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(2024) Frontiers in Immunology. 15, 1297473. Abstract
Recently, cancer immunotherapy has revolutionized cancer treatment. Various forms of immunotherapy have a manageable safety profile and result in prolongation of overall survival in patients with solid tumors, but only in a proportion of patients. Various factors in the tumor microenvironment play critical roles and may be responsible for this lack of therapeutic response. Signaling lymphocytic activation molecule family (SLAMF) members are increasingly being studied as factors impacting the tumor immune microenvironment. SLAMF members consist of nine receptors mainly expressed in immune cells. However, SLAMF receptors have also been detected in cancer cells, and they may be involved in a spectrum of anti-tumor immune responses. Here, we review the current knowledge of the expression of SLAMF receptors in solid tumors and tumor-infiltrating immune cells and their association with patient outcomes. Furthermore, we discuss the therapeutic potential of targeting SLAMF receptors to improve outcomes of cancer therapy in solid tumors. We believe the research on SLAMF receptor-targeted strategies may enhance anti-cancer immunity in patients with solid tumors and improve clinical outcomes.
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(2023) JCI insight. 8, 14, Abstract[All authors]
The authors recently became aware of inadvertent errors in Figure 1F. In the original version, the representative flow plots provided for CD14– HD-PD and CD14+ HD-PD were incorrect.
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Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry(2023) Journal of the American Chemical Society. 145, 6, p. 3346-3360 Abstract[All authors]
Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.
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(2022) Cell reports (Cambridge). 41, 5, 111572. Abstract
CD74 is receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF binding to CD74 induces a signaling cascade resulting in the release of its cytosolic intracellular domain (CD74-ICD) that serves as a transcriptional regulator in chronic lymphocytic leukemia (CLL) cells. In the current study, we investigated the transcriptional and regulatory function of CD74-ICD in normal B cells. We show that following activation, CD74-ICD forms a complex in the cytosol with transcription factors, like PAX5, and binds the chromatin at a significantly higher number of sites compared with its binding in CLL cells. The expression of a major portion of these bound genes is shut down in the malignant cells. The CD74-ICD:PAX5 complex binds the promoter areas of a tumor-suppressor gene, DMTF1, and downregulates its expression through inhibition of transcription. These findings can help identify novel therapeutic pathways that are regulated during oncogenic transformation and are targets for future treatments.
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(2022) Experimental Hematology. 115, p. 30-43 Abstract[All authors]
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of small, mature CD5+ B lympho-cytes in the blood, marrow, and lymphoid organs. Cell survival depends on interaction with the leukemic microenvironment. However, the mechanisms controlling CLL cell survival are still incompletely understood. Macrophage migration-inhibitory factor (MIF), a pro-inflammatory and immunoregulatory chemokine-like cytokine, interacts with CXCR4, a major chemokine receptor, as well as with CD74/invariant chain, a single -pass type II receptor. In this study, we analyzed the roles of CXCR4, CD74, and MIF in CLL. Mononuclear cells from patients with hematological malignancies were analyzed for coexpression of CXCR4 and CD74 by flow cytometry. Strong co-and overexpression of CXCR4 and CD74 were observed on B cells of CLL patients (n = 10). Survival and chemotaxis assays indicated that CXCR4 and CD74 work together to enhance the survival and migration of malignant cells in CLL. Blockade of the receptors, either individually or in com-bination, promoted cell death and led to an abrogation of MIF-driven migration responses in murine and human CLL cells, suggesting that joint activation of both receptors is crucial for CLL cell survival and mobility. These findings indicate that the MIF/CXCR4/CD74 axis represents a novel therapeutic target in CLL. (c) 2022 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.
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(2022) eLife. 11, e77714. Abstract
The transformation of normal to malignant cells is accompanied by substantial changes in gene expression programs through diverse mechanisms. Here, we examined the changes in the landscape of transcription start sites and alternative promoter (AP) usage and their impact on the translatome in TCL1-driven chronic lymphocytic leukemia (CLL). Our findings revealed a marked elevation of APs in CLL B cells from Eµ-Tcl1 transgenic mice, which are particularly enriched with intra-genic promoters that generate N-terminally truncated or modified proteins. Intra-genic promoter activation is mediated by (1) loss of function of \u2018closed chromatin\u2019 epigenetic regulators due to the generation of inactive N-terminally modified isoforms or reduced expression; (2) upregulation of transcription factors, including c-Myc, targeting the intra-genic promoters and their associated enhancers. Exogenous expression of Tcl1 in MEFs is sufficient to induce intra-genic promoters of epigenetic regulators and promote c-Myc expression. We further found a dramatic translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing metabolic stress. These findings uncovered the role of Tcl1 oncogenic function in altering promoter usage and mRNA translation in leukemogenesis.
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(2021) PLoS Biology. 19, 3, 3001121. Abstract[All authors]
Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.
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(2021) JCI insight. 6, 4, 141683. Abstract[All authors]
Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) within the BM. The BM microenvironment supports survival of the malignant cells and is composed of cellular fractions that foster myeloma development and progression by suppression of the immune response. Despite major progress in understanding the biology and pathophysiology of MM, this disease is still incurable and requires aggressive treatment with significant side effects. CD84 is a self-binding immunoreceptor belonging to the signaling lymphocyte activation molecule (SLAM) family. Previously, we showed that CD84 bridges between chronic lymphocytic leukemia cells and their microenvironment, and it regulates T cell function. In the current study, we investigated the role of CD84 in MM. Our results show that MM cells express low levels of CD84. However, these cells secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to an elevation of expression of genes regulating differentiation to monocytic/granulocytic-myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively) and upregulation of PD-L1 expression on MDSCs, which together suppress T cell function. Downregulation of CD84 or its blocking reduce MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that CD84 might serve as a novel therapeutic target in MM.
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(2020) Oncogene. 39, 9, p. 1997-2008 Abstract[All authors]
Chronic lymphocytic leukemia (CLL) is a malignancy of mature B lymphocytes. The microenvironment of the CLL cells is a vital element in the regulation of the survival of these malignant cells. CLL cell longevity is dependent on external signals, originating from cells in their microenvironment including secreted and surface-bound factors. Dendritic cells (DCs) play an important part in tumor microenvironment, but their role in the CLL bone marrow (BM) niche has not been studied. We show here that CLL cells induce accumulation of bone marrow dendritic cells (BMDCs). Depletion of this population attenuates disease expansion. Our results show that the support of the microenvironment is partly dependent on CD84, a cell surface molecule belonging to the Signaling Lymphocyte Activating Molecule (SLAM) family of immunoreceptors. Our results suggest a novel therapeutic strategy whereby eliminating BMDCs or blocking the CD84 expressed on these cells may reduce the tumor load.
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(2020) Nature Communications. 11, 409. Abstract[All authors]
The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.
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(2020) Molecular Cell. 78, 3, p. 434-444.e5 Abstract[All authors]
Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m(6)A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.
