Regulator Of Antigen Receptor Signaling Research Articles

Abstract C127: Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas

Abstract MALT1 is a key regulator of antigen receptor signaling in B and T cells. MALT1 partners with BCL10 and CARMA1 to form the CBM complex in which protease activity of MALT1 cleaves the negative regulators (A20, CYLD, RelB) leading to activation of canonical NF-kB pathway. Constitutive activation of NF-kB signaling is a key driver in B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). Therapeutic targeting of MALT1 protease activity can therefore be a useful approach for the treatment of B-cell lymphomas with dysregulated NF-kB signaling. We sought to discover and develop MALT1 inhibitors with the “best-in-class” profile with an emphasis on lowering plasma protein binding (PPB) to achieve improved pharmacological outcome. After extensive SAR efforts utilizing various computational methods, AUR-112 has been identified as a potential development candidate. The allosteric inhibitor AUR-112 exhibited potent and selective inhibition of MALT1 protease activity in both biochemical and cellular assays. Inhibition of MALT1 correlated with potent antiproliferative activity in an ABC-DLBCL cell line. The mechanistic activity of AUR-112 is supported by the inhibition of IL-10 in a cell line and IL-2 in human whole blood. Consistent with our objective, AUR112 showed low plasma protein binding across multiple species, which translated to greater potency in whole blood compared to published MALT1 inhibitors, positioning it as a best-in-class contender. AUR-112 has excellent oral bioavailability across species and exhibited desirable selectivity profile in kinase, protease and CEREP safety screen panels. Oral administration of AUR-112 led to dose dependent anti-tumor activity including durable tumor regression in cell line and patient derived xenograft models. Treatment with AUR-112 led to potent pharmacodynamic modulation in xenograft models as measured by significant and sustained reduction in serum IL-10 levels that correlated with the compelling efficacy. AUR-112 also demonstrated dose dependent inhibition of T-reg cells in vitro and in vivo supporting the immune modulatory role of MALT1 inhibition. AUR-112 is identified as a development candidate with potentially best-in-class physico-chemical properties resulting in a high free fraction for treatment of ABC-DLBCL patients. IND enabling studies are currently ongoing to support the clinical development of AUR-112. Citation Format: Wesley Roy Balasubramanian, Sammeta Srinivasa Raju, Vasantha K.Y, Archana Bhumi Reddy, Janith Mary Maben, Sandeep Sadashiv Patil, Raghavendra N.R, Kiran Aithal B, Charamanna K.B, Dinesh Chikkanna, Kavitha Nellore, Samiulla D.S, Girish Daginakatte, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of AUR-112, a novel MALT1 protease inhibitor for the treatement of B cell lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C127.

A novel class of oxazepine-based anti-cancer agents induces cell death in primary human CLL cells and efficiently reduces tumor growth in Eμ-TCL1 mice through the JNK/STAT4/p66Shc axis

Survival and expansion of malignant B cells in chronic lymphocytic leukemia (CLL) are highly dependent both on intrinsic defects in the apoptotic machinery and on the interactions with cells and soluble factors in the lymphoid microenvironment. The adaptor protein p66Shc is a negative regulator of antigen receptor signaling, chemotaxis and apoptosis whose loss in CLL B cells contributes to their extended survival and poor prognosis. Hence, the identification of compounds that restore p66Shc expression and function in malignant B cells may pave the way to a new therapeutic approach for CLL. Here we show that a novel oxazepine-based compound (OBC-1) restores p66Shc expression in primary human CLL cells by promoting JNK-dependent STAT4 activation without affecting normal B cells. Moreover, we demonstrate that the potent pro-apoptotic activity of OBC-1 in human leukemic cells directly correlates with p66Shc expression levels and is abrogated when p66Shc is genetically deleted. Preclinical testing of OBC-1 and the novel analogue OBC-2 in Eμ-TCL1 tumor-bearing mice resulted in a significantly longer overall survival and a reduction of the tumor burden in the spleen and peritoneum. Interestingly, OBCs promote leukemic cell mobilization from the spleen to the blood, which correlates with upregulation of sphingosine-1-phosphate receptor expression. In summary, our work identifies OBCs as a promising class of compounds that, by boosting p66Shc expression through the activation of the JNK/STAT4 pathway, display dual therapeutic effects for CLL intervention, namely the ability to mobilize cells from secondary lymphoid organs and a potent pro-apoptotic activity against circulating leukemic cells.

