scholarly journals Evaluation of the Functional Landscape of Systemic Immunity in Classical Hodgkin Using a Novel Single Cell Platform (Isolight)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3980-3980
Author(s):  
Catherine Diefenbach ◽  
Linda Lam ◽  
Bruce G. Raphael ◽  
Kenneth B. Hymes ◽  
Michael L. Grossbard ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cells ◽  
Research Funding ◽  
Treatment Time ◽  
Post Treatment ◽  
Cytokine Secretion ◽  
Systemic Immunity ◽  
Bayer Corporation ◽  
Cytokine Levels ◽  
The Impact

Background: Classical Hodgkin lymphoma (HL) has a unique histopathology, with rare malignant Hodgkin/Reed Sternberg (HRS) cells surrounded by a strong inflammatory cellular component in the tumor microenvironment (TME). Although extensive studies describe the interdependence of the HRS cells and the TME, the impact of HL on systemic immunity has not been well described. Here, we develop a new approach, employing a recently commercialized single cell cytokine secretion platform (IsoLight) to assess, precisely and comprehensively, the function of peripheral blood mononuclear cells (PBMCs) in HL patients. Methods: Cells were selected from 4 HL patients: 2 newly diagnosed who had a complete response (CR) to therapy standard first line therapy, and 2 relapsed patients who progressed on second line chemotherapy (PD). Cryopreserved PBMCs from a pre-treatment and post-treatment time point for each patient were thawed, rested overnight, stimulated with PMA/ionomycin and loaded into the IsoLight single cell cytokine secretion system. IsoLight captures single cells in microwells; as cytokines are secreted, they are bound by antibodies lining the microwell cover. Bound cytokines are then revealed by fluorescent secondary antibodies and photos are taken at various time points to assess fluorescence intensity, which corresponds to the relative amount of each cytokine secreted. Twenty thousand cells can be assayed per sample simultaneously. Results: The percentage of cytokine-secreting cells varied dramatically by donor (12%-48%), with monofunctional cells making only TNFa, MIP1b, or IL-15 dominating the functional landscape. Polyfunctional cells, capable of making three or more cytokines simultaneously represented only 0.1-7% of the cells in each sample, but there were more of these cells, and each secreted higher levels of cytokines, in individuals who responded to therapy with a CR. Responders also secreted higher levels of IL2, Perforin, IL4, IL12, MIP1a, and TNFb (p values ranging from 0.005 to 0.03), and lower levels of IL9 and IL22 (p=0.0028 and 0.021, respectively), compared to non-responders at diagnosis. Responders lost expression of IL4, IL7, and MIP1a over the course of treatment (pre- vs post-treatment, p=0.01 to 0.05), while non-responders gained cells that expressed IL4, IL5, IL10, IL17, and TNFb from diagnosis to end of treatment (p=0.001 to 0.05). Conclusion: This work represents an important methodological advance in immune monitoring for hematologic malignancies. Single cell cytokine secretion technology measures more cytokines simultaneously than flow cytometry, providing a sample-sparing and comprehensive overview of the functional landscape of immune cells in a patient. Moreover, the technology provides cell-by-cell information about cytokine secretion, unlike Luminex. Our work represents the first application of this technology to HL, which we use to define, for the first time, the particular combinations of 32 cytokines that can be secreted by individual immune cells. We also identify candidate cytokines whose frequency at diagnosis may predict treatment outcome, and reveal changes in cytokine levels over treatment time that may distinguish patients destined to relapse. Immunotherapy may impact PBMC function differently, this may partially explain the high efficacy of this therapy in the relapsed population. The impact of immunotherapy on cytokine levels is currently under investigation by our group in a larger study. Other important questions which are under investigation include the impact of prior chemotherapy on cytokine profiles in relapsed patients, and whether certain cytokines which increase during treatment may be a surrogage for tumor bulk in patients with PD. Cytokines elevated in patients with poor responses to treatment include IL9, IL10, IL17, and IL22, which may present attractive drug targets if validated in our larger ongoing follow-up study. Disclosures Diefenbach: Bristol-Myers Squibb: Consultancy, Research Funding; MEI: Research Funding; Denovo: Research Funding; Genentech: Consultancy, Research Funding; Incyte: Research Funding; LAM Therapeutics: Research Funding; Merck: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Trillium: Research Funding; Millenium/Takeda: Research Funding. Hymes:Celgene: Consultancy. Martin:Janssen: Consultancy; Sandoz: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Teneobio: Consultancy; I-MAB: Consultancy. Ruan:Celgene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie company: Research Funding; Juno: Consultancy; Kite: Consultancy. Leonard:Miltenyi: Consultancy; Akcea Therapeutics: Consultancy; Sandoz: Consultancy; AstraZeneca: Consultancy; Bayer Corporation: Consultancy; Epizyme, Inc: Consultancy; BeiGene: Consultancy; Miltenyi: Consultancy; ADC Therapeutics: Consultancy; Akcea Therapeutics: Consultancy; Sandoz: Consultancy; Celgene: Consultancy; Epizyme, Inc: Consultancy; Karyopharm Therapeutics: Consultancy; AstraZeneca: Consultancy; Bayer Corporation: Consultancy; Celgene: Consultancy; Sutro Biopharma: Consultancy; Merck: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Gilead: Consultancy; Karyopharm Therapeutics: Consultancy; Sutro Biopharma: Consultancy; Nordic Nanovector: Consultancy; ADC Therapeutics: Consultancy; MorphoSys: Consultancy; Gilead: Consultancy; Nordic Nanovector: Consultancy; BeiGene: Consultancy; Merck: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; MorphoSys: Consultancy.

