Inflammasome-Primed Myeloid Cells Maintain a Pro-Tumor Microenvironment in Multiple Myeloma

Background: Multiple myeloma (MM) disease progression is influenced by signals from the bone marrow (BM) microenvironment. Recently, we showed that the MM BM is characterized by inflammatory mesenchymal stromal cells (iMSCs) that transcribe MM survival factors and are predicted to recruit proliferating myeloma cells via CCL2-CCR2 interactions (de Jong et al. Nat Immunol. 2021). iMSCs also transcribed high levels of chemokines that can bind to CXCR1 and 2. Myeloid cells are known to express CXCR1/2, and have been implicated in both pro- and anti-tumor responses in various malignancies. Therefore, we hypothesized that iMSCs attract and influence myeloid populations in the MM BM. Results: Using flow cytometry, we verified expression of CXCR1/2 on myeloid cell populations in the BM of 5 newly diagnosed MM (NDMM) patients. CD15 + neutrophils were the most dominant population expressing these receptors, as 22.4% (± 9.8%) of cells expressed CXCR2 alone, and 72.6% (± 8.0%) expressed both CXCR1 and CXCR2. CD14 + monocytes only expressed CXCR2 (86.9% ± 15.8%). Importantly, less than 1% of myeloma cells expressed these receptors (n = 17 NDMM). As these findings suggested neutrophils and monocytes as potential targets of iMSC-mediated chemotaxis, we set out to identify MM-associated alterations in this population by performing single cell RNA sequencing of the full neutrophilic and monocytic lineages (n = 5 NDMM and 2 controls). In line with our flow cytometric data, CXCR1 transcripts were absent in monocytes, while CXCR2 was transcribed by classical monocytes of both myeloma patients and controls. Interestingly, CXCR1 and CXCR2 transcription was increased in mature neutrophils of MM patients compared to controls. Additionally, both mature classical monocytes as well as mature neutrophils of MM patients had an activated transcriptome as defined by increased transcription of C3AR1, SLPI, and IL6R, the plasma cell supportive factor TNFSF13B (encoding BAFF), and the inflammatory cytokines IL1B and IL18. Transcription of IL1B and IL18 can be regulated by pattern-recognition receptors (PRRs) binding damage-associated molecular patterns (DAMPs) resulting from e.g. matrix breakdown. Transcription of PRRs as TLR1, 2 and 4 was increased in mature neutrophils and classical monocytes of MM patients compared to controls. Secretion of IL-1β and IL-18 relies on the cleavage of pro-forms of these cytokines by the inflammasome, a multiprotein complex that is assembled in response to alarmins. Transcription of inflammasome components PYCARD, NLRP3 and CASP1 was increased in mature neutrophils and classical monocytes of patients with MM. Additionally, protein levels of both IL-18 and IL-1β are increased in BM plasma from MM patients, implicating activated neutrophils and monocytes as a potential sources of these cytokines. Conclusion: In MM, mature neutrophils and classical monocytes are activated and might interact with iMSCs via CXCR1 and/or 2. Moreover, these myeloid cells are inflammasome-primed and are likely to be sources of the increased IL-1β levels in the MM BM. Therefore, myeloid cells and iMSCs may form a feed-forward loop in which myeloid cells contribute to a pro-MM environment by maintaining iMSC and by directly providing BAFF to tumor cells. Disclosures Broyl: Celgene/BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Application of AI-Based Analytical Approaches for Multiplex Immunophenotyping of SF3B1-Mutant Myelodysplastic Syndrome Subtype

