Journal articles on the topic 'CD93 signaling'
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Barbera, Stefano, Luisa Raucci, Roberta Lugano, Gian Marco Tosi, Anna Dimberg, Annalisa Santucci, Federico Galvagni, and Maurizio Orlandini. "CD93 Signaling via Rho Proteins Drives Cytoskeletal Remodeling in Spreading Endothelial Cells." International Journal of Molecular Sciences 22, no. 22 (November 17, 2021): 12417. http://dx.doi.org/10.3390/ijms222212417.
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During angiogenesis, cell adhesion molecules expressed on the endothelial cell surface promote the growth and survival of newly forming vessels. Hence, elucidation of the signaling pathways activated by cell-to-matrix adhesion may assist in the discovery of new targets to be used in antiangiogenic therapy. In proliferating endothelial cells, the single-pass transmembrane glycoprotein CD93 has recently emerged as an important endothelial cell adhesion molecule regulating vascular maturation. In this study, we unveil a signaling pathway triggered by CD93 that regulates actin cytoskeletal dynamics responsible of endothelial cell adhesion. We show that the Src-dependent phosphorylation of CD93 and the adaptor protein Cbl leads to the recruitment of Crk, which works as a downstream integrator in the CD93-mediated signaling. Moreover, confocal microscopy analysis of FRET-based biosensors shows that CD93 drives the coordinated activation of Rac1 and RhoA at the cell edge of spreading cells, thus promoting the establishment of cell polarity and adhesion required for cell motility.
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Riether, Carsten, Ramin Radpour, Chantal L. Bachmann, Christian M. Schürch, Miroslav Arambasic, Gabriela M. Baerlocher, and Adrian F. Ochsenbein. "CD93-Signaling Regulates Self-Renewal and Proliferation of Chronic Myeloid Leukemia Stem Cells in Mice and Humans and Might be a Promising Target for Treatment." Blood 134, Supplement_1 (November 13, 2019): 187. http://dx.doi.org/10.1182/blood-2019-127864.
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Background: The introduction of BCR/ABL-specific tyrosine kinase inhibitors (TKIs) more than two decades ago revolutionized chronic myelogenous leukemia (CML) therapy. The majority of CML patients treated with TKIs obtain durable cytogenetic and molecular responses. However, only a subgroup of these patients can successfully discontinue TKI therapy and maintain a treatment-free remission (Laneuville et al. 2011). TKI-resistant leukemia stem cells (LSCs) persist in the majority of patients at low levels over a prolonged period. These quiescent, self-renewing LSCs in the BM are the major cause of relapse after drug discontinuation (Holyoake et al, 2017). The selective elimination of LSCs requires the definition of unique signaling pathways that promote self-renewal of LSCs but not of normal HSCs. Based on the documented expression of CD93 on LSCs (Kinstrie et al, 2015), the aim of the present study was to investigate the role of the cell surface receptor CD93 in the regulation of self-renewal of human and murine CML LSCs and its contribution to disease development and progression. Methods and Results: We found CD93 expression on LSCs and leukemia progenitor cells but not on more differentiated leukemia granulocytes in a murine retroviral lineage-negative Sca-1+ c-kit+ (LSK) transduction/transplantation CML model. Next-generation sequencing analysis revealed that Cd93-/- LSCs have a silenced gene expression signature particularly in genes involved in the regulation of gene expression, stem cell maintenance and proliferation. Out of the 1120 genes differentially expressed between BL/6 and Cd93-/- LSCs, 1108 genes were down-regulated. In contrast, naïve BL/6 and Cd93-/- hematopoietic stem cells (HSCs) did not display a dysregulation in these pathways. Functionally, CD93-deficiency in LSCs resulted in impaired self-renewal, reduced LSC frequencies in vitro (at least by a factor of 100, P<0.001) and in the incompetence to induce and propagate CML in mice. To study whether CD93-signaling in LSCs relies on ligand-binding to the extracellular domain of CD93, we generated an extracellular domain deletion mutant of CD93 (mCd93intra). Comparable to transduction with full-length mCd93, the expression of Cd93intra restored colony formation of Cd93-/- LSCs in vitro, suggesting that the maintenance of LSC self-renewal is independent of ligand-binding to the extracellular domain of CD93. Furthermore, analysis of the sub-cellular localization of CD93 in CML cells using a lentiviral expression vector encoding for AcGFP1-N1-Cd93 demonstrated nuclear localization of the CD93 intracellular domain (ICD). SCY1 like pseudokinase 1 (SCYL1), a regulator of gene transcription, directly interacts with the highly charged juxta membrane domain of the cytoplasmic tail of CD93 (Bohlson et al, 2005). Silencing of Scyl1 significantly reduced colony formation of BL/6 but not Cd93-/- LSCs in vitro suggesting that the ICD of CD93 regulates gene transcription via Scyl1 in CML LSCs. To discover compounds that affect LSC function similarly as genetic CD93 blockade, we performed a compound screen using the FDA approved drug library V2. The antiemetic agent metoclopramide, which is widely used in clinical routine to reduce nausea in cancer patients, was one very promising candidate identified in the screen. Metoclopramide treatment reduced clonogenic potential of CD93-competent LSCs to comparable levels as CD93-deficient LSCs in vitro without further affecting colony formation of CD93-deficient LSCs. Analysis of LSCs from newly diagnosed CML patients similarly demonstrated that CD93-signaling induces the expression of genes associated with proliferation and stemness, resulting in an increased clonogenic potential in vitro. In addition, colony formation and re-plating capacity in semisolid cultures of human CD34+CD38- LSCs was significantly impaired by metoclopramide at a pharmacological concentration of 0.1mM compared to control treatment. Conclusions: Overall, these results indicate that CD93-siganling is an important regulator of stemness and proliferation of human and murine CML LSCs. Furthermore, this study identifies expression of CD93 by LSCs as promising novel target for the treatment of CML. Disclosures Baerlocher: Novartis: Research Funding.
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Carroll, Virginia A., Mark K. Lafferty, Luigi Marchionni, Joseph L. Bryant, Robert C. Gallo, and Alfredo Garzino-Demo. "Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice." Proceedings of the National Academy of Sciences 113, no. 46 (October 31, 2016): 13168–73. http://dx.doi.org/10.1073/pnas.1615258113.
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HIV-1 infection is associated with increased risk for B-cell lymphomas. How HIV infection promotes the development of lymphoma is unclear, but it may involve chronic B-cell activation, inflammation, and/or impaired immunity, possibly leading to a loss of control of oncogenic viruses and reduced tumor immunosurveillance. We hypothesized that HIV structural proteins may contribute to lymphomagenesis directly, because they can persist long term in lymph nodes in the absence of viral replication. The HIV-1 transgenic mouse Tg26 carries a noninfectious HIV-1 provirus lacking part of the gag-pol region, thus constituting a model for studying the effects of viral products in pathogenesis. Approximately 15% of Tg26 mice spontaneously develop leukemia/lymphoma. We investigated which viral proteins are associated with the development of leukemia/lymphoma in the Tg26 mouse model, and performed microarray analysis on RNA from spleen and lymph nodes to identify potential mechanisms of lymphomagenesis. Of the viral proteins examined, only expression of HIV-1 matrix protein p17 was associated with leukemia/lymphoma development and was highly expressed in bone marrow before disease. The tumor cells resembled pro-B cells, and were CD19+IgM−IgD−CD93+CD43+CD21−CD23−VpreB+CXCR4+. Consistent with the pro-B-cell stage of B-cell development, microarray analysis revealed enrichment of transcripts, including Rag1, Rag2, CD93, Vpreb1, Vpreb3, and Igll1. We confirmed RAG1 expression in Tg26 tumors, and hypothesized that HIV-1 matrix protein p17 may directly induce RAG1 in B cells. Stimulation of human activated B cells with p17 enhanced RAG1 expression in three of seven donors, suggesting that intracellular signaling by p17 may lead to genomic instability and transformation.
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Hsu, Hui-Chen, Jennie A. Hamilton, Qi Wu, PingAr Yang, Bao Luo, Shutao Xie, Shanrun Liu, Jun Li, and John D. Mountz. "IL-17 receptor A signaling impedes NF-κB p50/p50 repressor and subverts B-cell anergy in BXD2 mice." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 210.7. http://dx.doi.org/10.4049/jimmunol.196.supp.210.7.
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Abstract B-cell fate decision checkpoint 2 at the transitional T2 stage represents a critical checkpoint defect in the development of autoimmune disease. We have found that there is a failure of anergy induction in the CD93+IgM+CD23+ transitional T2 B cells in the spleen of lupus prone BXD2 mice, which is associated with B-cell tolerance loss to anti-IgM or TLR7 in vitro. Interestingly, there was an enhanced BXD2-Il17ra−/− T2 B cell anergy phenotype in vivo, which is associated with a strong B cell anergic response to BCR or TLR7 stimulation in vitro. Abnormal survival of T2 B cells is thought to be regulated through a strong BAFF-R signaling. Surprisingly, despite normal expression of BAFF-R, BlyS cannot reverse the anergic response of BXD2-Il17ra−/− B cells to BCR stimulation. Analysis of the expression of IL-17RA in the B-cell subsets in BXD2 mice shows that only T2 and germinal center (PNA+Fas+) B cells, but not T1, T3 or mature (CD93−) B cells express surface IL-17RA. Single cell analysis of T2 B cells reveals the co-expression of Il17ra with B-cell activation gene, Bclxl. The strong anergic phenotype of BXD2-Il17ra−/− mouse B cells is associated with a dramatically enhanced nuclear expression of NF-κB1 (p50) and down-modulation of NF-κB phospho-p65. Our results suggest that, in BXD2-Il17ra−/− B cells, the anergy phenotype is established at the T2 stage. In these B cells, the stimulus-specific transcription repressor p50/p50 homodimer may act as a master transcriptional regulator to counteract pro-activation/survival NF-κB signaling provided by other major B-cell stimulators to enforce B cell anergy. Reagents that can promote the NF-κB p50/p50 repressome complex may be a novel strategy to enhance B-cell tolerance for autoimmunity.
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Riether, Carsten, Ramin Radpour, Nils M. Kallen, Damian T. Bürgin, Chantal Bachmann, Christian M. Schürch, Ursina Lüthi, et al. "Metoclopramide treatment blocks CD93-signaling-mediated self-renewal of chronic myeloid leukemia stem cells." Cell Reports 34, no. 4 (January 2021): 108663. http://dx.doi.org/10.1016/j.celrep.2020.108663.
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Zhang, Hui, Zhaohui Zhu, and Gary Meadows. "Effects of chronic alcohol consumption on B cells in B16BL6 melanoma-bearing mice (66.24)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 66.24. http://dx.doi.org/10.4049/jimmunol.186.supp.66.24.
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Abstract We previously found that chronic alcohol consumption inhibits CD8+ T cell immunity in B16BL6 melanoma-bearing mice and also decreases their survival. Recent reports indicate that lack of B cells, especially mature B cells, compromises CD8+ T cell anti-tumor immunity. It is unknown if alcohol consumption affects B cells in tumor-bearing hosts. Herein, we studied the effects of chronic alcohol consumption on B cell phenotypes in B16BL6 melanoma-bearing mice. Alcohol consumption did not alter the number of B cells in the bone marrow (BM), spleen or lymph nodes, but decreased B cells in the peritoneal cavity (PC) and blood. Notably, the B cell number decreased 3-4 fold in blood. The percentage of CD19+IgMhiIgDlo immature B cells slightly increased in the BM. Alcohol decreased marginal zone B cells and increased CD23-CD93+ immature B cells, and did not alter follicular B cells in the spleen. The percentage of CD19+CD5+ cells increased in the PC and CD19+CD5- cells decreased. The percentage of CD21+CD23-CD19+ B cells increased 2-fold in blood while CD19+CD23+ cells decreased. Alcohol consumption altered the gene expression of sphingosine-1-phosphate (S1P) receptor-1 (SIPR1) and S1P lyase. Collectively, these data indicate that alcohol decreases mature B cells in the blood and PC by impairing B cell circulation in melanoma-bearing mice. Altered S1P/S1PR1 signaling could contribute to impaired B cell circulation.
