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1
Rivière, Elodie, Juliette Pascaud, Nicolas Tchitchek, Saida Boudaoud, Audrey Paoletti, Bineta Ly, Anastasia Dupré, et al. "Salivary gland epithelial cells from patients with Sjögren’s syndrome induce B-lymphocyte survival and activation." Annals of the Rheumatic Diseases 79, no. 11 (August 25, 2020): 1468–77. http://dx.doi.org/10.1136/annrheumdis-2019-216588.
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ObjectivePrimary Sjögren's syndrome (pSS) is characterised by chronic hyperactivation of B lymphocytes. Salivary gland epithelial cells (SGECs) could play a role in promoting B-lymphocyte activation within the target tissue. We aimed to study the interactions between SGECs from patients with pSS or controls and B lymphocytes.MethodsPatients had pSS according to 2016 European League Against Rheumatism/American College of Rheumatology criteria. Gene expression analysis of SGECs and B lymphocytes from pSS and controls isolated from salivary gland biopsies and blood was performed by RNA-seq. SGECs from pSS and controls were cocultured with B-lymphocytes sorted from healthy donor blood and were stimulated. Transwell and inhibition experiments were performed.ResultsGene expression analysis of SGECs identified an upregulation of interferon signalling pathway and genes involved in immune responses (HLA-DRA, IL-7 and B-cell activating factor receptor) in pSS. Activation genes CD40 and CD48 were upregulated in salivary gland sorted B lymphocytes from patients with pSS. SGECs induced an increase in B-lymphocyte survival, which was higher for SGECs from patients with pSS than controls. Moreover, when stimulated with poly(I:C), SGECs from patients with pSS induced higher activation of B-lymphocytes than those from controls. This effect depended on soluble factors. Inhibition with anti-B-cell activating factor, anti-A proliferation-inducing ligand, anti-interleukin-6-R antibodies, JAK1/3 inhibitor or hydroxychloroquine had no effect, conversely to leflunomide, Bruton's tyrosine kinase (BTK) or phosphatidyl-inositol 3-kinase (PI3K) inhibitors.ConclusionsSGECs from patients with pSS had better ability than those from controls to induce survival and activation of B lymphocytes. Targeting a single cytokine did not inhibit this effect, whereas leflunomide, BTK or PI3K inhibitors partially decreased B-lymphocyte viability in this model. This gives indications for future therapeutic options in pSS.
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Ilangumaran, Subburaj, Anne Briol, and Daniel C. Hoessli. "CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes." Blood 91, no. 10 (May 15, 1998): 3901–8. http://dx.doi.org/10.1182/blood.v91.10.3901.
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Abstract CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.
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Ilangumaran, Subburaj, Anne Briol, and Daniel C. Hoessli. "CD44 Selectively Associates With Active Src Family Protein Tyrosine Kinases Lck and Fyn in Glycosphingolipid-Rich Plasma Membrane Domains of Human Peripheral Blood Lymphocytes." Blood 91, no. 10 (May 15, 1998): 3901–8. http://dx.doi.org/10.1182/blood.v91.10.3901.3901_3901_3908.
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CD44 is the major cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is implicated in a variety of biological events that include embryonic morphogenesis, lymphocyte recirculation, inflammation, and tumor metastasis. CD44 delivers activation signals to T lymphocytes, B lymphocytes, natural killer cells, polymorphonuclear leukocytes, and macrophages by stimulating protein tyrosine phosphorylation and calcium influx. The mechanism of signal transduction via CD44 remains undefined, although CD44 was shown to physically associate with intracellular protein tyrosine kinase Lck in T lymphocytes. In the present report, we show that a significant proportion of CD44 in human peripheral blood T lymphocytes and endothelial cells is associated with low-density plasma membrane fractions that represent specialized plasma membrane domains enriched in glycosphingolipids and glycosylphosphatidylinositol (GPI)-anchored proteins. CD44 and the GPI-anchored CD59 do not appear to directly interact in the low-density membrane fractions. In human peripheral blood T lymphocytes, 20% to 30% of the Src family protein tyrosine kinases, Lck and Fyn, are recovered from these fractions. CD44-associated protein kinase activity was selectively recovered from the low-density membrane fractions, corresponding to glycosphingolipid-rich plasma membrane microdomains. Reprecipitation of the in vitro phosphorylated proteins showed that CD44 associates not only with Lck but also with Fyn kinase in these membrane domains. Our results suggest that cellular stimulation via CD44 may proceed through the signaling machinery of glycosphingolipid-enriched plasma membrane microdomains and, hence, depend on the functional integrity of such domains.
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Shimonaka, Mika, Koko Katagiri, Toshinori Nakayama, Naoya Fujita, Takashi Tsuruo, Osamu Yoshie, and Tatsuo Kinashi. "Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow." Journal of Cell Biology 161, no. 2 (April 21, 2003): 417–27. http://dx.doi.org/10.1083/jcb.200301133.
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Chemokines arrest circulating lymphocytes within the vasculature through the rapid up-regulation of leukocyte integrin adhesive activity, promoting subsequent lymphocyte transmigration. However, the key regulatory molecules regulating this process have remained elusive. Here, we demonstrate that Rap1 plays a pivotal role in chemokine-induced integrin activation and migration. Rap1 was activated by secondary lymphoid tissue chemokine (SLC; CCL21) and stromal-derived factor 1 (CXCL4) treatment in lymphocytes within seconds. Inhibition of Rap1 by Spa1, a Rap1-specific GTPase-activating protein, abrogated chemokine-stimulated lymphocyte rapid adhesion to endothelial cells under flow via intercellular adhesion molecule 1. Expression of a dominant active Rap1V12 in lymphocytes stimulated shear-resistant adhesion, robust cell migration on immobilized intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, and transendothelial migration under flow. We also demonstrated that Rap1V12 expression in lymphocytes induced a polarized morphology, accompanied by the redistribution of CXCR4 and CD44 to the leading edge and uropod, respectively. Spa1 effectively suppressed this polarization after SLC treatment. This unique characteristic of Rap1 may control chemokine-induced lymphocyte extravasation.
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Mun, Yeung-Chul, Kyoung-Eun Lee, Jung Mi Kwon, Seung-Hyun Nam, Eun Sun Yoo, Yun-Kyung Bae, Seung-Eun Lee, et al. "Establishment of Effective B Lymphocyte Ex Vivo Expansion on Human Cord Blood Using TPO, SCF, FL, IL-4, IL-10, and CD40L." Blood 104, no. 11 (November 16, 2004): 2882. http://dx.doi.org/10.1182/blood.v104.11.2882.2882.
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Abstract In respect to B lymphocyte-mediated immunity, characteristics of human cord blood are low counts of mature B lymphocytes, deficient expression of CD40L and cytokine production in CD4+ T lymphocytes, defect in the isotype switch of immunoglobulin and the activation of B lymphocytes, and low IgG production of B lymphocytes. These characteristics of the B lymphocyte from human cord blood lead to a delayed B lymphocyte-mediated immune reconstitution and an increased susceptibility to infections after a cord blood transplantation. The mechanism of immunological recostitution after cord blood transplantation has been examined from a variety of viewpoints in experimental models as well as clinical studies. However, problems of sustained immunodeficiency after cord blood transplantation remain to be resolved. The aim of the present study is to establish culture conditions that support the effective B lymphocyte expansion of human cord blood using IL-4, IL-10, and CD40L, to which cytokines are defected in B lymphocyte of human cord blood, and established conditions are compared to previously established cytokine combinations, TPO+SCF+FL in our Lab (Br J Haematol 107:176–185, 1999 & Stem Cells 21:228–235, 2003). To elucidate the effective B lymphocyte-mediated immune reconstitution of cord blood after ex vivo expansion, mononuclear cells, separated from density gradient of Ficoll system, and CD34+ purified cells, isolated from immunomicrobead(MiniMACS) system, were cultured with various combinations of cytokines (TPO+FL+SCF and/or IL-4, IL-10 and CD40L) for 2 weeks or 4 weeks. This then allowed for cytometric analysis after immunofluorescence stain with CD34, CD38 (for HSC analysis) and CD19, IgG and IgM (for B lymphocyte-mediated immune reconstitution) and CD4 (for T helper cell) and CD25 (for lymphocyte activation assay) to be performed. In the B lymphocyte expansion aspect, the immunoglobulin expression, and functional activity, expansion with the TPO+FL+SCF+IL-4+IL-10 combination showed best results in the expression of CD19, CD25, IgG, and IgM. However, the addition of CD40L to those culture condition did not increase expression of CD19, CD25, IgG, and IgM after the expansion of human cord blood. Expansion of CD34+ purified cells was superior to MNCs in the expression of CD19, CD25, IgG, and IgM. In consideration for the duration of cultures, the 2 week culture was superior to the 4 week culture with respect to graft stemness (CD34+CD38- fraction). Our data suggests most superior results were observed from the ex vivo expansion of CD34+ purified cells cultured for 2 weeks with TPO+FL+SCF+IL-4+IL-10, in the B lymphocyte-mediated immune reconstitution and graft stemness aspect. The results of this study warrant further investigation on effective B lymphocyte-mediated immune reconstitution after cord blood transplantation in vivo using ex vivo expanded cord blood.
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Zhang, Jinyi, Amro Shehabeldin, Luis A. G. da Cruz, Jeffrey Butler, Ally-Khan Somani, Mary McGavin, Ivona Kozieradzki, et al. "Antigen Receptor–Induced Activation and Cytoskeletal Rearrangement Are Impaired in Wiskott-Aldrich Syndrome Protein–Deficient Lymphocytes." Journal of Experimental Medicine 190, no. 9 (November 1, 1999): 1329–42. http://dx.doi.org/10.1084/jem.190.9.1329.
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The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele (WAS−/−). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44−CD25+ to the CD44−CD25− stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-ζ, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH2-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS−/− cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS−/− lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS−/− neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.
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Zöller, M. "CD44, metastatic tumor spread, lymphocyte activation." Biomedicine & Pharmacotherapy 47, no. 4 (January 1993): 174. http://dx.doi.org/10.1016/0753-3322(93)90013-b.
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Maltzman, J. S., J. A. Carman, and J. G. Monroe. "Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes." Molecular and Cellular Biology 16, no. 5 (May 1996): 2283–94. http://dx.doi.org/10.1128/mcb.16.5.2283.
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The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.