Background: Somatic mutations in splicing factor 3B subunit 1 (SF3B1) occur in 25% of all myelodysplastic syndromes (MDS) cases and identify a condition characterized by ring sideroblasts, ineffective erythropoiesis, and indolent disease course in lower-risk (LR) MDS. The impact of SF3B1 mutations on erythroid dysregulation has become apparent but little is known about cellular immune phenotypes. Identification of patient characteristics downstream of the pathogenic mutations in SF3B1 may reveal immunological determinants of disease phenotype and response to therapeutic interventions (e.g. luspatercept). In the present study, we applied multiplex immunophenotyping technologies in combination with artificial intelligence (AI)-based analytical approaches to identify genotype-immunophenotype correlations that may affect disease course and clinical outcomes in the SF3B1-MDS subtype. Methods: We performed transcriptomic immune profiling on bone marrow (BM) mononuclear cells (MNCs) from 12 SF3B1-MUT and 10 SF3B1-WT LR-MDS patients using the NanoString nCounter PanCancer Immune Profiling Panel. Besides, viably frozen PBMC samples (8 SF3B1-MUT; 4 SF3B1-WT) were thawed and stained for CyTOF with a 35-marker MaxPar Immune Profiling Panel (Fluidigm) for deep immune profiling. CyTOF data were analyzed with an in-house developed pipeline (ImmunoCluster, Opzoomer et al. 2021) incorporating the FlowSOM algorithm for unsupervised clustering (K=70). We also retrieved flow cytometric data on fresh BM and peripheral blood (PB) samples acquired independently as part of the diagnostic work-up and re-analyzed them using the T-REX pipeline (Barone and Paul et al. 2020). Results: On the transcriptomic level, the SF3B1-MUT subtype showed a distinct inflammatory signature compared to SF3B1-WT (59 DE genes at FDR < 0.05, Fig. 1A & B). GO/KEGG pathway analysis indicated that downregulated genes in SF3B1-MUT patients are involved in antigen processing/presentation (HLA class II transcript, CD8a), regulation of proinflammatory interleukin-1 beta secretion (IL1B, NLRP3), and cellular response to interferon-gamma (HLA class II transcripts, IRF4/8, CCL5, CCL20). Genes that were upregulated in SF3B1-MUT BM MNCs are involved in cell cycle (CDK1, CCND3), proliferation/survival (AXL), iron homeostasis (TFRC), and erythroid differentiation (TAL1). Unbiased interrogation of cellular phenotypes in PB through FlowSOM analysis of CyTOF data revealed an increased abundance of a CD4 + T cell metacluster resembling a central memory (CM) phenotype in SF3B1-MUT compared to SF3B1-WT PBMCs (7.9 vs 4.5% of CD45 +). Independent automated T-REX-based analysis of clinical flow data supported this finding and further indicated expansion of a cluster resembling CM CD8 + T cells in SF3B1-MUT PB. Lower IL1B expression in SF3B1-MUT and the implication of the IL-1/IL-1RAP axis in the inflammatory leukemic niche (De Boer et al. 2020) prompted us to re-analyze clinical flow cytometric data on IL-1RAP expression using the T-REX pipeline. T-REX revealed differences in IL-1RAP-expressing clusters between SF3B1-MUT and SF3B1-WT BM samples (9 SF3B1-MUT; 9 SF3B1-WT). Specifically, we noticed a cluster of IL-1RAP + CD34 - cells (HLA-DR + CD123 +) present in SF3B1-WT but greatly contracted in SF3B1-MUT MDS (Fig. 1C & D). Our data suggest that proinflammatory signaling via the IL-1/IL-1RAP axis is reduced in SF3B1-MUT BM. Aberrant marker expression on SF3B1-MUT monocytes has been reported (Duetz et al. 2020). T-REX analysis of BM CD14 + cells revealed distinct monocyte clusters with differential expression of CD123, CD11b, and HLA-DR. Our initial analysis showed that SF3B1-MUT-specific clusters displayed lower expression of CD123 and CD11b, suggesting that mutations in SF3B1 directly or indirectly affect monocyte phenotypes and most likely function. Conclusions: We provide evidence that the SF3B1-MDS subtype is associated with a distinct inflammatory signature characterized by lower IL1B expression, changed IL-1RAP + clusters, distinct monocyte phenotypes, and preservation of T cell homeostasis. Further investigation of the immune signature in SF3B1-mutated MDS subtype may lead to using personalized immunotherapy strategies in the future. This work also exemplifies the potential of unsupervised strategies for interrogating cellular immune phenotypes in MDS subtypes within clinical flow cytometry datasets. Figure 1 Figure 1. Disclosures Harrison: BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sierra Oncology: Honoraria; Constellation Pharmaceuticals: Research Funding; AOP Orphan Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Promedior: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Galacteo: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Geron: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Keros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte Corporation: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Platzbecker: Celgene/BMS: Honoraria; Janssen: Honoraria; Novartis: Honoraria; AbbVie: Honoraria; Geron: Honoraria; Takeda: Honoraria. Kordasti: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Alexion: Honoraria; Beckman Coulter: Honoraria.