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Mountz, John D., Jennie Ann Hamilton, Qi Wu, PingAr Yang, Bao Luo, Shanrun Liu, Jun Li, and Hui-Chen Hsu. "Endogenous dsRNA and dsDNA sensing is increased in transitional B cells in BXD2 autoimmune-prone mice." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 47.2. http://dx.doi.org/10.4049/jimmunol.196.supp.47.2.
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Abstract Enhanced nucleic acid-sensing (NS) pathways has been associated with development of autoimmune disease. Increased type I IFN signaling is identified in lupus prone BXD2 mouse B cells and there is a predominant production of autoantibodies against RNA binding autoantigens in BXD2 mice. Determination of the expression of Usp18 and Oasl2 in early CD93+ transitional B cells (T1 and T2) revealed a surprising finding that early transitional B cell exhibited elevated type I IFN signature gene expression, compared to CD93− mature B cells. Consistent with this, there is also elevated expression of NS pathway genes in early transitional B cells. The most highly expressed were the dsRNA sensing Rig1 and transcription factors Irf3 and Irf7, which were expressed at higher levels compared to pDCs. Interestingly, although Usp18 and Oasl2 levels were undetectable in BXD2-Ifnar−/− transitional B cells, dsRNA sensing gene expression were highly expressed suggesting an IFNAR-independent up-regulation of dsRNA NS pathway in transitional B cells. There was lower expression of Mda5 and Pkr5, and genes involved in DNA sensing including Zbp1 and Irf9. Stimulation of B cells with a RIG1 agonist, poly(I:C)-LMW/LyoVec leads to significantly elevated induction of NS pathway genes in both WT and IFNAR deficient transitional B cells from BXD2 mice. These results suggest that transitional B cells have an intrinsic enhanced nucleic acid-sensing pathway, allowing them to rapidly react to nucleic acid stimulation. Dysregulation of this pathway as a result of apoptotic cell clearance defects can act together with type I IFN stimulation to lead to survival and maturation of B cells that by-pass tolerance checkpoint 2 and produce anti-nucleic acid autoantibodies in lupus.
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Sanchez-Aguilera, Abel, Jose Cancelas, and David A. Williams. "RhoH-Deficient Mice Show Altered B Cell Populations In Vivo." Blood 110, no. 11 (November 16, 2007): 2307. http://dx.doi.org/10.1182/blood.v110.11.2307.2307.
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Abstract RhoH is a GTPase-deficient, hematopoietic-specific member of the family of Rho GTPases (Li et al, 2002). RhoH has been described as regulating proliferation and engraftment of hematopoietic progenitor cells (Gu et al, 2005) and integrin-mediated adhesion in T cells (Cherry et al, 2004). Additionally, RhoH plays a critical role in T-cell development and T-cell receptor signaling (Gu et al, 2006; Dorn et al, 2007). However, the potential role of RhoH in the differentiation and biological functions of B cells are unknown. To answer these questions, we analyzed the B-cell phenotype of RhoH−/− mice and the in vitro properties of RhoH-deficient splenic B cells compared to their wild-type counterparts. RhoH−/− mice showed increased B-cell numbers in the bone marrow, mainly due to an increase in the number of pro-B, pre-B and immature B cells. In the spleen, lymph nodes and peripheral blood, RhoH−/− mice showed a significant decrease in the number of follicular (B-2) cells (B220+ CD93– IgDhigh CD21low). The number of splenic marginal zone B cells (B220+ CD93– IgDlow CD21high), plasma cells (CD93– CD38+ CD138+) in bone marrow and spleen, and B-1 cells (IgM+ CD5+) in peritoneal cavity were not significantly different from those in wild-type animals. These alterations have functional significance, since the serum concentrations of IgM and IgG1 were significantly lower in RhoH−/− mice. However, splenic B cells isolated from RhoH−/− mice did not show any significant differences in their in vitro activation by anti-IgM, CD40 ligation or IL-4 stimulation, nor did they differ in their proliferative response to lipopolysaccharide. In vitro migration of RhoH-deficient B cells in response to CXCL12 or CXCL13 was similar to that of wild-type B cells. Given the important role of RhoH in signal transduction downstream the T cell receptor, we investigated the possible role of RhoH in B cell receptor signaling. Although total splenic B cells from RhoH−/− mice showed markedly increased phosphorylation of SYK and ERK after anti-IgM stimulation compared to wild-type B cells, sorted populations of splenic B-2 and marginal zone B cells from RhoH−/− and wild-type animals did not differ in the activation of these kinases, suggesting that the observed difference can be attributed to the different cellular composition of the B cell compartment (i.e. B-2 vs marginal zone B cells) in RhoH−/− mice. These data imply that the phenotype observed in RhoH−/− mice may not reflect an intrinsic defect in B cells but may be attributed to crosstalk between B cells and other hematopoietic cell populations. Composition of B cell subsets in wild-type and RhoH−/− mice (total cell number ×106, ± standard deviation, N=9) Bone marrow Spleen (*) indicates p<0.05; (**), p<0.01; (***), p<0.005 RhoH+/+ RhoH−/− RhoH+/+ RhoH−/− total B cells 7.8±1.8 11.0±2.4 (**) total B cells 31.7±10.1 25.4±8.8 pro-B 0.12±0.03 0.15±0.04 (*) transitional 8.7±1.2 8.6±2.8 pre-B 2.6±0.6 3.8±0.8 (***) B-2 11.6±4.1 7.6±2.5 (*) immature 1.5±0.4 2.1±0.5 (*) marginal 3.2±1.1 3.9±1.6 mature 1.4±0.7 1.7±0.9
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Marsay, Katherine S., Sarah Greaves, Harsha Mahabaleshwar, Charmaine Min Ho, Henry Roehl, Peter N. Monk, Tom J. Carney, and Lynda J. Partridge. "Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration." PLOS ONE 16, no. 11 (November 30, 2021): e0260372. http://dx.doi.org/10.1371/journal.pone.0260372.
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Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.
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Aaron, Tonya, and David R. Fooksman. "Tumor Necrosis Factor Alpha inhibits humoral immunity by regulating the antibody secreting cell bone marrow niche." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 96.02. http://dx.doi.org/10.4049/jimmunol.206.supp.96.02.
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Abstract Humoral immunity is a critical component of the immune memory, and its longevity is partly dependent on the survival of antibody-secreting cells (ASC). Previous studies have shown that several types of infections can deplete pre-existing antibody titers, but the exact mechanism of this process is unclear. Most, if not all, forms of infection induce inflammation including Tumor Necrosis Factor Alpha (TNFa). Here we show that TNFa-mediated inflammation depletes pre-existing antibody titers by limiting ASC retention in the bone marrow (BM) survival niche. Acute recombinant TNFa (rTNFa) treatment induces ASC egress from the BM into the blood and limits engraftment of adoptively transferred ASCs into the BM of recipient mice. To determine if the TNFa-mediated ASC egress from the BM was cell-intrinsic or –extrinsic, we generated BM chimeras with wild-type and TNFa receptor knockout mice. We found that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells induces ASC egress from the BM in a cell-extrinsic manner. Treatment with rTNFa did not induce ASC death in the BM, but it increased glucose uptake and expression of CXCR4, a receptor involved in ASC retention in the BM. In contrast, mobilized ASCs in the blood had reduced glucose uptake, CD98 expression, and CD93 expression suggesting that ASC metabolism is highly dependent on the BM niche. Our findings demonstrate that inflammation alters ASC physiology in both cell-intrinsic and extrinsic manners, leading to reduced antibody titers and long-term immune memory.
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S. Kirshenbaum, Arnold, Yuzhi Yin, J. Bruce Sundstrom, Geethani Bandara, and Dean D. Metcalfe. "Description and Characterization of a Novel Human Mast Cell Line for Scientific Study." International Journal of Molecular Sciences 20, no. 22 (November 6, 2019): 5520. http://dx.doi.org/10.3390/ijms20225520.
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Background: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. Methods: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (β-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. Results: LADR cells were larger and more granulated as seen with Wright–Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. Conclusions: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.
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Qiu, Yuanyuan, Leah A. Marquez-Curtis, and Anna Janowska-Wieczorek. "Mesenchymal Stem Cells Express the Surface Receptor Calreticulin (cC1qR) and Complement C1q Chemoattracts Them." Blood 116, no. 21 (November 19, 2010): 3855. http://dx.doi.org/10.1182/blood.v116.21.3855.3855.
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Abstract Abstract 3855 Complement cleavage fragments play an important role in the trafficking of hematopoietic stem/progenitor cells as reported by us (Blood 2003;101:3784; Exp Hematol 2010;38:321; Transfusion Sept 2010), as well as of mesenchymal stem cells (MSC) as reported by others (J Immunol 2009;182:3827). Because MSC have a great potential for tissue regeneration and cellular therapies, in this study we investigated the mechanisms of migration of MSC to injured sites. As complement cascade is activated upon tissue injury we examined whether complement component 1 subcomponent q (C1q), the initiator of the classical pathway of complement activation, plays a role in the migration of MSC, and which C1q receptors (CR1, gC1qR, calreticulin (cC1qR), CD93) are expressed on MSC and could be involved in their migration. We previously reported that matrix metalloproteinases (MMPs), especially membrane type 1 (MT1)-MMP and MMP-2, regulate the migration of MSC (Stem Cells, 2006, 24:1254); therefore in this study we examined the effects of C1q on MMP expression and migration of MSC. Human MSC isolated from cord blood and bone marrow were maintained for 3–6 passages and characterized by adipocyte and osteoblast differentiation. We used chemoinvasion across the reconstituted basement membrane Matrigel to evaluate the migration of MSC, RT-PCR and flow cytometry to determine the expression of C1q receptors and, upon stimulation with C1q, zymography and Western blot to examine the expression of MMPs and ERK1/2 and PI3K/Akt signaling pathways. We found that MSC were chemoattracted by a C1q gradient in a dose-dependent manner, and MSC expressed transcripts for gC1qR, CD93 and cC1qR, but only calreticulin protein was detected on the surface of MSC. Specific antibody against calreticulin (anti-cC1qR antibody) inhibited the trans-Matrigel chemoinvasion of MSC towards C1q. Moreover, stimulation of MSC with C1q (10 μ g/mL) increased MT1-MMP expression, but no changes in MMP-2 secretion were observed. Trans-Matrigel migration of MSC towards C1q was also reduced by the MT1-MMP inhibitor EGCG. Further, the ERK1/2 inhibitor PD98059 and the PI3K inhibitor Ly294002 decreased the chemoinvasion of MSC towards C1q. In conclusion, this study indicates that: 1) C1q exerts chemoattractant activity towards MSC through its surface receptor calreticulin; 2) MT1-MMP regulates the C1q-induced trans-Matrigel migration of MSC; and 3) both the ERK1/2 and PI3K/Akt signaling pathways are involved in this process. These findings demonstrate a broader regulatory role for C1q, which functions as a chemotactic factor for MSC through its binding to surface calreticulin, and suggest a newly-found mechanism for the migration of MSC which may be important for its clinical use. Disclosures: No relevant conflicts of interest to declare.
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Märklin, Melanie, Stefanie Bugl, Marina Bechtel, Alexandra Poljak, Hans-Georg Kopp, Lothar Kanz, Anjana Rao, Stefan Wirths, and Martin R. Müller. "Ca2+/NFAT Signaling Regulates the Expression CD38 and ZAP70 in Murine B Cells and Controls B1a Cell Homeostasis." Blood 118, no. 21 (November 18, 2011): 183. http://dx.doi.org/10.1182/blood.v118.21.183.183.
