40 agonist antibodies have shown promising preclinical activity but limited clinical success thus far, likely owing to a suboptimal pharmacological profile, inappropriate dosing regimen, and lack of a biomarker strategy for patient selection. HFB301001 is a novel human IgG1 agonist antibody that binds to a unique epitope on OX40 allowing for agonistic activity without competing with the endogenous OX40 ligand, inducing minimal OX40 downregulation upon co-stimulation of T cells. Also, HFB301001 can both enhance effector T cells and deplete regulatory T cells. It demonstrated more potent in vivo anti-tumor activity than a benchmark OX40 agonist, suggesting potentially superior patient benefit compared to first generation OX40 antibodies. HFB301001 is being evaluated in a first-in-human, open-label, multi-center, dose escalation and expansion study in adult patients with advanced solid tumors. It is hypothesized that high levels of OX40 expression associated with effector T cells or T regulatory cells in solid tumors may represent a tumor microenvironment more sensitive to OX40 agonism. Based on this, the following cancer indications have been prioritized using DIS (Drug Intelligence Science is a HiFiBiO Inc. trademark): soft tissue sarcoma (STS), uterine carcinosarcoma (UCS), renal cell carcinoma (RCC), head and neck squamous cell carcinoma (HNSCC), and hepatocellular carcinoma (HCC). The dose escalation portion of the study explores increasing doses in four cohorts of up to six patients, utilizing an mTPI-2 design to determine recommended dose(s) for expansion (RDE(s)). Once the RDE(s) is determined, expansion of up to 5 cohorts is planned to determine the recommended phase 2 dose (RP2D). The primary objective is to characterize safety and tolerability of HFB301001, and to determine RDE(s) and RP2D. Secondary objectives include pharmacokinetic parameters, preliminary evidence of anti-tumor efficacy, and pharmacodynamic evaluation in blood and tumor. Furthermore, a potential predictive biomarker signature derived based on the DIS™ single-cell immune profiling platform will be investigated. NCT05229601. HiFiBiO Inc.

Research progress on application of single-cell TCR/BCR sequencing technology to the tumor immune microenvironment, autoimmune diseases, and infectious diseases.

Single-cell omics is the profiling of individual cells through sequencing and other technologies including high-throughput analysis for single-cell resolution, cell classification, and identification as well as time series analyses. Unlike multicellular studies, single-cell omics overcomes the problem of cellular heterogeneity. It provides new methods and perspectives for in-depth analyses of the behavior and mechanism of individual cells in the cell population and their relationship with the body, and plays an important role in basic research and precision medicine.Single-cell sequencing technologies mainly include single-cell transcriptome sequencing, single-cell assay for transposase accessible chromatin with high-throughput sequencing, single-cell immune profiling (single-cell T-cell receptor [TCR]/B-cell receptor [BCR] sequencing), and single-cell transcriptomics. Single-cell TCR/BCR sequencing can be used to obtain a large amount of single-cell gene expression and immunomics data at one time, and combined with transcriptome sequencing and TCR/BCR diversity data, can resolve immune cell heterogeneity. This paper summarizes the progress in applying single-cell TCR/BCR sequencing technology to the tumor immune microenvironment, autoimmune diseases, infectious diseases, immunotherapy, and chronic inflammatory diseases, and discusses its shortcomings and prospects for future application.

Dynamics of monocyte-derived macrophage diversity in experimental myocardial infarction.

Macrophages have a critical and dual role in post-ischaemic cardiac repair, as they can foster both tissue healing and damage. Multiple subsets of tissue resident and monocyte-derived macrophages coexist in the infarcted heart, but their precise identity, temporal dynamics, and the mechanisms regulating their acquisition of discrete states are not fully understood. To address this, we used multi-modal single-cell immune profiling, combined with targeted cell depletion and macrophage fate mapping, to precisely map monocyte/macrophage transitions after experimental myocardial infarction. We performed single-cell transcriptomic and cell-surface marker profiling of circulating and cardiac immune cells in mice challenged with acute myocardial infarction, and integrated single-cell transcriptomes obtained before and at 1, 3, 5, 7, and 11 days after infarction. Using complementary strategies of CCR2+ monocyte depletion and fate mapping of tissue resident macrophages, we determined the origin of cardiac macrophage populations. The macrophage landscape of the infarcted heart was dominated by monocyte-derived cells comprising two pro-inflammatory populations defined as Isg15hi and MHCII+Il1b+, alongside non-inflammatory Trem2hi cells. Trem2hi macrophages were observed in the ischaemic area, but not in the remote viable myocardium, and comprised two subpopulations sequentially populating the heart defined as Trem2hiSpp1hi monocyte-to-macrophage intermediates, and fully differentiated Trem2hiGdf15hi macrophages. Cardiac Trem2hi macrophages showed similarities to 'lipid-associated macrophages' found in mouse models of metabolic diseases and were observed in the human heart, indicating conserved features of this macrophage state across diseases and species. Ischaemic injury induced a shift of circulating Ly6Chi monocytes towards a Chil3hi state with granulocyte-like features, but the acquisition of the Trem2hi macrophage signature occurred in the ischaemic tissue. In vitro, macrophages acquired features of the Trem2hi signature following apoptotic-cell efferocytosis. Our work provides a comprehensive map of monocyte/macrophage transitions in the ischaemic heart, constituting a valuable resource for further investigating how these cells may be harnessed and modulated to promote post-ischaemic heart repair.