Integrative Study of Genotypic and Phenotypic Diversity in the Eurasian Orchid Genus Neotinea

Knowledge of population variation across species’ ranges is a prerequisite for correctly assessing the overall variability of any group of organisms and provides an invaluable basis for unraveling evolutionary history, optimizing taxonomy and devising effective conservation strategies. Here, we examine the genus Neotinea, which represents a relatively recently delimited monophyletic genus of orchids, for which a detailed study of its overall variability was lacking. We applied a suite of biosystematic methods, consisting of flow cytometry, multivariate and geometric morphometrics, and analysis of genomic SNP data, to identify phylogenetic lineages within the genus, to delineate phenotypic variation relevant to these lineages, and to identify potential cryptic taxa within lineages. We found clear differentiation into four major lineages corresponding to the groups usually recognized within the genus: Neotinea maculata as a distinct and separate taxon, the Neotinea lactea group comprising two Mediterranean taxa N. lactea and Neotinea conica, the Neotinea ustulata group comprising two phenologically distinct varieties, and the rather complex Neotinea tridentata group comprising two major lineages and various minor lineages of unclear taxonomic value. N. conica constitutes both a monophyletic group within N. lactea and a distinct phenotype within the genus and merits its proposed subspecies-level recognition. By contrast, the spring and summer flowering forms of N. ustulata (var. ustulata and var. aestivalis) were confirmed to be distinct only morphologically, not phylogenetically. The most complex pattern emerged in the N. tridentata group, which splits into two main clades, one containing lineages from the Balkans and eastern Mediterranean and the other consisting of plants from Central Europe and the central Mediterranean. These individual lineages differ in genome size and show moderate degrees of morphological divergence. The tetraploid Neotinea commutata is closely related to the N. tridentata group, but our evidence points to an auto- rather than an allopolyploid origin. Our broad methodological approach proved effective in recognizing cryptic lineages among the orchids, and we propose the joint analysis of flow cytometric data on genome size and endopolyploidy as a useful and beneficial marker for delineating orchid species with partial endoreplication.

TAS-Seq: a robust and sensitive amplification method for beads-based scRNA-seq

Single-cell RNA-sequencing (scRNA-seq) is valuable for analyzing cellular heterogeneity. Cell composition accuracy is critical for analyzing cell-cell interaction networks from scRNA-seq data. We developed terminator-assisted solid-phase cDNA amplification and sequencing (TAS-Seq), a scRNA-seq method relying on a terminator, terminal transferase, and nanowell/beads-based scRNA-seq platform that could acquire scRNA-seq data, is highly correlated with flow-cytometric data, has gene-detection sensitivity, and is more robust than widely-used methods.

POS0007 LOSS OF BALANCE BETWEEN PROTECTIVE AND PRO-INFLAMMATORY SYNOVIAL TISSUE T CELL POLYFUNCTIONALITY PREDATES CLINICAL ONSET OF RHEUMATOID ARTHRITIS