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Abstract Abstract 183 NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated and relocated to the cutoplasm by a network of several kinases including CK-1, GSK-3 and DYRK. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells. Here we have analyzed the role of NFAT2 in B cell development. Analysis of the role of this family member in hematopoiesis has been complicated by the fact that deletion of this gene is embryonic lethal around ED 13 because of defects in heart valve development. To circumvent this problem we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific cd19 promoter. B cells from these mice were isolated using CD19-labeled magnetic beads and subjected to analysis by flow cytometry. While CD19+ splenocytes from conditional NFAT2 knock-out mice occurred in normal numbers, these cells showed significantly reduced expression of CD38 and ZAP70 upon stimulation with anti-IgM antibody as compared to CD19+ splenocytes from wild-type controls. The reduction of these proteins could also be detected in B cells isolated from the peripheral blood and from bone marrow and was confirmed by western blotting and quantitative RT-PCR. CD38 and ZAP70 are well characterized prognostic factors in chronic lymphocytic leukemia (CLL) and their increased expression has been shown to correlate with poor patient survival. Our data indicate that the expression of these markers is at least in part regulated by Ca2+/NFAT signaling and that deregulation of this pathway can contribute to their overexpression in disease. Next we analyzed bone marrow and peritoneal lavages from conditional NFAT knock-out mice by flow cytometry. While we found no significant differences in the abundance of B cell subpopulations in bone marrow, we detected an almost complete absence of CD5+CD43+ B1a cells in the peritoneal cavity, clearly demonstrating the requirement of NFAT2 in the development of this subclass. B1a cells are a phenotypically and functionally distinct population of B cells which are long-lived and typically express CD5, CD43 and high levels of surface IgM together with low surface IgD and CD45 (B220). A human B cell equivalent of the murine B1a cell has been suggested as the leukemic precursor cell in chronic lymphocytic leukemia (CLL). To further delineate the role of NFAT2 in the development of B1a cells we determined the abundance of B1 progenitor cells (B1P) in bone marrow and spleen by FACS analysis. In NFAT2 knock-out mice we observed a significant reduction of the frequency of B220- CD19+ CD93+ B1P cells in bone marrow (0.8% vs. 4.7%) and spleen (0.16% vs. 0.82%) demonstrating that NFAT2 is essential for normal development of this precursor cell population. In summary, our data provide strong evidence that NFAT2 is critical for the expression of CD38 and ZAP70 in B cells and substantially controls B1a cell homeostasis implicating Ca2+/NFAT signaling as a potential target for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.
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Hamilton, Jennie, Jun Li, Qi Wu, PingAr Yang, Bao Luo, Hao Li, John Bradley, et al. "Increased PKC signaling in lupus La reactive B cells promotes development of the transitional T3 population and memory B cells in autoimmune BXD2 mice (BA3P.108)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 46.5. http://dx.doi.org/10.4049/jimmunol.194.supp.46.5.
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Abstract We recently identified that lupus prone BXD2 mice exhibited defective clearance of apoptotic debris. In vivo administration of excessive autologous apoptotic cells lead to elevation of IgG anti-La13-27 responses in BXD2 mice but not normal B6 mice, suggesting chronic self-antigen stimulation plus B-cell tolerance defects may be related to the IgG anti-La13-27 responses. To interrogate if there was abnormal maturation of anti-La13-27 B cells in naïve BXD2 mice, a recently developed La13-27 tetramer was used with CD80 and PD-L2 co-staining to reveal that there was a significantly higher frequency of memory CD80+PD-L2+ La13-27+ B cells in BXD2 (17%), compared to that in B6 (3%) mouse spleens. Tetramer analysis of CD93+ transitional B cells showed that there was an abnormal skewing of the La13-27+ T3 population, but not T1 or T2 B cells, in BXD2 (40%) compared to that in B6 (18%) mouse spleens. Survival of anti-La13-27 producing B cells in BXD2 but not B6 mice was associated with increased protein kinase C (PKC) activation, as stimulation of B cells with PMA+ionomycin which promotes survival of BCR stimulated transitional B cells resulted in an increase in IgG. As T3 B cells are considered anergic B cells maintaining self-tolerance through rapid turnover in vivo, the abnormal expansion of these B cells coupled with increased BCR signaling response in BXD2 mice may present a risk for B-cell tolerance loss to La13-27, leading to maturation into IgG secreting cells.
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Muller, Laurent, Masato Mitsuhashi, Edwin Jackson, and Theresa Whiteside. "Tumor-derived exosomes differentially modulate the adenosine pathway in human resting vs activated regulatory T cells (Treg) (TUM4P.927)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 138.28. http://dx.doi.org/10.4049/jimmunol.192.supp.138.28.
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Abstract Tumor-derived exosomes (TEX) suppress functions of human immune cells, while up-regulating functions of others. Here, we show that TEX isolated from supernatants of human cultured tumor cells or plasma of cancer patients differentially modify gene and protein expression of CD39, CD73 and adenosine receptors (ADORs) in Treg, influencing production of suppressive nucleosides. Resting or pre-activated (anti-CD3/CD28 Abs) CD4+CD39+ Treg were co-incubated overnight with TEX. Cellular mRNA was analyzed by qRT-PCR for expression of CD39, CD73, A1R, A2aR, A2bR and A3R genes. Protein expression was studied by flow cytometry and western blots. Nucleoside production by Treg ± TEX in the presence of 20µM ATP was measured by mass spectrometry. In resting Treg + TEX, mRNA expression of CD39/CD73 and ADORs was reduced relative to no TEX baseline (>1 log) except for A1R mRNA expression which was elevated (> 3 logs). CD39 protein expression was up-regulated, suggesting enhanced translation and signaling via A1R. Treg + TEX + ATP produced inosine only (7900ng/mL) and no 5’-AMP or ADO. In activated Treg +TEX, CD39/CD73 mRNA expression and protein levels were elevated (p< 0.05) as was mRNA expression for all ADORs, especially A2aR, and ADO was produced. Thus, TEX differentially regulated gene expression and protein levels of the ADO pathway components in resting vs activated Treg. The TEX-induced shift from inosine to ADO production implies changes in Treg-mediated immunosuppression.
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Silverman, L. B., R. C. Wong, E. Remold-O'Donnell, D. Vercelli, J. Sancho, C. Terhorst, F. Rosen, R. Geha, and T. Chatila. "Mechanism of mononuclear cell activation by an anti-CD43 (sialophorin) agonistic antibody." Journal of Immunology 142, no. 12 (June 15, 1989): 4194–200. http://dx.doi.org/10.4049/jimmunol.142.12.4194.
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Abstract CD43 (sialophorin, gpL115) is a sialoglycoprotein expressed on a wide variety of blood cells including lymphocytes, monocytes, neutrophils, and platelets. L10, an anti-CD43 mAb, has been shown to induce monocyte-dependent activation and proliferation of human T lymphocytes. We have studied the signaling mechanism involved in this activation process. Treatment of PBMC and purified populations of T cells and monocytes with L10 induced the hydrolysis of phosphoinositides with the resultant generation of the phosphoinositide-derived second messengers diacylglycerol and inositol phosphates. This was associated with the translocation of protein kinase C from cytosol to membrane fractions and an increase in free intracellular Ca2+ in treated cells. In human leukemic T cell lines, the magnitude of signaling via CD43 did not correlate with the density of the TCR/CD3 surface expression nor with the intensity of signaling via the TCR/CD3. Moreover, a mutant derived from the leukemic T cell line HPB-ALL that was severely defective in TCR/CD3 surface expression and signaling nevertheless had normal CD43 surface expression and signaling compared with the parent cell line. It is concluded that CD43 is functionally coupled to the phospholipase C/phosphoinositides signaling pathway. In human T cells, signaling via CD43 proceeds independently of TCR/CD3. The widespread expression of CD43 suggests a potentially important role for this molecule in orchestrating the activation of multiple cell types.
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Coy, Shannon, Rumana Rashid, Sylwia Stopka, Jia-Ren Lin, Philipp Euskirchen, Jaeho Hwang, Prasidda Khadka, et al. "TAMI-45. PHENOGENOMIC CHARACTERIZATION OF IMMUNOMODULATORY PURINERGIC SIGNALING IN GLIOBLASTOMA." Neuro-Oncology 22, Supplement_2 (November 2020): ii222—ii223. http://dx.doi.org/10.1093/neuonc/noaa215.932.
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Abstract INTRODUCTION Purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within tumors such as glioblastoma, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate immunosuppressive adenosine-rich environments. However, the composition, architecture, and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype are poorly characterized. METHODS We quantified CD73 expression by immunohistochemistry in a cohort of CNS tumors [meningiomas(n=222), gliomas(n=244), ependymomas(n=44), medulloblastomas(n=24), and craniopharyngiomas(n=38)]. We used publicly-available single-cell RNA-seq data and 36-marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastoma resections to characterize CD39 and CD73-expressing populations, define the immune architecture and tumor cell-states at single cell resolution, and identify markers of clinical outcome. We used mass spectrometry imaging (MALDI-MSI) to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (n=10). RESULTS CD73 exhibited strong expression in a subset of gliomas and meningiomas but was typically not expressed in ependymomas or medulloblastomas. CD73 expression correlated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq and t-CyCIF in glioblastoma showed CD73 expression in tumor cells, and CD39 expression in macrophages and endothelial cells. MALDI-MSI showed significantly greater adenosine concentrations (3.5-fold;p=0.04) in glioblastomas with high CD73 expression. scRNA-seq showed direct correlations between stem-like mRNA expression, proliferation, and CD73 expression in DIPG. CD73 expression significantly correlated with EGFR amplification, interferon signaling, and PD-L1 expression in glioblastoma. CONCLUSIONS Phenogenomic analysis of purinergic immunomodulatory signaling revealed significant interplay between CD73 activity and genotype, adenosine concentration, differentiation-state, clinical outcome, and possible interaction between CD39-positive macrophages and CD73-positive neoplastic cells. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling.
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Metcalf, Talibah, Peter Wilkinson, Anne Wertheimer, Janko Nikolich-Zugich, and Elias Haddad. "Global analyses of monocyte subsets revealed age-related alternations after stimulation of pathogen recognition receptors." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 60.20. http://dx.doi.org/10.4049/jimmunol.196.supp.60.20.
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Abstract In the aging population, intricate interactions between the innate and adaptive immune response is not as effective compared to younger adults. Monocytes play an important role in defense against microbes and express an array of pattern recognition receptors (PRRs). We applied a comprehensive approach to evaluate the effects of a broad range of innate immune agonists (LPS, CLO97, and 5′pppRNA) on monocyte subsets isolated from healthy non-frail adults and old subjects. We analyzed transcriptome data and measured cytokine and chemokine, ROS, and NO production. Under ex vivo conditions, we observed a larger proportion of genes were specifically upregulated in the classical (CD14+CD16−) and non-classical (CD14dimCD16+) monocytes while the intermediate (CD14+CD16+) monocytes showed a smaller proportion of upregulated genes. Distinct genes observed across both age groups for CD16+ monocytes included: CX3CR1, IL21R, IFITM1/2/3, SIGLEC10, and SOD1; whereas CD16− monocytes expressed CCR1/2, SELL, CD64, CD93, CD36, and CD14. Analysis of the transcriptional profiles elicited by innate agonists revealed age-related alternations in functional pathways. After stimulation of RIGI with 5′pppRNA, we observed that classical monocytes isolated from adults uniquely expressed the RIGI MDA5 mediated induction of IFNα and IFNβ pathway, as well as the IFNα transcript, which corresponded to higher levels of IFNα production. In addition, agonists elicited higher levels of IFNγ from non-classical monocytes isolated from adults. Our findings represent a comprehensive analysis of the influence of human aging on PRRs signaling and have implications for strategies to enhance the immune response in the context of infection or immunization.
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Bareche, Yacine, Sandra Pommey, Mayra Carneiro, Laurence Buisseret, Isabelle Cousineau, Pamela Thebault, Pavel Chrobak, et al. "High-dimensional analysis of the adenosine pathway in high-grade serous ovarian cancer." Journal for ImmunoTherapy of Cancer 9, no. 3 (March 2021): e001965. http://dx.doi.org/10.1136/jitc-2020-001965.
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BackgroundHydrolysis of extracellular ATP to adenosine (eADO) is an important immune checkpoint in cancer immunology. We here investigated the impact of the eADO pathway in high-grade serous ovarian cancer (HGSC) using multiparametric platforms.MethodsWe performed a transcriptomic meta-analysis of eADO-producing CD39 and CD73, an eADO signaling gene signature, immune gene signatures and clinical outcomes in approximately 1200 patients with HGSC. Protein expression, localization and prognostic impact of CD39, CD73 and CD8 were then performed on approximately 1000 cases on tissue microarray, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry and single-cell RNA sequencing on a subset of patients.ResultsConcomitant CD39 and CD73 gene expression, as well as high levels of an eADO gene signature, were associated with worse prognosis in patients with HGSC, notably in the immunoregulatory molecular subtype, characterized by an immune-active microenvironment. CD39 was further associated with primary chemorefractory and chemoresistant human HGSC and platinum-based chemotherapy of murine HGSC was significantly more effective in CD39-deficient mice. At protein level, CD39 and CD73 were predominantly expressed by cancer-associated fibroblasts, and CD39 was expressed on severely exhausted, clonally expanded and putative tissue-resident memory TILs.ConclusionsOur study revealed the clinical, immunological, subtype-specific impacts of eADO signaling in HGSC, unveiled the chemoprotective effect of CD39 and supports the evaluation of eADO-targeting agents in patients with ovarian cancer.