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(2019) Journal of Immunology. 203, 7, p. 1857-1866 Abstract
Multiple sclerosis is an inflammatory disease of the CNS characterized by neurologic impairment resulting from primary demyelination and axonal damage. The pathogenic mechanisms of disease development include Ag-specific T cell activation and Th1 differentiation, followed by T cell and macrophage migration into the CNS. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. We previously demonstrated that picomolar levels of CCL2 strongly restrict the development of inflammation in models of inflammatory bowel disease. Moreover, CCR2 deficiency in T cells promotes a program inducing the accumulation of Foxp3(+) regulatory T cells while decreasing the levels of Th17 cells in vivo. In the current study, the effect of picomolar levels of CCL2 on the autoimmune inflammatory response associated with a multiple sclerosis-like disease in mice was analyzed. We found that low dosages of CCL2 were effective in suppressing MOG-induced experimental autoimmune encephalomyelitis (EAE), and they downregulated chronic EAE. The modulation of EAE by CCL2 was associated with downregulation of Th1/Th17 cells and upregulation of TGF-13 and induction of regulatory CD4(+)Foxp3 T cells. Most strikingly, these low levels of CCL2 induced formation of highly functional regulatory T cells. Thus, this study strongly supports the potential use of CCL2 as a regulatory mediator for treating inflammatory autoimmune diseases.
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(2019) Clinical Immunology. 204, p. 23-30 Abstract
The Signaling Lymphocyte Activation Molecule family (SLAMF) is a collection of nine surface receptors expressed mainly on hematopoietic cells, and was found to modulate the behavior of immune cells. SLAMF receptors are expressed on B cells in health and disease. Each SLAM receptor has a unique differential expression pattern during the development and activation of B cells. Furthermore, recent findings have revealed a principal role for this family of receptors in B cell malignancies, emphasizing their importance in the control of malignant cell survival, cell to cell communication within the tumor microenvironment, retention in the supporting niches and regulation of T cell anti-tumor response. This review summarizes the latest studies regarding SLAMF expression and behavior in B cells and in B cell pathologies, and discusses the therapeutic potential of these receptors.
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(2019) Proceedings of the National Academy of Sciences of the United States of America. 116, 33, p. 16489-16496 Abstract
SLAMF9 belongs to the conserved lymphocytic activation molecule family (SLAMF). Unlike other SLAMs, which have been extensively studied, the role of SLAMF9 in the immune system remained mostly unexplored. By generating CRISPR/Cas9 SLAMF9 knockout mice, we analyzed the role of this receptor in plasmacytoid dendritic cells (pDCs), which preferentially express the SLAMF9 transcript and protein. These cells display a unique capacity to produce type I IFN and bridge between innate and adaptive immune response. Analysis of pDCs in SLAMF9(-/-) mice revealed an increase of immature pDCs in the bone marrow and enhanced accumulation of pDCs in the lymph nodes. In the periphery, SLAMF9 deficiency resulted in lower levels of the transcription factor SpiB, elevation of pDC survival, and attenuated IFN-alpha and TNF-alpha production. To define the role of SLAMF9 during inflammation, pDCs lacking SLAMF9 were followed during induced experimental autoimmune encephalomyelitis. SLAMF9(-/-) mice demonstrated attenuated disease and delayed onset, accompanied by a prominent increase of immature pDCs in the lymph node, with a reduced costimulatory potential and enhanced infiltration of pDCs into the central nervous system. These results suggest the crucial role of SLAMF9 in pDC differentiation, homeostasis, and function in the steady state and during experimental autoimmune encephalomyelitis.
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(2018) Journal of Clinical Investigation. 128, 12, p. 5465-5478 Abstract[All authors]
Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.
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(2018) Cellular Signalling. 46, p. 32-42 Abstract
Macrophage migration inhibitory factor (MIF) is a chemokine-like inflammatory cytokine, which plays a pivotal role in the pathogenesis of inflammatory and cardiovascular diseases as well as cancer. We previously identified MIF as a novel B cell chemokine that promotes B cell migration through non-cognate interaction with the CXC chemokine receptor CXCR4 and CD74, the surface form of MHC class II invariant chain. In this study, we have analyzed the regulation of the MIF receptors under inflammatory conditions by investigating the impact of lipopolysaccharide (LPS), tumor necrosis factor-a (TNF-alpha) and interleukin-1 beta (IL-1 beta) on CD74 and CXCR4 expression in B lymphocytes. We found that both LPS and TNF-a stimulation of primary B cells and the human B myeloma cell line RPMI-8226 enhanced protein expression as well as mRNA levels of CD74 in a time-and dose dependent manner. By contrast, no effect on CXCR4 expression was observed. Selective inhibition of I kappa B alpha phosphorylation significantly attenuated LPS-induced expression of CD74, suggesting the contribution of NF-kappa B signaling pathways to the regulation of CD74 expression. Importantly, individual or simultaneous blockade of MIF or CD74 using specific neutralizing antibodies markedly affected B cell proliferation after LPS exposure. Taken together, our findings unveil a connection between the pro-proliferative activity of MIF/CD74 signaling in B cells and inflammation, offering novel target mechanisms in inflammatory cardiovascular or autoimmune pathogenesis.
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(2017) Journal of Immunology. 198, 12, p. 4659-4671 Abstract[All authors]
Chemokines and chemokine receptors establish a complex network modulating immune cell migration and localization. These molecules were also suggested to mediate the differentiation of leukocytes; however, their intrinsic, direct regulation of lymphocyte fate remained unclear. CCR2 is the main chemokine receptor inducing macrophage and monocyte recruitment to sites of inflammation, and it is also expressed on T cells. To assess whether CCR2 directly regulates T cell responses, we followed the fates of CCR2-/- T cells in T cell-specific inflammatory models. Our in vitro and in vivo results show that CCR2 intrinsically mediates the expression of inflammatory T cell cytokines, and its absence on T cells results in attenuated colitis progression. Moreover, CCR2 deficiency in T cells promoted a program inducing the accumulation of Foxp3+ regulatory T cells, while decreasing the levels of Th17 cells in vivo, indicating that CCR2 regulates the immune response by modulating the effector/regulatory T ratio.
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(2017) Proceedings of the National Academy of Sciences of the United States of America. 114, 3, p. 562-567 Abstract[All authors]
CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74-ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74-ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.
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(2017) Oncogene. 36, 5, p. 628-638 Abstract[All authors]
Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Signals from the CLL microenvironment promote progression of the disease and induce drug resistance. This phenomenon is largely dependent on direct contact between the malignant B cells and stromal cells. CD84 belongs to the signaling lymphocyte activation molecule family of immunoreceptors, which self-associates, forming an orthogonal homophilic dimer. We therefore hypothesized that CD84 may bridge between CLL cells and their microenvironment, promoting cell survival. Our in vitro results show that CD84 expressed on CLL cells interact with CD84 expressed on cells in their microenvironment, inducing cell survival in both sides. Blocking CD84 in vitro and in vivo disrupt the interaction of CLL cells with their microenvironment, resulting in induced cell death. Thus, our findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.
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(2017) MIF Family Cytokines in Innate Immunity and Homeostasis. p. 1-20 Abstract
CD74 is a type II cell surface protein that was previously shown to play a role in antigen presentation and as a receptor for the cytokine macrophage migration inhibitory factor (MIF). Studies from recent years demonstrate an important role for CD74 in maintenance of innate and adaptive immune cells. This chapter discusses the MIF/CD74-dependent role in regulating cell survival, metabolism, adhesion, and response to hypoxia in health and disease.