Serum levels of soluble PD1 in pemphigus vulgaris: A useful marker for disease severity.

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease targeting the skin and mucous membranes. Programmed cell death protein 1 (PD1) is an immune checkpoint which binds to two ligands, PDL1 and PDL2 resulting in negative regulation of antigen receptor signaling, thus, play a critical role in the immune regulation of autoimmune diseases. In this work we aimed to assess serum levels of soluble PD1 (sPD1) in patients with active PV and in patients in remission in an attempt to evaluate its effect on disease severity. In this case-control study, 60 pemphigus vulgaris patients (30 clinically active and 30 in a clinical remission) and 30 age matched healthy control subjects were included. Severity of PV was assessed using pemphigus disease area index (PDAI) score. Serum levels of sPD1 were measured by ELISA for both patients and healthy control. Serum levels of sPD1 were significantly lower in PV patients than in controls (P<.001) and significantly lower in patients with active disease than in those in remission (P<.001). Serum sPD1 correlated negatively with the severity of the disease (P<.001, r=-0.4). A defect in PD1 pathway is suggested in PV patients, and this defect plays a substantial role in determining the severity of the disease. Thus, sPD1 could be considered a useful marker for disease severity and targeting PD1 pathway could be a potential aim for future therapies of PV.

Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation.

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.

Clinical and pathologic features of lung cancer expressing programmed cell death ligand 1 (PD-L1)

BackgroundProgrammed cell death 1 (PD-1) negatively regulates antigen receptor signaling upon binding by either of its ligands, programmed cell death ligand 1 or 2 (PD-L1/2). Blockade of this interaction with either PD-1 or PD-L1 antibodies has been successful in the treatment of human cancer, especially melanoma and non-small cell lung cancer. PD-L1 expression has been proposed as a predictor of tumor response. However, the relationships between PD-L1 expression and various clinicopathological characteristics remain unclear. Materials and methodsPD-L1 expression was examined in 220 non-small cell lung cancer specimens that were consecutively resected at our hospital after validating the E1L3N antibody immunohistochemical assay by comparing IHC and RT-PCR data for lung cancer cell lines. We evaluated the relationships between PD-L1 positivity, several clinical factors and the immunohistochemical expression of epithelial-mesenchymal transition (EMT), cancer stem cell and proliferative markers. ResultsPD-L1 was expressed in 22% of lung adenocarcinomas and 60% of squamous cell lung cancers. There was no significant association between PD-L1 expression and clinicopathological features in squamous cell lung cancer. However, in patients with lung adenocarcinoma, PD-L1 expression was significantly correlated with solid subtype histology, vimentin expression, increased Ki-67 labeling index and poor prognosis by multivariate analysis. ConclusionPD-L1 expression was associated with high proliferative activity and the EMT phenotype in adenocarcinoma but not in squamous cell carcinoma of the lung. PD-L1 expression was a significant poor prognostic factor in patients with lung adenocarcinoma.

Abstract LB-217: PD-1 suppresses antibody responses to Tn+ tumors

Abstract Cancer cells often have aberrant glycosylation of surface molecules leading to glycan antigens that are not typically found on normal cells. These antigens are referred to as tumor associated carbohydrate antigens (TACAs) and are a promising target for anti-cancer vaccines. One such TACA is the Thomsen-nouvelle (Tn) antigen, which is expressed by up to 90% of adenocarcinomas. The mechanisms regulating the B cell response to Tn and other TACAs are not well understood. This knowledge is essential for the development of vaccines to elicit protective immune responses to tumors bearing TACAs. PD-1 (programmed cell death 1) is a member of the B7:CD28 superfamily of receptors found on antigen-activated B and T cells. It negatively regulates antigen receptor signaling and thereby limits adaptive immune responses, including the anti-tumor T cell response. However, virtually nothing is known about the role of PD-1 in regulating B cell responses to tumors. In this study, we tested the hypothesis that PD-1 suppresses B cell production of Tn-specific antibodies and thereby suppresses protective humoral immunity against Tn+ tumors. Our results show that PD-1−/− mice immunized with a Tn-bearing mucin (desialyated bovine submaxillary mucin) have increased Tn+ tumor-reactive IgM and IgG antibodies, relative to wild type mice. PD-1−/− mice immunized with the Tn+ mucin had significantly increased survival relative to wild type mice following challenge with a Tn+ bearing mammary tumor (TA3-Ha). Survival in PD-1−/− mice was dependent on the presence of B cells. Therefore, our results show that PD-1 plays a critical role in suppressing the protective anti-tumor B cell response elicited by Tn-bearing antigen immunization. This work was supported by NIH/NCI F31 CA183567-01 fellowship to M.A.L. and an American Cancer Society Research Scholar Grant (RSG-12-170-01-LIB) to K.M.H. Citation Format: Marcela A. Leyva, Karen M. Haas. PD-1 suppresses antibody responses to Tn+ tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-217. doi:10.1158/1538-7445.AM2015-LB-217