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Forster, R., T. Emrich, E. Kremmer, and M. Lipp. "Expression of the G-protein--coupled receptor BLR1 defines mature, recirculating B cells and a subset of T-helper memory cells." Blood 84, no. 3 (August 1, 1994): 830–40. http://dx.doi.org/10.1182/blood.v84.3.830.bloodjournal843830.
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The G-protein-coupled receptor BLR1 related to receptors for chemokines and neuropeptides has been identified as the first lymphocyte-specific member of the gene family characterized by seven transmembrane-spanning regions. Using a high-affinity anti-BLR1 monoclonal antibody (MoAb) and three-color flow cytometry it is shown that BLR1 expression on peripheral blood cells is limited to B cells and to a subset of CD4+ (14%) and CD8+ (2%) lymphocytes. T cells expressing BLR1 were positive for CD45R0, were negative for interleukin-2 receptors, show high levels of CD44, and show low levels of L-selectin. The majority of CD4+ cells originating from secondary lymphatic tissue, but none of cord blood- derived T cells, express BLR1. These observations suggest that BLR1 is a marker for memory T cells. Furthermore, BLR1 expression was detected on all CD19+ peripheral or tonsillar B lymphocytes, but only on a fraction of cord blood cells and bone marrow cells expressing CD19, sIgM, or sIgD. Interestingly, activation of both mature B and T cells by CD40 MoAb and CD3 MoAb, respectively, led to complete downregulation of BLR1. These data suggest that the G-protein-coupled receptor BLR1 is involved in functional control of mature recirculating B cells and T- helper memory cells participating in cell migration and cell activation.
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Murakami, S., and H. Okada. "Lymphocyte-Fibroblast Interactions." Critical Reviews in Oral Biology & Medicine 8, no. 1 (January 1997): 40–50. http://dx.doi.org/10.1177/10454411970080010201.
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Chronic inflammatory reactions are usually characterized by inflammatory cell accumulation in the extravascular connective tissue. In such sites, inappropriate activation of circulating or resident lymphocytes becomes self-perpetuating and can lead to chronic tissue destruction. In addition to that, the locally infiltrated lymphocytes should have an opportunity to interact directly with fibroblasts composing the connective tissue. The direct interactions of those different cell types seem to play important roles in lymphocyte lodging and retention in such sites. Thus, for clarification of the immunopathogenesis of the chronic inflammatory diseases, including periodontitis, it is important that the molecular mechanisms involved in the heterotypic cell-cell interactions be revealed. In fact, it has been demonstrated that lymphocytes interact with various non-hematopoietic cells, such as epithelial cells and endothelial cells. Regarding interactions with fibroblasts, it has been shown that IFNγ-stimulated fibroblasts can regulate the proliferative responses of T-lymphocytes both positively and negatively. Furthermore, activated lymphocytes have demonstrated strong binding ability to various fibroblast cell lines. Blocking experiments utilizing monoclonal antibodies specific to various cell adhesion molecules revealed that very late antigen (VLA) integrins, lymphocyte-function-associated antigen (LFA-1)/intercellular adhesion molecule-1 (ICAM-1), CD44/hyarulonate are, at least in part, involved in lymphocyte-fibroblast interactions. In addition, recent findings raised the possibility that the adhesive interactions between lymphocytes and fibroblasts influenced the various cellular functions of each cell type. In fact, it was recently demonstrated that the adhesive interactions stimulated fibroblasts to increase expression of inflammatory cytokine mRNA. These results strongly suggest that fibroblasts are not merely innocent bystanders but actively participate in local inflammatory reactions by directly interacting with locally infiltrated lymphocytes.
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Forster, R., T. Emrich, E. Kremmer, and M. Lipp. "Expression of the G-protein--coupled receptor BLR1 defines mature, recirculating B cells and a subset of T-helper memory cells." Blood 84, no. 3 (August 1, 1994): 830–40. http://dx.doi.org/10.1182/blood.v84.3.830.830.
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Abstract The G-protein-coupled receptor BLR1 related to receptors for chemokines and neuropeptides has been identified as the first lymphocyte-specific member of the gene family characterized by seven transmembrane-spanning regions. Using a high-affinity anti-BLR1 monoclonal antibody (MoAb) and three-color flow cytometry it is shown that BLR1 expression on peripheral blood cells is limited to B cells and to a subset of CD4+ (14%) and CD8+ (2%) lymphocytes. T cells expressing BLR1 were positive for CD45R0, were negative for interleukin-2 receptors, show high levels of CD44, and show low levels of L-selectin. The majority of CD4+ cells originating from secondary lymphatic tissue, but none of cord blood- derived T cells, express BLR1. These observations suggest that BLR1 is a marker for memory T cells. Furthermore, BLR1 expression was detected on all CD19+ peripheral or tonsillar B lymphocytes, but only on a fraction of cord blood cells and bone marrow cells expressing CD19, sIgM, or sIgD. Interestingly, activation of both mature B and T cells by CD40 MoAb and CD3 MoAb, respectively, led to complete downregulation of BLR1. These data suggest that the G-protein-coupled receptor BLR1 is involved in functional control of mature recirculating B cells and T- helper memory cells participating in cell migration and cell activation.
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Grossman, William J., James W. Verbsky, Benjamin L. Tollefsen, Claudia Kemper, John P. Atkinson, and Timothy J. Ley. "Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells." Blood 104, no. 9 (November 1, 2004): 2840–48. http://dx.doi.org/10.1182/blood-2004-03-0859.
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Abstract Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.1,2 Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56+8- NK cells, and approximately half of circulating CD8+ T lymphocytes, coexpressed both granzymes A and B. In contrast, few circulating CD4+ T lymphocytes expressed granzymes A or B. Activation of CD8+ T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4+ T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of granzyme B, but not granzyme A. Naive CD4+CD45RA+ cells stimulated with antibodies to CD3/CD46 strongly expressed granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that granzyme B-expressing CD4+ Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different granzymes may play unique roles in immune system responses and regulation.
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Fatmawati, Fatmawati, Ellyza Nasrul, Nasrul Zubir, and Ferry Sandra. "Programmed Cell Death Protein 1-overexpressed CD8+ T Lymphocytes Play a Role in Increasing Chronic Hepatitis B Disease Progression." Indonesian Biomedical Journal 13, no. 3 (September 9, 2021): 310–5. http://dx.doi.org/10.18585/inabj.v13i3.1601.
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BACKGROUND: T lymphocyte activation depends on the balance of co-stimulatory and co-inhibitory signals determined by Cluster of Diffrentiation (CD)28 and Programmed Cell Death Protein 1 (PD-1) expression. Alteration in CD28 and PD-1 expression might affect the progression of chronic hepatitis B (CHB). Current study was conducted to evaluate the correlations of the CD28 and PD-1 expressions of T lymphocytes and CHB progression.METHODS: Subjects were recruited, selected and divided into 3 groups, inactive CHB, active CHB and CHB with End-Stage Liver Disease (ESLD). HBeAg was determined by using Enzyme-Linked Fluorescence Assay while HBV-DNA was carried out by the RT-PCR method. Numbers of T lymphocytes expressing CD3, CD4, CD8, CD45, CD28 and PD-1 molecules were determined by flowcytometry. RESULTS: There was no significant difference in the expression of CD28 by CD4+ and CD8+ T lymphocytes of inactive CHB, active CHB and CHB with ESLD subjects. There was also no significant difference in the expression of PD-1 in CD4+ lymphocytes of inactive CHB, active CHB and ESLD subjects. In contrast there was a significant increase in the expression of PD-1 in CD8+ T lymphocytes of ESLD subjects.CONCLUSION: CD28 expression among CHB subjects was within normal range and not related to disease progression, but PD-1 expression of CD8+ T lymphocyte was increased along with disease progression, especially in CHB subjects with ESLD. This suggests that PD-1-overexpressed CD8+ T lymphocyte play a role in increasing CHB disease progression.KEYWORDS: chronic hepatitis B, CD28, PD-1, T lymphocyte, disease progression
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Perico, N., D. Ostermann, M. Bontempeill, M. Morigi, C. S. Amuchastegui, C. Zoja, E. Akalin, M. H. Sayegh, and G. Remuzzi. "Colchicine interferes with L-selectin and leukocyte function-associated antigen-1 expression on human T lymphocytes and inhibits T cell activation." Journal of the American Society of Nephrology 7, no. 4 (April 1996): 594–601. http://dx.doi.org/10.1681/asn.v74594.
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Colchicine, which inhibits cell microtubule assembly by preventing polymerization of tubulin monomers, inhibits cell-mediated immune responses and promotes long-term survival of major histocompatibility complex-incompatible renal allografts in rats. Here we evaluated the effect of blocking cell microtubule assembly by colchicine on T cell and endothelial cell adhesion receptors involved in transducing signals for T cell activation. By using immunofluorescence flow cytometry analysis, evidence is presented that colchicine, in a dose-dependent fashion, downregulated L-selectin and leukocyte function-associated antigen-1, but not CD2 and CD44 on the surface of naive human peripheral blood lymphocytes. This effect was confirmed in two subsets of T lymphocytes, namely, CD45RA- and CD45RO-positive cells. However, colchicine did not influence the rapid shedding of L-selectin from T lymphocytes exposed to activating stimuli. Colchicine inhibited expression of interleukin-2 receptor on activated T lymphocytes. This effect was observed when T lymphocytes were stimulated with both anti-CD3 and anti L-selectin monoclonal antibodies. Colchicine also inhibited lymphocyte function in vitro as documented by inhibition of the human mixed lymphocyte response in a dose-dependent fashion. Moreover, colchicine downregulated surface expression of intercellular adhesion molecule-1 and E-selectin on activated human umbilical vein endothelial cells. These results indicate that blocking cell microfubule assembly inhibits surface expression of adhesion molecules on T cells and endothelial cells, and provides insights into the complex mechanisms of the action of colchicine in vivo.
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DeGrendele, H. C., P. Estess, L. J. Picker, and M. H. Siegelman. "CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte-endothelial cell primary adhesion pathway." Journal of Experimental Medicine 183, no. 3 (March 1, 1996): 1119–30. http://dx.doi.org/10.1084/jem.183.3.1119.
