scholarly journals Single-Cell Mass Cytometry on Peripheral Blood Identifies Immune Cell Subsets Associated with Primary Biliary Cholangitis

2020 ◽  
Author(s):  
Jin Sung Jang ◽  
Brian Juran ◽  
Kevin Y. Cunningham ◽  
Vinod K. Gupta ◽  
YoungMin Son ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Single Cell ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Memory B Cells ◽  
Primary Biliary Cholangitis ◽  
Mass Cytometry ◽  
Peripheral Immune System

AbstractThe relationship between Primary Biliary Cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n=33) and age-/sex-matched healthy controls (n=33) to obtain immune cell abundance and marker expression profiles. Hiearchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3+TCRgd+), CD8+ T cells (CD3+CD8+CD161+PD1+), and memory B cells (CD3-CD19+CD20+CD24+CD27+) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naïve B cells were observed in PBC compared to control. Furthermore, several naïve B cell (CD3-CD19+CD20+CD24-CD27-) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of CD8+CD161+ T cells and memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.

scholarly journals Single-cell mass cytometry on peripheral blood identifies immune cell subsets associated with primary biliary cholangitis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jin Sung Jang ◽  
Brian D. Juran ◽  
Kevin Y. Cunningham ◽  
Vinod K. Gupta ◽  
Young Min Son ◽  
...  
Keyword(s):  
Single Cell ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Cell Mass ◽  
Primary Biliary Cholangitis ◽  
Mass Cytometry ◽  
Immune Cell Subsets

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 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.

scholarly journals Differential immune cell infiltrations between healthy periodontal and chronic periodontitis tissues

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Wei Li ◽  
Zheng Zhang ◽  
Zuo-min Wang
Keyword(s):  
T Cells ◽  
B Cells ◽  
Mast Cells ◽  
Plasma Cells ◽  
Immune Cell ◽  
Memory T Cells ◽  
Host Immunity ◽  
Memory B Cells ◽  
Helper T Cells ◽  
Follicular Helper

Abstract Background Host immunity plays an important role against oral microorganisms in periodontitis. Methods This study assessed the infiltrating immune cell subtypes in 133 healthy periodontal and 210 chronic periodontitis tissues from Gene Expression Omnibus (GEO) datasets using the CIBERSORT gene signature files. Results Plasma cells, naive B cells and neutrophils were all elevated in periodontitis tissues, when compared to those in healthy controls. In contrast, memory B cells, resting dendritic, mast cells and CD4 memory cells, as well as activated mast cells, M1 and M2 macrophages, and follicular helper T cells, were mainly present in healthy periodontal tissues. Furthermore, these periodontitis tissues generally contained a higher proportion of activated CD4 memory T cells, while the other subtypes of T cells, including resting CD4 memory T cells, CD8 T cells, follicular helper T cells (TFH) and regulatory T cells (Tregs), were relatively lower in periodontitis tissues, when compared to healthy tissues. The ratio of dendritic and mast cells and macrophages was lower in periodontitis tissues, when compared to healthy tissues. In addition, there was a significant negative association of plasma cells with most of the other immune cells, such as plasma cells vs. memory B cells (γ = − 0.84), plasma cells vs. resting dendritic cells (γ = − 0.64), plasma cells vs. resting CD4 memory T cells (γ = 0.50), plasma cells versus activated dendritic cells (γ = − 0.46), plasma cells versus TFH (γ = − 0.46), plasma cells versus macrophage M2 cells (γ = − 0.43), or plasma cells versus macrophage M1 cells (γ = − 0.40), between healthy control and periodontitis tissues. Conclusion Plasma cells, naive B cells and neutrophils were all elevated in periodontitis tissues. The infiltration of different immune cell subtypes in the periodontitis site could lead the host immunity against periodontitis.

scholarly journals In VitroMeasles Virus Infection of Human Lymphocyte Subsets Demonstrates High Susceptibility and Permissiveness of both Naive and Memory B Cells