Single Cell Profiling Reveals Unique CXCL13 Positive T Cell Subsets in the Tumor Microenvironment of Lymphocyte Rich Classic Hodgkin Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Elizabeth Chavez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Single Cell ◽  
Immune Cells ◽  
Research Funding ◽  
Immune Cell ◽  
Expression Patterns ◽  
The Other ◽  
Tfh Cells

Introduction: Classic Hodgkin lymphoma (CHL) features a unique crosstalk between malignant cells and different types of normal immune cells in the tumor-microenvironment (TME). On the basis of histomorphologic and immunophenotypic features of the malignant Hodgkin and Reed-Sternberg (HRS) cells and infiltrating immune cells, four histological subtypes of CHL are recognized: Nodular sclerosing (NS), Mixed cellularity, Lymphocyte-rich (LR) and Lymphocyte-depleted CHL. Recently, our group described the high abundance of various types of immunosuppressive CD4+ T cells including LAG3+ and/or CTLA4+ cells in the TME of CHL using single cell RNA sequencing (scRNAseq). However, the TME of LR-CHL has not been well characterized due to the rarity of the disease. In this study, we aimed at characterizing the immune cell profile of LR-CHL at single cell resolution. METHODS: We performed scRNAseq on cell suspensions collected from lymph nodes of 28 primary CHL patients, including 11 NS, 9 MC and 8 LR samples, with 5 reactive lymph nodes (RLN) serving as normal controls. We merged the expression data from all cells (CHL and RLN) and performed batch correction and normalization. We also performed single- and multi-color immunohistochemistry (IHC) on tissue microarray (TMA) slides from the same patients. In addition, an independent validation cohort of 31 pre-treatment LR-CHL samples assembled on a TMA, were also evaluated by IHC. Results: A total of 23 phenotypic cell clusters were identified using unsupervised clustering (PhenoGraph). We assigned each cluster to a cell type based on the expression of genes described in published transcriptome data of sorted immune cells and known canonical markers. While most immune cell phenotypes were present in all pathological subtypes, we observed a lower abundance of regulatory T cells (Tregs) in LR-CHL in comparison to the other CHL subtypes. Conversely, we found that B cells were enriched in LR-CHL when compared to the other subtypes and specifically, all four naïve B-cell clusters were quantitatively dominated by cells derived from the LR-CHL samples. T follicular helper (TFH) cells support antibody response and differentiation of B cells. Our data show the preferential enrichment of TFH in LR-CHL as compared to other CHL subtypes, but TFH cells were still less frequent compared to RLN. Of note, Chemokine C-X-C motif ligand 13 (CXCL13) was identified as the most up-regulated gene in LR compared to RLN. CXCL13, which is a ligand of C-X-C motif receptor 5 (CXCR5) is well known as a B-cell attractant via the CXCR5-CXCL13 axis. Analyzing co-expression patterns on the single cell level revealed that the majority of CXCL13+ T cells co-expressed PD-1 and ICOS, which is known as a universal TFH marker, but co-expression of CXCR5, another common TFH marker, was variable. Notably, classical TFH cells co-expressing CXCR5 and PD-1 were significantly enriched in RLN, whereas PD-1+ CXCL13+ CXCR5- CD4+ T cells were significantly enriched in LR-CHL. These co-expression patterns were validated using flow cytometry. Moreover, the expression of CXCR5 on naïve B cells in the TME was increased in LR-CHL compared to the other CHL subtypes We next sought to understand the spatial relationship between CXCL13+ T cells and malignant HRS cells. IHC of all cases revealed that CXCL13+ T cells were significantly enriched in the LR-CHL TME compared to other subtypes of CHL, and 46% of the LR-CHL cases showed CXCL13+ T cell rosettes closely surrounding HRS cells. Since PD-1+ T cell rosettes are known as a specific feature of LR-CHL, we confirmed co-expression of PD-1 in the rosetting cells by IHC in these cases. Conclusions: Our results reveal a unique TME composition in LR-CHL. LR-CHL seems to be distinctly characterized among the CHL subtypes by enrichment of CXCR5+ naïve B cells and CD4+ CXCL13+ PD-1+ T cells, indicating the importance of the CXCR5-CXCL13 axis in the pathogenesis of LR-CHL. Figure Disclosures Savage: BeiGene: Other: Steering Committee; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Scott:Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy. Steidl:AbbVie: Consultancy; Roche: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding.