Clinical significance of soluble programmed death-1(sPD-1) in rheumatoid arthritis patients: Relation to disease activity and functional status

BackgroundProgrammed cell death-1 (PD-1) is an immunoreceptor that negatively regulates antigen receptor signaling and plays a critical role in the immunoregulation of autoimmune diseases. Aim of the workThis study aimed to measure the plasma and synovial fluid levels of soluble programmed death-1(sPD-1) in rheumatoid arthritis (RA) patients and to correlate them with the clinical and laboratory characteristics, disease activity, functional status and radiological severity. Patients and methodsWe measured sPD-1 in the plasma (n=60) and synovial fluid (SF) samples (n=24) from 60 RA patients and in the plasma from healthy control (n=30). In the patients, disease activity score using 28 joint counts (DAS28) and the health assessment questionnaire (HAQ) score were assessed; immunoglobulin-M rheumatoid factor (IgM-RF) titer, anti-cyclic citrullinated peptide (anti-CCP) antibodies titer and C-reactive protein (CRP) levels were measured and total Sharp score calculated. ResultsIn RA patients both plasma and SF sPD-1 levels (1416.9±1037.9pg/ml and 1503.9±1129.48pg/ml respectively) were highly significantly increased compared to its plasma level in the healthy control (165±26.11pg/mL) (p<0.001). In RA patients, the plasma and SF levels of sPD-1 significantly correlated with DAS28 (r=0.52 and 0.58 respectively, p<0.05), HAQ scores (r=0.48 and 0.51 respectively, p<0.05) and anti-CCP titers (r=0.55 and 0.58 respectively, p<0.05). ConclusionsRheumatoid arthritis patients have significantly elevated plasma and synovial levels of sPD-1 that remarkably correlated with the DAS28 suggesting that it could be a useful marker to reflect RA disease activity. The considerable association of sPD-1 with autoantibodies production implies a possible role in the pathogenesis of RA.

Uncoupling Malt1 Threshold Function from Paracaspase Activity Results in Destructive Autoimmune Inflammation

The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-κB activation, and its proteolytic domain cleaves negative NF-κB regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration invivo. Paracaspase activity is essential for regulatory Tcell (Treg) and innate-like B cell development, but it is largelydispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-κB inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFNγ) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.

Negative regulation of chemokine receptor signaling and B-cell chemotaxis by p66Shc

Shc (Src homology 2 domain containing) adaptors are ubiquitous components of the signaling pathways triggered by tyrosine kinase-coupled receptors. In lymphocytes, similar to other cell types, the p52 and p66 isoforms of ShcA/Shc participate in a self-limiting loop where p52Shc acts as a positive regulator of antigen receptor signaling by promoting Ras activation, whereas p66Shc limits this activity by competitively inhibiting p52Shc. Based on the fact that many signaling mediators are shared by antigen and chemokine receptors, including p52Shc, we have assessed the potential implication of p66Shc in the regulation of B-cell responses to chemokines, focusing on the homing receptors CXCR4 (C-X-C chemokine receptor type 4) and CXCR5 (C-X-C chemokine receptor type 5). The results identify p66Shc as a negative regulator of the chemotactic responses triggered by these receptors, including adhesion, polarization and migration. We also provide evidence that this function is dependent on the ability of p66Shc to interact with the chemokine receptors and promote the assembly of an inhibitory complex, which includes the phosphatases SHP-1 (Src homology phosphatase-1) and SHIP-1 (SH2 domain-containing inositol 5'-phosphatase-1), that results in impaired Vav-dependent reorganization of the actin cytoskeleton. This function maps to the phosphorylatable tyrosine residues in the collagen homology 1 (CH1) domain. The results identify p66Shc as a negative regulator of B-cell chemotaxis and suggest a role for this adaptor in the control of B-cell homing.