2018 ◽  
Vol 92 (8) ◽  
pp. e00131-18 ◽  
Author(s):  
Brigitta M. Laksono ◽  
Christina Grosserichter-Wagener ◽  
Rory D. de Vries ◽  
Simone A. G. Langeveld ◽  
Maarten D. Brem ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Immune Suppression ◽  
Peripheral Blood ◽  
Measles Virus ◽  
Memory B Cells ◽  
T And B Cells ◽  
B Cell Subsets

ABSTRACTMeasles is characterized by a transient immune suppression, leading to an increased risk of opportunistic infections. Measles virus (MV) infection of immune cells is mediated by the cellular receptor CD150, expressed by subsets of lymphocytes, dendritic cells, macrophages, and thymocytes. Previous studies showed that human and nonhuman primate memory T cells express higher levels of CD150 than naive cells and are more susceptible to MV infection. However, limited information is available about the CD150 expression and relative susceptibility to MV infection of B-cell subsets. In this study, we assessed the susceptibility and permissiveness of naive and memory T- and B-cell subsets from human peripheral blood or tonsils toin vitroMV infection. Our study demonstrates that naive and memory B cells express CD150, but at lower frequencies than memory T cells. Nevertheless, both naive and memory B cells proved to be highly permissive to MV infection. Furthermore, we assessed the susceptibility and permissiveness of various functionally distinct T and B cells, such as helper T (TH) cell subsets and IgG- and IgA-positive memory B cells, in peripheral blood and tonsils. We demonstrated that TH1TH17 cells and plasma and germinal center B cells were the subsets most susceptible and permissive to MV infection. Our study suggests that both naive and memory B cells, along with several other antigen-experienced lymphocytes, are important target cells of MV infection. Depletion of these cells potentially contributes to the pathogenesis of measles immune suppression.IMPORTANCEMeasles is associated with immune suppression and is often complicated by bacterial pneumonia, otitis media, or gastroenteritis. Measles virus infects antigen-presenting cells and T and B cells, and depletion of these cells may contribute to lymphopenia and immune suppression. Measles has been associated with follicular exhaustion in lymphoid tissues in humans and nonhuman primates, emphasizing the importance of MV infection of B cellsin vivo. However, information on the relative susceptibility of B-cell subsets is scarce. Here, we compared the susceptibility and permissiveness toin vitroMV infection of human naive and memory T- and B-cell subsets isolated from peripheral blood or tonsils. Our results demonstrate that both naive and memory B cells are more permissive to MV infection than T cells. The highest infection levels were detected in plasma cells and germinal center B cells, suggesting that infection and depletion of these populations contribute to reduced host resistance.

Mezagitamab Induces Immunomodulatory Effect in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Michael Abadier ◽  
Jose Estevam ◽  
Deborah Berg ◽  
Eric Robert Fedyk
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Cell Populations ◽  
Landscape Changes ◽  
Ki 67 ◽  
Immune Cell Subsets