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The extravasation of leukocytes from the blood into tissues occurs as a multistep process: an initial transient interaction ("rolling"), generally thought to be mediated by the selectin family of adhesion molecules, followed by firm adhesion, usually mediated by integrins. Using a parallel plate flow chamber designed to approximate physiologic flow in postcapillary venules, we have characterized a rolling interaction between lymphoid cells and adherent primary and cultured endothelial cells that is not selectin mediated. Studies using blocking monoclonal antibodies indicate that this novel interaction is mediated by CD44. Abrogation of the rolling interaction could be specifically achieved using both soluble hyaluronate (HA) and treatment of the adherent cells with HA-reactive substances, indicating that HA is the ligand supporting this rolling interaction. Some B and T cell lines, as well as normal lymphocytes, either constitutively exhibit rolling or can be induced to do so by phorbol ester or in vivo antigen activation. These studies indicate that CD44 and its principal ligand hyaluronate represent another receptor/carbohydrate ligand pair mediating a novel activation-dependent pathway of lymphocyte/endothelial cell adhesion.
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Waters, W. R., T. E. Rahner, M. V. Palmer, D. Cheng, B. J. Nonnecke, and D. L. Whipple. "Expression of l-Selectin (CD62L), CD44, and CD25 on Activated Bovine T Cells." Infection and Immunity 71, no. 1 (January 2003): 317–26. http://dx.doi.org/10.1128/iai.71.1.317-326.2003.
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ABSTRACT Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4+ cells from infected animals. CD62L MFI on PPD- and PWM-stimulated γδ T-cell receptor-positive (TCR+) and CD8+ cells was also reduced compared to that of nonstimulated γδ TCR+ and CD8+ cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4+ cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.
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Sandmaier, BM, R. Storb, FR Appelbaum, and WM Gallatin. "An antibody that facilitates hematopoietic engraftment recognizes CD44." Blood 76, no. 3 (August 1, 1990): 630–35. http://dx.doi.org/10.1182/blood.v76.3.630.630.
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Abstract Pretreatment of recipients with the monoclonal antibody (MoAb) S5 facilitates engraftment of bone marrow from mismatched, unrelated donors in the canine transplantation model. In the direct comparisons reported here, the S5 glycoprotein (gp) was found to have structural homology to CD44 that in humans has been implicated in adhesive interactions of one type of effector cell, the lymphocyte. The S5 antigen and gp90Hermes-1 exhibited codistribution on canine peripheral blood cells. Both S5 and Hermes-1 (anti-CD44) MoAbs recognized 90-Kd species in radioimmune precipitations of 125I surface-labeled canine peripheral blood lymphocytes and bone marrow cells. Competitive antibody binding experiments showed that the epitope detected by S5 was distinct from that bound by Hermes-1 but overlapped with those defined by two other known anti-CD44 reagents, IM7 and Hutch-1. Sequential immunoprecipitation with S5 and Hermes-1 indicated that the two antibodies recognize the same or overlapping subsets of membrane gps. Tryptic digestion of S5 and anti-CD44 immunoprecipitates generated two major iodinated peptides of 27 and 35 Kd in both cases, a further indication of structural homology. Similarly, after N-glycanase digestion, S5 and CD44 immunoprecipitates were resolved to a single 68- Kd species. These findings suggest that CD44-mediated adhesive events may affect the fate of transplanted hematopoietic cells. The previous implications of this gp in T-lymphocyte activation and lymphocyte adhesion to endothelium thus provide useful paradigms to analyze its function in the bone marrow transplant setting.
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Sandmaier, BM, R. Storb, FR Appelbaum, and WM Gallatin. "An antibody that facilitates hematopoietic engraftment recognizes CD44." Blood 76, no. 3 (August 1, 1990): 630–35. http://dx.doi.org/10.1182/blood.v76.3.630.bloodjournal763630.
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Pretreatment of recipients with the monoclonal antibody (MoAb) S5 facilitates engraftment of bone marrow from mismatched, unrelated donors in the canine transplantation model. In the direct comparisons reported here, the S5 glycoprotein (gp) was found to have structural homology to CD44 that in humans has been implicated in adhesive interactions of one type of effector cell, the lymphocyte. The S5 antigen and gp90Hermes-1 exhibited codistribution on canine peripheral blood cells. Both S5 and Hermes-1 (anti-CD44) MoAbs recognized 90-Kd species in radioimmune precipitations of 125I surface-labeled canine peripheral blood lymphocytes and bone marrow cells. Competitive antibody binding experiments showed that the epitope detected by S5 was distinct from that bound by Hermes-1 but overlapped with those defined by two other known anti-CD44 reagents, IM7 and Hutch-1. Sequential immunoprecipitation with S5 and Hermes-1 indicated that the two antibodies recognize the same or overlapping subsets of membrane gps. Tryptic digestion of S5 and anti-CD44 immunoprecipitates generated two major iodinated peptides of 27 and 35 Kd in both cases, a further indication of structural homology. Similarly, after N-glycanase digestion, S5 and CD44 immunoprecipitates were resolved to a single 68- Kd species. These findings suggest that CD44-mediated adhesive events may affect the fate of transplanted hematopoietic cells. The previous implications of this gp in T-lymphocyte activation and lymphocyte adhesion to endothelium thus provide useful paradigms to analyze its function in the bone marrow transplant setting.
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Högerkorp, Carl-Magnus, Sven Bilke, Thomas Breslin, Sigurdur Ingvarsson, and Carl A. K. Borrebaeck. "CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analyzed by DNA microarrays." Blood 101, no. 6 (March 15, 2003): 2307–13. http://dx.doi.org/10.1182/blood-2002-06-1837.
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A number of studies have implicated a role for the cell surface glycoprotein CD44 in several biologic events, such as lymphopoiesis, homing, lymphocyte activation, and apoptosis. We have earlier reported that signaling via CD44 on naive B cells in addition to B-cell receptor (BCR) and CD40 engagement generated a germinal center–like phenotype. To further characterize the global role of CD44 in B differentiation, we examined the expression profile of human B cells cultured in vitro in the presence or absence of CD44 ligation, together with anti-immunoglobulin (anti-Ig) and anti-CD40 antibodies. The data sets derived from DNA microarrays were analyzed using a novel statistical analysis scheme created to retrieve the most likely expression pattern of CD44 ligation. Our results show that genes such as interleukin-6 (IL-6), IL-1α, and β2-adrenergic receptor (β2-AR) were specifically up-regulated by CD44 ligation, suggesting a novel role for CD44 in immunoregulation and inflammation.
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Bleul, Conrad C., Joachim L. Schultze, and Timothy A. Springer. "B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement." Journal of Experimental Medicine 187, no. 5 (March 2, 1998): 753–62. http://dx.doi.org/10.1084/jem.187.5.753.
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Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.
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Hertweck, Magdalena, Felix Erdfelder, Alexandra Filipovich, Sabrina Uhrmacher, Rajesh Kumar Gandhirajan, Iris Gehrke, Julian Paesler, et al. "Evidence for a Pathological Ratio of CD44s/CD44v6 in Chronic Lymphoctic Leukemia (CLL) Cells." Blood 114, no. 22 (November 20, 2009): 2628. http://dx.doi.org/10.1182/blood.v114.22.2628.2628.
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Abstract Abstract 2628 Poster Board II-604 Chronic Lymphocytic Leukemia (CLL) is the most common adult-onset leukemia in the western world. It is characterized by an accumulation of functionally incompetent monoclonal CD5+ B-lymphocytes and an increased resistance to apoptosis. CD44 is a cell surface transmembrane glycoprotein which has more than ten isoforms generated by alternative splicing. It is expressed on different cells as e.g. cells of lymphohematopoietic origin, epithelial cells and keratinocytes. CD44 is involved in lymphocyte activation, recirculation and homing. It acts as an adhesion molecule and plays an important role in angiogenesis, cell proliferation, differentiation and migration. One of these variants, CD44 variant 6 (CD44v6), is shown to be responsible for an increased apoptotic resistance in Jurkat cells. Our aim was to show that i) CD44 is overexpressed in CLL cells, ii) that this overexpression is regulated by the WNT pathway, which is known to be constitutively active in CLL cells, and iii) that this overexpression is a possible cause for the increased resistance to apoptosis in CLL cells. Peripheral blood of CLL patients was incubated with specific antibodies directed against CD5, CD19, CD44 standard (CD44s) and CD44v6 and measured by flow cytometry. We measured the mean fluorescence intensity of CD44s and CD44v6 on CD5/CD19 double positive CLL cells. The patient pool included patients with poor prognosis ( ZAP70+, CD38+) as well as patients having a good prognosis (ZAP70-, CD38-). CD19 positive B-cells from healthy volunteers, aged from 18 to 60, were analyzed for their expression of CD44s and CD44v6 as well. Expression levels of CD44s in 37 CLL samples and of CD44v6 in 29 CLL samples were measured and compared those of 22 healthy volunteers. We found a high expression of CD44s in CLL-cells (mean: 368.02) but an even higher expression in healthy B-cells (mean: 538.8; p<0.05). In contrast, the CD44 variant 6 was expressed five- to six-fold higher in CLL cells compared to healthy B cells (mean CLL cells: 20.0; mean healthy B cells: 3.6; p<0.001). Taken together, we found that there is a significantly altered ratio of CD44s/CD44v6 expression in CLL cells when compared to normal B cells. As CD44v6 is known to be oncogenic this phenomenon may contribute to the malignant phenotype of these cells. Currently, we are investigating the effect of WNT3a, a WNT signaling initiator, on the expression of CD44v6 in CLL cells and the effect of an anti human CD44v6 antibody on the apoptosis rate in CLL-cells. Disclosures: Hallek: BayerScheringAG: Honoraria, Research Funding.
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Greil, Johann, Tobias Rausch, Thomas Giese, Obul Reddy Bandapalli, Volker Daniel, Isabelle Bekeredjian-Ding, Adrian M. Stuetz, et al. "Whole-Exome Sequencing Links CARD11 Inactivation with SCID." Blood 120, no. 21 (November 16, 2012): 258. http://dx.doi.org/10.1182/blood.v120.21.258.258.