The Shc family protein adaptor, Rai, acts as a negative regulator of Th17 and Th1 cell development

Rai, a Shc adapter family member, acts as a negative regulator of antigen receptor signaling in T and B cells. Rai(-/-) mice develop lupus-like autoimmunity associated to the spontaneous activation of self-reactive lymphocytes. Here, we have addressed the potential role of Rai in the development of the proinflammatory Th1 and Th17 subsets, which are centrally implicated in the pathogenesis of a number of autoimmune diseases, including lupus. We show that Rai(-/-) mice display a spontaneous Th1/Th17 bias. In vitro polarization experiments on naive and effector/memory CD4(+) T cells demonstrate that Rai(-/-) favors the development and expansion of Th17 but not Th1 cells, indicating that Rai modulates TCR signaling to antagonize the pathways driving naive CD4(+) T cell differentiation to the Th17 lineage, while indirectly limiting Th1 cell development in vivo. Th1 and Th17 cell infiltrates were found in the kidneys of Rai(-/-) mice, providing evidence that Rai(-/-) contributes to the development of lupus nephritis, not only by enhancing lymphocyte activation but also by promoting the development and expansion of proinflammatory effector T cells. Interestingly, T cells from SLE patients were found to have a defect in Rai expression, suggesting a role for Rai in disease pathogenesis.

CIN85 is required for Cbl-mediated regulation of antigen receptor signaling in human B cells

AbstractThe aberrant regulation of B-cell receptor (BCR) signaling allows unwanted B cells to persist, thereby potentially leading to autoimmunity and B-cell malignancies. Casitas B-lineage lymphoma (Cbl) proteins suppress BCR signaling; however, the molecular mechanisms that control Cbl function in human B cells remain unclear. Here, we demonstrate that CIN85 (c-Cbl interacting protein of 85 kDa) is constitutively associated with c-Cbl, Cbl-b, and B-cell linker in B cells. Experiments using CIN85-overexpressing and CIN85-knockdown B-cell lines revealed that CIN85 increased c-Cbl phosphorylation and inhibited BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, whereas it did not affect BCR internalization. The Syk phosphorylation in CIN85-overexpressing and CIN85-knockdown cells was inversely correlated with the ubiquitination and degradation of Syk. Moreover, CIN85 knockdown in primary B cells enhanced BCR-induced survival and growth, and increased the expression of BcLxL, A1, cyclin D2, and myc. Following the stimulation of BCR and Toll-like receptor 9, B-cell differentiation- associated molecules were up-regulated in CIN85-knockdown cells. Together, these results suggest that CIN85 is required for Cbl-mediated regulation of BCR signaling and for downstream events such as survival, growth, and differentiation of human B cells.

Quantitative differences in CD45 expression unmask functions for CD45 in B-cell development, tolerance, and survival

The receptor-like tyrosine phosphatase CD45 positively regulates antigen receptor signaling by dephosphorylating the inhibitory tyrosine of the src family kinases. CD45-deficient mice fail to fully unmask the role of CD45 in B cells because of the expression of a partially redundant tyrosine phosphatase, CD148. However, mice that are doubly deficient in CD45 and CD148 exhibit a very early block in B-cell development, thereby obscuring later roles for CD45. To overcome these limitations, here we take advantage of an allelic series of mice in which CD45 expression is titrated broadly (0-180%). Although high expression of CD45 inhibits T-cell receptor (TCR) signaling, we show that CD45 plays a purely positive regulatory role during B-cell receptor (BCR) signaling. In concert with exaggerated BCR signaling, increasing CD45 expression drives enhanced receptor editing in the bone marrow and profound loss of follicular and marginal zone B cells in the spleen. In the context of the IgHEL/sHEL model of B-cell tolerance, such high CD45 expression transforms anergy into deletion. Unexpectedly, elimination of the autoantigen sHEL in this model system in order to block clonal deletion fails to rescue survival of mature B cells. Rather, high CD45 expression reduces B-cell activating factor receptor (BAFFR) expression and inhibits B-cell activating factor (BAFF)-induced B-cell survival in a cell-intrinsic manner. Taken together, our findings reveal how CD45 function diverges in T cells and B cells, as well as how autoreactive B cells are censored as they transit development.