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(2017) Journal of Immunology. 199, 8, p. 2745-2757 Abstract[All authors]
The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, proliferation, and apoptosis. In this study, we focused on the role of T cells in the maintenance/survival of the mature naive peripheral B cell population. We show that naive B and T cells interact via the signaling lymphocyte activation molecule (SLAM) family receptor, SLAMF6. This interaction induces cell type-specific signals in both cell types, mediated by the SLAM-associated protein (SAP) family of adaptors. This signaling results in an upregulation of the expression of the cytokine migration inhibitory factor in the T cells and augmented expression of its receptor CD74 on the B cell counterparts, consequently enhancing B cell survival. Furthermore, in X-linked lymphoproliferative disease patients, SAP deficiency reduces CD74 expression, resulting in the perturbation of B cell maintenance from the naive stage. Thus, naive T cells regulate B cell survival in a SLAMF6- and SAP-dependent manner.
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(2017) Oncogene. 36, 26, p. 3686-3694 Abstract
The tyrosine phosphatase PTPROt is a suggested tumor suppressor (TS) in B-cell chronic lymphocytic leukemia (CLL), and its expression is reduced in this disease. In order to examine how reduced PTPROt expression affects CLL in vivo we induced CLL in PTPROt-targeted mice. Unexpectedly, loss of both Ptprot alleles delayed disease detection and progression and lengthened survival relative to mice carrying two intact alleles, indicating that PTPROt fulfills a novel tumor-promoting role in CLL. Tumor cells from mice lacking PTPROt exhibited reduced B-cell receptor (BCR)-induced signaling, as well as increased apoptosis and autophagy. Inhibition of BCR/Src signaling in CLL cells induced their apoptosis, indicating that these findings are linked causally. These results suggest a cell-autonomous mechanism for the weakened CLL phenotype of PTPROt-deficient mice and uncover non-redundant roles for PTPROt in support of BCR signaling and survival of CLL cells. In contrast, loss of only one Ptprot allele induced earlier detection and progression of CLL and reduced survival, consistent with a tumor-suppressing role for PTPROt. Tumor cells from mice lacking one or both Ptprot allele exhibited increased interleukin-10 (IL-10) expression and signaling, factors known to support CLL; cells lacking one Ptprot alleles exhibited normal BCR signaling and cell death rates. We conclude that loss of one Ptprot allele promotes CLL, most likely by activating IL-10 signaling. Loss of both Ptprot alleles also reduces BCR signaling and increases cell death rates, offsetting the IL-10 effects and reducing the severity of the disease. PTPROt thus functions as an obligate haploinsufficient TS in CLL, where its expression levels determine its role as a promoter or inhibitor of the tumorigenic process in mice. Partial loss of PTPROt generates the strongest disease phenotype, suggesting that its intermediate expression levels in CLL are selected for.
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(2016) Inflammatory Bowel Diseases. 22, 2, p. 257-267 Abstract
The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease. CD151 formed a cell surface complex with VLA-4 and LFA-1 integrins, and its activation led to enhanced migration of T cells. Picomolar levels of CCL2 that were previously shown to inhibit T-cell migration to lymph nodes suppressed CD151 expression and dissociated CD151-integrin complexes in T lymphocytes, resulting in attenuated migration toward T-cell attractant chemokines. To directly inhibit CD151 function, a truncated CD151 peptide fragment mimicking of the CD151 extracellular loop was designed. CD151 extracellular loop inhibited T-cell migration in vitro and in vivo and attenuated the development of dextrane sulfate sodium-induced colitis. Thus, CD151 is a key orchestrator of T cell motility; interference with its proper function results in attenuated progression of inflammatory bowel disease.
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(2016) Oncotarget. 7, 7, p. 7550-7562 Abstract[All authors]
Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression. The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG. Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.
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(2014) Oncogene. 33, 8, p. 1006-1016 Abstract
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow. The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the signaling lymphocyte activating molecule family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 messenger RNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.
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(2014) British Journal of Pharmacology. 171, 4, p. 888-895 Abstract
In healthy individuals, the pool of peripheral lymphocytes is constant in size. The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, survival and proliferation. Survival factors have been shown to play a critical role in maintaining the correct size of lymphocyte populations. Midkine, a heparin-binding cytokine was recently shown to be involved in cell proliferation, differentiation and apoptosis in various cell types including normal and malignant B cells. This review focuses on the role of midkine in the regulation of peripheral B cell survival in health and disease.
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(2014) Journal of Immunology. 192, 11, p. 5273-5284 Abstract
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with chemokine-like functions that plays a pivotal role in the pathogenesis of inflammatory diseases by promoting leukocyte recruitment. We showed that MIF promotes the atherogenic recruitment of monocytes and T cells through its receptors CXCR2 and CXCR4. Effects of MIF on B cell recruitment have not been addressed. In this study, we tested the involvement of MIF in B cell chemotaxis and studied the underlying mechanism. We show that MIF promotes primary murine B cell chemotaxis in a dose-dependent manner, comparable to the B cell chemokines CXCL13 and CXCL12. Splenic B cells express CXCR4 and the receptor CD74 but not CXCR2. Inhibition of CXCR4 or CD74 or a genetic deficiency of Cd74 in primary B cells fully abrogated MIF-mediated B cell migration, implying cooperative involvement of both receptors. MIF stimulation of B cells resulted in a rapid increase in intracellular Ca2+ mobilization and F-actin polymerization. Intriguingly, the tyrosine kinase ZAP-70 was activated upon MIF and CXCL12 treatment in a CXCR4- and CD74-dependent manner. Pharmacological inhibition of ZAP-70 resulted in abrogation of primary B cell migration. Functional involvement of ZAP-70 was confirmed by small interfering RNA-mediated knockdown in Ramos B cell migration. Finally, primary B cells from ZAP-70 gene-deficient mice exhibited ablated transmigration in response to MIF or CXCL12. We conclude that MIF promotes the migration of B cells through a ZAP-70-dependent pathway mediated by cooperative engagement of CXCR4 and CD74. The data also suggest that MIF may contribute to B cell recruitment in vivo (e.g., in B cell-related immune disorders). The Journal of Immunology, 2014, 192: 5273-5284.
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The Integral Role of CD74 in Antigen Presentation, MIF Signal Transduction, and B Cell Survival and Homeostasis(2014) Mini-Reviews in Medicinal Chemistry. 14, 14, p. 1132-1138 Abstract
Molecules that link innate and adaptive immunity and regulate immune response in health and disease are now the focus of many studies. This review discusses CD74 and its ligand macrophage migration inhibitory factor (MIF), and their central position in linking these two components of the immune response by controlling survival of macrophages and B cells.
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(2013) Journal of Allergy and Clinical Immunology. 131, 2, p. 477-485.e1 Abstract[All authors]
Background: Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. Objective: We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. Methods: Molecular, immunologic, and functional assays were performed. Results: The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. Conclusion: Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects.