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Abstract Abstract 258 Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and the adaptive immune response and are per se clinically highly relevant, because in SCID patients infections by opportunistic pathogens are typically life-threatening early in life. We identified an infant of consanguineous parents suffering from a novel form of SCID, who presented with a life-threatening Pneumocystis jirovecii pneumonia. This entity was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T- and B-lymphocytes. Lymphocyte proliferation was strongly inhibited after stimulation of PBMCs with T-cell mitogens such as PHA, Con A, or anti-CD3 monoclonal antibody. The expression of several T-cell response associated cytokines upon stimulation with PMA/ionomycin was dramatically reduced in comparison to normal controls. By contrast, proliferation induced by the classical B-cell mitogen PWM was almost comparable to healthy controls. Immunophenotyping revealed a predominantly naïve phenotype (CD45RA+ CCR7+) in CD4+ and CD8+ T-lymphocytes, whereas central memory T-lymphocytes (CD45RA− CCR7+) were nearly absent. B-lymphocytes from peripheral blood were mainly naïve B-cells (CD27−) with a uniformly immature transitional B-lymphocyte phenotype (CD24++, CD38++). Patient B-lymphocytes retained the ability to proliferate and differentiate in response to BCR-independent stimuli, while their response to BCR activation was defective. Our findings thus revealed a combined defect of TCR-mediated T-lymphocyte functions and BCR-mediated B-lymphocyte functions but did not enable us to link the immunological phenotype with one of the known molecularly defined categories of SCID. Diagnostic whole-exome sequencing and systematic variant categorization revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. CARD11 is a scaffold protein that is known to be required for the assembly and activation of the NF-kB complex. In reconstitution assays we demonstrated that the patient derived truncated CARD11 protein is defective in antigen receptor signaling and NF-kB activation. Several lines of evidence substantiate the involvement of the identified CARD11 mutation in the new form of SCID that we report here. First, PCR and Sanger re-sequencing validated the truncating CARD11 mutation to be homozygous in the patient and heterozygous in the parents, in agreement with the recessive transmission of the mutation through the healthy consanguineous parents. Second, CARD11 is a scaffold protein required for TCR- and BCR-induced NF-kB activation as well as lymphocyte activation and proliferation, which is specifically expressed in hematopoietic cells, consistent with a causative role of CARD11 mutations in the context of an immune disorder. Third, the GUK domain of CARD11, which is missing in the mutated form of CARD11 due to truncation, was previously reported to be necessary for NF-kB activation by PMA/ionomycin treatment, further supporting the presumed damaging nature of the homozygous CARD11 mutation observed in the female patient reported here. Finally, the immunological findings in this patient are compatible with the phenotype of a previously described Card11 −/− k.o. mouse, which shows a selective defect in NF-κB activation leading to diminished antigen receptor or PKC mediated proliferation and defective cytokine production in T-cells and B-cells. Thus, we have identified an inactivating CARD11 mutation linking defective NF-kB signaling with a novel cause of autosomal recessive SCID, which must be considered in the diagnostic assessment of patients with suspected SCID but with quantitatively normal T-cells. Disclosures: No relevant conflicts of interest to declare.
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Hoshino, Yoshihiko, Koh Nakata, Satomi Hoshino, Yoshihiro Honda, Doris B. Tse, Tatsuo Shioda, William N. Rom, and Michael Weiden. "Maximal HIV-1 Replication in Alveolar Macrophages during Tuberculosis Requires both Lymphocyte Contact and Cytokines." Journal of Experimental Medicine 195, no. 4 (February 18, 2002): 495–505. http://dx.doi.org/10.1084/jem.20011614.
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HIV-1 replication is markedly upregulated in alveolar macrophages (AM) during pulmonary tuberculosis (TB). This is associated with loss of an inhibitory CCAAT enhancer binding protein β (C/EBPβ) transcription factor and activation of nuclear factor (NF)-κB. Since the cellular immune response in pulmonary TB requires lymphocyte–macrophage interaction, a model system was developed in which lymphocytes were added to AM. Contact between lymphocytes and AM reduced inhibitory C/EBPβ, activated NF-κB, and enhanced HIV-1 replication. If contact between lymphocytes and macrophages was prevented, inhibitory C/EBPβ expression was maintained and the HIV-1 long terminal repeat (LTR) was not maximally stimulated although NF-κB was activated. Antibodies that cross-linked macrophage expressed B-7, and vascular cell adhesion molecule and CD40 were used to mimic lymphocyte contact. All three cross-linking antibodies were required to abolish inhibitory C/EBPβ expression. However, the HIV-1 LTR was not maximally stimulated and NF-κB was not activated. Maximal HIV-1–LTR stimulation required both lymphocyte-derived soluble factors, and cross-linking of macrophage expressed costimulatory molecules. High level HIV-1–LTR stimulation was also achieved when IL-1β, IL-6, and TNF-β were added to macrophages with cross-linked costimulatory molecules. Contact between activated lymphocytes and macrophages is necessary to down-regulate inhibitory C/EBPβ, thereby derepressing the HIV-1 LTR. Lymphocyte-derived cytokines activate NF-κB, further enhancing the HIV-1 LTR.
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Maltzman, J. S., J. A. Carmen, and J. G. Monroe. "Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: role for transcription factor EGR1." Journal of Experimental Medicine 183, no. 4 (April 1, 1996): 1747–59. http://dx.doi.org/10.1084/jem.183.4.1747.
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Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.
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Qiao, Zhenhua, Fang Ye, Tao Yang, Li Zhang, and Ning Jia. "The Effect of Mesenchymal Stem Cells on Preventing Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation—the Effect of Mesenchymal Stem Cells on Active T Lymphocytes(in vitro)." Blood 116, no. 21 (November 19, 2010): 5182. http://dx.doi.org/10.1182/blood.v116.21.5182.5182.
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Abstract Abstract 5182 Objective To study the immune regulatory effects of human bone marrow Mesenchymal stem cells(MSCs) on active T lymphocytes in vitro and to explore the new strategy to prevent Graft-Versus-Host Disease(GVHD) in allogeneic hematopoietic stem cell transplantation(allo-HSCT). Methods 1. Mononuclear cells from human peripheral blood cells were isolated and cultured in the presence of phytohemagglutinin (PHA) (final concentration was 10μ g/ml) for different time (24hours, 36hours, 48hours, 72hours). 2. The ability of T lymphocyte proliferation and activation was measured by 3H-Thydimine incorporation. 3. MSCs from human bone marrow cells were isolated and cultured.The purity of MSCs were identified with morphological characterization by microphotograph and the phenotypes were tested by flow cytometry (FCM). 4.MSCs were planted in 6-well plates (2×104/well for group B,4×104/well for group C and 8×104/well for group D) and cocultured for 72 hours with T cells activated as the control group (group A). CD3+CD4+, CD3+CD8+, CD4+CD25+ and CD4+CD152+ expressed on T cells were analyzed by FCM after coculture with MSCs for 72 hours respectively.T lymphocyte proliferation after cocultured with MSCs was evaluated by 3H-Thydimine incorporation. The expressions of IL-2, sIL-2R and TGF-β1 proteins in these four groups was detected by ELISA. Results 1. The ability of T lymphocyte proliferation in the same PHA concentration increased with the time changing. Its ability was strongest when culturing for 48 hours (P<0.01)and had no difference between 48 hours and 72 hours (P>0.05). 2. The phenotypes of MSCs were measured by FCM:they were positive on the expression of CD44, CD105, CD29 and FIK1 and negtive on the expression of CD33, CD34, CD45 and HLA-DR. 3. MSCs inhibited T lymphocyte proliferation and the inhibitory effect depended on the amount of MSCs:CD3+CD8+, CD4+CD25+ and CD4+CD152+ T cells cocultured with bone marrow MSCs (group B, group C, group D) increased obviously and a significant decrease of CD3+CD4+ T cell proliferation as compared with control group (T lymphocytes uncocultured with MSCs) (P<0.01), and there were no difference between group C and group D (P>0.05). In group B, C and D, IL-2 and sIL-2R levels were lower than control group (P<0.01) and TGF-β1 level was higher obviously than group A (P<0.01). Every of IL-2, sIL-2R and TGF-β1 expressions has difference during group B, C and D (P<0.05). Conclusions 1. Mitogen PHA could increase proliferation function of T lymphocytes and this function was strongest when cultured for 48 hours in same concentration of PHA. 2. MSCs inhibit T lymphocyte proliferation and perform their immunosuppressive function by up-regulation of CD3+CD8+ T cells, CD4+CD25+ Tcells and CD4+CD152+ Tcells. MSCs decreased IL-2 and sIL-2R levels and increased TGF-β1 level in PHA-stimulated peripheral blood lymphocytes. 3. MSCs play a critical immune negative regulatory effect on preventing GVHD. MSCs pretreatment may be useful in the prevention of GVHD in allo-HSCT and provides a new strategy to induce transplantation tolerance. Disclosures: No relevant conflicts of interest to declare.
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Serrador, Juan M., José L. Alonso-Lebrero, Miguel A. del Pozo, Heinz Furthmayr, Reinhard Schwartz-Albiez, Javier Calvo, Francisco Lozano, and Francisco Sánchez-Madrid. "Moesin Interacts with the Cytoplasmic Region of Intercellular Adhesion Molecule-3 and Is Redistributed to the Uropod of T Lymphocytes during Cell Polarization." Journal of Cell Biology 138, no. 6 (September 22, 1997): 1409–23. http://dx.doi.org/10.1083/jcb.138.6.1409.
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During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.
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Billard, Christian, Stéphanie Delaire, Emmanuel Raffoux, Armand Bensussan, and Laurence Boumsell. "Switch in the protein tyrosine phosphatase associated with human CD100 semaphorin at terminal B-cell differentiation stage." Blood 95, no. 3 (February 1, 2000): 965–72. http://dx.doi.org/10.1182/blood.v95.3.965.003k39_965_972.
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Human CD100, the first semaphorin identified in the immune system, is a transmembrane protein involved in T-cell activation. In the present study, we showed that activation of peripheral blood or tonsillar B lymphocytes induced the expression of CD100 in CD38+CD138− cell populations, including in CD148+ subpopulations, thus expressing a memory B-cell–like phenotype. Using an in vitro enzymatic assay, we found that protein tyrosine phosphatase (PTP) activities were immunoprecipitated with CD100 in these cell populations, which were isolated by cell sorting, as well as in most B-cell lines representing various stages of B-cell differentiation. Immunodepletion and Western blotting experiments demonstrated that CD45 was the PTP associated with CD100 in cell lines displaying pre-B, activated B, and pre-plasma cell phenotypes. CD45 also accounted for PTP activity immunoprecipitated with CD100 in CD38+CD138− cells sorted after activation of peripheral blood or tonsillar B lymphocytes. In contrast, no CD100-CD45 association was observed in plasma cell lines corresponding to the terminal B-cell differentiation stage. CD148, the other transmembrane PTP known to be implicated in lymphocyte signaling pathways, was either only partly involved in the CD100-associated PTP activity or not expressed in plasma cell lines, indicating the association of CD100 with another main PTP. Our data show that CD100 is differentially expressed and can functionally associate with distinct PTPs in B cells depending on their activation and maturation state. They also provide evidence for a switch in the CD100-associated PTP at terminal stage of B-cell differentiation.