scholarly journals Single-Cell RNA-Seq Reveals Naïve B cells Associated with Better Prognosis of HCC

2019 ◽  
Author(s):  
Jian He ◽  
Yingxin Lin ◽  
Xianbin Su ◽  
Qing Luo ◽  
Shila Ghazanfar ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
B Cells ◽  
Single Cell ◽  
Solid Tumor ◽  
Immune Cells ◽  
High Morbidity ◽  
Malignant Solid Tumor ◽  
Functional States ◽  
Interaction Map ◽  
The Impact

AbstractHepatocellular Carcinoma (HCC) is a type of malignant solid tumor, causing high morbidity and mortality around the world and the major portion of HCC patients is from China. Cancer immunotherapies have shown some clinical responses in treating some types of cancer but did not shown significant efficiency in HCC treatment. This in part due to the impact of immune cells in the tumor microenvironment. It is commonly believed that HCC is a heterogeneous solid tumor and the microenvironment of HCC plays an important role in tumorgenesis and development. Currently, the residents of the microenvironment of HCC is not well-defined and clarification, especially the immune cells, which we believe that paly pivotal roles in tumorgenesis and development. To depict the landscape of the composition, lineage and functional states of the immune cells in HCC, we performed single-cell RNA sequencing on Diethylnitrosamine (DEN)-induced mouse HCC model. We observed heterogeneity within the immune and hepatocytes both in the precancerous condition of tumorigenesis and cancerous condition of HCC. In this study we found that the disease-associated changes appeared early in pathological progression and were highly cell-type specific. Specific subsets of T and B cells preferentially enriched in HCC, and we identified signature genes for each subset. Additionally, we mapped this group of specific cells to the human TCGA database. We found a cluster of naïve B cells characterized by high expression of CD38 associated with better prognosis of human HCC. Our study demonstrates signaling interaction map based on receptor-ligand bonding on the single-cell level could broaden our comprehending of cellular networks in varies status. Our finding provides a new approach for patient stratification and will help further understand the functional states, dynamics and signaling interaction of B cells in hepatocellular carcinoma, and may provide a novel insight and therapeutics for the HCC.