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(2013) Journal of Leukocyte Biology. 93, 1, p. 51-61 Abstract
Cytokines and chemokines are secreted, small cell-signaling protein molecules, whose receptors are expressed on immune cells. These factors play a critical role in immune cell differentiation, migration, and polarization into functional subtypes and in directing their biological functions. Much attention has been devoted to exploring the role of key inflammatory cytokines and promigratory chemokines in autoimmune, autoinflammatory, and allergic diseases, leading to development of therapeutic strategies that are based on their targeted neutralization. Recent studies, including those coming from our groups, show that several major proinflammatory cytokines and chemokines, including IFN-γ, IL-2, CCL2, and CXCL12, may also function as anti-inflammatory mediators and therefore, may have potential as anti-inflammatory drugs. Likewise, major anti-inflammatory mediators, such as TGF- β, may under certain conditions, in combination with other cytokines, exhibit proinflammatory function and direct the polarization of the highly inflammatory CD4+ Th17 cells. We show here that the biological function of pro- and antiinflammatory cytokines is dependent on three key parameters: the local concentration of a given cytokine, the stage of disease in which it is administered, and its combination with other cytokines. The therapeutic implications of these findings are discussed, including two very recent studies summarizing clinical trials, in which low-dose administration of IL-2 was used to successfully suppress HCV and GVHD.
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(2012) Cytokine. 60, 1, p. 13-22 Abstract
Adaptive immunity depends on the production and maintenance of a pool of mature peripheral lymphocytes throughout life. The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Lasting B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. Cytokines have been shown to play a critical role in maintaining lymphocyte homeostasis. This review focuses on the role of cytokines and their receptors in the regulation of peripheral B cell survival, with an emphasis on those that have received relatively less attention in the literature.
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(2012) FEBS Letters. 586, 18, p. 2906-2910 Abstract
Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world, is characterized by the progressive accumulation of small mature CD5 +B lymphocytes in the peripheral blood, lymphoid organs, and bone marrow (BM). The main feature of the disease is decreased apoptosis, resulting in the pathologic accumulation of these malignant cells. Appropriate cellular responses to changes in oxygen tension during normal development or pathological processes, such as cardiovascular disease and cancer, are ultimately regulated by the transcription factor, hypoxia-inducible factor (HIF). Unlike their normal counterparts, CLL cells express HIF-1α even under normoxia. In addition, overexpression of HIF-1α has been observed in leukemic cells in BM specimens from CLL patients. The HIF transcription factor has been implicated in controlling the expression of a wide variety of genes implicated in apoptosis, angiogenesis, invasion, and metastasis. This review describes pathways regulating CLL survival with a focus on HIF-1α and its target genes, MIF and Midkine (MK), and the potential cross-talk between these factors.
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(2012) Journal of Immunology. 188, 1, p. 259-269 Abstract[All authors]
Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.
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(2011) Immunology. 132, 1, p. 87-95 Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice. In the present study, the effect of treatment with hCDR1 on the CD74/macrophage migration inhibitory factor (MIF) pathway was studied. We report here that B lymphocytes from SLE-afflicted mice express relatively elevated levels of CD74, compared with B cells from healthy mice. CD74 is a receptor found in complex with CD44, and it binds the pro-inflammatory cytokine MIF. The latter components were also up-regulated in B cells from the diseased mice, and treatment with hCDR1 resulted in their down-regulation and in reduced B-cell survival. Furthermore, up-regulation of CD74 and CD44 expression was detected in brain hippocampi and kidneys, two target organs in SLE. Treatment with hCDR1 diminished the expression of those molecules to the levels determined for young healthy mice. These results suggest that the CD74/MIF pathway plays an important role in lupus pathology.
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(2011) Leukemia & Lymphoma. 52, 8, p. 1446-1454 Abstract
This review deals with the cytokine macrophage migration inhibitory factor (MIF) and its receptor, CD74. MIF and CD74 have been shown to regulate peripheral B cell survival and were associated with tumor progression and metastasis. CD74 expression has been suggested to serve as a prognostic factor in many cancers, with higher relative expression of CD74 behaving as a marker of tumor progression. In chronic lymphocytic leukemia (CLL) cells, binding of MIF to CD74 induces nuclear factor-κB (NF-κB) activation and up-regulation of TAp63 expression, resulting in the secretion of interleukin 8 (IL-8), which in turn promotes cell survival. In addition, TAp63 expression elevates expression of the integrin VLA-4, particularly during the advanced stage of the disease. Blocking of CD74, TAp63, or VLA-4 inhibits the in vivo homing of CLL cells to the BM. Thus, CD74 and its target genes, TAp63 and VLA-4, facilitate migration of CLL cells back to the BM, where they interact with the supportive BM environment that helps rescue them from apoptosis. These results are expected to pave the way toward novel therapeutic strategies aimed at interrupting this survival pathway. One such agent, the monocolonal antibody milatuzumab directed at CD74, is already being studied in early clinical trials.
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(2010) Inflammatory Bowel Diseases. 16, 9, p. 1496-1504 Abstract
Background: Chemokines regulate the pathways that restrict homing of specific subsets of immune cells, and thereby fine tune the immune response at specific lymphoid and peripheral tissues. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. Previously, we demonstrated that pM levels of CCL2 dramatically inhibit migration of T cells. The aim was to test whether subphysiological doses of CCL2 can ameliorate murine colitis and inflammation-induced colorectal cancer. Methods: TNBS (2,4,6 trinitrobenzene sulfonic acid) colitis and dextran sodium sulfate (DSS) colitis were induced in Balb/c and C57BL/6 mice, respectively. Mice were treated daily with intraperitoneal CCL2 injections. Disease activity was assessed clinically, histologically, and by measuring inflammatory cytokine levels. In addition, an inflammatory cancer model was induced by azoxymethane-DSS (AOM-DSS) in Balb/c mice. Mice were treated daily with CCL2 for 11 weeks and then assessed for number of tumors in the colons. Results: Daily administration of CCL2 (60-120 ng) significantly decreased the development of TNBS- and DSS-induced colitis. In a DSS-AOM model, CCL2-treated mice developed significantly fewer tumors (P
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(2010) World Journal of Gastroenterology. 16, 26, p. 3258-2366 Abstract
AIM: To investigate the expression and function of CD74 in normal murine colon epithelial cells (CEC) and colon carcinoma cells. METHODS: Expression of CD74 mRNA and protein were measured by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and fluorescence- activated cell sorter (FACS). The effect of migration inhibitory factor (MIF) on the survival of normal CEC from C57BL/6, NOD/SCID, and CD74 deficient mice both in vitro and in vivo, and on the CT26 carcinoma cell line was analyzed by (quantitative) qRT-PCR, RTPCR, Western blotting and FACS. RESULTS: CD74 was found to be expressed on normal CEC. Stimulation of CD74 by MIF induced a signaling cascade leading to up-regulation of Bcl-2 expression, resulting in a significant increased survival of CEC. CD74 was also expressed on the CT26 colon carcinoma cell line and its stimulation by MIF resulted in enhanced cell survival, up-regulation of Akt phosphorylation and Bcl-2 expression. CONCLUSION: CD74 is expressed on CEC and colon carcinoma cells and serves as a survival receptor in these cells. These results may have implications on colorectal cancer research.
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(2010) Molecular Biology of the Cell. 21, 11, p. 1864-1871 Abstract
Immature B cells migrate to the spleen where they differentiate into mature cells. This final maturation step is crucial to enable B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that Id2 acts as a negative regulator of the differentiation of immature B cells occurring in the spleen. Id2 expression has been found to depend on Myc-Max-Mad transcriptional complexes in mammary epithelial cells. Nearly all studies to date have shown that Mad proteins inhibit proliferation, presumably by antagonizing the function of Myc proteins. In the current study, we followed the Mad family members during peripheral B cell differentiation. We show that Mad3 actively regulates B cell differentiation. Our results demonstrate that high expression levels of Mad3 in immature B cells induce Id2 expression, which inhibits transcription of genes essential for B cell differentiation. During their differentiation to mature cells, B cells reduce their Mad3 expression, enabling the maturation process to occur.