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Galibert, L., N. Burdin, C. Barthélémy, G. Meffre, I. Durand, E. Garcia, P. Garrone, F. Rousset, J. Banchereau, and Y. J. Liu. "Negative selection of human germinal center B cells by prolonged BCR cross-linking." Journal of Experimental Medicine 183, no. 5 (May 1, 1996): 2075–85. http://dx.doi.org/10.1084/jem.183.5.2075.
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The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.
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Rafi, Asimah, Mitzi Nagarkatti, and Prakash S. Nagarkatti. "Hyaluronate-CD44 Interactions Can Induce Murine B-Cell Activation." Blood 89, no. 8 (April 15, 1997): 2901–8. http://dx.doi.org/10.1182/blood.v89.8.2901.
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Abstract CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. In the current study, we investigated whether HA or monoclonal antibodies (MoAbs) against CD44 would induce a proliferative response in mouse lymphocytes. Spleen cells from normal and nude, but not severe combined immunodeficient mice, exhibited strong proliferative responsiveness to stimulation with soluble HA or anti-CD44 MoAbs. Furthermore, purified B cells, but not T cells, were found to respond to HA. HA was unable to stimulate T cells even in the presence of antigen presenting cells (APC) and was unable to act as a costimulus in the presence of mitogenic or submitogenic concentrations of anti-CD3 MoAbs. In contrast, stimulation of B cells with HA in vitro, led to B-cell differentiation as measured by production of IgM antibodies in addition to increased expression of CD44 and decreased levels of CD45R. The fact that the B cells were responding directly to HA through its binding to CD44 and not to any contaminants or endotoxins was demonstrated by the fact that F(ab)2 fragments of anti-CD44 MoAbs or soluble CD44 fusion proteins could significantly inhibit the HA-induced proliferation of B cells. Also, HA-induced proliferation of B cells was not affected by the addition of polymixin B, and B cells from lipopolysaccharide (LPS)-unresponsive C3H/HeJ strain responded strongly to stimulation with HA. Furthermore, HA, but not chondroitin-sulfate, another major component of the ECM, induced B-cell activation. It was also noted that injection of HA intraperitoneally, triggered splenic B cell proliferation in vivo. Together, the current study demonstrates that interaction between HA and CD44 can regulate murine B-cell effector functions and that such interactions may play a critical role during normal or autoimmune responsiveness of B cells.
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Krinzman, S. J., G. T. De Sanctis, M. Cernadas, L. Kobzik, J. A. Listman, D. C. Christiani, D. L. Perkins, and P. W. Finn. "T cell activation in a murine model of asthma." American Journal of Physiology-Lung Cellular and Molecular Physiology 271, no. 3 (September 1, 1996): L476—L483. http://dx.doi.org/10.1152/ajplung.1996.271.3.l476.
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To determine the mechanisms by which inhaled antigens produce pulmonary inflammation and bronchial hyperreactivity, we have developed a murine model of asthma. BALB/c mice are sensitized and challenged with ovalbumin (OVA). Compared with mice treated with phosphate-buffered saline (PBS), OVA-treated mice developed increased lung resistance, decreased dynamic compliance, and greater methacholine reactivity. Bronchoalveolar lavage fluid revealed significant increases in the proportion of neutrophils and eosinophils. Tissue sections of OVA-treated mice demonstrated goblet cell metaplasia and focal perivascular and peribronchial infiltrates composed of lymphocytes, neutrophils, and eosinophils. Analysis of thoracic lymphocytes via flow cytometry revealed an expansion of both CD4+ and B cell populations, with increased expression of interleukin-2 receptor on CD4+ T cells, indicated increased activation. There was also increased expression of CD44 on CD4+ and CD8+ lymphocytes, suggesting an expansion of the local memory cell population. These findings support the hypothesis that activation of T lymphocytes mediates allergic pulmonary inflammation and bronchial reactivity in asthma.
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Nakayama, K., K. Nakayama, L. B. Dustin, and D. Y. Loh. "T-B cell interaction inhibits spontaneous apoptosis of mature lymphocytes in Bcl-2-deficient mice." Journal of Experimental Medicine 182, no. 4 (October 1, 1995): 1101–9. http://dx.doi.org/10.1084/jem.182.4.1101.
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Bcl-2 expression is tightly regulated during lymphocyte development. Mature lymphocytes in Bcl-2-deficient mice show accelerated spontaneous apoptosis in vivo and in vitro. Stimulation of Bcl-2-deficient lymphocytes by anti-CD3 antibody inhibited the spontaneous apoptosis not only in T cells but also in B cells. The rescue of B cells was dependent on the presence of T cells, mainly through CD40L and interleukin (IL)-4. Furthermore, we generated Bcl-2-deficient mice transgenic for a T cell receptor or an immunoglobulin, both specific for chicken ovalbumin, to test for antigen-specific T-B cell interaction in the inhibition of the spontaneous apoptosis. The initial T cell activation by antigenic peptides presented by B cells suppressed apoptosis in T cells. Subsequently, T cells expressed CD40L and released ILs, leading to the protection of B cells from spontaneous apoptosis. These results suggest that the antiapoptotic signaling via CD40 or IL-4 may be largely independent of Bcl-2. Engagement of the Ig alone was not sufficient for the inhibition of B cell apoptosis. Thus, the physiological role of Bcl-2 in mature lymphocytes may be to protect cells from spontaneous apoptosis and to extend their lifespans to increase the opportunity for T cells and B cells to interact with each other and specific antigens in secondary lymphoid tissues. Bcl-2, however, appears to be dispensable for survival once mature lymphocytes are activated by antigen-specific T-B cell collaboration.
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Ravanel, Kissia, Claire Castelle, Thierry Defrance, T. Fabian Wild, Dominique Charron, Vincent Lotteau, and Chantal Rabourdin-Combe. "Measles Virus Nucleocapsid Protein Binds to FcγRII and Inhibits Human B Cell Antibody Production." Journal of Experimental Medicine 186, no. 2 (July 21, 1997): 269–78. http://dx.doi.org/10.1084/jem.186.2.269.
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Despite the development of an efficient specific immune response during measles virus (MV) infection, an immunosuppression occurs contributing to secondary infections. To study the role of nucleocapsid protein (NP) in MV-induced immunosuppression, we produced recombinant MV NP. Purified recombinant NP exhibited biochemical, antigenic, and tridimensional structure similar to viral NP. By flow cytometry, we showed that viral or recombinant NP bound to human and murine B lymphocytes, but not to T lymphocytes. This binding was specific, independent of MHC class II expression, and dependent of the B lymphocyte activation state. The murine IIA1.6 B cell line, deficient in the Fc receptor for IgG (FcγRII) expression, did not bind NP efficiently. Transfected IIA1.6 cells expressing either murine FcγRIIb1 or b2, or human FcγRIIa, b1*, or b2 isoforms efficiently bound NP. Furthermore, this binding was inhibited up to 90% by monoclonal antibodies 2.4G2 or KB61 specific for murine and human FcγRII, respectively. Finally, the in vitro Ig synthesis of CD40- or Ig-activated human B lymphocytes in the presence of interleukin (IL)-2 and IL-10 was reduced by 50% in the presence of recombinant NP. These data demonstrate that MV NP binds to human and murine FcγRII and inhibits in vitro antibody production, and therefore suggests a role for NP in MV-induced immunosuppression.
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Zanetta, J. P., J. Wantyghem, S. Kuchler-Bopp, A. Badache, and M. Aubery. "Human lymphocyte activation is associated with the early and high-level expression of the endogenous lectin CSL at the cell surface." Biochemical Journal 311, no. 2 (October 15, 1995): 629–36. http://dx.doi.org/10.1042/bj3110629.
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Lymphocytes undergo activation in response to antigens, cytokines, lectins and antibodies interacting with specific cell-surface molecules or through substances influencing signal transduction pathways. This study shows that human T- and B-cells stimulated using phorbol esters or plant lectins express early (2 h using phorbol esters and 24 h using plant lectins) a high level of a polyvalent carbohydrate-binding protein, the cerebellar soluble lectin (CSL), which is in part externalized. The lectin, immunologically related to CDw70, interacts with specific glycoprotein ligands of the lymphocyte surface, including CD3 on T-cells and CD24 on B-cells. Major changes in phosphorylations associated with activation appear as largely CSL-dependent since they are specifically inhibited by anti-CSL Fab fragments. It is suggested that the lectin induces the clustering of specific cell-surface glycoproteins and plays the role of an endogenous amplifier of activation signals.
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Galli, Grazia, Sandra Nuti, Simona Tavarini, Luisa Galli-Stampino, Claudia De Lalla, Giulia Casorati, Paolo Dellabona, and Sergio Abrignani. "CD1d-restricted Help To B Cells By Human Invariant Natural Killer T Lymphocytes." Journal of Experimental Medicine 197, no. 8 (April 14, 2003): 1051–57. http://dx.doi.org/10.1084/jem.20021616.
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Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen α-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of α-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4−CD8−), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.
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Rossini, Aldo A., David C. Parker, Nancy E. Phillips, Fiona H. Durie, Randolph J. Noelle, John P. Mordes, and Dale L. Greiner. "Induction of Immunological Tolerance to Islet Allografts." Cell Transplantation 5, no. 1 (January 1996): 49–52. http://dx.doi.org/10.1177/096368979600500109.
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T-cell dependent activation of resting B cells involves the interaction of gp39 on T cells with its receptor, CD40, on B cells. We administered either a combination of T-cell-depleted splenic lymphocytes and anti-gp39 monoclonal antibody or antibody alone to establish islet allografts in mice without continuous immunosuppression. Fully allogeneic H-2q FVB islets were permanently accepted by chemically diabetic H-2b C57BL/6 mice provided that the recipients were pretreated with both T-cell-depleted donor spleen cells and anti-gp39 antibody. Antibody alone was less effective in prolonging allograft survival, but we did observe that anti-gp39 mAb alone can exert an independent, primary effect on islet allograft survival that was dose dependent. Targeting gp39, in combination with lymphocyte transfusion, might prove suitable for tolerance induction and allotransplantation without immunosuppression.