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Tiwari-Heckler, Silpa, Maria Serena Longhi, James Harbison, Leo E. Otterbein, Carl J. Hauser, and Simon C. Robson. "Enhanced adenosinergic signaling and immune cell exhaustion after trauma." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 182.43. http://dx.doi.org/10.4049/jimmunol.202.supp.182.43.
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Abstract Trauma is currently the leading cause of death in young adults, in some cases due to infection as a consequence of acquired immune suppression. The pathogenesis for this is poorly understood. Extracellular ATP released from cells during injury serves as a danger molecule, but is rapidly converted to AMP by the ectonucleotidase CD39; AMP is then hydrolyzed to immunosuppressive adenosine by CD73 ecto-5’-nucleotidase. Here, we studied the impact of purinergic signaling in trauma patients over time (day 0/1 after trauma (D1) vs day 2 after trauma (D2) vs controls undergoing elective surgery). We noted higher expression of Adenosine A2A- and A2B- receptor mRNA levels in blood PBMCs on D2 and later, when compared to D1 trauma patients and control samples. Analyses of BAL-derived mononuclear cells revealed decreases in CD39 activity in D1 trauma patients and marked decreases in CD73 activity in D2 trauma patients, when compared to controls. Notably, there was increased CD39 and CD73 expression in PBMC-derived CD8 T-cells of trauma patients, suggesting these cells as the prominent subset involved in the purinergic signaling during trauma. Importantly, CD39+CD8+cells from trauma patients displayed features of “immune exhaustion” phenotype (i.e. high PD-1 and Tim-3 levels) and exhibited less cytotoxicity. We conclude that the early phase after trauma is initially characterized by heightened systemic inflammation with boosted ATP-mediated signaling, linked with loss of CD39 activity. This is followed by an immunosuppressive phase characterized by enhanced adenosinergic signaling and by increase in exhausted CD39+CD8+cells, which might impair the innate immune responses and lead to increased susceptibility to infections in trauma patients.
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YAUCH, Robert L., and Martin E. HEMLER. "Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase." Biochemical Journal 351, no. 3 (October 24, 2000): 629–37. http://dx.doi.org/10.1042/bj3510629.
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In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63–PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF–PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling.
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Katsuta, Eriko, Tao Dai, Abhisha Sawant Dessai, and Subhamoy Dasgupta. "Abstract P5-06-12: Extracellular adenosine synthesis genes regulated by estrogen signaling are associated with cancer aggressiveness and poor prognosis in estrogen receptor (ER)-positive breast cancer." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–06–12—P5–06–12. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-06-12.
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Abstract Background: Accumulation of extracellular adenosine regulates tumor progression. Extracellular adenosine binds to adenosine receptors, which mediates signaling to induce angiogenesis and cell proliferation, as well as functioning as an immunosuppressive agent in the tumor microenvironment (TME). CD73 and CD39 are two cell surface enzymes that catalyze the synthesis of extracellular adenosine from AMP, ADP and ATP in the TME. However, the underlying mechanisms that regulate adenosine synthesis in the TME of ER-positive breast cancer remains unknown. Methods: In order to investigate the transcriptional regulation of CD73 and CD39 in ER-positive breast cancer, we treated ER-positive breast cancer cell line, MCF7 with estrogen and tamoxifen. We also investigated the clinical significance of CD73/39 expression in ER-positive patients by bioinformatical approach using TCGA and GEO breast cancer cohorts. Results: In TCGA cohort, higher CD73 expression was associated with worse overall survival in ER-positive tumors (p=0.003), but not in ER-negative tumors. Gene set enrichment analysis revealed that estrogen response gene sets (Early; p=0.043, Late; p=0.021) were significantly enriched in CD73 low expressing ER-positive tumors, suggesting estrogen signaling may inhibit CD73 expression. To test this hypothesis, we analyzed the expression of CD73 and CD39 in MCF7 cells treated with estrogen, tamoxifen, or vehicle control. Our data revealed that estrogen treatment suppressed CD73 and CD39 expression, however tamoxifen treatment significantly enhanced expression of the genes. These findings suggest that tamoxifen treatment can induce the expression of CD73 and CD39 in ER-positive breast tumors, by removing the repressive effect of hormone signaling. Additionally, gene set enrichment analysis revealed that CD73-high ER-positive tumor was significantly associated with cancer aggressiveness characteristics, such as epithelial-mesenchymal transition (EMT) (p<0.001) and angiogenesis (p<0.001). On the other hand, CD73-high ER-positive tumor have significantly less infiltrating CD8-positive T cells, memory B cells and plasma cells in silico analysis, implying that CD73-high tumors have immunosuppressive environment. Further, we found that CD73 expression was significantly elevated post-chemotherapy as compared to the tumors prior to the treatment (p=0.007), and CD73 high expressing patients demonstrated worse relapse-free survival in the neoadjuvant chemotherapy cohort (p=0.003). Conclusion: Our molecular findings indicate that expression of CD73 and CD39 are transcriptionally repressed by estrogen signaling, however tamoxifen treatment reverses the effect. Increased expression of CD73 significantly correlates with worse outcomes in ER-positive breast cancer patients which may be due to immunosuppressive tumor microenvironment created by extracellular adenosine driving a pro-metastatic phenotype. Our data indicate an intriguing mechanism which could be therapeutically exploited by targeting CD73/39 to reverse ‘immune-cold’ microenvironment for the treatment of recurrent and metastatic ER-positive breast cancers. Citation Format: Eriko Katsuta, Tao Dai, Abhisha Sawant Dessai, Subhamoy Dasgupta. Extracellular adenosine synthesis genes regulated by estrogen signaling are associated with cancer aggressiveness and poor prognosis in estrogen receptor (ER)-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-12.
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Adamiak, Mateusz, Kamila Bujko, Anna Lenkiewicz, Magdalena Kucia, Janina Ratajczak, and Mariusz Z. Ratajczak. "Novel Evidence That the Ectonucleotidases CD39 and CD73, Which Are Expressed on Hematopoietic Stem/Progenitor Cells (HSPCs), Regulate Mobilization and Homing - Studies in CD39-/- and CD73-/- Mice and with Small-Molecule CD39 and CD73 Inhibitors." Blood 132, Supplement 1 (November 29, 2018): 2060. http://dx.doi.org/10.1182/blood-2018-99-110631.
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Abstract Background . Adenosine triphosphate (ATP) is an important nucleotide involved in intracellular energy transfer, but it is also released from activated cells into the extracellular space as a crucial component of the purinergic signaling network. Purinergic signaling is an ancient form of extracellular signaling that is mediated by extracellular nucleotides (EXNs), including most importantly the purine ATP and its nucleoside metabolite, adenosine. Purinergic receptors for EXNs are expressed on the surface of all cells in the body; are represented by several families of P1, P2X, and P2Y receptors; and are among the most abundant receptors in living organisms. ATP, the most important EXN, is secreted from cells through pannexin and connexin channels and is processed in the extracellular space to ADP, AMP and finally adenosine by the cell-surface-expressed ectonucleotidases CD39 and CD73. We recently reported that the ATP-adenosine axis regulates the mobilization of hematopoietic stem progenitor cells (HSPCs) (Leukemia 2018, in press,doi: 10.1038/s41375-018-0122-0). Hypothesis. We hypothesized that the ectonucleotidases CD39 and CD73 are important modulators of the ATP-adenosine axis and are new and underappreciated modulators, not only of mobilization but also of bone marrow (BM) homing of HSPCs. Materials and Methods. To shed more light on the role of purinergic signaling in the trafficking of HSPCs, we i) phenotyped murine and human HSPCs for expression of CD39 and CD73 ectonucleotidases by FACS, ii) performed mobilization and homing studies in CD39-/- and CD73-/- mice, and iii) blocked CD39 and CD73 activity by employing the small-molecule inhibitors ARL 67156 and AMP-CP, respectively. CD39- and CD73-deficient mice were mobilized with G-CSF or AMD3100, and wild type (WT) animals were exposed to CD39 and CD73 inhibitors in addition to mobilizing agents. Following mobilization, we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. To address the involvement of CD39 and CD73 expressed on the surface of HSPCs or in the hematopoietic microenvironment, we created irradiation chimeras that were subsequently mobilized with G-CSF or AMD3100. Bone marrow (BM) transplantation studies were performed by transplanting WT mice with WT BM cells or BM cells from ectonucleotidase-deficient mice. Homing efficiency was evaluated by determining i) the number of fluorochrome-labeled cells in BM 24 hours after transplantation ii) the number of CFU-S and CFU-GM progenitors that had engrafted in BM on day 12, and iii) the recovery of peripheral blood counts. Results. We found that CD39 and CD73 are expressed by murine and human HSPCs and process ATP to adenosine, which as we observed, is a novel and potent inhibitor of HSPC trafficking. In support of this finding, CD73-/- and CD39-/- mice, with reduced extracellular adenosine levels, mobilize HSPCs much better than their normal control littermates. A similar effect was observed in WT mice exposed to CD39 and CD73 small-molecule inhibitors during mobilization. Studies in irradiation chimeras revealed that this effect depended on expression of CD39 and CD73 on the surface of HSPCs and not in the BM microenvironment. Moreover, adenosine also inhibited the homing of HSPCs to BM, as cells from CD39-/- or CD73-/- mice engrafted better than WT cells. Furthermore, homing was also inhibited in the presence of exogenously added adenosine. That ectonucleotidase-deficient mice engrafted BM cells from WT littermates as efficiently as did WT recipients indicates the involvement of HSPC-expressed CD39 and CD73 in regulating the homing process. Conclusions. We demonstrate for the first time that HSPC-expressed CD39 and CD39 ectonucleotidases modulate the trafficking of HSPCs. By processing ATP to adenosine, these enzymes negatively affect both the mobilization and the homing processes. Based on this finding, future strategies employing small-molecule inhibitors of both ectonucleotidases could find practical application in improving mobilization in poor mobilizers and accelerating the engraftment of HSPCs after transplantation. Disclosures No relevant conflicts of interest to declare.
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Elsaghir, Alaa, Ehsan MW El-Sabaa, Abdulrahman K. Ahmed, Sayed F. Abdelwahab, Ibrahim M. Sayed, and Mohamed A. El-Mokhtar. "The Role of Cluster of Differentiation 39 (CD39) and Purinergic Signaling Pathway in Viral Infections." Pathogens 12, no. 2 (February 8, 2023): 279. http://dx.doi.org/10.3390/pathogens12020279.
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CD39 is a marker of immune cells such as lymphocytes and monocytes. The CD39/CD73 pathway hydrolyzes ATP and adenosine, which has a potent immunosuppressive effect. CD39 regulates the function of a variety of immunologic cells through the purinergic signaling pathways. CD39+ T cells have been implicated in viral infections, including Human Immunodeficiency Virus (HIV), Cytomegalovirus (CMV), viral hepatitis, and Corona Virus Disease 2019 (COVID-19) infections. The expression of CD39 is an indicator of lymphocyte exhaustion, which develops during chronicity. During RNA viral infections, the CD39 marker can profile the populations of CD4+ T lymphocytes into two populations, T-effector lymphocytes, and T-regulatory lymphocytes, where CD39 is predominantly expressed on the T-regulatory cells. The level of CD39 in T lymphocytes can predict the disease progression, antiviral immune responses, and the response to antiviral drugs. Besides, the percentage of CD39 and CD73 in B lymphocytes and monocytes can affect the status of viral infections. In this review, we investigate the impact of CD39 and CD39-expressing cells on viral infections and how the frequency and percentage of CD39+ immunologic cells determine disease prognosis.