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(2010) Blood. 116, 26, p. 5907-5918 Abstract[All authors]
Talin1 is a key integrin coactivator. We investigated the roles of this cytoskeletal adaptor and its target integrins in B-cell lymphogenesis, differentiation, migration, and function. Using CD19 Cre-mediated depletion of talin1 selectively in B cells, we found that talin1 was not required for B-cell generation in the bone marrow or for the entry of immature B cells to the white pulp of the spleen. Loss of talin1 also did not affect B-cell maturation into follicular B cells but compromised differentiation of marginal zone B cells. Nevertheless, serum IgM and IgG levels remained normal. Ex vivo analysis of talin1-deficient spleen B cells indicated a necessary role for talin1 in LFA-1 and VLA-4 activation stimulated by canonical agonists, but not in B-cell chemotaxis. Consequently, talin1 null B splenocytes could not enter lymph nodes nor return to the bone marrow. Talin1 deficiency in B cells was also impaired in the humoral response to a T cell-dependent antigen. Collectively, these results indicate that talin1 is not required for follicular B-cell maturation in the spleen or homeostatic humoral immunity but is critical for integrin-dependent B lymphocyte emigration to lymph nodes and optimal immunity against T-dependent antigens.
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(2010) Journal of Immunology. 184, 9, p. 4761-4769 Abstract
The hallmark of chronic lymphocytic leukemia (CLL) is the relentless accumulation of mature lymphocytes, mostly due to their decreased apoptosis. CD74 was recently shown to serve as a survival receptor on CLL cells. In this study, we show that stimulation of CD74 with its natural ligand, migration inhibitory factor, initiates a signaling cascade that results in upregulation of TAp63, which directly regulates CLL survival. In addition, TAp63 expression elevates the expression of the integrin VLA-4, particularly during the advanced stage of the disease. Blocking of CD74, TAp63, or VLA-4 inhibits the in vivo homing of CLL cells to the bone marrow (BM). Thus, CD74 and its target genes TAp63 and VLA-4 facilitate migration of CLL cells back to the BM, where they interact with the supportive BM environment that rescues them from apoptosis. These results could form the basis of novel therapeutic strategies aimed at blocking homing of CLL cells in their return to the BM and attenuating their survival.
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(2009) Annals of Neurology. 66, 4, p. 521-531 Abstract
Objective: Myasthenia gravis (MG), a neuromuscular disease mediated by anti-acetylcholine receptor (AChR) autoantibodies, is associated with thymic hyperplasia characterized by ectopic germinal centers that contain pathogenic antibody-producing B cells. Our thymic transcriptome study demonstrated increased expression of CCL21, a recruiter of immune cells. Accordingly, we are investigating its implication in MG pathogenesis. Methods: The expression of CCL21 and its CCR7 receptor was analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting, respectively. Chemotaxis of T and B cells to CCL21 was measured by transwell assay. The nature of the thymic cells overexpressing CCL21 was investigated by immunochemistry and laser-capture microdissection combined with real-time PCR. Results: We demonstrate that CCL21 is overexpressed specifically in hyperplastic MG thymuses, whereas there is no variation in CCR7 levels on blood cells. We show that although CCL21 attracts both human T and B cells, it acts more strongly on naive B cells. CCL21 overexpression is normalized in corticoid-treated MG patients, suggesting that targeting this chemokine could represent a new selective treatment, decreasing the abnormal peripheral lymphocyte recruitment. Moreover, we locate protein and messenger RNA overexpression of CCL21 to specific endothelial vessels. Investigation of the nature of these vessels demonstrated different angiogenic processes in MG thymuses: high endothelial venule angiogenesis and lymphangiogenesis. Unexpectedly, CCL21 overexpression originates from afferent lymphatic endothelial vessels. Interpretation: We postulate that thymic overexpression of CCL21 on specialized lymphatic vessels results in abnormal peripheral lymphocyte recruitment, bringing naive B cells in contact with the inflammatory environment characteristic of MG thymuses, where they can be sensitized against AChR.
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(2009) Cancer Research. 69, 7, p. 3121-3130 Abstract[All authors]
Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We LFA-I and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-I due to low ß2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL,2. CLL cells failed to transmigrate across VCAM-1-expressing, ICAM-I-expressing, and CXCL,2-expressing endothelium, whereas when LFA-I expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1-dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1-independent and VLA-4-independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen.
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(2008) Blood. 112, 13, p. 5016-5025 Abstract
Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1 - and LFA-1mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemo-kines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.
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(2008) Blood. 111, 1, p. 50-59 Abstract
To complete their maturation and participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or to sites of inflammation, enabling their targeting to the spleen for final maturation. This inhibition is mediated by IFN-γ, which is transcribed and secreted at low levels by these immature B cells; IFN-γ expression is extinguished following B-cell maturation. Stimulation of the MHC class I receptor, Ly49D, triggers a signaling cascade that increases transcription of both IL-12 (p40) and IL-18; these, in turn, induce the secretion of IFN-γ. In the present study, we demonstrate that Ly49D-dependent secretion of IL-12 and IL-18 induces IL-15 expression by immature B cells, and that these 3 factors together regulate IFN-γ production that inhibits their ability to home to the lymph nodes or to sites of inflammation. Thus, IL-15 controls immature B-cell homing, resulting in shaping the B-cell repertoire to enable an efficient immune response.
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(2008) Journal of Biological Chemistry. 283, 5, p. 2784-2792 Abstract
Macrophage migration inhibitory factor (MIF) is an upstream activator of innate immunity that regulates subsequent adaptive responses. It was previously shown that in macrophages, MIF binds to a complex of CD74 and CD44, resulting in initiation of a signaling pathway. In the current study, we investigated the role of MIF in B cell survival. We show that in B lymphocytes, MIF initiates a signaling cascade that involves Syk and Akt, leading to NF-κB activation, proliferation, and survival in a CD74-and CD44-dependent manner. Thus, MIF regulates the adaptive immune response by maintaining the mature B cell population.
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(2008) Nature Immunology. 9, 4, p. 388-395 Abstract
Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.
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(2007) Blood. 110, 13, p. 4303-4311 Abstract
Most mature follicular B cells circulate within the periphery in a quiescent state, without actively contributing to an acute immune response. Lasting B-cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. We recently demonstrated that cell surface CD74 controls mature B-cell survival. Stimulation of cell surface CD74 leads to NF-κB activation, which enables entry of the stimulated B cells into the S phase, induction of DNA synthesis, and cell division, and augments the expression of survival genes. In the present study, we investigated CD74 target genes to determine the identities of the molecules whose expression is modulated by CD74, thereby regulating B-cell survival. We report that CD74 activates the p65 member of the NF-κB family, which in turn up-regulates the expression of p53-related TAp63 proteins. TAp63 then binds and transactivates the Bcl-2 gene and induces the production of Bcl-2 protein, thereby providing the cells with increased survival capacity. Thus, the CD74/NF-κB/TAp63 axis defines a novel antiapoptotic pathway in mature B cells, resulting in the shaping of both the B-cell repertoire and the immune response.