Pivotal Advance: CD45RB glycosylation is specifically regulated during human peripheral B cell differentiation

A screen of cell surface markers differentially expressed during peripheral B cell differentiation identified that the CD45RB epitope detected by the mAb MEM-55 was highly expressed on CD27(+) memory B cells and absent on CD27(-) naïve B cells. IgG(+)CD27(-) memory and a previously unacknowledged CD27(-) population in blood also expressed high levels of CD45RB(MEM55). Naïve and memory B cells from tonsils followed the pattern observed in blood, and CD38(high) B cells had a bimodal expression pattern when analyzed using flow cytometry. No CD38(high) GC B cells, however, expressed the CD45RB(MEM55) epitope when assayed using immunohistochemistry. Rather, CD38(high)CD45RB(MEM55high) B cells had a distinct cellular phenotype and were localized outside of GCs. CD45RB epitopes, detected by other antibody clones, were expressed at high levels through B cell differentiation, and no changes in splicing of the CD45RB exon were observed during B cell differentiation. Instead, B cells regulated their expression of the CD45RB(MEM55) epitope through site-specific modifications of an O-linked glycochain. CD4(+) T cells differentially spliced CD45 but did not vary the glycosylation of the CD45RB(MEM55) epitope, and CD8(+) cells modified CD45RB(MEM55) expression in a similar manner as B cells. Monocytes expressed the CD45RB exon but not the CD45RB(MEM55) epitope. As CD45 is a highly expressed tyrosine phosphatase that regulates antigen receptor signaling strength in lymphocytes, we conclude that regulated O-linked glycosylation of CD45RB can be used to follow B cell differentiation and that this regulation may be involved in fine-tuning antigen signaling in the cell.

Positive and negative regulation of antigen receptor signaling by the Shc family of protein adapters

The Shc adapter family includes four members that are expressed as multiple isoforms and participate in signaling by a variety of cell-surface receptors. The biological relevance of Shc proteins as well as their variegated function, which relies on their highly conserved modular structure, is underscored by the distinct and dramatic phenotypic alterations resulting from deletion of individual Shc isoforms both in the mouse and in two model organisms, Drosophila melanogaster and Caenorhabditis elegans. The p52 isoform of ShcA couples antigen and cytokine receptors to Ras activation in both lymphoid and myeloid cells. However, the recognition of the spectrum of activities of p52ShcA in the immune system has been steadily expanding in recent years to other fundamental processes both at the cell and organism levels. Two other Shc family members, p66ShcA and p52ShcC/Rai, have been identified recently in T and B lymphocytes, where they antagonize survival and attenuate antigen receptor signaling. These developments reveal an unexpected and complex interplay of multiple Shc proteins in lymphocytes.

PD-1 and PD-1 ligands: from discovery to clinical application

Programmed cell death-1 (PD-1, Pdcd1), an immunoreceptor belonging to the CD28/CTLA-4 family negatively regulates antigen receptor signaling by recruiting protein tyrosine phosphatase, SHP-2 upon interacting with either of two ligands, PD-L1 or PD-L2. Because of the wide range of ligand distribution in the body, its biological significance pervades almost every aspect of immune responses including autoimmunity, tumor immunity, infectious immunity, transplantation immunity, allergy and immunological privilege. In this review, we would like to summarize the history of PD-1 research since its discovery and recent findings that suggest promising future for the clinical application of PD-1 agonists and antagonists to various human diseases.

Leupaxin binds to PEST domain tyrosine phosphatase PEP

PEST domain tyrosine phosphatase (PEP) is an intracellular protein tyrosine phosphatase and characterized by PEST motifs and proline-rich domains in the carboxyl terminal half. PEP is primarily expressed in hematopoietic cells, and together with PEP-binding Csk, may act as a negative regulator of antigen receptor signaling in lymphocytes. Here, we show the binding capability of PEP for leupaxin, which is preferentially expressed in hematopoietic cells and a comparatively new member of the paxillin family characterized by two protein-protein interaction modules, LIM domains and LD motifs. These results suggested that leupaxin might participate in the regulation of the signaling cascade through the binding to PEP in lymphocytes.