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

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

Abstract 3392: Rapid, scalable isolation of human tumor nuclei for single cell genomics

Abstract Sample preparation remains a significant roadblock to the implementation and scaling of single cell technologies in clinical and translational research. Time consuming, custom dissociation protocols performed on fresh tissue by experts are often utilized to obtain high quality single cell data. However, these complex protocols result in low sample throughput and high variability driven by logistical challenges and user variability. Additionally, many samples that are of interest for clinical and translational studies arrive in research labs snap frozen, leaving nuclei extraction as the only viable path to generate single cell/nuclei data. To overcome these challenges, we present a scalable, easy to use, spin column-based nuclei isolation platform that generates single nuclei that are ready for input into downstream 10x Genomics single cell assays in under an hour. We processed eight unique human tumors in parallel (skin melanoma, breast, ovary, prostate, lung, kidney, colorectal, and pancreas). Starting from snap frozen tissue, we were able to generate single nuclei suspensions for all samples, in parallel, within 90 minutes. The resulting single nuclei were run through the 10x Genomics Single Cell Gene Expression, Single Cell Immune Profiling, and Single Cell ATAC workflows. Single cell gene expression data retained high data complexity across all tumor types tested. Expected cell type clusters, including tissue-specific tumor cells, surrounding/infiltrating immune cells and stromal cells were easily identified and profiled via gene expression analysis. Even fragile cell types often missing in single-cell data (e.g. mature adipocytes) were preserved and profiled in this experiment. Additionally, epigenetic changes as measured by Single Cell ATAC profiling were consistent with both general and tissue-specific epigenetics of each cancer type analyzed. In summary, this platform streamlines sample preparation for single cell studies from snap frozen tissue, enabling these samples to be used much more routinely and unlock biobanked sample cohorts. The findings presented here demonstrate that this fast and easy to use nuclei isolation system provides a consistent platform for generating high quality single nuclei data at scale for clinical, translational and basic research studies alike. Citation Format: Michael Gibbons, Alaina Puleo, Jill Herschleb, Sarah Taylor. Rapid, scalable isolation of human tumor nuclei for single cell genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3392.

Abstract 5782: Sample management for enabling successful single-cell RNAseq processing

Abstract Background: Single cell analysis allows the profiling of biomolecules on a cell-by-cell basis, revealing insights into how cellular heterogeneity impacts biological mechanisms. Drug discovery and development have greatly benefited from the comparative, longitudinal, and causal information single cell analysis provides, transforming our ability to understand disease. Many preclinical and clinical studies leveraging single cell resolution have already played a crucial role in target identification and biomarker discovery. From collection through analysis, proper sample management is a crucial and challenging aspect of large-scale clinical research studies and directly influences specimen quality and the ability to interrogate specific cell populations of interest. Methods: We sought to determine the effect of collection and storage conditions for PBMCs enriched from whole blood prior to downstream processing using our scRNAseq workflow. PBMCs were freshly isolated from whole blood collected in EDTA tubes or following storage at various timepoints and temperatures until PBMC isolation. Samples were then cryopreserved after the conclusion of each isolation stage and later processed to assess impact of cryopreservation. All PBMC samples were processed using the Chromium Single Cell Immune Profiling kit to compare gene expression and immune repertoire profiling across all storage conditions. Results: We found that gene expression data were highly correlated across all storage conditions tested. We also found that the clustering and annotation of PBMC libraries were comparable across all conditions tested, but certain subsets were more sensitive to these conditions, indicated by an elevation in cell stress and apoptosis markers. Conclusions: When planning future studies, it is imperative to consider collection and storage conditions in the workflow of processing for enabling high quality samples for single cell analysis, especially with regard to sensitive and rare cell types of interest. Citation Format: Priya J. Halvorsen, Caitlin W. Britton, Hiba Shaban, Fernando Torres, Douglas Wilson, Joseph Modarelli. Sample management for enabling successful single-cell RNAseq processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5782.