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(2007) Journal of Immunology. 178, 1, p. 163-171 Abstract
The ability to recover from CNS injuries is strain dependent. Transgenic mice that weakly express the p41 CD74 isoform (an integral membrane protein functioning as a MHC class II chaperone) on an I-Ab genetic background have normal CD4+ T cell populations and normal surface expression of MHC class II, but their B cell development is arrested while the cells are still immature. After a CNS injury, these mice recover better than their matched wild-type controls. We generated p41-transgenic mice on an I-Ad background (p41-I-Ad mice), and found that their recovery from CNS injuries was worse than that of controls. A correlative inverse effect was seen with respect to the kinetics of T cell and B cell recruitment to the injured CNS and the expression of insulin-like growth factor at the lesion site. These results, besides verifying previous findings that B cells function in the damaged CNS, demonstrate that the outcome of a particular genetic manipulation may be strain dependent.
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(2007) European Journal of Immunology. 37, 7, p. 1996-2007 Abstract
In order to complete their maturation and participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to LN or to sites of inflammation, enabling their targeting to the spleen. This inhibition is mediated by IFN-γ, which is transcribed and secreted at low levels by these immature B cells; its expression is subsequently down-regulated following B cell maturation. The activating and inhibitory MHC class I receptors, Ly49D and Ly49G2, regulate IFN-γ secretion in B cells, preventing their migration to antigen-enriched sites and their premature encounter with an antigen, while enabling their entry into the LN when mature. In the present study, we elucidate the pathways by which the Ly49 receptors regulate IFN-γ levels. We show that Ly49D stimulation triggers a signaling cascade that increases transcription of both IL-12B and IL-18; these, in turn, can interact with their specific receptors, which are expressed at elevated levels on immature B cells. Ligation of the IL-12B and IL-18 receptors induces the secretion of IFN-γ, thereby regulating their cytoskeleton rearrangement and migration.
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(2007) Proceedings of the National Academy of Sciences of the United States of America. 104, 33, p. 13408-13413 Abstract
Chronic lymphocytic leukemia (CLL) is a malignant disease of small mature lymphocytes. Previous studies have shown that CLL B lymphocytes express relatively large amounts of CD74 mRNA relative to normal B cells. In the present study, we analyzed the molecular mechanism regulated by CD74 in B-CLL cells. The results presented here show that activation of cell-surface CD74, expressed at high levels from an early stage of the disease by its natural ligand, macrophage migration-inhibition factor (MIF), initiates a signaling cascade that contributes to tumor progression. This pathway induces NF-kappa B activation, resulting in the secretion of IL-8 which, in turn, promotes cell survival. Inhibition of this pathway leads to decreased cell survival. These findings could form the basis of unique therapeutic strategies aimed at blocking the CD74-induced, IL-8- dependent survival pathway.
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(2006) Blood. 107, 12, p. 4807-4816 Abstract
CD74 is an integral membrane protein that was thought to function mainly as an MHC class II chaperone. However, CD74 was recently shown to have a role as an accessory-signaling molecule. Our studies demonstrated that CD74 regulates B-cell differentiation by inducing a pathway leading to the activation of transcription mediated by the NF-κB p65/RelA homodimer and its coactivator, TAFII105. Here, we show that CD74 stimulation with anti-CD74 antibody leads to an induction of a signaling cascade resulting in NF-κB activation, entry of the stimulated cells into the S phase, elevation of DNA synthesis, cell division, and augmented expression of BCL-XL. These studies therefore demonstrate that surface CD74 functions as a survival receptor.
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(2006) Journal of Immunology. 176, 8, p. 4716-4729 Abstract
The mechanism(s) that regulates NK cell mobilization and the significance of this process to NK cell activity are unknown. After Con A-induced hepatitis, NK cells are mobilized from the spleen and bone marrow into the periphery in an IFN-γ-dependent fashion. Intraperitoneal administration of IFN-γ stimulates the mobilization of NK cells into the circulation, but not their cell death or proliferation. Increased number of circulating NK cells was coupled with their accumulation in the peritoneum, liver, and tumor-bearing lung tissue. Furthermore, increased number of NK cells in the lung reduced metastasis of Lewis lung carcinoma cells (3LL cell line) resulting in significantly extended NK-dependent survival. Mobilization of NK cells was specific and required the presence of T cells. Moreover, mobilization and migration of spleen NK cells in response to IFN-γ treatment is dependent on the chemokine receptor CXCR3. Mechanistic insights regarding the role of IFN-γ in the regulation of NK cell mobilization and their accumulation at sites of tumor metastasis may lead to the development of novel immunotherapy for cancer.
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(2005) Molecular Biology of the Cell. 16, 11, p. 5061-5069 Abstract
Quite a few regulatory proteins, including transcription factors, are normally maintained in a dormant state to be activated after internal or environmental cues. Recently, a novel strategy, requiring proteolytic cleavage, was described for the mobilization of dormant transcription factors. These transcription factors are initially synthesized in an inactive form, whereas "nesting" in integral membrane precursor proteins. After a cleavage event, these new active factors are released from the membrane and can migrate into the nucleus to drive regulated gene transcription. This mechanism, regulated intramembrane proteolysis (RIP), controls diverse biological processes in prokaryotes and eukaryotes in response to a variety of signals. The MHC class II chaperone, CD74 (invariant chain, Ii), was previously shown to function as a signaling molecule in several pathways. Recently, we demonstrated that after intramembranal cleavage, the CD74 cytosolic fragment (CD74-ICD) is released and induces activation of transcription mediated by the NF-κB p65/RelA homodimer and the B-cell-enriched coactivator, TAFII105. Here, we add CD74 to the growing family of RIP-processed proteins. Our studies show that CD74 ectodomain must be processed in the endocytic compartments to allow its intramembrane cleavage that liberates CD74 intracellular domain (CD74-ICD). We demonstrate that CD74-ICD translocates to the nucleus and induces the activation of the p65 member of NF-κB in this compartment.
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(2005) Applied Physics Letters. 87, 25, Abstract
A slab waveguide was fabricated in a potassium lithium tantalate niobate crystal by the implantation of He2+ ions at 2.26 MeV. The waveguide profile and loss were evaluated by measuring the dark mode TE spectrum using the prism coupling method at lambda=1.3 mu m. The implantation generated amorphous cladding layer 5 mu m below the surface of the crystal with a refractive index lower by 3.9% then that of the substrate. The propagation loss of the waveguided modes was found to be 0.1-0.2 dB/cm. Thermal stability of the waveguide was obtained by isothermal annealing at 351 and 446 degrees C. Following the annealing the waveguide index profile remained unchanged when subjected to annealing at 150 degrees C for one week. (c) 2005 American Institute of Physics.