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Imbert, V., J. F. Peyron, D. Farahi Far, B. Mari, P. Auberger, and B. Rossi. "Induction of tyrosine phosphorylation and T-cell activation by vanadate peroxide, an inhibitor of protein tyrosine phosphatases." Biochemical Journal 297, no. 1 (January 1, 1994): 163–73. http://dx.doi.org/10.1042/bj2970163.
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Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in the transduction of activation signals to T-lymphocytes. The regulatory role of protein tyrosine phosphatases (PTPases) in this process was explored by studying the effects of a powerful PTPase inhibitor, vanadate peroxide (pervanadate), on the activation cascade of Jurkat human leukaemic T-cells. Pervanadate induced activation of the tyrosine kinases lck and fyn (4- and 3-fold respectively) and a dramatic increase in tyrosine phosphorylation of cellular proteins, notably phospholipase C gamma 1. After this event, we observed a rise in intracellular Ca2+ concentration, corresponding to an influx. This effect required surface expression of the CD45 PTPase and was not observed in CD45-deficient variants of Jurkat cells. In the CD45-negative variant, the effect of pervanadate on tyrosine phosphorylation was globally decreased and some phosphorylated substrates were specifically missing. Pervanadate also stimulated transcription of the c-fos gene and accumulation of its mRNA as well as several other hallmarks of T-lymphocyte activation such as surface expression of the CD69 antigen and the interleukin 2 receptor alpha-chain (CD25). Pervanadate synergized with signals delivered by T-cell antigen receptor engagement or by a phorbol ester to induce interleukin 2 production. Pervanadate activated NF-kappa B, as shown by an increase in DNA-binding activity of this transcription factor. We thus conclude that PTPases play a crucial role in the negative regulation of signal transduction culminating in T-lymphocyte activation. Moreover, induction of tyrosine phosphorylation appears sufficient per se to initiate a complete activation programme.
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van der Voort, Robbert, Robert M. J. Keehnen, Esther A. Beuling, Marcel Spaargaren, and Steven T. Pals. "Regulation of Cytokine Signaling by B Cell Antigen Receptor and Cd40-Controlled Expression of Heparan Sulfate Proteoglycans." Journal of Experimental Medicine 192, no. 8 (October 9, 2000): 1115–24. http://dx.doi.org/10.1084/jem.192.8.1115.
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Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell–derived factor (SDF)-1α, even though the affinities of HGF and SDF-1α for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.
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Balakhonov, S. V., V. I. Dubrovina, V. V. Voitkova, K. M. Korytov, N. L. Barannikova, V. B. Nikolaev, and T. T. Shkaruba. "Immunophenotyping of blood cells of experimental animals immunized with Brucella abortus thermoextracts." Journal of microbiology epidemiology immunobiology, no. 4 (September 2, 2019): 25–31. http://dx.doi.org/10.36233/0372-9311-2019-4-25-31.
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Aim. To study the subpopulational structure of blood cells of the experimental animals immunized with thermoextracts (TE) of Brucella abortus in L- and S-form. Materials and methods. Total 100 certified («Vector», Novosibirsk) outbred mice were immunized with B. abortus I-206 TE in L- and S-form in 20 μg protein dose. After 1, 3, 7, 14 and 21 days of observation the phenotypes (CD45, CD3, CD4, CD8, CD19, CD69) of blood cells were detected. Results. General regularities were revealed after injection of the experimental preparations. So, B. abortus TE in L- and S-form caused the immune response that increased granulocyte number and expression of early activation marker CD69 by T- and B-lymphocytes of blood in early period of observation (1-3 days), decrease in general B-lymphocyte content in late periods of observation (7-21 days). Thus, mice received B. abortus ТE in L-form demonstrated authentically higher CD69 expression of blood lymphocyte subpopulations than mice received B. abortus ТE in S-form. Distinctions in formation of humoral immune response were revealed that probably was connected with alteration of Brucella chemical composition in the course of L-transformation. Conclusion. The investigation established that B. abortus TE in L- or S-form caused immunological reorganization in the experimental animal organisms. On the basis of the fin
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Maynadie, Marc M., Romain Casey, Karine Piazzon, Jean Claude Capiod, and Paule-Marie Carli. "Peripheral Blood Lymphocytes Subpopulations and Apoptotic Markers in Patients with Lymphoid Malignancies and in Controls: An Epidemiologic Case-Control Study." Blood 104, no. 11 (November 16, 2004): 3858. http://dx.doi.org/10.1182/blood.v104.11.3858.3858.
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Abstract Few references ranges of normal peripheral blood lymphocytes subpopulations are available in the literature and fewer data were available regarding activation, proliferation and apoptosis antigen expression on such populations. We studied these parameters in patients included in an epidemiologic case-control study on risk factors of lymphoid malignancies conducted within European countries. Cell surface staining of peripheral blood lymphocyte antigens were analysed by multicolour flow cytometry in 300 cases and 300 controls. We determined CD3+, CD3+/CD4+, CD3+/CD8+, CD3−/CD56+CD16+, CD19+, CD19+/CD5+, CD19+/CD5− and CD57+ populations. Expression of CD25, CD16, CD40, CD154, CD95 and CD178 were studied on these populations. CD expressions were compared by multiple regressions between controls and diseases, after stratification on circulating phase. In controls we observed a significant decrease of B cells and an increase of NK cells with age. No difference was found according to sex, smoking status and Body Mass Index. In Follicular Lymphoma, Diffuse Large B Cell Lymphoma and Marginal Zone Lymphoma (MZL) without circulating phase cases, we observed a decrease of the B cell subset and an increase of the NK cell subset instead of only a trend was found in Multiple Myeloma, Mycosis Fongoides, Hodgkin Disease and Lymphoplasmacytic Lymphoma. CD95 expression was increased in HD, DLBCL, MZL without circulating phase and Hairy Cell Leukemia but decreased in B-cell Chronic Lymphocytic Leukemia and MZL with circulating phase. CD40 was decreased on B cells in HD, FL, MZL, B-ALL and CLL. This study was one of the most important, in term of number of patients included, particularly concerning data on activation, proliferation and apoptosis markers in normal subjects but also in several lymphoid malignancies.
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Cooke, M. P., A. W. Heath, K. M. Shokat, Y. Zeng, F. D. Finkelman, P. S. Linsley, M. Howard, and C. C. Goodnow. "Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells." Journal of Experimental Medicine 179, no. 2 (February 1, 1994): 425–38. http://dx.doi.org/10.1084/jem.179.2.425.
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The specificity of antibody (Ab) responses depends on focusing helper T (Th) lymphocyte signals to suitable B lymphocytes capable of binding foreign antigens (Ags), and away from nonspecific or self-reactive B cells. To investigate the molecular mechanisms that prevent the activation of self-reactive B lymphocytes, the activation requirements of B cells specific for the Ag hen egg lysozyme (HEL) obtained from immunoglobulin (Ig)-transgenic mice were compared with those of functionally tolerant B cells isolated from Ig-transgenic mice which also express soluble HEL. To eliminate the need for surface (s)Ig-mediated Ag uptake and presentation and allow the effects of sIg signaling to be studied in isolation, we assessed the ability of allogeneic T cells from bm12 strain mice to provide in vivo help to C57BL/6 strain-transgenic B cells. Interestingly, non-tolerant Ig-transgenic B cells required both allogeneic Th cells and binding of soluble HEL for efficient activation and Ab production. By contrast, tolerant self-reactive B cells from Ig/HEL double transgenic mice responded poorly to the same combination of allogeneic T cells and soluble HEL. The tolerant B cells were nevertheless normally responsive to stimulation with interleukin 4 and anti-CD40 Abs in vitro, suggesting that they retained the capacity to respond to mediators of T cell help. However, the tolerant B cells exhibited a proximal block in the sIg signaling pathway which prevented activation of receptor-associated tyrosine kinases in response to the binding of soluble HEL. The functional significance of this sIg signaling defect was confirmed by using a more potent membrane-bound form of HEL capable of triggering sIg signaling in tolerant B cells, which markedly restored their ability to collaborate with allogeneic Th cells and produce Ab. These findings indicate that Ag-specific B cells require two signals for mounting a T cell-dependent Ab response and identify regulation of sIg signaling as a mechanism for controlling self-reactive B cells.
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Kazantseva, E. V., N. V. Dolgushina, and P. P. Tereshkov. "FAS-receptor expression in peripheral blood lymphocytes and monocytes in pregnant women with fetal growth restriction." Kazan medical journal 95, no. 4 (August 15, 2014): 511–15. http://dx.doi.org/10.17816/kmj1832.
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Aim. To study the expression of Fas-receptor on peripheral blood monocytes and lymphocytes in women who have given birth to children with low birth weight. Methods. Population-based study recruited 242 women who have given birth at the gestation term of more than 35 weeks. Group 1 (n=108) included mother-newborn pairs with low birth weight of newborns, group 2 (n=134) - mother-newborn pairs with normal birth weight for the gestational age. Peripheral blood lymphocytes and monocytes total count and subpopulations, CD95 (Fas-receptor) apoptosis receptor expression level (using four- and five-parametric phenotypic test) were determined, using a combination of monoclonal antibodies to differentiation and activation markers. Results. CD3+CD95+ cells levels were significantly higher in women who delivered of low birth weight children. In T-cell subpopulation of group 1 women, there was a statistically significant increase in relative values of Fas-receptor expression on T-helpers and T-cytotoxic lymphocytes - by 1.6 (p 0.001) and 6.3 (p 0.001) times, respectively, and by 2.9 (p 0.001) and 2.6 (p 0.001) times, respectively, in absolute values. There was a reduction in absolute counts of CD3+CD4+ and CD3+CD8+ T-lymphocyte subpopulations in women who delivered of low birth weight children compared to controls. The ratio of CD95+-expressing T-lymphocyte subpopulations in group 1 women was the following: CD4+CD95+/CD8+CD95+ ratio was 4.9 times higher compared to the second group (p 0.001). Increased absolute and relative Fas-receptor expression on B-lymphocytes [by 63.9% (p 0.001) and 33.3% (p=0.002), respectively] was found in women who delivered of low birth weight children compared to women who delivered of children with normal birth weight. CD14+CD95+ expression analysis revealed increased absolute and relative counts of Fas-receptor expressing monocytes. Conclusion. Marked expression of CD95+ in circulating monocytes and raised CD4+CD95+/CD8+CD95+ ratio in women who delivered of low birth weight children may be a laboratory test for detecting higher chance for fetal growth restriction.
