scholarly journals Single-cell profiling reveals the importance of CXCL13/CXCR5 axis biology in lymphocyte-rich classic Hodgkin lymphoma

2021 ◽  
Vol 118 (41) ◽  
pp. e2105822118
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Ashley Marshall ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Hodgkin Lymphoma ◽  
Immune Cell ◽  
Cell Subset ◽  
Progression Free Survival ◽  
Tissue Microarrays ◽  
Classic Hodgkin Lymphoma ◽  
Reactive Lymph Node ◽  
Technical Advances

Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4+ helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1+CXCL13+ T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46% of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5+ normal B cells in close proximity to CXCL13+ T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1+CXCL13+ T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL (P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1+CXCL13+ T cells as a treatment target in LR-CHL.

It’s all about the CD3+ T-cells: how circulating immune cell subset analyses can predict early relapse in Hodgkin lymphoma

2019 ◽  
Vol 60 (10) ◽  
pp. 2345-2347
Author(s):  
Jonathan Moreira ◽  
Leonidas C. Platanias ◽  
Kehinde U. A. Adekola
Keyword(s):  
T Cells ◽  
Hodgkin Lymphoma ◽  
Immune Cell ◽  
Cell Subset ◽  
Immune Cell Subset ◽  
Early Relapse

Multiparametric Analysis of Intra-Tumoral T-Cells in Hodgkin's Lymphoma Using Mass Cytometry (CyTOF)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1438-1438
Author(s):  
Jose Villasboas Bisneto ◽  
Stephen M Ansell
Keyword(s):  
T Cells ◽  
Data Analysis ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Progression ◽  
Single Cell ◽  
Hodgkin Lymphoma ◽  
Cell Subset ◽  
Mass Cytometry ◽  
Mean Frequency

Abstract Classic Hodgkin lymphoma (cHL) is characterized by a rich non-malignant immune infiltrate. T-cells are key components of the antitumoral immune response and studies characterizing subsets in cHL have yielded conflicting results. Initial studies suggested a predominance of TH2-polarized CD4+ T-cells, thought to allow tumor progression due to exhaustion and hypofunctionality. More recent data contest these findings, supporting the theory of tumor progression through evasion from a TH1-rich infiltrate that is potentially functional. The role of tumor evasion in cHL has been highlighted by compelling early clinical data with the use of PD-1 blockade in patients with advanced disease. A similar trial in patients with non-Hodgkin lymphoma (NHL) yielded far more modest results. Intrinsic differences in T-cell subpopulations in the tumor microenvironment may correlate to response to immune checkpoint inhibitor therapy. CyTOF or mass cytometry is a platform able to evaluate more than 45 simultaneous parameters on a single-cell level using nonradioactive nonbiological isotopes tagged to monoclonal antibodies. Measurements are made based on mass spectrometry, avoiding the hurdles of interference and spectral overlap experienced with fluorochromes. This constitutes an ideal tool for the study of the tumor microenvironment given its ability to assess a large number of parameters and resolve differences in a heterogeneous population. We hypothesize that the phenotype of intratumoral lymphocytes in cHL identifies T-cells that can effectively eradicate malignant cells. To test this hypothesis, we compared the phenotype of intratumoral T-cells in cHL to that of NHL and nodular lymphocyte-predominant Hodgkin Lymphoma (nlpHL). Tonsil and hyperplastic lymph node (LN) tissues were used as normal controls. Single-cell suspensions created from tumor specimens were stained with a metal-tagged antibody panel containing 31 surface markers and acquired on CyTOF. Multiparametric data analysis was performed on Cytobank using spanning-tree progression analysis of density-normalized events (SPADE) and t-Distributed Stochastic Neighbor Embedding (viSNE) algorithms. Inferential statistical analyses were performed with JMP®, Version 10.0.0 (SAS Institute Inc., Cary, NC, 1989-2007) using two-tailed tests and a 95% confidence interval. Cell subsets are expressed as percentages of parent population (CD45+CD3+CD19-). A total of 10 samples were studied (4 cHL, 1 nlpHL, 3 NHL, 1 tonsil, 1 LN). The total T-cell population ranged from 30.52 to 67.05% in cHL and 15.36 to 47% in NHL compared to 4.02% and 24.58% in tonsil and LN respectively. The CD4+ T-cell subset ranged from 58.05 to 35.3% in cHL, 50.03 to 82.61% in NHL and corresponded to 82.74% and 87.07% in tonsil and LN respectively. SPADE analysis identified two areas of asymmetric frequency of events amongst samples (figure 1 and 2). The CD4+ Tnaive subset (CD4+CD45RA+CCR7+) ranged from 7.8 to 31.2% of total T-cells in cHL compared to 10.7% in nlpHL, 0.17 to 3.02% in NHL and 6.2 to 6.7% in controls. The pooled mean frequency of CD4+ Tnaive subset was significantly higher in HL (cHL + nlpHL) compared to NHL (14.3% vs. 1.55%; p<0.05; figure 3A). The regulatory T-cell subset (Treg; CD25+CCR4+) ranged from 0.49 to 1.84% of total T-cells in HL compared to 9.3 to 21.04% in NHL, and 4.45 to 8.28% in controls. The pooled mean frequency of the Treg subset was significantly smaller in HL compared to NHL (1.28% vs. 16.23%; p<0.05; figure 3B). Our data supports the use of mass cytometry as a platform to study the tumor microenvironment in B-cell lymphomas. Multiparametric data analysis revealed significant differences in the intratumoral T-cell population between HL and NHL samples, namely in the CD4+ Tnaive and Treg subsets. Further validation in a larger sample is underway and will include panels to evaluate intracellular cytokine production and cell signaling pathways. Correlation between specific intratumoral T-cell phenotypic signatures and clinical outcomes may identify prognostic and predictive characteristics and provide insight to mechanisms of resistance to immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD16ahigh NK cell infiltration and spatial relationships with T cells and macrophages can predict improved progression-free survival in high grade ovarian cancer