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(2005) Journal of Immunology. 175, 8, p. 5034-5042 Abstract
To complete their maturation and to participate in the humoral immune response, immature B cells that leave the bone marrow are targeted to specific areas in the spleen, where they differentiate into mature cells. Previously, we showed that immature B cells actively down-regulate their integrin-mediated migration to lymph nodes or sites of inflammation, enabling their targeting to the spleen to allow their final maturation. This inhibition is mediated by IFN-γ, which is transcribed and secreted at low levels by these immature B cells and is down-regulated at the mature stage. The activating MHC class I receptor, Ly49D, which is expressed at high levels on immature B cells, stimulates this IFN-γ secretion. In this study we show that B cells coexpress the inhibitory MHC class I receptor, Ly49G2. In addition, we demonstrate a tight regulation in the expression of the Ly49 family members on B cells that depends on their cell surface levels. High levels of Ly49G2 have a dominant inhibitory effect on Ly49D expressed at low levels on immature bone marrow and mature B cells, resulting in inhibition of IFN-γ secretion. However, low levels of the inhibitory receptor, Ly49G2, coexpressed with high levels of the activating receptor, Ly49D, on the immigrating immature B cells enable the secretion of specific low levels of IFN-γ. This expression pattern insures the inhibitory control of peripheral immature B cell to prevent premature encounter with an Ag while enabling entry to the lymph nodes during the mature stage.
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(2004) Blood. 104, 4, p. 933-941 Abstract
Immature B cells are targeted to specific areas in the spleen, where a fraction of these cells receive signals that induce them to mature and participate in the immune response. In this study, we show that the C-C chemokine receptor 2 (CCR2) is transcribed in immature B cells, while its message is dramatically down-regulated at the mature stage. CCR2-deficient cells exhibit upregulation of chemokine-induced actin polymerization, migration, and homing to the lymph nodes of immature B cells. In addition, we demonstrate that control of homing by CCR2 is mediated by its ligand, CCL2/JE, which is secreted by B cells and down-regulates the stromal derived factor-1 (SDF-1) signaling cascade. Thus, this study describes an additional, previously uncharacterized, role for CCR2 and its ligand as negative regulators of the homing of immature B cells.
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(2004) Leukemia. 18, 12, p. 1948-1950 Abstract
Grb7, a noncatalytic intracellular adaptor protein involved in cell migration, is overexpressed in certain invasive and metastatic solid tumors. We found a highly significant difference in the level of expression of Grb7 between chronic lymphocytic leukemia (CLL) cells obtained from stage I and stage IV patients ( P
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(2004) European Journal of Immunology. 34, 4, p. 1068-1076 Abstract
The fat soluble vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its nuclear receptor play an important role in regulating immune responses. While 1,25(OH 2D3 is known to inhibit transcription of cytokine genes that are required for Th1 differentiation or are products of differentiated Th1 cells, its role in regulating differentiation of Th2 cells is less clear. In this study, we show that 1,25(OH 2D3 has anti-inflammatory effects in an in vivo Th2-dependent asthma model. In addition, we demonstrate that 1,25(OH 2D3 down-regulates the cytoskeleton rearrangement required for promoting integrin-mediated adhesion of naive and effector CD4+ T cells. Finally, 1,25(OH)2D3 inhibits chemokine-induced migration of naive cells and their homing to the lymph nodes. Thus, in addition to its regulation of cytokine transcription, 1,25(OH)2D3 regulates migration of cells and thus controls the skewing of various Th subsets in the secondary lymphoid organs and inhibits Th function at sites of inflammation.
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(2003) Trends in Immunology. 24, 5, p. 264-268 Abstract
Invariant chain (Ii) is a type II integral membrane protein, which acts as a chaperone for MHC class II protein expression and facilitates antigen presentation. Recently, an additional role for Ii in the differentiation of immature to mature B cells has been described. These studies showed that Ii acts as a signaling molecule; its cytosolic domain induces B-cell maturation by activation of transcription mediated by the p65 member of the NF-κB family, a process that requires the B-cell-enriched coactivator, TAFII105. NF-κB activation is mediated by the cytosolic region of Ii, which is liberated from the membrane. The process of intramembrane cleavage followed by nuclear translocation and transcriptional activation is reminiscent of regulated intramembrane cleavage (RIP). In this Review we suggest that the behavior of Ii shows remarkable similarities to the function of proteins processed by RIP and propose that the roles of Ii as a chaperone and as a signaling molecule are intertwined.
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(2003) Journal of Immunology. 171, 9, p. 4630-4638 Abstract
Low levels of IFN-γ secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN-γ secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN-γ transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN-γ gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN-γ and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation.
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(2002) Journal of Immunology. 168, 8, p. 3707-3711 Abstract
Activation of naive T and B cells occurs only within the context of organized lymphoid tissue. Thus, the continuous recirculation of mature lymphocytes is crucial for the development of primary immune response to foreign Ags. We have previously shown that low levels of IFN-γ inhibit homing of B cells to the secondary lymphoid organs. In this study, we demonstrate that similarly low doses of IFN-γ down-regulate integrin-mediated adhesion and migration of naive T and Th2 cells, and have a profound effect on the in vivo homing of naive T cells to the lymph nodes. Moreover, we show that these low doses of IFN-γ have anti-inflammatory effects in an in vivo asthma model. Thus, in contrast to the proinflammatory effects of IFN-γ at relatively high concentrations, low dose IFN-γ appears to exert global suppressory effects on T cell trafficking and may have clinical application as an anti-inflammatory agent.
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(2002) Journal of Biological Chemistry. 277, 20, p. 17821-17829 Abstract
The general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 12-14 TBP-associated factors (TAF(II)s). Some TAF(II)s act as bridges between transcription activators and the general transcription machinery through direct interaction with activation domains. Although TAF-mediated transcription activation has been established, there is little genetic evidence connecting it to binding of an activator. TAF(II)105 is a substoichiometric subunit of transcription factor IID highly expressed in B lymphocytes. In this study, we examined the physiological role of TAF(II)105 and its mechanism of action in vivo by expressing two forms of dominant-negative mutant TAF(II)105 in mice. We show that TAF(II)105 has a pro-survival role in B and T lymphocytes, where the native protein is expressed. In addition, TAF(II)105 is important for T cell maturation and for production of certain antibody isotypes. These phenotypic alterations were absent in mice expressing a dominant-negative mutant that lacks one of the domains mediating p65/RelA binding in vitro. These findings provide support to the notion that interaction between the activator and TAF is important for their function in vivo.
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(2002) Journal of Immunology. 168, 11, p. 5507-5513 Abstract
Early stages of B cell development occur in the bone marrow, resulting in formation of immature B cells. These immature cells migrate to the spleen where they differentiate into mature (B2 or marginal zone (MZ)) cells. This final maturation step is crucial for B cells to become responsive to Ags and to participate in the immune response. Id2 is a helix-loop-helix protein that lacks a DNA-binding region; and therefore, inhibits basic helix-loop-helix functions in a dominant negative manner. In this study, we show that Id2 expression is down-regulated during differentiation of immature B cells into mature B2 and MZ B cells. The high levels of Id2 expressed in the immature B cells result in inhibition of E2A binding activity to an E2 box site. Moreover, mice lacking Id2 show an elevation in the proportion of mature B2 cells in the spleen, while the MZ population in these mice is almost absent. Thus, Id2 acts as a regulator of the differentiation of immature B cells occurring in the spleen, it negatively controls differentiation into mature B2 cells while allowing the commitment to MZ B cells. In the absence of Id2 control, the unregulated differentiation is directed toward the mature B2 population.