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Devergne, O., E. Hatzivassiliou, K. M. Izumi, K. M. Kaye, M. F. Kleijnen, E. Kieff, and G. Mosialos. "Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation." Molecular and Cellular Biology 16, no. 12 (December 1996): 7098–108. http://dx.doi.org/10.1128/mcb.16.12.7098.
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The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.
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Réthi, Bence, Péter Gogolák, Istvan Szatmari, Ágota Veres, Erika Erdôs, Laszlo Nagy, Éva Rajnavölgyi, Cox Terhorst, and Árpád Lányi. "SLAM/SLAM interactions inhibit CD40-induced production of inflammatory cytokines in monocyte-derived dendritic cells." Blood 107, no. 7 (April 1, 2006): 2821–29. http://dx.doi.org/10.1182/blood-2005-06-2265.
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AbstractSignaling lymphocyte activation molecule (SLAM, CD150, or SLAMF1) is a self-ligand receptor on the surface of activated T- and B-lymphocytes, macrophages, and dendritic cells (DCs). Here we examine the effect of SLAM/SLAM interactions on CD40L-induced CD40 signaling pathways in human DCs. CD40L-expressing L929 cells induced DCs to produce interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-12, which was strongly inhibited by coexpression of SLAM on the surface of the L929 cells. Similarly, transfection of DCs with SLAM strongly reduced CD40L-induced IL-12 production. Furthermore, the negative effect of SLAM/SLAM interactions on CD40L-induced DC activation was also detected in the presence of lipopolysaccharide (LPS). LPS-induced IL-12 secretion, however, was not inhibited by SLAM engagement. CD40L-activated DCs affected by exposure to SLAM/SLAM engagement were impaired in their ability to induce differentiation of naive T lymphocytes into interferon-γ (IFN-γ)–producing T-helper 1 (Th1) effector cells. These inhibitory effects were not the result of a general unresponsiveness of DCs to CD40L, as SLAM/SLAM interactions did not prevent CD40L-induced up-regulation of CD83, CD86, or human leukocyte antigen (HLA)–DQ on the surface of DCs. Taken together, the results indicate that SLAM/SLAM interactions inhibit CD40-induced signal transduction in monocyte-derived dendritic cells, an effect that was not detectable in earlier studies using anti-SLAM monoclonal antibodies.
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Lee, Jong-Hwan, Tomoya Katakai, Takahiro Hara, Hiroyuki Gonda, Manabu Sugai, and Akira Shimizu. "Roles of p-ERM and Rho–ROCK signaling in lymphocyte polarity and uropod formation." Journal of Cell Biology 167, no. 2 (October 25, 2004): 327–37. http://dx.doi.org/10.1083/jcb.200403091.
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Front–rear asymmetry in motile cells is crucial for efficient directional movement. The uropod in migrating lymphocytes is a posterior protrusion in which several proteins, including CD44 and ezrin/radixin/moesin (ERM), are concentrated. In EL4.G8 T-lymphoma cells, Thr567 phosphorylation in the COOH-terminal domain of ezrin regulates the selective localization of ezrin in the uropod. Overexpression of the phosphorylation-mimetic T567D ezrin enhances uropod size and cell migration. T567D ezrin also induces construction of the CD44-associated polar cap, which covers the posterior cytoplasm in staurosporine-treated, uropod-disrupted EL4.G8 cells or in naturally unpolarized X63.653 myeloma cells in an actin cytoskeleton–dependent manner. Rho-associated coiled coil–containing protein kinase (ROCK) inhibitor Y-27632 disrupts the uropod but not the polar cap, indicating that Rho–ROCK signaling is required for posterior protrusion but not for ERM phosphorylation. Phosphorylated ezrin associates with Dbl through its NH2-terminal domain and causes Rho activation. Moreover, constitutively active Q63L RhoA is selectively localized in the rear part of the cells. Thus, phosphorylated ERM has a potential function in establishing plasma membrane “posteriority” in the induction of the uropod in T lymphocytes.
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Rohon, Peter, Kimmo Porkka, and Satu Mustjoki. "Differential Effects of Imatinib and Dasatinib On Immune Effector Cells in Patients with Chronic Myeloid Leukemia (CML)." Blood 114, no. 22 (November 20, 2009): 3285. http://dx.doi.org/10.1182/blood.v114.22.3285.3285.
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Abstract Abstract 3285 Poster Board III-1 Introduction. Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL oncoprotein are the current standard care of patients with CML. They also inhibit off-target kinases (e.g. c-KIT, TEC, BTK, PDGFR, SRC), some of which may have important physiological functions in immune responses. Recently, several papers have indicated a significant suppressive effect of TKIs on T- and NK-cell activation and cytotoxicity in vitro. The aim of this study was to assess the effects of TKIs on immune effector cells in patients with CML in chronic phase. Patients and methods. We collected 88 peripheral blood (PB) and 73 bone marrow (BM) samples from 54 CML patients at the time of diagnosis (n=19/17; PB/BM), during imatinib (n=40/39), and dasatinib (n=29/17) therapies. In addition, 16/16 (PB/BM) samples were analyzed from 17 healthy controls. The median time on imatinib or dasatinib therapy was 11 months in both groups (ranges 4-29 and 3-42 months, respectively). Lymphocyte subtypes (T, B, NK and NKT-cells) and regulatory cells (regulatory T-cells and dendritic cells) were analyzed with an extensive flow cytometry panel including key lymphocyte markers for activation (CD57, HLA-DR), proliferation (Ki67), differentiation (CD4, CD8, CD19, CD34, TCR alpha/beta, TCRgamma/delta) and memory status (CD45RA, CD45RO, CD62L). Results. At CML diagnosis, the characteristic findings were a lower B-cell (immature CD34+CD19+ B-cells in particular) and increased NKT proportion of lymphocytes in the BM. Further, a larger proportion of BM CD3+ T-cells expressed CD57. Both in PB and BM the proportions of myeloid and plasmacytoid dendritic cells were decreased at diagnosis. There were no significant differences in proportion of regulatory T-cells (Tregs) from CD4+ cells (all compared to healthy controls, p<0.05). During imatinib therapy, all these changes normalized. The distribution of lymphocyte subclasses and the proliferation and activation status of lymphocytes in imatinib patients did not differ from healthy controls. The only imatinib-specific aberrations observed were a lower number of PB T-cells carrying the gamma-delta T-cell receptor (TCR) chain and a larger amount of CD45RO positive CD4+ T-cells in PB as compared to controls. Dasatinib treated patients differed more markedly from healthy controls and from imatinib. The absolute numbers and proportions of monocytes were increased both in PB and BM, proportions of B-cells were decreased in PB and BM, and proportions and absolute numbers of CD8+ T-cells expressing activation markers HLA-DR and CD57 were increased in PB (p<0.05). When compared to imatinib patients, dasatinib use was associated with a significantly lower Treg proportion in PB (5.7 vs. 2.7% of CD4+) and BM (5.5 vs. 2.8%, p<0.001 for both). Remarkably, dasatinib patients were clearly divided into two distinct equal-sized groups: the other group (“normal”) resembled that of imatinib patients and healthy controls while the other group (“immunoactivated”) was characterized by elevated CD8+ lymphocyte, NK and NKT cell counts in PB (fig.a). Lymphocytes (CD4+, CD8+) of the latter group strongly expressed CD57+ (fig.b), HLA-DR and CD45RO and had lower levels of CD62L antigen conforming to a phenotype of a late memory cytotoxic lymphocyte characteristic of e.g. latent CMV infection. No clinical signs of immunosuppression, such as opportunistic bacterial, viral or fungal infections, were observed in any of the patients. Conclusions. TKIs had a significant and differential off-target effect on the numbers and proportions of immune effector cells. No signs of TKI-therapy related immunosuppression was seen and in particular, a subgroup of patients on dasatinib therapy displayed an immune-activated cellular profile putatively linked to Treg deficiency, unknown individual (genetic) host factors and recurrent antigen exposure. These results underline the importance of careful, long-term surveillance for adverse or off-target effects, which could be restricted only to a subgroup of patients. In addition, our data suggests that TKIs could be used to modulate immune responses in various malignant and non-malignant disorders related to immune dysfunction. Disclosures: Porkka: BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:BMS: Honoraria.
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Boumedine, Radia Sidi, Gorazd Krosl, Marc Vaillancourt, Claude Perreault, and Denis-Claude Roy. "Specific Elimination of Alloreactive T Lymphocytes Using Photodynamic Therapy Prevents GVHD and Enables Rapid Immune Reconstitution." Blood 104, no. 11 (November 16, 2004): 4987. http://dx.doi.org/10.1182/blood.v104.11.4987.4987.
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Abstract Graft-versus-host disease (GVHD) and impaired immune reconstitution are the primary obstacles limiting the efficacy of allogeneic stem cell transplantation (SCT). Graft-versus-host disease (GVHD) and impaired immune reconstitution are the primary obstacles limiting the efficacy of allogeneic stem cell transplantation (SCT). The purpose of this study was to determine whether selective depletion of donor alloantigen-specific T lymphocytes using photodynamic therapy (PDT) would prevent GVHD and enable immune reconstitution in the context of MHC-mismatched SCT. This question was addressed in an MHC-incompatible mouse model of GVHD. The donor (C57BL/6; H-2b) derived spleen cells were first activated against C3H/HeJ (H-2k) host spleen cells in a one-way mixed lymphocyte culture and then exposed to photodynamic treatment, using dibromorhodamine methyl ester (TH9402) as a photosensitizer. Activated T cells showed preferential retention of this photosensitizer compared to resting lymphocytes. In addition, in vitro experiments revealed that PDT eradicated a significantly higher proportion of activated than resting T cells. When lethally irradiated H-2k mice (C3H/HeJ and B10BR) were transplanted with C57BL/6 derived T cell-depleted bone marrow cells supplemented with C57BL/6 derived spleen cells activated with C3H/HeJ targets, they rapidly succumbed to acute GVHD (within 10–47 days). In contrast, both mouse strains receiving histoincompatible C57BL/6 T cells previously exposed to PDT after activation against C3H/HeJ survived until the end of the observation period (>100 days)(p<0.0001). Additionally, transplantation of treated T cells induced lethal GVHD in C57BL/6 histoincompatible strains of mice (third party), suggesting PDT specifically eradicated activated T cells while sparing most resting T lymphocytes. Analysis of immune recovery, evaluating T and B cell populations in thymus and spleen, activated T cells components (CD44, CD62L, CD69), proliferative responses (anti-CD3 and conA), and Vβ repertoire indicated that T and B cell reconstitution in MHC-mismatched mice transplanted with treated primed cells was similar to that of mice transplanted with treated or control autologous cells indicating that immune cells were preserved and functional. These results demonstrate that PDT can selectively eliminate alloreactive T cells and prevent the development of GVHD, while sparing T cells reactive against non-host antigens, thus offering protection against infection and disease relapse.