CD45 Phosphatase Is Involved in Motility and Development of Hematopoietic Stem and Maturing Cells by the Regulation of Cell Adhesion and Cytokine Signaling.

The phosphatase CD45 is a key regulator of antigen receptor signaling in lymphocytes. Still, CD45 is highly expressed in all hematopoietic lineages at most stages of development, suggesting that this phosphatase also regulates other cells and processes. During development, as well as in clinical transplantation, hematopoietic stem cells (HSCs) migrate through the circulation to the bone marrow (BM) and repopulate it. Migration and development of HSCs are multi-step processes, which are tightly regulated by interplays between cytokines, chemokines, adhesion molecules and proteolytic enzymes; however, not all the related key players have been fully identified.In this study we explored the involvement of CD45 in hematopoietic cell motility and development, its role in cytokine signaling and adhesion interactions. The roles of CD45 were tested by either blocking the function of CD45 expressed on human and murine HSCs by neutralizing antibodies, or by utilizing CD45 knockout (KO) mice.Our results show that blocking CD45 completely prevented homing of human CD34+ enriched progenitors to the murine BM, consequently abrogating repopulation in transplanted NOD/SCID mice. In addition, CD45 neutralization impaired the capacity of human progenitors to migrate in-vitro towards a gradient of SDF-1, suggesting a cross-talk between SDF-1 and CD45 signaling. Furthermore, blocking CD45 on human G2 cells (pre-B ALL line) activated signaling pathways, including an increase in phosphorylation of MAP kinase and the tyrosine kinase Pyk2, which are involved in cell adhesion and migration. This activation enhanced cell adhesion to stromal and endothelial cell lines in-vitro. Importantly, blockage of CD45 in human progenitors resulted in cell aggregation, which inhibited cell proliferation and impaired the capacity to form colonies in-vitro. In an additional set of experiments we tested the role of CD45 in cell mobilization. In-vivo studies in normal mice demonstrated that neutralization of CD45 function inhibited the release of mature leukocytes and progenitor cells from the BM to the circulation both under steady state conditions and in stress-induced recruitment by stimulation with G-CSF or LPS. More importantly, BM derived mononuclear cells from CD45KO mice displayed a significant reduction in in-vivo homing and in-vitro migration compared to their wild type counterparts. Furthermore, G-CSF induced mobilization was impaired in CD45KO mice, and accompanied by a reduction in MMP-9 secretion from blood-derived leukocytes. Unexpectedly, the ability of CD45KO progenitors to form colonies in-vitro was impaired in the absence of in-vivo BM environment, documenting a crucial role for phosphatases such as CD45 in stem cell differentiation.Taken together, our findings demonstrate that functional CD45 is essential for human and murine hematopoietic cell migration and development (both homing and mobilization) by the regulation of adhesion and cytokine-induced signaling machineries. We suggest that in these cells CD45 may act as a negative regulator of major signaling pathways controlling adhesion properties and maintaining the balance between anchorage and release. We reveal a novel and dual role for CD45 in regulation of hematopoietic cell trafficking in general and progenitor cell motility and development in particular.

Differential Regulation of CXCR4-mediated T-cell Chemotaxis and Mitogen-activated Protein Kinase Activation by the Membrane Tyrosine Phosphatase, CD45