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Kuptsova, D. G., T. V. Radigina, S. V. Petrichuk, N. N. Murashkin, A. A. Khotko, and R. A. Ivanov. "Assessment of CD4<sup>+</sup> cells subpopulations with the expressing CD39 and CD73 ectonucleotidases in children with psoriasis." Medical Immunology (Russia) 24, no. 3 (July 13, 2022): 587–96. http://dx.doi.org/10.15789/1563-0625-aoc-2487.
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Purinergic signaling modulates systemic and local inflammatory responses in immune-mediated and autoimmune diseases, including psoriasis. Extracellular ATP is an important factor of purinergic regulation, and its levels are regulated by catalytic effects of CD39 and CD73 ectonucleotidases. The aim of the present study was to estimate the number of regulatory T cells (Tregs), activated T-helper cells (Thact), T-helper type 17 (Th17) expressing CD39 and CD73 ectonucleotidases in children with psoriasis vulgaris, depending on age, disease duration and severity of the pathological process. We have examined a total of 114 children with psoriasis vulgaris (70 girls and 44 boys) and 41 healthy children serving as a comparison group (25 girls and 16 boys). The age of children with psoriasis was 12.5 (10.1-15.8) years, and 12.4 (7.4-16.1) years for the comparison group. The severity of psoriasis was assessed by the PASI and BSA indices. The number of cells with CD39 and CD73 expression on Tregs, Thact and Th17 was estimated by flow cytofluorimetry. The highest number of CD39-expressing cells was found in the Tregs and CD73-expressing cells in Thact, both in children with psoriasis and in the comparison group. The number of CD39+Th17 was lower in children with psoriasis, but CD39+CD73+Thact and CD39+CD73+Th17 were higher than in comparison group (p < 0.05). There was a decreased number of CD73+Tregs, CD39+Thact, CD39+Th17, CD39+CD73+Thact and CD39+CD73+Th17 with age in healthy children (p < 0.05). In patients with psoriasis, the number of CD73+Th17 increased with age. A decrease in CD73+Th17, and an increase in CD39+CD73+ Tregs with higher PASI and BSA indices were detected. An increased PASI (> 10) showed patients with both high and low CD39+Tregs, with CD39+Tregs being reduced in 48% of cases, increased in 35% and normal values in only 17% of cases. Monitoring the numbers of Tregs, Thact and Th17 cells expressing CD39 and CD73 in children with psoriasis may be used to evaluate chronic inflammation, given the role of CD39 and CD73 ectonucleotidases in shaping the immune response in immune-mediated diseases,
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Lee, Kyoung Jin, Yeon Ho Yoo, Min Seo Kim, Birendra Kumar Yadav, Yuri Kim, Dongyoung Lim, Cheol Hwangbo, et al. "CD99 inhibits CD98-mediated β1 integrin signaling through SHP2-mediated FAK dephosphorylation." Experimental Cell Research 336, no. 2 (August 2015): 211–22. http://dx.doi.org/10.1016/j.yexcr.2015.07.010.
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McMillan-Ward, Eileen, and Sara Israels. "Platelet tetraspanin complexes and their association with lipid rafts." Thrombosis and Haemostasis 98, no. 11 (2007): 1081–87. http://dx.doi.org/10.1160/th06-08-0455.
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SummaryTetraspanins are a superfamily of integral membrane proteins that facilitate the organization of membrane and intracellular signaling molecules into dynamic signaling microdomains, tetraspaninenriched microdomains (TEMs). Four tetraspanin family members have been identified in platelets: CD9, CD151 and TSSC6, which are constitutively associated with αIIb3, and CD63, which is present on granule membranes in resting platelets and associates with αIIbβ3-CD9 following platelet activation. CD63 and CD9 associate with a type II phosphatidylinositol 4-kinase, PI4K55, in both resting and activated platelets. Immunoelectron microscopic studies showed co-localization of CD63 and PI4K55 on internal membranes of resting platelets and on the filopodia of thrombin-activated platelets. Because TEMs in malignant cell lines appear to be distinct from prototypic lipid rafts, this study examined whether CD63-PI4K55 and CD9-PI4K55 complexes were resident in platelet-lipid rafts, or formed distinct microdomains. CD63, CD9 and PI4K55 were recovered from low-density membrane fractions (LDMFs) of sucrose gradients following platelet lysis in Brij 35, but unlike lipidraft proteins were not insoluble in Triton X-100, being absent from LDMFs of platelets lysed with Triton. In cubation of platelets with methyl-β-cyclodextrin, to deplete cholesterol and disrupt lipid rafts, shifted the complexes to higher density sucrose gradient fractions, but did not disrupt the tetraspanin-PI4K55 complexes. These results demonstrate that tetraspanin complexes in platelets form cholesterol-associated microdomains that are distinct from lipid rafts. It is probable thatTEMs and lipid rafts associate under certain conditions, resulting in the close proximity of distinct sets of signaling molecules, facilitating signal transduction.
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Israels, Sara J., and Eileen M. McMillan-Ward. "Palmitoylation Augments the Association of Tetraspanin CD63 with the αIIbβ3-CD9 Complex and the Actin Cytoskeleton in Thrombin-Activated Platelets." Blood 112, no. 11 (November 16, 2008): 5370. http://dx.doi.org/10.1182/blood.v112.11.5370.5370.
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Abstract CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. In resting platelets, CD63 is located in the membranes of lysosomes and dense granules, and CD9 complexes with the αIIbβ3 integrin on the platelet surface. Following platelet activation and granule exocytosis, CD63 translocates to the plasma membrane, where it co-localizes with the αIIbβ3-CD9 complex and is incorporated in the Triton-insoluble actin cytoskeleton. Tetraspanin complexes cluster dynamically in unique cholesterol-rich membrane microdomains (tetraspanin-enriched microdomains, TEMs) that differ from prototypic lipid rafts. The assembly and maintenance of TEMs depends on the palmitoylation of both tetraspanins and some of their partner proteins. Protein palmitoylation most commonly involves the thioester linkage of palmitate to a cysteine residue and, because the process is reversible, can regulate cellular functions. In cell lines, palmitoylation of tetraspanin juxta-membrane cysteine residues affects subcellular distribution, complex stability, cellular signaling and motility. Recently we demonstrated that tetraspanins and their partner proteins form TEMs in platelets, however the role of palmitoylation in platelet TEMs assembly and maintenance has not been studied. 3[H]-palmitate-labeled, washed human platelets were studied at rest, or following activation with thrombin (0.1U/ml). CD63 and CD9 were isolated by immunoprecipitation, separated by density gradient centrifugation, and 3[H]-palmitate quantitated. Palmitate levels increased in all fractions however the relative inter-fraction distribution did not change, consistent with previous results showing that the distribution of CD63 and CD9 does not change following platelet activation. 2-bromopalmitate (2- BP), which blocked palmitoylation as demonstrated by decreased 3[H]-palmitate-labeling of platelets, inhibited both thrombin-induced platelet aggregation and platelet spreading on immobilized fibrinogen, in a dose and time-dependent manner. 2-BP also inhibited the activation-dependent association of CD63 with CD9 and the incorporation of CD63 into the Triton-insoluble actin cytoskeleton in thrombin-activated platelets. In contrast 2-BP had minimal effect on either the integrity of the α IIb β3-CD9 complex or its agonist-induced association with the cytoskeleton. In summary, inhibition of palmitoylation blocked the activation-dependent association of CD63 with the αIIbβ3-CD9 complex and with the actin cytoskeleton but did not alter the tetraspanin-integrin association present in resting platelets. The effect of 2-BP on platelet spreading on fibrinogen mirrored that previously seen with anti-CD63 MoAbs, and supports the hypothesis that the association of CD63 with αIIbβ3-CD9 modulates outside-in signaling in adherent platelets.
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Israels, Sara J., and Eileen M. McMillan-Ward. "Platelet Tetraspanin Complexes and Their Relation to Lipid Rafts." Blood 108, no. 11 (November 16, 2006): 1530. http://dx.doi.org/10.1182/blood.v108.11.1530.1530.
Full textAbstract:
Abstract CD63 and CD9 are members of the tetraspanin superfamily of integral membrane proteins that function as organizers of multi-molecular signaling complexes involved in cell morphology, motility and proliferation. CD63 is located in the membranes of lysosomes and dense granules in resting platelets. Following platelet activation and granule exocytosis, CD63 is expressed on the platelet plasma membrane and co-localizes with the αIIbβ3-CD9 complex. D545, a monoclonal antibody (MoAb) directed at the second extracellular loop of CD63, inhibits activated platelet spreading on immobilized fibrinogen and FAK phosphorylation in the adherent platelets. To identify CD63-associated signaling enzymes that could be involved in the signaling complex, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase with the enzymatic properties of PI4-kinase type II, confirmed by re-precipitation and immunoblotting with 4C5G (MoAb specific for the 55kDa PI4-kinase, PI4K55). The CD63-PI4K55 complex could be co-precipitated from both resting and activated platelets using anti-CD63 MoAb, and co-localized on the filopodia of thrombin-activated platelets using immuno-electron microscopy. Previous studies have demonstrated that tetraspanins associate with cholesterol-enriched membrane domains in a variety of cells including platelets. There is evidence, however, that these tetraspanin-enriched microdomains (TEMs) can be distinguished from prototypic lipid rafts on the basis of detergent solubility and protein composition. To investigate the association of the CD63-PI4K55 complex with lipid rafts in platelets, resting and thrombin-activated platelets were lysed in buffer containing either 1% Brij 35, or Triton X-100, the low- and high-density membrane fractions separated by isopycnic sucrose gradient centrifugation, and the identification of the low-density membrane fractions (LDMF) confirmed by the presence of LAT. CD63, CD9 and PI4K55 were present in the LDMF of platelets lysed in Brij 35 but not in Triton X-100; they were also present in the denser membrane fractions. CD63 and CD9 associated with cholesterol, as demonstrated by recovery of these proteins in the pellet following centrifugation of platelets lysed with 1% digitonin(a cholesterol-precipitating reagent), but not from lysates made with Brij 35/Triton X-100. Incubation of platelets with methyl-β-cyclodextrin(mβCD) to partially deplete cholesterol and disrupt the lipid rafts shifted LAT, CD63, CD9 and PI4K55 to denser fractions within the gradient. Immunoprecipitation of mβCD-treated platelets with anti-PI4K55 MoAb co-precipitated CD63 and CD9, demonstrating that the complexes were not dependent on residence within LDMFs, but remained intact in the denser fractions and pellet. Platelet tetraspanin complexes associate with cholesterol-enriched domains under conditions of mild detergent extraction. The maintenance of the complexes, however, was not dependent on their residence within lipid rafts, as the complexes remained intact following cholesterol depletion. Their presence in LDMF suggests that tetraspanin complexes may associate with platelet lipid rafts under some conditions, which could bring tetraspanin protein partners into proximity with raft residents, and facilitate the assembly and interaction of signaling complexes following platelet activation.
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Allard, David, Pavel Chrobak, Yacine Bareche, Bertrand Allard, Priscilla Tessier, Marjorie A. Bergeron, Nathalie A. Johnson, and John Stagg. "CD73 Promotes Chronic Lymphocytic Leukemia." Cancers 14, no. 13 (June 26, 2022): 3130. http://dx.doi.org/10.3390/cancers14133130.
Full textAbstract:
The ecto-nucleotidase CD73 is an important immune checkpoint in tumor immunity that cooperates with CD39 to hydrolyze pro-inflammatory extracellular ATP into immunosuppressive adenosine. While the role of CD73 in immune evasion of solid cancers is well established, its role in leukemia remains unclear. To investigate the role of CD73 in the pathogenesis of chronic lymphocytic leukemia (CLL), Eµ-TCL1 transgenic mice that spontaneously develop CLL were crossed with CD73−/− mice. Disease progression in peripheral blood and spleen, and CLL markers were evaluated by flow cytometry and survival was compared to CD73-proficient Eµ-TCL1 transgenic mice. We observed that CD73 deficiency significantly delayed CLL progression and prolonged survival in Eµ-TCL1 transgenic mice, and was associated with increased accumulation of IFN-γ+ T cells and effector-memory CD8+ T cells. Neutralizing IFN-γ abrogated the survival advantage of CD73-deficient Eµ-TCL1 mice. Intriguingly, the beneficial effects of CD73 deletion were restricted to male mice. In females, CD73 deficiency was uniquely associated with the upregulation of CD39 in normal lymphocytes and sustained high PD-L1 expression on CLL cells. In vitro studies revealed that adenosine signaling via the A2a receptor enhanced PD-L1 expression on Eµ-TCL1-derived CLL cells, and a genomic analysis of human CLL samples found that PD-L1 correlated with adenosine signaling. Our study, thus, identified CD73 as a pro-leukemic immune checkpoint in CLL and uncovered a previously unknown sex bias for the CD73-adenosine pathway.