scholarly journals Single-Cell Multi-Omics Defines the Cell-Type Specific Impact of SF3B1 Splicing Factor Mutations on Hematopoietic Differentiation in Human Clonal Hematopoiesis and Myelodysplastic Syndromes

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 145-145
Author(s):  
Federico Gaiti ◽  
Allegra Hawkins ◽  
Paulina Chamely ◽  
Ariel Swett ◽  
Xiaoguang Dai ◽  
...  
Keyword(s):  
Single Cell ◽  
Research Funding ◽  
Mrna Translation ◽  
Splicing Factor ◽  
Cell Type ◽  
Advisory Committees ◽  
Oxford Nanopore ◽  
Cell Type Specific ◽  
The Impact ◽  
Oxford Nanopore Technologies

Abstract Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype. The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C). We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mutvs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H). Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wtEP cells (Fig. K). Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O). In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. Figure 1 Figure 1. Disclosures Dai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Single-cell RNA-sequencing reveals profound changes in circulating immune cells in patients with heart failure

2020 ◽  
Author(s):  
Wesley T Abplanalp ◽  
David John ◽  
Sebastian Cremer ◽  
Birgit Assmus ◽  
Lena Dorsheimer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Flow Cytometry ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cells ◽  
Healthy Subjects ◽  
Patients With Heart Failure ◽  
Single Cell Rna Sequencing ◽  
The Impact ◽  
Classical Monocytes

Abstract Aims Identification of signatures of immune cells at single-cell level may provide novel insights into changes of immune-related disorders. Therefore, we used single-cell RNA-sequencing to determine the impact of heart failure on circulating immune cells. Methods and results We demonstrate a significant change in monocyte to T-cell ratio in patients with heart failure, compared to healthy subjects, which were validated by flow cytometry analysis. Subclustering of monocytes and stratification of the clusters according to relative CD14 and FCGR3A (CD16) expression allowed annotation of classical, intermediate, and non-classical monocytes. Heart failure had a specific impact on the gene expression patterns in these subpopulations. Metabolically active genes such as FABP5 were highly enriched in classical monocytes of heart failure patients, whereas β-catenin expression was significantly higher in intermediate monocytes. The selective regulation of signatures in the monocyte subpopulations was validated by classical and multifactor dimensionality reduction flow cytometry analyses. Conclusion Together this study shows that circulating cells derived from patients with heart failure have altered phenotypes. These data provide a rich source for identification of signatures of immune cells in heart failure compared to healthy subjects. The observed increase in FABP5 and signatures of Wnt signalling may contribute to enhanced monocyte activation.

scholarly journals Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells

PLoS ONE ◽  
2017 ◽  
Vol 12 (8) ◽  
pp. e0181904 ◽  
Author(s):  
Xingyue An ◽  
Victor G. Sendra ◽  
Ivan Liadi ◽  
Balakrishnan Ramesh ◽  
Gabrielle Romain ◽  
...  
Keyword(s):  
Single Cell ◽  
Immune Cells ◽  
Cytokine Secretion ◽  
Single Cell Profiling

scholarly journals Dynamic Immune Surveillance in Durable Clinical Response to Combined BTK and BCL2 Inhibition in MCL at Longitudinal Single-Cell Resolution

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1323-1323
Author(s):  
Kevin Wang ◽  
Maurizio Di Liberto ◽  
Yang Hu ◽  
Xiangao Huang ◽  
Rachel M Koldej ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Single Cell ◽  
Clinical Response ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Immune Surveillance ◽  
Mantle Cell