2021 ◽  
Author(s):  
Sarah Nersesian ◽  
Stacey N Lee ◽  
Stephanie Grantham ◽  
Liliane Meunier ◽  
Laudine Communal ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Immune Cell ◽  
Nk Cell ◽  
Cell Subset ◽  
Progression Free Survival ◽  
High Grade ◽  
Free Survival ◽  
Adaptive Immune ◽  
Adaptive Immune Cell

Background: High grade serous cancer (HGSC) remains a highly fatal malignancy with less than 50% of patients surviving 5 years after diagnosis. Despite its high mutational burden, HGSC is relatively refractory to checkpoint immunotherapy, suggesting that additional features of the cancer and its interactions with the immune system remain to be understood. Natural killer (NK) cells may contribute to HGSC control, but the role(s) of this population or its subsets in this disease are poorly understood. Methods: We used a TMA containing duplicate treatment-naive tumors from 1145 patients with HGSC and a custom staining panel to simultaneously measure macrophages, T cells and NK cells, separating NK cells based on CD16a expression. Using pathologist-validated digital pathology, machine learning, computational analysis and Pearsons correlations, we quantitated infiltrating immune cell density, co-infiltration and co-localization with spatial resolution to tumor region. We compared the prognostic value of innate, general, and adaptive immune cell neighborhoods to define characteristics of HGSC tumors predictive for progression-free survival and used flow cytometry to define additional features of the CD16adim NK cell subset. Results: NK cells were observed in >95% of tumor cores. Intrastromal localization of CD16alow and CD16ahigh NK cells was associated with shorter and longer progression-free survival, respectively. CD16ahigh NK cells most frequently co-localized with T cells and macrophages; their proximity was termed an adaptive neighborhood. We find that tumors with more area represented by adaptive immune cell neighborhoods corresponded to superior progression free survival. In contrast, CD16alow NK cells did not co-infiltrate with other immune cell types, and expressed the ectonucleotidases, CD39 and CD73, which have been previously associated with poor prognosis in patients with HGSC. Conclusions: Progression-free survival for patients with HGSC may be predicted by the subset of NK cells within the tumor infiltrate (i.e. CD16ahigh vs. CD16alow). NK cell subtypes were associated predictable co-infiltrating and co-localizing leukocyte subsets, suggesting that their presence and activity may influence, or be influenced by the tumor microenvironment. Our data suggest that immunotherapeutic strategies for HGSC should consider the constitution of NK cell subsets and may benefit from mobilizing and activating CD16high NK cells.