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Adamiak, Mateusz, Kamila Bujko, Katarzyna Brzezniakiewicz-Janus, Magda Kucia, Janina Ratajczak, and Mariusz Z. Ratajczak. "The Inhibition of CD39 and CD73 Cell Surface Ectonucleotidases by Small Molecular Inhibitors Enhances the Mobilization of Bone Marrow Residing Stem Cells by Decreasing the Extracellular Level of Adenosine." Stem Cell Reviews and Reports 15, no. 6 (September 13, 2019): 892–99. http://dx.doi.org/10.1007/s12015-019-09918-y.
Full textAbstract:
Abstract We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.
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Walker, Janek S., Casey B. Cempre, Jordan N. Skinner, Brandi R. Walker, John C. Byrd, and Rosa Lapalombella. "Simultaneous Disruption of XPO1 and A20 in Murine B Cells Influences Both B and T Cell Repertoire." Blood 138, Supplement 1 (November 5, 2021): 1542. http://dx.doi.org/10.1182/blood-2021-152777.
Full textAbstract:
Abstract Introduction & Objectives: Efforts to characterize the heterogeneity of advanced hematologic malignancies using large-scale genomic studies have identified recurrent monoallelic mutations affecting the E571 residue of the essential nuclear exporter, Exportin-1 (XPO1; E571K in ~80% of cases, E571G in ~15% of cases). E571-XPO1 mutations alter the charge and structural basis of the cargo-binding region, disrupting critical biophysical interactions between XPO1 and its' cargos. Enriched in hematologic malignancies, E571-XPO1 mutations are predominantly reported in chronic lymphocytic leukemia (CLL; 5-10% of cases), classical Hodgkin's lymphoma (~25% of cases), and primary mediastinal B cell lymphoma (PMBCL; 25-30% of cases). The subsequent change in XPO1-cargo localization alters the transcriptional profile and overall phenotype of the leukemic cell, with evidence suggesting hyper-active NF-κB and NFAT signaling pathways as leading leukemogenic mechanisms. Moreover, while overall immune dysfunction in CLL leads to infections as a major cause of morbidity and mortality, CLL patients with E571-XPO1 mutations are more susceptible to death by infection, suggesting these mutations may exacerbate the leukemia-induced immunosuppressive phenotype. Similarly, inactivating mutations/deletions to A20 (TNFAIP3 gene), the master regulator of NF-κB, are recurrently reported in several B cell malignancies but most frequently observed in PMBCL (~30% of cases). E571-XPO1 mutations and TNFAIP3 deletions/mutations have been found as co-occurring genetic abnormalities in PMBCL, and while TNFAIP3 mutations in CLL are rare, functional convergence on NF-κB and immune signaling suggests altered XPO1 and A20 activity may have unreported pathogenic significance in CLL. Thus, we aimed to explore the oncogenic and immunologic consequence of co-occurring XPO1 and A20 abnormalities by evaluating a novel in-vivo model recapitulating this scenario. Methods: To explore concurrent aberrations to XPO1 and A20, we developed a novel mouse model to recapitulate this event (Eµ-XPO1xA20 KO). This model was generated by crossing the Eµ-XPO1 transgenic mouse - which overexpresses wildtype (WT), E571K, or E571G-XPO1 under control of a VH promoter-IgH-Eµ enhancer to target transgene expression to immature and mature B cells - with a B cell-specific A20 inactivation mouse (A20 KO) - which lacks functional A20 as a result of Cre recombinase-mediated excision of TNFAIP3 exon 3 via loxP recombination sites flanking this region and Cre recombinase expressed under CD19 promoter/enhancer elements. Eµ-XPO1 and Eµ-XPO1xA20 KO mice were aged and followed, and their B and T cell repertoire was assessed via flow cytometry. Results & Conclusion: We previously demonstrated Eµ-XPO1 mice develop a CLL-like disease (CD19+/CD5+/B220dim B lymphocytes), but leukemia development is significantly delayed - evident between 20-30 months of age. Preliminary analysis in adolescent animals revealed irregular lymphocyte populations as early as 6 months of age in the blood and spleen of Eµ-XPO1xA20 KO mice when compared to non-transgenic and Eµ-XPO1 mice; highlighted by elevated populations of CD93+/CD23+ transitional B cells and CD3+ T cells, and reduced populations of CD21+/IgM+ marginal zone B cells. Moreover, development of a circulating CLL-like disease accompanied by palpable lymphadenopathy and splenomegaly was observed in Eµ-XPO1xA20 KO mice as early as 17-20 months of age, again presenting a distinct immunophenotype inconsistent with that observed in Eµ-XPO1 mice. Additionally, progressive accumulation of CD3+/CD19- T cell leukemia-like populations were observed in a subset of Eµ-XPO1xA20 KO and A20 KO mice, indicating these aberrations may further disrupt and stimulate uncontrolled proliferation affecting the overall immune repertoire. Significance: We report that simultaneous disruption of essential regulators XPO1 and A20 in murine B cells encourages development of irregular B and T cell populations, and can stimulate a progressive CLL-like or T cell leukemia-like expansion. Continued investigation with these models can further our understanding of the relationship between overall immune function and these critical regulatory molecules, and can provide considerable insight to identifying pathways for selective targeting as a personalized therapy in several high-risk cancer types. Disclosures Byrd: AstraZeneca: Consultancy; Takada: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Syndex: Consultancy; Trillium: Consultancy; Vincera Pharmaceuticals: Current equity holder in publicly-traded company.
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Mills, Jeffrey H., Cynthia Mueller, and Margaret S. Bynoe. "The key to the blood brain door: Differential signaling of the adenosine receptor subtypes regulates lymphocyte entry into the central nervous system at the choroid plexus (95.12)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 95.12. http://dx.doi.org/10.4049/jimmunol.182.supp.95.12.
Full textAbstract:
Abstract Lymphocyte entry into the central nervous system (CNS) is a highly regulated process which serves to promote tissue repair (when required) while protecting against potentially fatal inflammation. As the formation of extracellular adenosine (catalyzed from ATP by CD39 and CD73) during inflammation serves as a negative-feedback signal to prevent excessive cell damage, our results suggest that adenosine receptor (AR) signaling also regulates CNS lymphocyte infiltration. We have recently shown that CD73 and AR signaling are required for CNS lymphocyte entry during experimental autoimmune encephalomyelitis (EAE), the animal model of autoimmune inflammatory disease Multiple Sclerosis. We now show that CD39, CD73, and the A1- and A2A-AR subtypes are found clustered in the CNS only at the choroid plexus, a structure which regulates lymphocyte migration from the blood to the cerebrospinal fluid. Moreover, while A1- and A2A-AR activation have functionally opposing cellular effects (cAMP studies), mice given A2AAR antagonists are protected against EAE and CNS lymphocyte infiltration while A1AR-/- mice develop severe EAE. Additionally, AR signaling can induce lymphocyte transmigration across an in vitro choroid plexus transwell barrier while A2AAR activation promotes the upregulation of ICAM-1. Therefore, we conclude that AR signaling at the choroid plexus mediates CNS lymphocyte entry. AI 57854 and NS 063011
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Zhang, Bin, YinWei Ho, Tinisha McDonald, Allen Lin, David S. Snyder, Vu N. Ngo, Tessa L. Holyoake, and Ravi Bhatia. "Role of Enhanced Microenvironmental Interleukin-1 (IL-1) Expression and Increased IL-1 Responsiveness in Persistence of Leukemia Stem Cells in TKI Treated CML Patients." Blood 124, no. 21 (December 6, 2014): 4357. http://dx.doi.org/10.1182/blood.v124.21.4357.4357.
Full textAbstract:
Abstract BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in the treatment of chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. In previous studies we have shown that altered expression of inflammatory cytokines in the CML bone marrow (BM) microenvironment provides a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) (Cancer Cell 2012, 21:577). Our studies suggest an important role for the pivotal pro-inflammatory cytokine Interleukin-1α/β (IL-1α/β) in selectively promoting growth of CML LTHSC. Using a transgenic BCR-ABL mouse model of CML and human CML and normal CD34+CD38- cells, we showed that inhibition of IL-1 signaling using recombinant IL-1 receptor antagonist (IL-1RA) in combination with nilotinib (NIL) resulted in significantly greater inhibition of CML LSC, compared with NIL alone (Blood 2013, abstract 512). To further investigate the mechanisms underlying increased IL-1 sensitivity of CML stem cells, we evaluated expression of the IL-1 receptor components, IL-1 receptor-associated protein (IL-1RAP) and IL-1R1, on CML and normal stem cells using flow cytometry. Expression of both IL-1RAP and IL-1R1 were increased on primary CML CD34+CD38-CD90+ cells compared to their normal counterparts (n=5, p<0.05). Exposure to IL-1α (10ng/ml) resulted in increased expression of p-NF-kB (p65), p-p38 MAPK and p-JNK in CML compared to normal CD34+CD38-CD90+ cells as evaluated by flow cytometry, indicating enhanced sensitivity to IL-1 induced signaling (n=5, p<0.05). The expression of p-NF-kB(p65), p-p38 MAPK and p-JNK in CML CD34+CD38-CD90+ cells cultured in CML BM conditioned medium (CM) was reduced after treatment with NIL or IL-1RA, and further reduced by the combination of NIL and IL-1RA (n=4, p<0.05). Immunohistochemistry (IHC) analysis showed that nuclear NF-κB p65 protein was reduced in NIL and IL-1RA treated CML CD34+CD38-CD90+ cells compared with controls. Treatment with NIL and IL-1RA also significantly reduced expression of the NF-κB target genes NFκB1A, BCL2L1, BIRC3 and CD83 (n=6, p<0.001), and of the inflammatory cytokines IL6, CXCL1, CXCL2, CCL2, CCL3, CCL4 and TNF-α (n=6, p<0.05), as assessed by Q-RT-PCR. We evaluated IL-1 expression in BM samples from CML patients with undetectable minimal residual disease (UMRD) using Q-RT-PCR. Interestingly IL-1α, but not IL-1β, expression was increased in BM samples from CML patients with UMRD compared to normal BM samples (n=12, p<0.05). To evaluate the source of increased IL-1α expression we analyzed selected monocyte (CD45+CD14+), non-monocytic myeloid cell (CD45+CD14-CD33+), T cell (CD45+CD14-CD33-CD3+), B cell (CD45+CD14-CD33-CD19+), endothelial cell (CD45-GPA-CD31+) and mesenchymal cell (CD45-GPA-CD31-) populations from BM samples obtained from CML patients with UMRD and from normal healthy controls. These studies revealed significantly elevated IL-1α expression in BM CD14+ monocytic and CD31+ endothelial cells from CML patients with UMRD compared to normal controls (n=12, p<0.05). Our studies indicate that CML LSC demonstrate increased IL-1 receptor expression and IL-1 induced NF-kB, p38 MAPK and JNK signaling. We also observe enhanced IL-1α expression in BM endothelial and monocytic cells from CML patients achieving UMRD, indicating persistence of an inflammatory microenvironment that may contribute to persistence of residual LSC. Our studies provide a strong rationale for the application of anti-IL-1 directed strategies to inhibit inflammatory signaling and enhance LSC elimination in TKI treated CML patients. Disclosures No relevant conflicts of interest to declare.
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Roliano, Gabriela Gonçalves, Juliana Hofstätter Azambuja, Veronica Toniazzo Brunetto, Hannah Elizabeth Butterfield, Antonio Nochi Kalil, and Elizandra Braganhol. "Colorectal Cancer and Purinergic Signalling: An Overview." Cancers 14, no. 19 (October 6, 2022): 4887. http://dx.doi.org/10.3390/cancers14194887.