The chemokine receptor CXCR4 and its cognate ligand, stromal cell-derived factor-1alpha (CXCL12), regulate lymphocyte trafficking and play an important role in host immune surveillance. However, the molecular mechanisms involved in CXCL12-induced and CXCR4-mediated chemotaxis of T-lymphocytes are not completely elucidated. In the present study, we examined the role of the membrane tyrosine phosphatase CD45, which regulates antigen receptor signaling in CXCR4-mediated chemotaxis and mitogen-activated protein kinase (MAPK) activation in T-cells. We observed a significant reduction in CXCL12-induced chemotaxis in the CD45-negative Jurkat cell line (J45.01) as compared with the CD45-positive control (JE6.1) cells. Expression of a chimeric protein containing the intracellular phosphatase domain of CD45 was able to partially restore CXCL12-induced chemotaxis in the J45.01 cells. However, reconstitution of CD45 into the J45.01 cells restored the CXCL12-induced chemotaxis to about 90%. CD45 had no significant effect on CXCL12 or human immunodeficiency virus gp120-induced internalization of the CXCR4 receptor. Furthermore, J45.01 cells showed a slight enhancement in CXCL12-induced MAP kinase activity as compared with the JE6.1 cells. We also observed that CXCL12 treatment enhanced the tyrosine phosphorylation of CD45 and induced its association with the CXCR4 receptor. Pretreatment of T-cells with the lipid raft inhibitor, methyl-beta-cyclodextrin, blocked the association between CXCR4 and CD45 and markedly abolished CXCL12-induced chemotaxis. Comparisons of signaling pathways induced by CXCL12 in JE6.1 and J45.01 cells revealed that CD45 might moderately regulate the tyrosine phosphorylation of the focal adhesion components the related adhesion focal tyrosine kinase/Pyk2, focal adhesion kinase, p130Cas, and paxillin. CD45 has also been shown to regulate CXCR4-mediated activation and phosphorylation of T-cell receptor downstream effectors Lck, ZAP-70, and SLP-76. Our results show that CD45 differentially regulates CXCR4-mediated chemotactic activity and MAPK activation by modulating the activities of focal adhesion components and the downstream effectors of the T-cell receptor.

Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56lck

AbstractPlatelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin gene superfamily that is expressed on the surface of platelets, endothelial cells, myeloid cells, and certain lymphocyte subsets. PECAM-1 has recently been shown to contain functional immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic domain, and co-ligation of PECAM-1 with the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of PECAM-1, recruitment of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2), and attenuation of TCR-mediated cellular signaling. To determine the molecular basis of PECAM-1 inhibitory signaling in lymphocytes, the study sought to (1) establish the importance of the PECAM-1 ITIMs for its inhibitory activity, (2) determine the relative importance of SHP-2 versus SHP-1 in mediating the inhibitory effect of PECAM-1, and (3) identify the protein tyrosine kinases required for PECAM-1 tyrosine phosphorylation in T cells. Co-ligation of wild-type PECAM-1 with the B-cell antigen receptor expressed on chicken DT40 B cells resulted in a marked reduction of calcium mobilization—similar to previous observations in T cells. In contrast, co-ligation of an ITIM-less form of PECAM-1 had no inhibitory effect. Furthermore, wild-type PECAM-1 was unable to attenuate calcium mobilization in SHP-2–deficient DT40 variants despite abundant levels of SHP-1 in these cells. Finally, PECAM-1 failed to become tyrosine phosphorylated in p56lck-deficient Jurkat T cells. Together, these data provide important insights into the molecular requirements for PECAM-1 regulation of antigen receptor signaling.

A novel B lymphocyte-associated adaptor protein, Bam32, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase.

We have identified and characterized a novel src homology 2 (SH2) and pleckstrin homology (PH) domain-containing adaptor protein, designated Bam32 (for B cell adaptor molecule of 32 kD). cDNAs encoding the human and mouse Bam32 coding sequences were isolated and the human bam32 gene was mapped to chromosome 4q25-q27. Bam32 is expressed by B lymphocytes, but not T lymphocytes or nonhematopoietic cells. Human germinal center B cells show increased Bam32 expression, and resting B cells rapidly upregulate expression of Bam32 after ligation of CD40, but not immunoglobulin M. Bam32 is tyrosine-phosphorylated upon B cell antigen receptor (BCR) ligation or pervanadate stimulation and associates with phospholipase Cgamma2. After BCR ligation, Bam32 is recruited to the plasma membrane through its PH domain. Membrane recruitment requires phosphatidylinositol 3-kinase (PI3K) activity and an intact PI(3,4, 5)P(3)-binding motif, suggesting that membrane association occurs through binding to 3-phosphoinositides. Expression of Bam32 in B cells leads to a dose-dependent inhibition of BCR-induced activation of nuclear factor of activated T cells (NF-AT), which is blocked by deletion of the PH domain or mutation of the PI(3,4,5)P(3)-binding motif. Thus, Bam32 represents a novel B cell-associated adaptor that regulates BCR signaling downstream of PI3K.