540 Transcriptionally defined immune landscape in human gliomas

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A576-A576
Author(s):  
Pravesh Gupta ◽  
Minghao Dang ◽  
Krishna Bojja ◽  
Huma Shehwana ◽  
Tuan Tran ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Wild Type ◽  
Cell Clusters ◽  
Md Anderson ◽  
Antigen Presenting

BackgroundBrain immunity is largely myeloid cell dominated rather than lymphoid cells in healthy and diseased state including malignancies of glial origins called as gliomas. Despite this skewed myeloid centric immune contexture, immune checkpoint and T cell based therapeutic modalities are generalizably pursued in gliomas ignoring the following facts i) T cells are sparse in tumor brain ii) glioma patients are lymphopenic iii) gliomas harbor abundant and highly complex myeloid cell repertoire. We recognized these paradoxes pertaining to fundamental understanding of constituent immune cells and their functional states in the tumor immune microenvironment (TIME) of gliomas, which remains elusive beyond a priori cell types and/or states.MethodsTo dissect the TIME in gliomas, we performed single-cell RNA-sequencing on ~123,000 tumor-derived sorted CD45+ leukocytes from fifteen genomically classified patients comprising IDH-mutant primary (IMP; n=4), IDH-mutant recurrent (IMR; n=4), IDH-wild type primary (IWP; n=3), or IDH-wild type recurrent (IWR; n=4) gliomas (hereafter referred as glioma subtypes) and two non-glioma brains (NGBs) as controls.ResultsUnsupervised clustering analyses delineated predominant 34-myeloid cell clusters (~75%) over 28-lymphoid cell clusters (~25%) reflecting enormous heterogeneity within and across glioma subtypes. The glioma immune diversity spanned functionally imprinted phagocytic, antigen-presenting, hypoxia, angiogenesis and, tumoricidal myeloid to classical cytotoxic lymphoid subpopulations. Specifically, IDH-mutant gliomas were predominantly enriched for brain-resident microglial subpopulations in contrast to enriched bone barrow-derived infiltrates in IDH-wild type especially in a recurrent setting. Microglia attrition in IWP and IWR gliomas were concomitant with invading monocyte-derived cells with semblance to dendritic cell and macrophage like transcriptomic features. Additionally, microglial functional diversification was noted with disease severity and mostly converged to inflammatory states in IWR gliomas. Beyond dendritic cells, multiple antigen-presenting cellular states expanded with glioma severity especially in IWP and IWR gliomas. Furthermore, we noted differential microglia and dendritic cell inherent antigen presentation axis viz, osteopontin, and classical HLAs in IDH subtypes and, glioma-wide non-PD1 checkpoints associations in T cells like Galectin9 and Tim-3. As a general utility, our immune cell deconvolution approach with single-cell-matched bulk RNA sequencing data faithfully resolved 58-cell states which provides glioma specific immune reference for digital cytometry application to genomics datasets.ConclusionsAltogether, we identified prognosticator immune cell-signatures from TCGA cohorts as one of many potential immune responsiveness applications of the curated signatures for basic and translational immune-genomics efforts. Thus, we not only provide an unprecedented insight of glioma TIME but also present an immune data resource that can be exploited for immunotherapy applications.Ethics ApprovalThe brain tumor/tissue samples were collected as per MD Anderson internal review board (IRB)-approved protocol numbers LAB03-0687 and, LAB04-0001. One non-tumor brain tissue sample was collected from patient undergoing neurosurgery for epilepsy as per Baylor College of Medicine IRB-approved protocol number H-13798. All experiments were compliant with the review board of MD Anderson Cancer Center, USA.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal

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 Sequencing Reveals Immunosuppressive Myeloid Cell Diversity and Restricted Cytotoxic Effector Cell Trafficking and Activation During Malignant Progression in Glioma

2021 ◽  
Author(s):  
Sakthi Rajendran ◽  
Clayton Peterson ◽  
Alessandro Canella ◽  
Yang Hu ◽  
Amy Gross ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Immune Cell ◽  
Myeloid Cell ◽  
Malignant Progression ◽  
Low Grade ◽  
Cell Trafficking ◽  
High Grade ◽  
Single Cell Rna Sequencing

Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

scholarly journals Single Cell Transcriptome Analysis Reveals Disease-Defining T Cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 547-547
Author(s):  
Tomohiro Aoki ◽  
Lauren C. Chong ◽  
Katsuyoshi Takata ◽  
Katy Milne ◽  
Monirath Hav ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Research Funding ◽  
Cell Clusters ◽  
Mhc Ii ◽  
Spatial Profiling ◽  
Classic Hodgkin Lymphoma ◽  
Cellular Components

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (&gt; 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

scholarly journals Single-Cell Analysis of the Neonatal Immune System Across the Gestational Age Continuum

2021 ◽  
Vol 12 ◽  
Author(s):  
Laura S. Peterson ◽  
Julien Hedou ◽  
Edward A. Ganio ◽  
Ina A. Stelzer ◽  
Dorien Feyaerts ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
Single Cell ◽  
Gestational Age ◽  
Immune Cell ◽  
Single Cell Analysis ◽  
Inflammatory Diseases ◽  
Interferon Α ◽  
Cytotoxic Lymphocytes ◽  
Neonatal Immune System