Abstract 4248: HFB200603, a novel anti-BTLA monoclonal antibody that provides therapeutic potential for immune escape and synergizes with anti-PD-1 treatment

Abstract BTLA is a co-inhibitory immune checkpoint molecule sharing sequence and structural homology with PD-1 and CTLA4. Ligation of HVEM with BTLA induces recruitment of SHP1 and SHP2, and triggers inhibition of T cell proliferation and cytokine production. In melanoma, HVEM is described to have a broader expression than PD-L1 and constituted a negative prognostic marker; in PD-L1 negative NSCLC, the expression of HVEM has been shown to contribute to immune escape. Therefore BTLA-HVEM axis could play an important role in the immune escape, thus BTLA blockade in combination with PD-1/PD-L1 blockade could represent an effective therapeutic option. Using our proprietary microfluidic-based single cell platform CelliGO™, we identified a series of anti-human BTLA antibodies that were characterized for their binding affinity, cross-reactivity, selectivity and functional activity. Amongst them, HFB200603 was identified as a single-digit nanomolar binder to human and cynomolgus BTLA, capable of reversing HVEM-mediated immune suppression in a BTLA-HVEM reporter system and in a primary CD4+ T cell proliferation assay. HFB200603 showed synergistic effect with anti-PD-1 to enhance IFN-γ production in an MLR assay, and demonstrated favorable developability and pharmacokinetic profiles. Profiling of tumor infiltrating lymphocytes demonstrated that BTLA+PD-1+ T cells are present in melanoma, NSCLC, and HCC. Blockade of the BTLA-HVEM interaction with HFB200603 alone or in combination with anti-PD-1 led to increases in IFN-γ, CXCL9, IP-10 and other proinflammatory cytokines in primary dissociated tumor cultures. Based on its favorable pharmacological activity and excellent developability, HFB200603 is currently being developed as a potential novel immunotherapy coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Juying Li, Qian Zhang, Bingqing Shen, Manan Shah, Mingjie Chen, Sharon Li, Ling Dong, Francisco Adrian, Liang Schweizer. HFB200603, a novel anti-BTLA monoclonal antibody that provides therapeutic potential for immune escape and synergizes with anti-PD-1 treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4248.

Abstract 2899: Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors

Abstract Regulatory T cells (Tregs) contribute to immunosuppression within the tumor microenvironment and have been associated with poor outcomes in a range of cancers. Targeted depletion of tumor-infiltrating Tregs (TITRs) is an attractive therapeutic strategy with the potential to enhance antitumor immunity in patients who do not respond to current treatments. However, such approaches have been limited to date by a lack of markers that are highly specific for TITRs. CCR8 is a chemokine G protein-coupled receptor (GPCR) that has recently been shown to be specifically expressed on TITRs as compared to Tregs in the periphery or other T cells within tumors. Here, we report the development and characterization of HFB101110, a humanized monoclonal antibody against CCR8 with potent and specific antibody-dependent cellular cytotoxicity (ADCC) activity. HFB101110 specifically recognizes an epitope on the N-terminus extracellular domain of CCR8 and does not recognize the closely related chemokine receptor CCR4. HFB101110 acts through a dual mechanism of action, by both depleting CCR8+ cells via ADCC and blocking binding of the CCL1 chemokine to its receptor CCR8. Blockade by HFB101110 inhibited calcium flux and chemotaxis induced by the interaction between CCL1 and CCR8. Furthermore, HFB101110 showed potent single agent anti-tumor activity associated with depletion of intratumoral Tregs in a human CCR8 knock-in mouse model. HFB101110 mediated specific ex vivo killing of Tregs from primary patient tumor samples both in the presence, and in some cases the absence, of allogeneic NK cells. HFB101110 showed favorable pharmacokinetic properties and a favorable developability profile, and was well-tolerated in both wild-type mice and cynomolgus monkeys. HFB101110 is currently being developed as a novel immunotherapy for the treatment of solid tumors coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Roshan M. Kumar, Julie Prigent, Hombline Poullain, Ayrin Kok, Carine George, Sami Ellouze, Yun-Yueh Lu, Rebecca Silver, Clarisse Monchecourt, Ross B. Fulton, Qian Zhang, Nicola Beltraminelli, Bernhard Moser, Francisco Adrian, Liang Schweizer. Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2899.