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(2002) Proceedings of the National Academy of Sciences of the United States of America. 99, 5, p. 3018-3023 Abstract
Early stages of B cell development take place in the bone marrow, resulting in formation of immature B cells, which migrate to the spleen for their final differentiation into mature cells. This final maturation step is essential for B cells to become responsive to antigens and to participate in the immune response. Previously, we showed that the MHC class II chaperone, invariant chain (Ii), controls the differentiation of B cells from the immature to the mature stage. In this study, by generating transgenic mice expressing truncated Ii lacking its luminal domain, we could dissect the chaperonin activity of Ii from its role in B cell maturation. We demonstrate in vivo that Ii N-terminal domain is directly involved in the maturation of B cells and is sufficient to promote B cell differentiation.
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(2002) Immunity. 17, 5, p. 549-560 Abstract
Immature B cells differentiate in the spleen into mature B cells, a process that is essential for their participation in the immune response. Previously, we showed that the MHC class II chaperone, invariant chain (Ii), controls this differentiation to the mature stage. Ii cytosolic domain-induced B cell maturation involves activation of transcription mediated by the NF-κB p65/RelA homodimer and requires the B cell enriched coactivator, TAFII105. In this study we show that the cytosolic region of Ii is cleaved within the plane of the membrane to generate a cytosolic fragment, which is essential for NF-κB activation and B cell differentiation. Our results suggest that Ii functions as a membrane-bound inactive inducer of NF-κB transcription that is activated by intramembrane proteolytic cleavage.
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(2002) Journal of Immunology. 168, 4, p. 1610-1617 Abstract
MHC class II molecules associate with the invariant chain (Ii) molecule during biosynthesis. Ii facilitates the folding of class II molecules, interferes with their peptide association, and is involved in MHC class II transport. In this study, we have investigated the in vitro and in vivo immune response of Ii-deficient mice (Ii-/-). Our results have demonstrated that CD4+ T cells from Ii-/- mice proliferate normally in vitro after in vivo immunization with protein Ags. However, cytokine secretion profiles of Ag-primed CD4+ T cells from Ii-/- mice differ from CD4+ T cells from wild-type mice. Whereas cells from wild-type mice secrete IFN-γ and IL-4, cells from Ii-/- mice secrete mostly IFN-γ. Moreover, Ii-/- mice exhibit a normal Th1 response in the delayed-type hypersensitivity and trinitrobenzene sulfonic acid colitis models; however, these mice lack an in vivo Th2 response, as demonstrated in the asthma model. Therefore, we suggest that defective Ag presentation in Ii-/- mice leads selectively to a Th1 effector response.
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(2002) Journal of Immunology. 169, 6, p. 2861-2865 Abstract
The resistance of rats or mice to glutamate-induced toxicity depends on their ability to spontaneously manifest a T cell-dependent response to the insult. Survival of retinal ganglion cells (RGCs) exposed to glutamate in BALB/c SCID mice (a strain relatively resistant to glutamate toxicity) was significantly worse than in the wild type. In the susceptible C57BL/6J mouse strain, however, significantly more RGCs survived among SCID mutants than in the matched wild type. RGC survival in the SCID mutants of the two strains was similar. These results suggest 1) that immunodeficiency might be an advantage in strains incapable of spontaneously manifesting protective T cell-dependent immunity and 2) that B cells might be destructive in such cases. After exposure of RGCs to toxic glutamate concentrations in three variants of B cell-deficient C57BL/6J mice, namely muMT-/- (B cell knockout mice) and Ii-/- mice reconstituted with transgenically expressed low levels of Ii p31 isoforms (p31 mice) or Ii p41 isoforms (p41 mice), significantly more RGCs survived in these mice than in the wild type. The improved survival was diminished by replenishment of the B cell-deficient mice with B cells derived from the wild type. It thus seems that B cells have an adverse effect on neuronal recovery after injury, at least in a strain that is unable to spontaneously manifest a T cell-dependent protective mechanism. These findings have clear implications for the design of immune-based therapies for CNS injury.
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(2001) Journal of Biological Chemistry. 276, 29, p. 27203-27206 Abstract
Early stages of B cell development occur in the bone marrow, resulting in formation of immature B cells. From there these immature cells migrate to the spleen where they differentiate to mature cells. This final maturation step is crucial for the B cells to become responsive to antigens and to participate in the immune response. Recently, invariant chain (Ii), a major histocompatibility complex class II chaperone, as well as the transcription factors c-Rel and p65/RelA, were found to play a role in the final antigen-independent differentiation stage of B cells in the spleen. In this study, we investigated a possible link between Ii-dependent B cell maturation and the NF-κB pathway. Our studies indicate that Ii-induced B cell maturation involves activation of transcription mediated by the NF-κB p65/RelA homodimer and requires the B cell-enriched coactivator TBP-associated factor II105.
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(2001) Journal of Biological Chemistry. 276, 50, p. 46701-46706 Abstract
In order to fully mature and participate in the humoral immune response, immature B cells must first migrate into specific areas in the spleen where they differentiate into mature cells. However, before their maturation in the spleen, immature B cells must be excluded from non-splenic secondary lymphoid organs where any antigen encounter would lead to the death of the cells because of the negative selection process. We have recently shown that immature B cells can actively exclude themselves from antigen-enriched sites by down-regulating their integrin-mediated adhesion in a process mediated by interferon-γ (IFN-γ). In this study, we followed the pathway by which IFN-γ regulates the homing of B cells. We show here that the inhibitory signal of IFN-γ is transmitted through the IFN-γ receptor whose engagement leads to the activation of PI3K. This PI3K activation subsequently leads to the inhibition of PKCα phosphorylation and cytoskeleton rearrangement required for promoting integrin-mediated adhesion and migration of B cells.
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(2000) Journal of Experimental Medicine. 192, 9, p. 1381-1387 Abstract
The mechanism by which immature B cells are sequestered from encountering foreign antigens present in lymph nodes or sites of inflammation, before their final maturation in the spleen, has not been elucidated. We show here that immature B cells fall to home to the lymph nodes. These cells can actively exclude themselves from antigen-enriched sites by downregulating their integrin-mediated adhesion to the extracellular matrix protein, fibronectin. This inhibition is mediated by interferon γ secretion. Perturbation of interferon γ activity in vivo leads to the homing of immature B cells to the lymph nodes. This is the first example of autocrine regulation of immune cell migration to sites of foreign antigen presentation.
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(2000) Journal of Experimental Medicine. 191, 6, p. 1057-1062 Abstract
The association of invariant (Ii) chain with major histocompatibility complex (MHC) class II dimers is required for proper antigen presentation to T cells by antigen-presenting cells. Mice lacking Ii chain have severe abnormalities in class II transport, T cell selection, and B cell maturation. We demonstrate here that H2-M, which is required for efficient class II antigenic peptide loading, is unexpectedly downregulated in splenocytes and mature dendritic cells (DCs) from Ii-(/)- mice. Downregulation reflects an increased rate of degradation in Ii-(/)- cells. Degradation apparently occurs within lysosomes, as it is prevented by cysteine protease inhibitors such as E64, but not by the proteasome inhibitor lactacystin. Thus, Ii chain may act as a lysosomal protease inhibitor in 13 cells and DCs, with its deletion contributing indirectly to the loss of H2-M.