Abstract Combined inhibition of BTK with ibrutinib and BCL2 with venetoclax is one of the most promising therapies for B cell malignancies, especially mantle cell lymphoma (MCL), where durable complete remission continued after therapy cessation in some patients (Tam et al, NEJM 2018, Handunnetti ASH 2019). The MCL-intrinsic and extrinsic mechanisms underlying this deep and durable clinical response are unknown, nor have resistance mechanisms been identified. Since BTK is expressed mainly in B lineage cells and venetoclax inhibits BCL2 universally, we hypothesize that BTK inhibition selectively primes MCL cells for vulnerability to BCL2 inhibition while maintaining immune cell homeostasis, leading to differential elimination of MCL cells through immune surveillance. To test this, we undertook integrative longitudinal single-cell RNA-sequencing analysis (scRNA-seq) of PBMCs from sequential tissue and blood specimens (n=32) of 8 MCL patients before and during ibrutinib-venetoclax combination therapy, after therapy cessation or progression, as well as 4 treatment-naïve MCL patients and 4 normal subjects as controls. High dimensional analysis using a unique MCL RNA reference library that we built from bulk RNA-seq data of MCL cells from 57 patients reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling and proinflammatory pathways; C3 represents non-proliferating, long-lived MCL cells that accumulate as disease progresses; and C4 is highly proliferative, expanding with disease progression in untreated patients or on therapy. Integrative analysis of scRNA-seq and CBC with differential showed that homeostasis of all immune cells was maintained throughout ibrutinib-venetoclax therapy and after therapy cessation in 6 MCL patients with a complete response (CR). CD8+T and NK cells were functional, evidenced by the expression of cytotoxic genes such as GNLY, FGFBP2, and GZMH. In contrast, these genes were profoundly suppressed in CD8+T cells that were rapidly depleted on MCL progression after transient response in 2 patients. NK cells were also depleted on progression. In one patient, this was preceded by suppression of cytotoxic genes and loss of MHC-I and MHC-II in MCL cells. Exhaustion did not appear to be the cause. Rather, TSC22D3 upregulation suggests that inhibition of TCR-induced IL2 and IL2R expression and NF-kB activation may underlie the loss of CD8+T and NK cells in ibrutinib-venetoclax resistance. To determine whether BTK inhibition primes MCL cells for subsequent venetoclax killing while sparing other immune cells in a clinical response to ibrutinib-venetoclax, we found that BCL2 expression was higher in MCL cells than in other immune cells pre-therapy. Ibrutinib selectively downregulated the anti-apoptotic MCL1 and upregulated PMAIP1 encoding the pro-apoptotic NOXA in MCL cells, concurrent with TSC22D3 induction and inhibition of the NF-kB signaling pathway. As expected from the lack of BTK expression, CD4+ and CD8+T cells and NK cells expressed MCL1 over PMAIP1 in CR patients, but greater PMAIP1 in patients who developed progressive MCL. Collectively, these data support the hypothesis that BTK inhibition selectively primes MCL cells for venetoclax sensitivity. scRNA-seq analysis further showed that B cells emerging after therapy cessation in CR patients were nearly identical to the bone marrow B cells pre-therapy, as quiescent B cells in cluster 1 and activated B cells in cluster 2. None were detected in clusters 3 or 4. The restoration of B cell immunity confirms CR at the single-cell transcriptome level, speaking to the power of dual BTK and BCL2 inhibition, and supporting the role of immune surveillance in clinical response in targeted therapy. In summary, by unbiased longitudinal scRNA-seq analysis of sequential patient specimens from the ibrutinib-venetoclax clinical trial, we have provided the first evidence that priming of MCL cells for venetoclax vulnerability by BTK inhibition cooperates with immune surveillance to determine the depth and durability in combined targeting of BTK and BCL2 in lymphoma in humans. These findings parallel our longitudinal scRNA-seq analysis of dual BTK and CDK4/6 inhibition, suggesting priming of cancer cells and cooperation with immune surveillance underpin targeted therapy. Disclosures Koldej: CRISPR Therapeutics: Research Funding. Ritchie: CRISPR Therapeutics: Research Funding; Takeda: Research Funding; BMS: Research Funding; Novartis: Honoraria; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria. Martin: ADCT: Consultancy. Elemento: One Three Biotech: Consultancy, Other: Current equity holder; Owkin: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Champions Oncology: Consultancy. Tam: Beigene: Research Funding; Loxo: Honoraria; Beigene: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Abbvie: Research Funding; Janssen: Research Funding. OffLabel Disclosure: Venetoclax is a BCL2 inhibitor FDA-approved for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and acute myeloid leukemia (AML). It was used off-label in combination with ibrutinib in a phase II clinical trial in patients with mantle cell lymphoma.