Phase I study of HFB200301, a first-in-class TNFR2 agonist monoclonal antibody in patients with solid tumors selected via Drug Intelligent Science (DIS).

TPS2670 Background: Tumor necrosis factor receptor-2 (TNFR2) is expressed on effector CD8+ T cells, CD4+ T cells, T regulatory cells, natural killer cells, and myeloid cells. Targeting TNFR2 is anticipated to yield effective anti-tumor immunity by stimulating T-cell and NK-cell activation and proliferation in the tumor microenvironment. HFB200301 is a first-in-class anti-TNFR2 agonistic monoclonal antibody that triggers both innate and adaptive immune stimulation by binding to a specific epitope on TNFR2. HFB200301 has demonstrated dose-dependent anti-tumor activity in human TNFR2 knock-in mice bearing MC38 and Hepa1-6 syngeneic tumors. Methods: HFB200301 is being evaluated in a first-in-human, open-label, multi-center, dose escalation and expansion study in adult patients with advanced solid tumors. A single-cell immune profiling platform, DIS, was deployed to identify unique tumor-infiltrating T cell signatures that could help optimize patient selection for HFB200301 treatment. It is hypothesized that the presence of an effector T cell subpopulation that express both TNFR2 and CD8A in solid tumors may represent a tumor microenvironment favorable to TNFR2 agonism. The following cancer indications have been identified based on the prevalence of a TNFR2 high/CD8 high signature: Epstein-Barr Virus positive (EBV+) gastric cancer, clear cell renal cell carcinoma (ccRCC), cutaneous melanoma, testicular germ cell tumor (TGCT), soft tissue sarcoma (STS), and PD-L1+ cancers: cervical cancer, pleural mesothelioma, lung adenocarcinoma, and head and neck squamous cell carcinoma (HNSCC). The escalation portion of the study explores increasing doses in cohorts of up to six patients, utilizing mTPI-2 design to determine recommended dose(s) for expansion (RDE(s)). Based on pharmacokinetic modeling to maximize HFB200301 activity, 60-minute intravenous infusions of HFB200301 are administered every 4 weeks. Once RDE(s) is determined, expansion into three indication-specific cohorts is planned to determine the recommended phase 2 dose (RP2D). Key eligibility criteria include histologically documented advanced or metastatic solid tumors in the above listed indications. Patient enrollment opened in February 2022 in the USA, with plans for additional clinical sites in Spain and China. The primary objective is to identify the RDE, characterize safety and tolerability of HFB200301, and determine RP2D. Secondary objectives include pharmacokinetic parameters, preliminary evidence of anti-tumor efficacy (e.g., ORR, DCR, DOR) and pharmacodynamic evaluation (e.g., T cell subsets) in the blood and in the tumor. Furthermore, a potential predictive biomarker signature derived based on the DIS single-cell immune profiling approach will be investigated retrospectively. Clinical trial information: NCT05238883.

Discovery of potently-neutralizing antibodies against SARS-CoV-2 using Barcode Enabled Antigen Mapping (BEAM)

Abstract Humans who have recovered from infectious disease possess a memory B cell pool that contains highly-potent antibodies against the cleared pathogenic agent. This pool is a valuable source of therapeutic antibodies but identifying these requires a method that can screen a large number of cells rapidly to identify promising candidates. We used Barcode Enabled Antigen Mapping (BEAM) to screen 100 million peripheral blood mononuclear cells (PBMCs) from a donor who had recovered from COVID-19. This method labels antigens of interest with unique reporter oligonucleotides and uses these labeled antigens to stain lymphocytes according to the binding specificity of their antigen receptors. Individual antigen-bound cells are then captured and analysed using the 10x Genomics Single Cell Immune Profiling Solution. This allows us to identify the antigen-specific cells while also generating transcriptomic profiles and natively paired, heavy and light chain full-length B-cell receptor sequences from each cell. We used BEAM to identify B cells that bound to the spike protein of SARS-CoV-2 and discovered 222 antibodies with high binding affinity confirmed by SPR. The majority of these antibodies bound to multiple variants of concern with some also recognising endemic coronaviruses. We then investigated the potential for these antibodies to neutralize live SARS-CoV-2 and confirmed that 55 exhibited potent neutralization activity. Finally, we performed epitope binning and discovered that the collection of antibodies bound to multiple different epitopes on the spike protein. Compared with hybridoma or other approaches, single cell methods have the potential to discover therapeutic antibodies more rapidly and with a higher diversity of leads.