Background Mezagitamab is a fully human immunoglobulin (Ig) G1 monoclonal antibody with high affinity to CD38 that depletes tumor cells expressing CD38 by antibody- and complement-dependent cytotoxicity. CD38 is a cell surface molecule that is highly expressed on myeloma cells, plasma cells, plasmablasts, and natural killer (NK) cells, and is induced on activated T cells and other suppressor cells including regulatory T (Tregs) and B (Bregs) cells. Data suggest that immune landscape changes in cancer patients and this may correlate with disease stage and clinical outcome. Monitoring specific immune cell subsets could predict treatment responses since certain cell populations either enhance or attenuate the anti-tumor immune response. Method To monitor the immune landscape changes in RRMM patients we developed a mass cytometry panel that measures 39-biomarkers to identify multiple immune cell subsets, including T cells (naïve, memory, effector, regulatory), B cells (naïve, memory, precursors, plasmablasts, regulatory), NK cells, NKT cells, gamma delta T cells, monocytes (classical, non-classical and intermediate), dendritic cells (mDC; myeloid and pDC; plasmacytoid) and basophils. After a robust analytical method validation, we tested cryopreserved peripheral blood and bone marrow mononuclear cells from 19 RRMM patients who received ≥ 3 prior lines of therapy. Patients were administered 300 or 600 mg SC mezagitamab on a QWx8, Q2Wx8 and then Q4Wx until disease progression schedule (NCT03439280). We compared the percent change in immune cell subsets at baseline versus week 4 and week 16. Results CD38 is expressed at different levels on immune cells and sensitivity to depletion by mezagitamab generally correlates positively with the density of expression. CD38 is expressed at high densities on plasmablasts, Bregs, NK-cells, pDC and basophils at baseline and this was associated with reductions in peripheral blood and bone marrow (plasmablasts, 95%, Bregs, 90%, NK-cells, 50%, pDC, 55% and basophils, 40%) at week 4 post treatment. In contrast, no changes occurred in the level of total T-cells and B-cells, which is consistent with low expression of CD38 on most cells of these large populations. Among the insensitive cell types, remaining NK-cells acquired an activated, proliferative and effector phenotype. We observed 60-150% increase in activation (CD69, HLA-DR), 110-200% increase in proliferation (Ki-67), and 40-375% increase in effector (IFN-γ) markers in peripheral blood and bone marrow. Importantly, NK-cells which did not express detectable CD38, also exhibited a similar phenotype possibly by a mechanism independent of CD38. Consistent with these data, the remaining CD4 and CD8 T-cell populations exhibited an activated effector phenotype as observed by 40-200% increase in activation, 60-200% increase in proliferation and 40-90% increase in effector markers in peripheral blood. A potential explanation for this acquisition of activated effector phenotypes could be a reduction in suppressive regulatory lymphocytes. Next, we measured levels of Tregs and Bregs, and observed that Bregs which are CD24hiCD38hi were reduced to 60-90% in peripheral blood and bone marrow. In contrast, total Tregs were reduced by only 5-25% because CD38 expression in Tregs appears as a spectrum where only ~10-20% are CD38+, and thus CD38+ Tregs were reduced more significantly (45-75%), reflecting the selectively of mezagitamab to cells expressing high levels of CD38. CD38+ Tregs are induced in RRMM patients, thus we looked at the phenotype of CD38-, CD38mid, and CD38high -expressing Tregs. We observed higher level of markers that correlate with highly suppressive Tregs such as Granzyme B, Ki-67, CTLA-4 and PD-1 in CD38high Tregs. Accordingly, the total Treg population exhibited a less active phenotype after exposure to mezagitamab, which selectively depleted the highly suppressive CD38+ Tregs. Conclusions Chronic treatment with mezagitamab is immunomodulatory in patients with RRMM, which is associated with reductions in tumor burden, subpopulations of B and T regulatory cells, and characterized by conventional NK and T cells exhibiting an activated, proliferative and effector phenotype. The immune landscape changes observed is consistent with the immunologic concept of converting the tumor microenvironment from cold-to-hot and highlights a key mechanistic effect of mezagitamab. Disclosures Berg: Takeda Pharmaceuticals Inc: Current Employment.

scholarly journals The Influence of Aging and Sex Hormones on Expression of Growth Hormone-Releasing Hormone in the Human Immune System1

2001 ◽  
Vol 86 (7) ◽  
pp. 3157-3161
Author(s):  
O. Khorram ◽  
M. Garthwaite ◽  
T. Golos
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Mrna Expression ◽  
Postmenopausal Women ◽  
Cell Line ◽  
Immune Cell ◽  
Hormone Replacement ◽  
Men And Women