Background:Effective treatment of Rheumatoid arthritis (RA) patients is achievable within a short window of opportunity following diagnosis. T-cells are early drivers of synovial inflammation of RA, therefore, identification of pathogenic T-cell subsets at the synovial tissue of pre-RA, arthralgia subjects, would greatly improve our understanding of disease pathogenesis. Comparative analysis of healthy control, arthralgia subject and RA-patient derived synovial tissue T-cell responses will lead to the identification of pathogenic as well as protective cytokine milieu, thus enabling the identification of early therapeutic targets to help steer the immune response towards resolution.Objectives:Characterization of T-cell polyfunctionality in the periphery and synovial tissue of ’at-risk; subjects (Arthralgia) RA-patients and healthy controls (HC).Identification of specific, pathogenic, synovial tissue T-cell subsets.Methods:Synovial biopsies from RA, AR and HC were obtained by arthroscopic surgery followed by RNAseq analysis (Guo et al., PLoS One, 2018). Single cell synovial tissue cell suspensions from RA, AR and HC and paired PBMC were stimulated in vitro and polyfunctional synovial T-cell subsets examined by flow cytometric analysis, SPICE visualization and FlowSom clustering. Flow-Imaging, was utilised to confirm specific T-cell cluster identification. Fluorescent Lifetime Imaging Microscopy (FLIM) was used to visualise metabolic status of specific T-cell populations.Results:T-cell associated pro-inflammatory gene pathways were increased in RNAseq analysis of RA-patient and arthralgia subject compared to HC synovial tissue biopsies. Flow cytometric analysis of pro-inflammatory cytokine (TNF-α, IFN-γ, IL-2, GM-CSF, IL-17A, IL-22) production and SPICE analysis of ex vivo stimulated T-cells revealed marked polyfunctionality of arthralgia subject synovial T-cells, thus providing evidence for a dysregulated synovial T-cell response that pre-dates clinical onset of disease. Importantly, HC synovial tissue harbours a small, albeit surprisingly polyfunctional, CD4 T-cell population characterised by significantly increased IL-4 and GM-CSF cytokine production compared to arthralgia subject (P<0.001 and P=0.01) and RA-patient (P<0.001 and P=0.004) synovial tissue. However, not all polyfunctional T-cells are equal in their pathogenic potential. Therefore, in order to identify highly pathogenic synovial T-cells, cluster analysis of flow cytometric data using the unsupervised algorithm FlowSom was performed and led to the identification of specific T-cell clusters with unique polyfunctionality characteristics. Specifically a cluster of CD4+CD8+ double positive (DP) T-cells with high polyfunctionality scores was identified. Hybrid flow cytometry and imaging technique confirmed the co-expression of CD4 and CD8 by a synovial T-cell population. DP T-cells are enriched in RA-patient synovial fluid and synovial tissue and arthralgia subject synovial tissue, but are absent from HC synovial tissue. Importantly, DP T-cell synovial accumulation strongly (P=0.002) correlates with DAS28(CRP) of RA-patients. Initial studies utilising the novel, non-invasive FLIM technique for visualisation of cellular NAD, revealed that DP T-cells have a metabolic profile indicative of activated memory T-cells.Conclusion:These data highlight a key early loss of balance between protective and pathogenic synovial T-cell polyfunctionality and the emergence of specific, highly polyfunctional and pathogenic T-cell clusters in RA.Figure 1.Identification of highly polyfunctional and pro-inflammatory synovial DP T-cells. A. Cluster analysis of RA-patient synovial tissue T-cells (asterisks indicate DP T-cell clusters). B. Flow imaging of CD4+, CD8+ and DP synovial T-cells. C. SPICE flow cytometric data visualization of DP arthralgia subject and RA-patient synovial T-cells. D. Correlation between the frequency of RA-patient synovial DP T-cells and disease severity.Disclosure of Interests:Achilleas Floudas: None declared, Nuno Neto: None declared, Mary Canavan: None declared, Trudy McGarry Employee of: Novartis, Vinod Krishna Employee of: Janssen, Sunil Nagpal Employee of: Janssen, GSK, Michael Monaghan: None declared, Douglas Veale Speakers bureau: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Consultant of: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Grant/research support from: Janssen, Abbvie, Pfizer, UCB, Ursula Fearon Speakers bureau: Abbvie, Grant/research support from: Janssen, Abbvie, Pfizer, UCB.

Systemic and mucosal mobilization of granulocyte subsets during lentivirus infection

Granulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentivirus infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver, and whole blood from naive, and chronically SHIVsf162p3-infected RM were analyzed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils - CD45+CD66+CD49d+; neutrophils - CD45+CD66+CD14+; and basophils - CD45+CD123+FcRε+. Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcRγII) but did not express CD16 (FcRγIII). Additionally, constitutive expression of CD64 (FcRγI) on neutrophils and FcRε on basophils, indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naive whole blood ranged 25.4-81.5% neutrophils, 0.59-13.3% eosinophils and 0.059-1.8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils, and colorectal eosinophils were all observed in chronic lentivirus disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils, and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentivirus infection, most notably in the gastrointestinal mucosae where significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.

Grape Seed Procyanidins Inhibit the Growth of Breast Cancer MCF-7 Cells by Down-Regulating the EGFR/VEGF/MMP9 Pathway

Breast cancer is the most common invasive cancer in women and the second leading cause of cancer death in women. However, it is not clear about its effective treatments. As a potential anticancer agent, grape seed procyanidins (GSPs) have been shown to inhibit the proliferation of various cancer cells in vitro and in vivo. In this study, it was shown that GSPs significantly inhibit MCF-7 cell proliferation in a concentration/time-dependent manner. The flow cytometric data clearly demonstrated that GSPs cause cell cycle arrest in the G2/M phase, followed by cell apoptosis. Moreover, it also confirmed that growth inhibition mediated by treatment with GSPs is related to the induction of apoptosis due to p53 elevation, purportedly by inhibition of the epidermal growth factor receptor (EGFR)/vascular endothelial growth factor (VEGF)/matrix metalloproteinase 9 (MMP9) pathway. Taken together, these findings suggest that GSPs inhibit MCF-7 cells proliferation and induce cell apoptosis by suppressing EGFR/VEGF/MMP9 pathway.