Full textAbstract:
Colorectal cancer (CRC) is among the most common cancers and exhibits a high fatality rate. Gut inflammation is related to CRC, with loss of homeostasis in immune cell activities. The cells of the innate and adaptive immune system, including macrophages, neutrophils, mast cells, and lymphocytes, are present in most solid tumors. Purinergic signaling allows for communication between immune cells within the tumor microenvironment (TME) and can alter the TME to promote tumor progression. This system is regulated by the availability of extracellular purines to activate purinoceptors (P1 and P2) and is tightly controlled by ectonucleotidases (E-NPP, CD73/CD39, ADA) and kinases, which interact with and modify nucleotides and nucleosides availability. In this review, we compiled articles detailing the relationship of the purinergic system with CRC progression. We found that increased expression of CD73 leads to the suppression of effector immune cell functions and tumor progression in CRC. The P1 family purinoceptors A1, A2A, and A2B were positively associated with tumor progression, but A2B resulted in increased cancer cell apoptosis. The P2 family purinoceptors P2X5, P2X7, P2Y2, P2Y6, and P2Y12 were factors primarily associated with promoting CRC progression. In summary, CD39/CD73 axis and the purinergic receptors exhibit diagnostic and prognostic value and have potential as therapeutic targets in CRC.
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Dianzani, U., V. Redoglia, M. Bragardo, C. Attisano, A. Bianchi, D. Di Franco, U. Ramenghi, H. Wolff, L. F. Thompson, and A. Pileri. "Co-stimulatory signal delivered by CD73 molecule to human CD45RAhiCD45ROlo (naive) CD8+ T lymphocytes." Journal of Immunology 151, no. 8 (October 15, 1993): 3961–70. http://dx.doi.org/10.4049/jimmunol.151.8.3961.
Full textAbstract:
Abstract CD73 is a molecule expressed by a subset of CD8+ human T lymphocytes and is involved in T cell activation. CD73 expression and function were analyzed in peripheral blood CD45RAhiCD45ROlo (naive) and CD45RAloCD45ROhi (memory) CD8+ cells. We found that CD73 was expressed by a majority of naive cells (74 +/- 12%), whereas fewer memory cells were CD73+ (29 +/- 10%). Moreover, CD73 was selectively expressed by the CD11b- subset of naive CD8+ cells, which were almost all CD73+. The same result was found on CD8+ cord blood lymphocytes, which prevalently display the naive phenotype. Naive CD8+ CD11b- cells were almost unresponsive to CD3 engagement, but this apparent anergy was completely overcome when CD3 and CD73 were simultaneously cross-linked by plastic-immobilized CD73 and CD3 mAb, showing that CD73 delivers an accessory signal that allows their activation via the CD3/TCR. This costimulatory signal was tenfold more potent than that induced by CD28 ligation. A phosphotyrosine analysis by Western blotting showed that cross-linking of CD73 induced the phosphorylation of two proteins with a molecular mass of approximately 28 and 100 kDa respectively, whereas ligation of CD3 induced phosphorylation of many substrates. When CD3 and CD73 were simultaneously triggered these substrates were hypophosphorylated. Because CD73 is linked to the cell surface by a GPI anchor, the transduction of this signal is probably mediated by a lateral interaction with transmembrane molecules. This hypothesis was assessed by cocapping, which showed that CD73 associates strongly with CD45RC, moderately with CD8, and weakly with CD3. These data suggest that CD73 signaling is coupled to both tyrosine kinase and phosphatase activities.
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Passarelli, Anna, Marco Tucci, Francesco Mannavola, Claudia Felici, and Francesco Silvestris. "The metabolic milieu in melanoma: Role of immune suppression by CD73/adenosine." Tumor Biology 41, no. 4 (April 2019): 101042831983713. http://dx.doi.org/10.1177/1010428319837138.
Full textAbstract:
The mechanisms leading to immune escape of melanoma have been largely investigated in relation to its tumour immunogenicity and features of inflamed microenvironment that promote the immune suppression during the disease progression. These findings have recently led to advantages in terms of immunotherapy-based approaches as rationale for overcoming the immune escape. However, besides immune checkpoints, other mechanisms including the adenosine produced by ectonucleotidases CD39 and CD73 contribute to the melanoma progression due to the immunosuppression induced by the tumour milieu. On the other hand, CD73 has recently emerged as both promising therapeutic target and unfavourable prognostic biomarker. Here, we review the major mechanisms of immune escape activated by the CD39/CD73/adenosine pathway in melanoma and focus potential therapeutic strategies based on the control of CD39/CD73 downstream adenosine receptor signalling. These evidences provide the basis for translational strategies of immune combination, while CD73 would serve as potential prognostic biomarker in metastatic melanoma.
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Carena, Ilaria, Abdijapar Shamshiev, Alena Donda, Marco Colonna, and Gennaro De Libero. "Major Histocompatibility Complex Class I Molecules Modulate Activation Threshold and Early Signaling of T Cell Antigen Receptor–γ/δ Stimulated by Nonpeptidic Ligands." Journal of Experimental Medicine 186, no. 10 (November 17, 1997): 1769–74. http://dx.doi.org/10.1084/jem.186.10.1769.
Full textAbstract:
Killer cell inhibitory receptors and CD94-NKG2-A/B heterodimers are major histocompatibility complex class I–specific inhibitory receptors expressed by natural killer cells, T cell antigen receptor (TCR)-γ/δ cells, and a subset of TCR-α/β cells. We studied the functional interaction between TCR-γ/δ and CD94, this inhibitory receptor being expressed on the majority of γ/δ T cells. When engaged by human histocompatibility leukocyte antigen class I molecules, CD94 downmodulates activation of human TCR-γ/δ by phosphorylated ligands. CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen. CD94 engagement has major effects on TCR signaling cascade. It facilitates recruitment of SHP-1 phosphatase to TCR–CD3 complex and affects phosphorylation of Lck and ZAP-70 kinase, but not of CD3 ζ chain upon TCR triggering. These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.
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Mills, Jeffrey, Leah Alabanza, Cynthia Mueller, and Margaret Bynoe. "Extracellular adenosine triggers lymphocyte entry into the central nervous system during experimental autoimmune encephalomyelitis by regulating chemokine and adhesion molecule expression in the brain (44.12)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 44.12. http://dx.doi.org/10.4049/jimmunol.184.supp.44.12.
Full textAbstract:
Abstract Lymphocyte entry into the central nervous system (CNS) is a highly regulated process which serves to promote tissue repair while protecting against potentially fatal inflammation. As the formation of extracellular adenosine (catalyzed from ATP by CD39 and CD73) during inflammation serves as a negative-feedback signal to prevent excessive cell damage, our results suggest that adenosine receptor (AR) signaling also regulates CNS lymphocyte infiltration. We have shown that extracellular adenosine is required for the progression experimental autoimmune encephalomyelitis (EAE), the animal model of autoimmune inflammatory disease Multiple Sclerosis. We now show that AR signaling induces the CNS expression of the chemokine CX3CL1 (fractalkine), which acts as a chemoattractant for monocytes, lymphocytes, and NK cells. In addition, CX3CL1 expression is up-regulated in the CNS immediately prior to EAE onset only in wild type, but not CD73-/- mice, which cannot produce extracellular adenosine, are protected from EAE, and have minimal CNS lymphocyte infiltration following EAE induction. Moreover, CD39, CD73, and ARs are found clustered in the brain only at the choroid plexus, a structure which is known to regulate lymphocyte migration into the CNS. As AR signaling can induce CX3CL1 and ICAM-1 expression and lymphocyte transmigration across an in vitro choroid plexus transwell barrier, we conclude that AR signaling regulates lymphocyte entry into the CNS.
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Yang, Xiuwei, Oleg V. Kovalenko, Wei Tang, Christoph Claas, Christopher S. Stipp, and Martin E. Hemler. "Palmitoylation supports assembly and function of integrin–tetraspanin complexes." Journal of Cell Biology 167, no. 6 (December 20, 2004): 1231–40. http://dx.doi.org/10.1083/jcb.200404100.
Full textAbstract:
As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (α3, α6, and β4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of β4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient β4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core α6β4–CD151 complex formation was unaltered. There is also a functional connection between CD9 and β4 integrins, as evidenced by anti-CD9 antibody effects on β4-dependent cell spreading. Notably, β4 palmitoylation neither increased localization into “light membrane” fractions of sucrose gradients nor decreased solubility in nonionic detergents—hence it does not promote lipid raft association. Instead, palmitoylation of β4 (and of the closely associated tetraspanin CD151) promotes CD151–α6β4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.
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Polancec, Zenic, Hudetz, Boric, Jelec, Rod, Vrdoljak, et al. "Immunophenotyping of a Stromal Vascular Fraction from Microfragmented Lipoaspirate Used in Osteoarthritis Cartilage Treatment and Its Lipoaspirate Counterpart." Genes 10, no. 6 (June 21, 2019): 474. http://dx.doi.org/10.3390/genes10060474.
Full textAbstract:
Osteoarthritis (OA) is a degenerative joint disease accompanied by pain and loss of function. Adipose tissue harbors mesenchymal stem/stromal cells (MSC), or medicinal signaling cells as suggested by Caplan (Caplan, 2017), used in autologous transplantation in many clinical settings. The aim of the study was to characterize a stromal vascular fraction from microfragmented lipoaspirate (SVF-MLA) applied for cartilage treatment in OA and compare it to that of autologous lipoaspirate (SVF-LA). Samples were first stained using a DuraClone SC prototype tube for the surface detection of CD31, CD34, CD45, CD73, CD90, CD105, CD146 and LIVE/DEAD Yellow Fixable Stain for dead cell detection, followed by DRAQ7 cell nuclear dye staining, and analyzed by flow cytometry. In SVF-LA and SVF-MLA samples, the following population phenotypes were identified within the CD45- fraction: CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells (SA-ASC). The immunophenotyping profile of SVF-MLA was dominated by a reduction of leukocytes and SA-ASC, and an increase in EP, evidencing a marked enrichment of this cell population in the course of adipose tissue microfragmentation. The role of EP in pericyte-primed MSC-mediated tissue healing, as well as the observed hormonal implication, is yet to be investigated.
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Waclavicek, Martina, Otto Majdic, Thomas Stulnig, Markus Berger, Raute Sunder-Plassmann, Gerhard J. Zlabinger, Thomas Baumruker, et al. "CD99 Engagement on Human Peripheral Blood T Cells Results in TCR/CD3-Dependent Cellular Activation and Allows for Th1-Restricted Cytokine Production." Journal of Immunology 161, no. 9 (November 1, 1998): 4671–78. http://dx.doi.org/10.4049/jimmunol.161.9.4671.
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Abstract We have assessed the functional effect of CD99 engagement on resting human peripheral blood (PB) T cells. CD99, as detected by the mAb 3B2/TA8, is constitutively expressed on all PB T cells and becomes further up-regulated upon cellular activation. In this study we demonstrate that cross-linking of the CD99 molecule with the agonistic mAb 3B2/TA8 cooperates with suboptimal TCR/CD3 signals, but not with phorbol ester, ionomycin, or CD28 mAb stimulation, to induce proliferation of resting PB T cells. Comparable stimulatory effects were observed with the CD99 mAb 12E7. Characterization of the signaling pathways involved revealed that CD99 engagement leads to the elevation of intracellular Ca2+, which is dependent on the cell surface expression of the TCR/CD3 complex. No CD99 mAb-induced calcium mobilization was observed on TCR/CD3-modulated or TCR/CD3-negative T cells. To examine the impact of CD99 stimulation on subsequent cytokine production by T cells, we cross-linked CD99 molecules in the presence of a suboptimal TCR/CD3 trigger followed by determination of intracellular cytokine levels. Significantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-α and IFN-γ after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-γ when stimulated in a similar fashion. We conclude that CD99 is a receptor that mediates TCR/CD3-dependent activation of resting PB T cells and specifically induces Th1-type cytokine production in polyclonally activated T cell lines, Th1 and Th0 clones.
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Klysz, Dorota, Meena Malipatlolla, Katherine Freitas, Malek Bashti, Louai Labanieh, Peng Xu, Cecilia Ramello, et al. "Abstract 1362: Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1362. http://dx.doi.org/10.1158/1538-7445.am2022-1362.