GHRH is a neuropeptide that has also been localized to the immune system. The physiological function of GHRH in the immune system has not been elucidated. This study was conducted to determine whether immune GHRH expression is altered in certain pathological states, such as immune cell tumors, and whether gender, aging, and alterations in the sex steroid milieu influence the expression of this peptide in immune cells. Using double color flow cytometry, GHRH protein was found to be expressed in less than 2% of peripheral blood mononuclear cells (PBMC). Monocytes and B and T cells all expressed GHRH protein, although a greater percentage of T cells compared with B cells and monocytes expressed GHRH (5- to 7-fold). Semiquantitative RT-PCR was used to quantify GHRH messenger ribonucleic acid (mRNA) in PBMC and several immune cell-derived tumors. PBMC and granulocytes expressed low levels of GHRH mRNA with relatively higher levels of expression in monocytes. The tumor cell lines CEMX 174 (B/T cells), HUT 78 (T cells), WIL2-N (B cells), U937 (monocytes/macrophages), and JM 1 (pre-B cell lymphoma) all showed greater expression of GHRH mRNA relative to PBMC. However, two cell lines, CCRF-SB, a B lymphoblastoid cell line, and HL-60, a promyelocytic cell line, expressed GHRH mRNA at similar levels as PBMC. A significant decrease in the percentage of lymphocytes (CD45+ cells) expressing GHRH protein was found in age-advanced men and women compared with young men and women. This decline was noted in B cells (CD20+) and monocytes (CD14+), but not in T cells (CD3+). GHRH mRNA expression in PBMC derived from postmenopausal women was lower than that from premenopausal women. However, no differences in PBMC GHRH mRNA expression were found in young and old men. Although in older men there were fewer peripheral lymphocytes that express GHRH protein, these cells secreted significantly more GHRH in vitro than cells from postmenopausal women with no hormone replacement therapy (HRT), but similar levels as cells from women receiving HRT. PBMC from women receiving HRT secreted more GHRH in vitro than cells from women receiving no hormone replacement. This study demonstrates that the expression of immune GHRH is dynamic, and therefore likely to be regulated. Increased expression of GHRH in certain immune tumors suggests that GHRH may be mitogenic under certain conditions and therefore play a role in the pathogenesis of select immune cell tumors. Collectively, these results suggest a role for GHRH as a local immune modulator and in the pathophysiology of immunosenescence and immune cell tumors.

scholarly journals SHIP1 Inhibition As Novel Therapeutic Approach in Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 894-894
Author(s):  
Veronika Ecker ◽  
Martina Braun ◽  
Tanja Neumayer ◽  
Markus Muschen ◽  
Jürgen Ruland ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
Immune Responses ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Immune Cell ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling

Abstract Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the Western world. Malignant B cells are blocked from differentiating into immunoglobulin producing-plasma cells and clonally accumulate in the spleen, lymph nodes, bone marrow and peripheral blood. CLL is characterized by immunosuppression throughout all disease stages, which is mediated by increased numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Jitschin and Braun et al., Blood 2014) and direct inhibitory effects of the malignant CLL cells on T cells (Christopoulos etal., Blood 2011). Over the past decade, significant improvement in understanding the pathogenesis of CLL has highlighted the importance of active B cell receptor (BCR) signaling. This has revealed promising targeted treatment options, including the small molecule inhibitors targeting the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Idelalisib and Duvelisib are under clinical investigation for CLL treatment, however, treatment-related toxicities are limiting their application and none of these approaches are curative, highlighting the importance of functional anti-tumor immune responses in CLL for prolonged treatment efficacy. Here, we are testing a novel approach that aims to selectively target CLL B cells and simultaneously restore an appropriate immune cell function. The phosphatase SH2-domain-containing inositol 5ʹ-phosphatase 1 (SHIP1) serves as negative feedback molecule and downregulates PI3K signaling in B cells by dephosphorylating the 5`phosphate of Phosphatidylinositol (3,4,5)-trisphosphate. We hypothesize that CLL cells rely on such negative regulators for optimal survival and can only tolerate a maximum signaling level. We are therefore testing whether SHIP1 inhibition induces hypersignaling and thereby CLL cell death. Furthermore, we are investigating whether SHIP1 inhibition simultanously stimulates immune responses, as it has been shown to induce expansion of murine hematopoietic and mesenchymal stem cell compartments (Brooks et al., Stem cells 2014). 3α-Aminocholestane (3AC) is a small molecule inhibitor of SHIP1 and can be used for pharmacological inhibition. First, we investigated the expression and phosphorylation levels of SHIP1 in CLL. We found SHIP1 to be expressed at various levels in CLL peripheral blood and strongly phosphorylated compared to age-matched healthy donors. Besides, SHIP1 transcription is upregulated in lymph nodes as compared to peripheral blood, which is in line with the assumption of increased BCR signaling in secondary lymphoid organs. We then set out to investigate the consequences of SHIP1 phosphatase inhibition. Similarly, to recent findings in acute lymphoblastic leukemia (Chen et al., Nature 2015), pharmacological inhibition of SHIP1 lead to rapid cell death of CLL cells. We further investigated the mode of cell death and observed specific features of apoptosis, namely caspase 3/7 activation and phosphatidylserine exposure on the outer cell membrane. This has been tested on primary CLL patient samples and T cell leukemia/lymphoma 1 (TCL1)-driven murine CLL cells and was not observed or significantly less pronounced in other lymphoma cell lines or healthy primary B cells. To confirm the specificity of the observed effects, we genetically activated AKT with a GFP reporter in the TCL1-driven mouse model in vivo and in vitro. By tracking GFP-expressing CLL cells, we observed an initial expansion followed by rapid cell death in vitro. When we induced AKT activation in vivo, GFP+ CLL cells were not detectable in the peripheral blood, total CLL count declined upon induction and we found decreased tumor burden in the secondary lymphoid organs, particularly in the lymph nodes. In addition to the direct effects on CLL cells, we sought to investigate the impact of SHIP1 inhibition on other immune cell functions. We observed that SHIP1 inhibition lowers the activity threshold of T cells: When we stimulated a reporter cell line with suboptimal doses of anti-CD3, 3AC treatment significantly enhanced the response rate. Therefore, we propose SHIP1 as a novel interesting target in CLL. In contrast to kinase inhibition and downregulation of the BCR signaling strength, phosphatase inhibition and BCR signaling overaction provides an attractive new treatment strategy for elimination of malignant CLL cells and stimulation of immune responses. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single cell transcriptome profiling of the Atlantic cod immune system

2020 ◽  
Author(s):  
Naomi Guslund ◽  
Monica Hongrø Solbakken ◽  
Kjetill S. Jakobsen ◽  
Shuo-Wang Qiao
Keyword(s):  
Immune System ◽  
Single Cell ◽  
Peripheral Blood ◽  
Bacterial Infections ◽  
Immune Cell ◽  
Atlantic Cod ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Gene Markers ◽  
Putative Gene

AbstractThe Atlantic cod’s unusual immune system, entirely lacking the Major Histocompatibility class II pathway, has prompted intriguing questions about what mechanisms are used to combat bacterial infections and how immunological memory is generated. Here, we examine the diversity of 8,180 spleen cells and peripheral blood leukocytes by single cell RNA sequencing. Unbiased transcriptional clustering revealed eleven distinct immune cell signatures. Resolution at the single cell level enabled characterisation of the major cell subsets including the cytotoxic T cells, B cells, erythrocytes, thrombocytes, neutrophils and macrophages. Further, we describe for the first time rare cell subsets which may represent dendritic cells, natural killer-like cells and a population of cytotoxic cells expressing GATA-3. We propose putative gene markers for each cluster and describe the relative proportions of each cell type in the spleen and peripheral blood leukocytes. By single cell analysis, this study provides the most detailed molecular and cellular characterization of the immune system of the Atlantic cod so far.

scholarly journals Potent Anti-Tumor Activity of Bcma CAR-T Therapy Against Heavily Treated Multiple Myeloma and Dynamics of Immune Cell Subsets Using Single-Cell Mass Cytometry

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1859-1859 ◽  
Author(s):  
Yongxian Hu ◽  
Zhang Yanlei ◽  
Guoqing Wei ◽  
Chang alex Hong ◽  
He Huang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Immune Cell ◽  
Cell Mass ◽  
Mass Cytometry ◽  
Car T Cells ◽  
Car T Cell ◽  
Immune Cell Subsets ◽  
Car T