scholarly journals Identifying Cytokine Biomarkers of Response to Pegylated-Interferon Therapy in Polycythemia Vera and Essential Thrombocythemia: Correlative Analysis from the MPN-RC 111/112 Trials

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1661-1661
Author(s):  
Andrew Dunbar ◽  
Heidi E. Kosiorek ◽  
Aishwarya Krishnan ◽  
Erin McGovern ◽  
Young Park ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Pegylated Interferon ◽  
Post Treatment ◽  
Serum Samples ◽  
Advisory Committees ◽  
Ifna Therapy ◽  
Cytokine Levels ◽  
Equity Ownership ◽  
Pre Treatment

Background: Pegylated Interferon-alpha (PEG-IFNa) improves hematologic parameters and reduces mutant allele fraction in patients with myeloproliferative neoplasms (MPNs); however, widespread clinical use is limited by frequent discontinuation due to side effects. Identifying biomarkers of response to PEG-IFNa therapy might identify patient subsets most likely to derive benefit from this therapy. Serum pro-inflammatory cytokine levels are increased in MPN patients and associated with adverse clinical outcomes. The effect of PEG-IFNa therapy on cytokine levels in MPN patients and correlation with clinical response has not been elucidated. We hypothesized that identification of cytokine signatures could predict for clinical/molecular response to PEG-IFNa in MPNs. Methods: Pre- and post-treatment serum samples were collected from available patients enrolled on the Myeloproliferative Disorders Research Consortium (MPD-RC) 111/112 trials-multicenter studies assessing efficacy of PEG-IFNa in patients with high-risk polycythemia vera/essential thrombocythemia either up front or in the setting of hydroxyurea failure/intolerance. Serum was prepared using the Millipore MAP Human 41-Cytokine Magnet Bead System (Millipore; Cat:HCYTOMAG-60K) and run on the Luminex FLEXMAP 3D platform. Data was analyzed using Luminex xPonent 4.3 software. Correlative mutational data on 156 genes implicated in myeloid malignancies was also available for all patients. Age-matched serum from healthy individuals (N=6) was obtained from AllCellsTM for use as wild-type (WT) controls. Baseline cytokine samples were compared between groups of interest by use of Wilcoxon rank-sum test. Change from baseline in cytokines for each group were compared to the null hypothesis of zero change. P values <0.05 were considered statistically significant. Results: Pre- and post-treatment serum samples were available on 29 MPN patients from the MPN-RC 111/112 cohorts. 21 patients were classified as complete responders (CRs) vs. 8 non-responders (NRs) using European LeukemiaNet criteria. Baseline cytokine levels were generally increased in both CRs and NRs compared to WT, including significant increases in Eotaxin (p=0.023), IL3 (p=0.048), IP10 (p=0.04), and TNFa (p=0.006) (Table 1), as well as a non-significant trend increase in IL8 (p=0.076). Notably, other cytokines were decreased at baseline in comparison to WT, including EGF (p<0.001), as well as MDC (p=0.040) and PDGFaabb (p=0.02), in line with previous studies. Compared to CRs, NRs displayed significantly lower levels of GRO (p=0.020), MDC (p=0.003), and PDGFaabb (p=0.026) at baseline along with elevated levels of IP10 (p=0.028) suggesting pre-treatment levels of these cytokines might predict response to therapy. Comparison of pre- and post- treatment samples revealed a general increase in median cytokine levels in CRs, with significant changes in GM-CSF (p=0.012), IP10 (p<0.001), MCP1 (p=0.015) and IFNa itself (p=0.010). Notably, median cytokine levels in NRs post-treatment, including IFNa, were relatively unchanged from baseline despite on-going PEG-IFNa therapy. Additional pre-treatment serum samples, as well as correlative analysis of cytokine changes with JAK-STAT driver mutant allele fraction and mutational profiling are underway and will be presented at the meeting. Conclusions: Preliminary findings suggest individual cytokines are affected by PEG-IFNa in MPNs. Furthermore, baseline levels of GRO, MDC, PDGFaabb, and IP10 correlate with response to PEG-IFNa. These data may identify MPN patients most likely to respond to PEG-IFNa therapy and those for whom this therapy may be most appropriate. Further analysis with a larger cohort of treatment-naïve patients is underway to validate these findings and identify additional molecular and cytokine signatures of response to PEG-IFNa therapy. Disclosures Yacoub: Agios: Speakers Bureau; Hylapharm: Equity Ownership; Dynavax: Equity Ownership; Cara: Equity Ownership; Ardelyx: Equity Ownership; Incyte: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Honoraria, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Mascarenhas:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Levine:Lilly: Honoraria; Qiagen: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Loxo: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Gilead: Consultancy; Amgen: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy. Hoffman:Merus: Research Funding. Rampal:Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy.