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Abstract Chimeric Antigen Receptor (CAR) T cell therapy has resulted in remarkable clinical outcomes in the context of acute and chronic lymphoblastic leukemia, but remains unsuccessful in the treatment of solid tumors. One reason for this failure is thought to be T cell dysfunction or exhaustion promoted by suppressive soluble factors within the tumor microenvironment (TME). High extracellular levels of the immunosuppressive factor adenosine (Ado) are generated in the TME via breakdown of ATP by ecto-enzymes CD39 and CD73 expressed on tumor-infiltrating immune cells. Binding of extracellular Ado to its receptor A2a on T cells results in inhibition of proliferation and effector function. Interestingly, CD39 has recently been described as a surrogate marker of exhaustion on human CAR-T cells and non-engineered T cells. Therefore, we hypothesized that CD39 expression on exhausted CAR-T cells promotes dysfunction through generation of extracellular adenosine. Using an in vitro model of T cell exhaustion, whereby human T cells express a CAR that tonically signals in an antigen-independent manner (HA CAR), we demonstrate that exhausted HA CAR T cells actively hydrolyze extracellular ATP via their elevated expression of CD39 and CD73. Moreover, exhausted CD39+ CAR T cells upregulate several genes associated with a Treg phenotype at the mRNA and protein levels, suggesting that this cell population might be suppressive. To assess whether CD39+/CD73+ CAR T cells exhibit suppressive functions, we co-cultured them with non-exhausted CD19-CAR T cells. Indeed, proliferation and secretion of IL-2 by CD19 CAR T cells were diminished when they were co-cultured with exhausted CD39+ CAR T cells, and that this suppression is dependent on the A2a receptor. Using this knowledge, we used gene-editing and overexpression approaches to engineer CAR-T cells with resistance to suppressive adenosine signaling. In contrast to genetic deletion of CD39 or CD73, which did not alleviate CAR T cell dysfunction, genetic deletion of adenosine receptor A2aR in exhausted CAR T cells resulted in phenotypic changes and a modest improvement in tumor-specific killing. Further, ectopic overexpression of adenosine deaminase (ADA) in CAR T cells led to decreased exhaustion marker expression and significantly enhanced effector function. These data indicate that ADA overexpression is an innovative approach to increase the functionality of CAR T cells through avoidance of suppressive adenosine signaling, and provides proof-of-concept that metabolic engineering of CAR-T cells can pave the way for responses in patients with solid tumors. Citation Format: Dorota Klysz, Meena Malipatlolla, Katherine Freitas, Malek Bashti, Louai Labanieh, Peng Xu, Cecilia Ramello, Amaury Lerust, Hyatt Balke Want, Kaithlen Zen Pacheco, Evan W. Weber, Shabnum Patel, Steven Feldman, Elena Sotillo, Crystal L. Mackall. Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1362.
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Zhong, Tingting, Zhaoliang Huang, Xinghua Pang, Na Chen, Xiaoping Jin, Yu Xia, Zhongmin Maxwell Wang, Baiyong Li, and Yu Xia. "702 Dual blockade of the PD-1 checkpoint pathway and the adenosinergic negative feedback signaling pathway with a PD-1/CD73 bispecific antibody for cancer immune therapy." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A744. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0702.
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BackgroundCD73 (ecto-5’-nucleotidase) is an ecto-nucleotidase that dephosphorylate AMP to form adenosine. Activation of adenosine signaling pathway in immune cells leads to the suppression of effector functions, down-regulate macrophage phagocytosis, inhibit pro-inflammatory cytokine release, as well as yield aberrantly differentiated dendritic cells producing pro-tumorigenic molecules.1 In the tumor microenvironment, adenosinergic negative feedback signaling facilitated immune suppression is considered an important mechanism for immune evasion of cancer cells.2 3 Combination of CD73 and anti-PD-1 antibody has shown promising activity in suppressing tumor growth. Hence, we developed AK119, an anti- human CD73 monoclonal antibody, and AK123,a bi-specific antibody targeting both PD-1 and CD73 for immune therapy of cancer.MethodsAK119 is a humanized antibody against CD73 and AK123 is a tetrameric bi-specific antibody targeting PD-1 and CD73. Binding assays of AK119 and AK123 to antigens, and antigen expressing cells were performed by using ELISA, Fortebio, and FACS assays. In-vitro assays to investigate the activity of AK119 and AK123 to inhibit CD73 enzymatic activity in modified CellTiter-Glo assay, to induce endocytosis of CD73, and to activate B cells were performed. Assay to evaluate AK123 activity on T cell activation were additionally performed. Moreover, the activities of AK119 and AK123 to mediate ADCC, CDC in CD73 expressing cells were also evaluated.ResultsAK119 and AK123 could bind to its respective soluble or membrane antigens expressing on PBMCs, MDA-MB-231, and U87-MG cells with high affinity. Results from cell-based assays indicated that AK119 and AK123 effectively inhibited nucleotidase enzyme activity of CD73, mediated endocytosis of CD73, and induced B cell activation by upregulating CD69 and CD83 expression on B cells, and showed more robust CD73 blocking and B cell activation activities compared to leading clinical candidate targeting CD73. AK123 could also block PD-1/PD-L1 interaction and enhance T cell activation.ConclusionsIn summary, AK119 and AK123 represent good preclinical biological properties, which support its further development as an anti-cancer immunotherapy or treating other diseases.ReferencesDeaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007; 204:1257–65.Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood. 1997; 90:1600–10.Novitskiy SV, Ryzhov S, Zaynagetdinov R, Goldstein AE, Huang Y, Tikhomirov OY, Blackburn MR, Biaggioni I,Carbone DP, Feoktistov I, et al. Adenosine receptors in regulation of dendritic cell differentiation and function. Blood 2008; 112:1822–31.
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Ryzhov, Sergey V., Michael W. Pickup, Anna Chytil, Agnieszka E. Gorska, Qinkun Zhang, Philip Owens, Igor Feoktistov, Harold L. Moses, and Sergey V. Novitskiy. "Role of TGF-β Signaling in Generation of CD39+CD73+ Myeloid Cells in Tumors." Journal of Immunology 193, no. 6 (August 15, 2014): 3155–64. http://dx.doi.org/10.4049/jimmunol.1400578.
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Massaia, M., L. Perrin, A. Bianchi, J. Ruedi, C. Attisano, D. Altieri, G. T. Rijkers, and L. F. Thompson. "Human T cell activation. Synergy between CD73 (ecto-5'-nucleotidase) and signals delivered through CD3 and CD2 molecules." Journal of Immunology 145, no. 6 (September 15, 1990): 1664–74. http://dx.doi.org/10.4049/jimmunol.145.6.1664.
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Abstract Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.
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Dragić, Milorad, Nataša Mitrović, Marija Adžić, Nadežda Nedeljković, and Ivana Grković. "Microglial- and Astrocyte-Specific Expression of Purinergic Signaling Components and Inflammatory Mediators in the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration." ASN Neuro 13 (January 2021): 175909142110448. http://dx.doi.org/10.1177/17590914211044882.
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The present study examined the involvement of purinergic signaling components in the rat model of hippocampal degeneration induced by trimethyltin (TMT) intoxication (8 mg/kg, single intraperitoneal injection), which results in behavioral and neurological dysfunction similar to neurodegenerative disorders. We investigated spatial and temporal patterns of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5′ nucleotidase (eN/CD73) activity, their cell-specific localization, and analyzed gene expression pattern and/or cellular localization of purinoreceptors and proinflammatory mediators associated with reactive glial cells. Our study demonstrated that all Iba1+ cells at the injured area, irrespective of their morphology, upregulated NTPDase1/CD39, while induction of eN/CD73 has been observed at amoeboid Iba1+ cells localized within the hippocampal neuronal layers with pronounced cell death. Marked induction of P2Y12R, P2Y6R, and P2X4-messenger RNA at the early stage of TMT-induced neurodegeneration might reflect the functional properties, migration, and chemotaxis of microglia, while induction of P2X7R at amoeboid cells probably modulates their phagocytic role. Reactive astrocytes expressed adenosine A1, A2A, and P2Y1 receptors, revealed induction of complement component C3, inducible nitric oxide synthase, nuclear factor-kB, and proinflammatory cytokines at the late stage of TMT-induced neurodegeneration. An increased set of purinergic system components on activated microglia (NTPDase1/CD39, eN/CD73, and P2X7) and astrocytes (A1R, A2AR, and P2Y1), and loss of homeostatic glial and neuronal purinergic pathways (P2Y12 and A1R) may shift purinergic signaling balance toward excitotoxicity and inflammation, thus favoring progression of pathological events. These findings may contribute to a better understanding of the involvement of purinergic signaling components in the progression of neurodegenerative disorders that could be target molecules for the development of novel therapies.
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Festag, J., T. Thelemann, M. Schell, S. Raith, S. Michel, R. Klar, and F. Jaschinski. "P03.02 Suppression of T-cell proliferation and cytokine release by the adenosine axis are mediated by different mechanisms." Journal for ImmunoTherapy of Cancer 8, Suppl 2 (October 2020): A22.2—A23. http://dx.doi.org/10.1136/jitc-2020-itoc7.42.
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BackgroundThe so-called adenosine axis has emerged as a promising therapeutic target pathway as high adenosine levels in the tumor microenvironment contribute to the suppression of antitumor immune responses. The ectonucleotidases CD39 and CD73 act in concert to degrade extracellular immune-stimulating adenosine triphosphate (ATP) to immunosuppressive adenosine. According to the current model, subsequent suppression of effector immune cell function is caused by binding of adenosine to adenosine receptors like the A2a receptor (A2aR). The ectonucleotidases CD39 and CD73 as well as the A2aR have emerged as molecular targets within the adenosine axis with currently more than 20 clinical trials investigating antitumor effects of CD39-, CD73- or A2aR blockade. We aimed to perform a direct comparison of these targets with regard to their roles in regulating T-cell proliferation and IFN-γ secretion.Materials and MethodsCD39 and CD73 expression was suppressed using LNAplusTM antisense oligonucleotides (ASOs). ASOs were synthesized as gapmers with flanking locked nucleic acids (LNA) to increase stability and affinity to the target RNA, leaving a central gap for recruitment of the RNA-degrading enzyme RNaseH I. Knockdown efficacy of ASOs on mRNA and protein level was investigated in primary human T cells. Furthermore, the effects of ATP, AMP and adenosine analogues on T–cell proliferation and IFN–γ secretion were investigated. A2aR was blocked using small molecule inhibitors that are currently under clinical investigation.ResultsTreatment of human T cells with LNA-modified ASOs specific for human CD39 and CD73 resulted in potent target knockdown in vitro without the use of a transfection reagent. T-cell proliferation was reduced after addition of ATP to activated T cells that was completely reverted by ASO-mediated suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogues inhibited IFN–γ secretion of activated T cells, however, they did not suppress T-cell proliferation. Blockade of the adenosine kinase was able to revert the anti-proliferative effect of ATP degradation products, arguing for downstream metabolites of adenosine, but not A2aR signaling, being responsible for the suppression of T-cell proliferation.ConclusionsCytokine secretion and proliferation of T cells might be differentially regulated by the adenosine axis. Adenosine might primarily affect cytokine secretion via A2aR signaling, whereas adenosine metabolites might especially impair proliferation of activated T cells independent from A2aR signaling. Therefore, inhibition of CD39 and/or CD73 holds exceptional advantages over A2aR blockade as both, A2aR dependent and A2aR independent effects of ATP degradation products are targeted simultaneously.Disclosure InformationJ. Festag: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. T. Thelemann: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. M. Schell: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. S. Raith: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. S. Michel: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. R. Klar: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG. F. Jaschinski: A. Employment (full or part-time); Significant; Secarna Pharmaceuticals GmbH & Co. KG.
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Fulda, Simone, Gudrun Strauss, Eric Meyer, and Klaus-Michael Debatin. "Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells." Blood 95, no. 1 (January 1, 2000): 301–8. http://dx.doi.org/10.1182/blood.v95.1.301.
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Abstract Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)
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Fulda, Simone, Gudrun Strauss, Eric Meyer, and Klaus-Michael Debatin. "Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells." Blood 95, no. 1 (January 1, 2000): 301–8. http://dx.doi.org/10.1182/blood.v95.1.301.001k24_301_308.
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Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)