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Chiang, Elaine Y., Andres Hidalgo, Jungshan Chang, and Paul S. Frenette. "Real-Time Identification of Leukocyte Subsets and Cell Surface Receptor Microdomains in the Microvasculature of Wild-Type and Sickle Cell Mice In Vivo." Blood 108, no. 11 (November 16, 2006): 1229. http://dx.doi.org/10.1182/blood.v108.11.1229.1229.
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Abstract Intravital microscopy has contributed enormously to the recent mechanistic advances in leukocyte (WBC) trafficking. However, current methods have provided little knowledge about differential WBC subset behavior or the spatial redistribution of surface adhesion molecules during recruitment and activation. Here, we use digital high-speed multichannel fluorescence intravital videomicroscopy to identify WBCs and localize cell surface molecules. Adherent WBCs were ascertained using a series of near-simultaneous 4-channel images in randomly selected venular segments following the injection of low-dose (0.02–0.12 mg/kg) fluorescently labeled Mab against CD45, Gr-1, and F4/80. Preliminary experiments on FAC-sorted WBCs revealed that Gr–1+F4/80- cells were highly enriched in neutrophils (PMNs, ~99%), whereas the F4/80+ fraction was mostly monocytes (~70%) and the fraction negative for both markers was largely lymphocytes (~88%). In the cremaster muscle of C57BL/6 mice (n=6), 39 ± 4% of adherent CD45+ cells recruited in venules ~90 min following surgery were Gr–1+F4/80- (PMNs), and 45 ± 7% were F4/80+ (monocytes), while 18 ± 3% were Gr–1-F4/80- (lymphocytes). In contrast, the proportion of PMNs was significantly increased in cremasteric venules of sickle cell (SS) mice (Berkeley) (79 ± 7%, n=5; p<0.001 compared to C57BL/6). Our previous studies have revealed that the interactions between erythrocytes (RBCs) and adherent WBCs correlate with vascular occlusion and mortality in SS mice (Turhan et al, PNAS, 2002). To determine which WBC subset can capture RBCs in vivo, we monitored RBC-WBC interactions in high-resolution brightfield movies acquired immediately after the capture of fluorescence images. We found that the vast majority of interactions (~93%) occurred with CD45+Gr–1+F4/80- PMNs. The high proportion of lymphocytes in systemic venules was unexpected. We thus analyzed directly lymphocyte subclasses with antibodies specific for B and T cell markers (B220 and CD4/CD8). The proportion of B and T cells was consistent with the above results (total ~20%), similar between C57BL/6 and sickle cell mice, with B cells predominating in both groups (70–76% of all lymphocytes). Since the bone marrow (BM) is a target of SS disease, we examined differential WBC recruitment in this vascular bed. In contrast to the cremasteric microcirculation, CD45+Gr–1-F4/80- lymphocytes represented the major WBC subset in both C57BL/6 and SS venules (49 ± 2% and 63 ± 5%, respectively.) Adherent CD45+B220+ B lymphocytes were significantly more numerous than CD45+CD4/CD8+ T lymphocytes in C57BL/6 (6-fold ↑, p<0.001) and SS (9-fold ↑, p<0.005) mice. We next evaluated the spatial distribution of LFA-1 and PSGL–1 on Gr–1+ WBCs recruited in TNF-α-stimulated cremasteric venules. We observed that PSGL–1 distribution was polarized in the great majority of adherent Gr–1+ cells (93 – 97%), whereas neither LFA–1 nor Gr–1 was polarized. The majority of PSGL–1 clusters was luminal rather than adjacent to the endothelium in both stationary and slowly migrating cells. In stationary cells, PSGL–1 appeared random in relation to the direction of blood flow. By contrast, in migrating cells, PSGL–1 redistribution was strongly associated with the direction of cell migration, with PSGL–1 clusters being almost exclusively located at the trailing edge. In summary, our results reveal a profound difference in the WBC subsets recruited in the cremastric and BM venules, and a powerful method to analyze the formation of discrete microdomains in vivo during cell activation.
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del Pozo, M. A., P. Sánchez-Mateos, M. Nieto, and F. Sánchez-Madrid. "Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway." Journal of Cell Biology 131, no. 2 (October 15, 1995): 495–508. http://dx.doi.org/10.1083/jcb.131.2.495.
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Leukocyte recruitment is a key step in the inflammatory reaction. Several changes in the cell morphology take place during lymphocyte activation and migration: spheric-shaped resting T cells become polarized during activation, developing a well defined cytoplasmic projection designated as cellular uropod. We found that the chemotactic and proinflammatory chemokines RANTES, MCP-1, and, to a lower extent, MIP-1 alpha, MIP-1 beta, and IL-8, were able to induce uropod formation and ICAM-3 redistribution in T lymphoblasts adhered to ICAM-1 or VCAM-1. A similar chemokine-mediated effect was observed during T cells binding to the fibronectin fragments of 38- and 80-kD, that contain the binding sites for the integrins VLA-4 and VLA-5, respectively. The uropod structure concentrated the ICAM-3 adhesion molecule (a ligand for LFA-1), and emerged to the outer milieu from the area of contact between lymphocyte and protein ligands. In addition, we found that other adhesion molecules such as ICAM-1, CD43, and CD44, also redistributed to the lymphocyte uropod upon RANTES stimulation, whereas a wide number of other cell surface receptors did not redistribute. Chemokines displayed a selective effect among different T cell subsets; MIP-1 beta had more potent action on CD8+ T cells and tumor infiltrating lymphocytes (TIL), whereas RANTES and MIP-1 alpha targeted selectively CD4+ T cells. We have also examined the involvement of cAMP signaling pathway in uropod formation. Interestingly, several cAMP agonists were able to induce uropod formation and ICAM-3 redistribution, whereas H-89, a specific inhibitor of the cAMP-dependent protein kinase, abrogated the chemokine-mediated uropod formation, thus pointing out a role for cAMP-dependent signaling in the development of this cytoplasmic projection. Since the lymphocyte uropod induced by chemokines was completely abrogated by Bordetella pertussis toxin, the formation of this membrane projection appears to be dependent on G proteins signaling pathways. In addition, the involvement of myosin-based cytoskeleton in uropod formation and ICAM-3 redistribution in response to chemokines was suggested by the prevention of this phenomenon with the myosin-disrupting agent butanedione monoxime. Interestingly, this agent also inhibited the ICAM-3-mediated cell aggregation, but not the cell adhesion to substrata. Altogether, these results demonstrate that uropod formation and adhesion receptor redistribution is a novel function mediated by chemokines; this phenomenon may represent a mechanism that significantly contributes to the recruitment of circulating leukocytes to inflammatory foci.
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Hao, Jian-Jiang, Yin Liu, Michael Kruhlak, Karen E. Debell, Barbara L. Rellahan, and Stephen Shaw. "Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane." Journal of Cell Biology 184, no. 3 (February 9, 2009): 451–62. http://dx.doi.org/10.1083/jcb.200807047.
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Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.
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Gordeychuk, I. V., A. I. Tukhvatulin, S. P. Petkov, M. A. Abakumov, S. A. Gulyaev, N. M. Tukhvatulina, T. V. Gulyaeva, M. I. Mikhaylov, D. Y. Logunov, and M. G. Isaguliants. "Assessment of the Parameters of Adaptive Cell-Mediated Immunity in Naïve Common Marmosets (Callithrix jacchus)." Acta Naturae 10, no. 4 (December 15, 2018): 63–69. http://dx.doi.org/10.32607/20758251-2018-10-4-63-69.
Full textAbstract:
Common marmosets are small New World primates that have been increasingly used in biomedical research. This report presents efficient protocols for assessment of the parameters of adaptive cell-mediated immunity in common marmosets, including the major subpopulations of lymphocytes and main markers of T- and B-cell maturation and activation using flow cytometry with a multicolor panel of fluorescently labelled antibodies. Blood samples from eight common marmosets were stained with fluorescently labeled monoclonal antibodies against their population markers (CD45, CD3, CD20, CD4, CD8) and lymphocyte maturation and activation markers (CD69, CD62L, CD45RO, CD107a and CD27) and analyzed by flow cytometry. Within the CD45+ population, 22.75.5% cells were CD3- CD20+ and 67.66.3% were CD3+CD20-. The CD3+ subpopulation included 55.75.5% CD3+CD4+CD8- and 34.33.7% CD3+CD4-CD8+ cells. Activation and maturation markers were expressed in the following lymphocyte proportions: CD62L on 54.010.7% of CD3+CD4+ cells and 74.412.1% of CD3+CD8+ cells; CD69 on 2.71.2% of CD3+CD4+ cells and 1.20.5% of CD3+CD8+ cells; CD45RO on 1.60.6% of CD3+CD4+ cells and 1.80.7% of CD3+CD8+ cells; CD107a on 0.70.5% of CD3+CD4+ cells and 0.50.3% of CD3+CD8+ cells; CD27 on 94.62.1% of CD3+ cells and 8.93.9% CD20+ cells. Female and male subjects differed in the percentage of CD3+CD4+CD45RO+ cells (1.90.5 in females vs 1.10.2 in males; p 0.05). The percentage of CD20+CD27+ cells was found to highly correlate with animals age (r = 0.923, p 0.005). The basal parameters of adaptive cell-mediated immunity in nave healthy marmosets without markers of systemic immune activation were obtained. These parameters and the described procedures are crucial in documenting the changes induced in common marmosets by prophylactic and therapeutic immune interventions.