scholarly journals Redefining intestinal immunity with single-cell transcriptomics

2021 ◽  
Author(s):  
Kylie Renee James ◽  
Rasa Elmentaite ◽  
Sarah Amalia Teichmann ◽  
Georgina Louise Hold
Keyword(s):  
Complex System ◽  
Single Cell ◽  
Immune Cells ◽  
The Body ◽  
Intestinal Immunity ◽  
Open Questions ◽  
Intestinal Immune System ◽  
Gut Immunity ◽  
The Impact ◽  
Microbiota Diversity

AbstractThe intestinal immune system represents the largest collection of immune cells in the body and is continually exposed to antigens from food and the microbiota. Here we discuss the contribution of single-cell transcriptomics in shaping our understanding of this complex system. We consider the impact on resolving early intestine development, engagement with the neighbouring microbiota, diversity of intestinal immune cells, compartmentalisation within the intestines and interactions with non-immune cells. Finally, we offer a perspective on open questions about gut immunity that evolving single-cell technologies are well placed to address.

scholarly journals Hematopoietic Stem and Progenitor Cells Improve Survival from Sepsis By Boosting Immunomodulatory Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3261-3261
Author(s):  
Daniel Enrique Morales-Mantilla ◽  
Bailee Nicole Kain ◽  
Duy Le ◽  
Anthony R Flores ◽  
Silke Paust ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Immune Cells ◽  
Bacterial Load ◽  
Myeloid Cells ◽  
Lineage Tracing ◽  
Hematopoietic Stem ◽  
Pathogen Load ◽  
Cytokine Levels ◽  
Stem And Progenitor Cells ◽  
The Impact