Background BCMA CAR-T cells have demonstrated substantial clinical activity against relapsed/refractory multiple myeloma (RRMM). In different clinical trials, the overall response rate (ORR) varied from 50% to 100%. Complete remission (CR) rate varied from 20% to 80%. Here we developed a BCMA CAR-T cell product manufactured via lentiviral vector-mediated transduction of activated T cells to express a second-generation CAR with 4-1BB costimulatory domain and evaluated the efficacy and safety, moreover, dynamics of immune cell subsets using single-cell mass cytometry during treatment were analyzed. Methods Our trial (ChiCTR1800017404) is a phase 1, single-arm, open-label single center study to evaluate the safety and efficacy of autologous BCMA CAR-T treatment for RRMM. Patients were subjected to a lymphodepleting regimen with Flu and Cy prior to CAR-T infusion. BCMA CAR-T cells were administered as a single infusion at a median dose of 3.5 (1 to 6) ×106/kg. MM response assessment was conducted according to the International Uniform Response Criteria. Cytokine-release syndrome (CRS) was graded as Lee DW et al described (Blood.2014;124(2):188-195). Phenotypic analysis of peripheral blood mononuclear cells (PBMCs), frozen BCMA CAR-T aliquots, phenotype and in vivo kinetics of immune cell subsets after CAR-T infusion were performed by single-cell mass cytometry. Results As of the data cut-off date (August 1st, 2019), 33 patients, median age 62.5 (49 to 75) years old were infused with BCMA CAR-T cells. The median observation period is 8.0 (0.7 to 18) months. ORR was 100% (The patient who died of infection at 20 days after CAR-T infusion were excluded). All the 32 patients achieved MRD negative in bone marrow by flow cytometry in 2 weeks after CAR-T infusion. Partial response (4 PR, 12.1%), VGPR (7 VGPR, 21.2%), and complete response (21 CR, 63.6%) within 12 weeks post CAR-T infusion were achieved. Durable responses from 4 weeks towards the data cut-off date were found in 28/33 patients (84.8%) (Figure 1a). All patients had detectable CAR-T expansion by flow cytometry from Day 3 post CAR-T cell infusion. The peak CAR-T cell expansion in CD3+ lymphocytes of peripheral blood (PB) varied from 35% to 95% with a median percentage of 82.9%. CRS was reported in all the 33 patients, including 4 with Grade 1, 13 with Grade 2 and 16 with Grade 3. During follow-up, 1-year progression-free survival (PFS) was 70.7% (Figure 1b) and overall survival (OS) was 71.7% (Figure 1c). Multivariate analysis of patients with PR and patients with CR+VGPR revealed that factors including extramedullary infiltration, age>60 years old, high-risk cytogenetics, late stage and CAR-T cell dose were not associated with clinical response (P>0.05). Single-cell mass cytometry revealed that the frequency of total T cells, CD8+ T cells, NK cells and CD3+CD56+ NKT cells in PB was not associated with BCM CAR-T expansion or clinical response. CD8+ Granzyme B+ Ki-67+ CAR-T cells expanded prominently in CRS period. As serum cytokines increased during CRS, non-CAR-T immune cell subsets including PD1+ NK cells, CD8+ Ki-67+ ICOS+ T cells expanded dominantly implying that non-CAR-T cells were also activated after CAR-T treatment. After CRS, stem cell like memory CAR-T cells (CD45RO+ CCR7- CD28- CD95+) remain the main subtype of CAR-T cells (Figure 1d). Conclusions Our data showed BCMA CAR-T treatment is safe with prominent efficacy which can overcome the traditional high-risk factors. We also observed high expansion level and long-term persistence of BCMA CAR-T cells contribute to potent anti-myeloma activity. Stem cell like memory CAR-T cells might be associated with long-term persistence of BCMA CAR-T cells. These initial data provide strong evidence to support the further development of this anti-myeloma cellular immunotherapy. Figure 1. Disclosures No relevant conflicts of interest to declare.

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