Although most causes of death and morbidity in premature infants are related to immune maladaptation, the premature immune system remains poorly understood. We provide a comprehensive single-cell depiction of the neonatal immune system at birth across the spectrum of viable gestational age (GA), ranging from 25 weeks to term. A mass cytometry immunoassay interrogated all major immune cell subsets, including signaling activity and responsiveness to stimulation. An elastic net model described the relationship between GA and immunome (R=0.85, p=8.75e-14), and unsupervised clustering highlighted previously unrecognized GA-dependent immune dynamics, including decreasing basal MAP-kinase/NFκB signaling in antigen presenting cells; increasing responsiveness of cytotoxic lymphocytes to interferon-α; and decreasing frequency of regulatory and invariant T cells, including NKT-like cells and CD8+CD161+ T cells. Knowledge gained from the analysis of the neonatal immune landscape across GA provides a mechanistic framework to understand the unique susceptibility of preterm infants to both hyper-inflammatory diseases and infections.

Recovery of Paired T Cell Receptors from Single-cell Seq-Well Libraries

2019 ◽  
Author(s):  
Ang A. Tu ◽  
Todd M. Gierahn ◽  
Brinda Monian ◽  
Duncan M. Morgan ◽  
Naveen K. Mehta ◽  
...  
Keyword(s):  
T Cells ◽  
Food Allergy ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptors ◽  
Immune Cell ◽  
Cost Effective ◽  
Antigen Specificity ◽  
Activated T Cells ◽  
Cell Receptors

Abstract High-throughput 3’ single-cell RNA-Sequencing (scRNA-Seq) allows for cost-effective, detailed characterization of thousands of individual immune cells from healthy and diseased tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including the variable sequences of T cell receptors (TCRs) that confer antigen specificity in T cells. Here, we present an enrichment strategy that enables simultaneous analysis of TCR variable sequences and corresponding full transcriptomes from 3’ barcoded scRNA-Seq samples. This approach is compatible with common 3’ scRNA-Seq methods, and adaptable to processed samples post hoc. We applied the technique to resolve clonotype-to-phenotype relationships among antigen-activated T cells from immunized mice and from patients with food allergy. We observed diverse but preferential cellular phenotypes manifest among subsets of expanded clonotypes, including functional Th2 states associated with food allergy. These results demonstrate the utility of our method when studying complex diseases in which clonotype-driven immune responses are critical to understanding the underlying biology.

scholarly journals PD-1 and TIGIT coexpression identifies a circulating CD8 T cell subset predictive of response to anti-PD-1 therapy

2020 ◽  
Vol 8 (2) ◽  
pp. e001631
Author(s):  
Sylvain Simon ◽  
Valentin Voillet ◽  
Virginie Vignard ◽  
Zhong Wu ◽  
Camille Dabrowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Therapeutic Response ◽  
Immune Cell ◽  
Expression Profiles ◽  
Cell Subset ◽  
Phenotypic Characterization ◽  
Multiparameter Flow Cytometry ◽  
Specific Gene

BackgroundClinical benefit from programmed cell death 1 receptor (PD-1) inhibitors relies on reinvigoration of endogenous antitumor immunity. Nonetheless, robust immunological markers, based on circulating immune cell subsets associated with therapeutic efficacy are yet to be validated.MethodsWe isolated peripheral blood mononuclear cell from three independent cohorts of melanoma and Merkel cell carcinoma patients treated with PD-1 inhibitor, at baseline and longitudinally after therapy. Using multiparameter flow cytometry and cell sorting, we isolated four subsets of CD8+ T cells, based on PD-1 and TIGIT expression profiles. We performed phenotypic characterization, T cell receptor sequencing, targeted transcriptomic analysis and antitumor reactivity assays to thoroughly characterize each of these subsets.ResultsWe documented that the frequency of circulating PD-1+TIGIT+ (DPOS) CD8+ T-cells after 1 month of anti-PD-1 therapy was associated with clinical response and overall survival. This DPOS T-cell population was enriched in highly activated T-cells, tumor-specific and emerging T-cell clonotypes and T lymphocytes overexpressing CXCR5, a key marker of the CD8 cytotoxic follicular T cell population. Additionally, transcriptomic profiling defined a specific gene signature for this population as well as the overexpression of specific pathways associated with the therapeutic response.ConclusionsOur results provide a convincing rationale for monitoring this PD-1+TIGIT+ circulating population as an early cellular-based marker of therapeutic response to anti-PD-1 therapy.

Sign in / Sign up

Export Citation Format

Share Document