Abstract Sepsis is a dysregulated inflammatory syndrome that accounts for as many as 20% of deaths worldwide. Elevated production of pro-inflammatory cytokines during sepsis, such as IL-1, IL-6, interferons (IFNs), and tumor necrosis factor contribute to the development of fever, vasodilation, and multiorgan failure. Novel therapies to treat sepsis are urgently needed. Hematopoietic stem and progenitor cells (HSPC) are responsible for the day-to-day production of blood and immune cells. Recent work from our group and others indicates that during emergency hematopoiesis, inflammatory signals including cytokines, chemokines, and pathogen-derived molecules direct HSPCs to differentiate into effector immune cells. While these signals are essential for a proper immune response, excessive signaling in HSPCs can be detrimental and lead to their depletion. Thus, the interactions between HSPCs and their inflammatory environment may play a deterministic role in immune responses and sepsis. We used a mouse model of Group A Streptococcus (GAS) infection to examine the role of HSPCs in pathogenic infection and sepsis. GAS is a common pathogen that can cause a plethora of diseases from mild skin infections to life-threatening necrotizing fasciitis. We infected mice with 10 6 cfu GAS by intramuscular injection, which typically results in sepsis and death within 7 days, and examined the impact of this infection on peripheral blood (PB) and bone marrow (BM) populations. In just 24 hrs after GAS infection, BM myeloid and HSPC populations are significantly depleted, with myeloid cells being heavily trafficked into circulation following increased levels of monocyte chemoattractant protein-1 (MCP-1). Lineage tracing experiments using KRT18-CreERT2:Rosa26-lox-STOP-lox-TdTomato demonstrated that endogenous HSPCs differentiate toward the myeloid lineage after GAS infection. Based on these data, we hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation resulting in a depletion that could be rescued by the infusion of new HSPCs. To test this hypothesis, we infused GAS-infected mice with 10 4 naïve HSPCs (1.7x10 7 cells per m 2) and evaluated pathogen load and overall survival. This number of HSPCs infused is very low in comparison to the current granulocyte therapies that use ~10 10 cells per m 2 cells per infusion. BM and PB analysis showed that HSPC infusion restored HSPC levels and significantly increased myeloid progenitors and circulating myeloid cells. Strikingly, HSPC infusion in GAS-infected mice significantly increased survival, with 50-75% of mice surviving as opposed to 0-10% of controls. Despite the restoration of hematopoietic populations, surprisingly, GAS-infected mice infused with HSPCs did not show a reduction in pathogen load. Given that HSPC infusion significantly increased survival without impacting pathogen clearance, we sought to determine whether infused HSPCs served an immunomodulatory role. Analysis of BM and PB did not show any changes in lymphocyte populations, suggesting that Tregs and Bregs were not strongly affected. However, BM and PB MDSC populations were severely depleted during GAS sepsis, and HSPC infusion led to a dramatic restoration of these MDSC populations. Interestingly and in accordance with MDSC numbers, the overall cytokine levels of GAS-infected mice are lower after HSPC infusion. Notably, serum levels of cytokines known to drive the symptoms of sepsis, like TNF, IL-12, MIP-1a, IL-6, and IL-1b were dampened in HSPC-rescued mice. In conclusion, while HSPC infusion did not reduce bacterial load, it conferred a significant survival advantage to GAS-infected mice. Our data showing restoration of MDSCs and lower cytokine levels after HSPC infusion suggest that HSPC infusion supports the development of immunomodulatory cells that can prevent sepsis-related hyperinflammation and death. Current work is directed at defining specific HSPC subpopulations that mediate this effect. Importantly, the rescue potential of such low numbers of infused HSPCs highlights the feasibility of this technique and its potential applications. Overall, the information gained in this project may contribute to a new therapeutic strategy to use HSPCs to fight bacterial infections and sepsis where granulocyte infusions have so far produced only mixed results. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell RNA-seq Reveals Obesity-Induced Alterations in the Brca1-Mutated Mammary Gland Microenvironment

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2235
Author(s):  
Pang-Kuo Lo ◽  
Yuan Yao ◽  
Qun Zhou
Keyword(s):  
Breast Cancer ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cells ◽  
Experimental Studies ◽  
Cell Communication ◽  
Mammary Glands ◽  
Breast Tumorigenesis ◽  
Stromal Fibroblasts ◽  
The Impact

Clinical and experimental studies have shown that obesity increases the development and progression of breast cancer. The impact of obesity on the tumor microenvironment plays an important role in tumorigenesis, yet the precise mechanisms underlying obesity-mediated effects on cell-to-cell communication within the tumor microenvironment have been difficult to define. In this study, we conducted single-cell RNA sequencing (scRNA-seq) studies to investigate the impact of high-fat diet (HFD)-induced obesity on transcriptomic landscapes of stromal and immune cells in mammary glands of Brca1−/−; p53+/− mice, an animal breast cancer model. Hierarchical clustering and gene pathway enrichment analyses of scRNA-seq data showed that five different subtypes of stromal fibroblasts existed in mouse Brca1-mutated mammary glands. HFD-induced obesity led to upregulated expression of extracellular matrix (ECM) genes (Col3a1, Col6a3, Eln, and Sparc) and downregulated expression of immunoregulatory genes (Iigp1 and Cxcl10) in these stromal subtype cells. These findings, taken together, suggest that obesity alters the ECM composition and immune ecosystem through modulating the functionality of mammary stromal fibroblasts. Moreover, scRNA-seq analysis of mammary immune cells indicated that HFD-induced obesity promoted the generation and/or recruiting of pro-tumorigenic M2 macrophages in mammary glands. Our studies provide new insight into a mechanistic paradigm wherein obesity modulates the functions of stromal and immune cells to create the tumorigenic microenvironment for promoting breast tumorigenesis.

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