Thy-1抗原

Thy-1抗原(原称θ抗原)是小鼠T细胞的一个重要的表面抗原,同时又是区分小鼠T和B淋巴细胞的特异表面标记。研究表明,Thy-1抗原还广泛地存在于包括人类在内的多种动物的脑及其他一些组织中。目前,Thy-1抗原分子已从淋巴细胞和脑组织中提纯,并对其生化特性、化学组成以及结构与功能进行了深入的研究。Thy-1抗原在分布上以及化学结构上,具有许多其他细胞膜蛋白所没有的特点,这引起了许多学者的重视。本文简要地介绍有关Thy-1抗原研究的一些进展。一、Thy-1抗原的发现五十年代末,淋巴细胞的免疫学功能已逐步明确。1961年,胸腺功能的发现使人们开始认识到,淋巴细胞是一个不均一的细胞群,它至少可以分为二类:一类是B淋巴细胞,担负着机体的体液免疫功能(分泌抗体);另一类是T淋巴细胞,担负着机体的细胞免疫功能。 1963年,Boyse等在研究小鼠白血病细胞的表面抗原时偶然发现,一些小鼠的胸腺     

Thy-1抗原

Thy-1抗原(原称θ抗原)是小鼠T细胞的一个重要的表面抗原,同时又是区分小鼠T和B淋巴细胞的特异表面标记。研究表明,Thy-1抗原还广泛地存在于包括人类在内的多种动物的脑及其他一些组织中。目前,Thy-1抗原分子已从淋巴细胞和脑组织中提纯,并对其生化特性、化学组成以及结构与功能进行了深入的研究。Thy-1抗原在分布上以及化学结构上,具有许多其他细胞膜蛋白所没有的特点,这引起了许多学者的重视。本文简要地介绍有关Thy-1抗原研究的一些进展。     

Localization of Thy-1�Cpositive Cells in the Perichondrium During Endochondral Ossification

We elucidated the localization of Thy-1–positive cells in the perichondrium of fetal rat limb bones to clarify the distribution of osteogenic cells in the process of endochondral ossification. We also examined the formation of calcified bone-like matrices by isolated perichondrial cells in vitro. At embryonic day (E) 15.5, when the cartilage primodia were formed, immunoreactivity for Thy-1 was detected in cells of the perichondrium adjacent to the zone of hypertrophic chondrocytes. At E17.5, when the bone collar formation and the vascular invasion were initiated, fibroblast-like cells at the sites of vascular invasion, as well as in the perichondrium, showed Thy-1 labeling. Double immunostaining for Thy-1 and osterix revealed that Thy-1 was not expressed in the osterix-positive osteoblasts. Electron microscopic analysis revealed that Thy-1–positive cells in the zone of hypertrophic chondrocytes came in contact with blood vessels. Perichondrial cells isolated from limb bones showed alkaline phosphatase activity and formed calcified bone-like matrices after 4 weeks in osteogenic medium. RT-PCR demonstrated that Thy-1 expression decreased as calcified nodules formed. Conversely, the expression of osteogenic marker genes Runx2, osterix, and osteocalcin increased. These results indicate that Thy-1 is a good marker for characterizing osteoprogenitor cells. (J Histochem Cytochem 58:455–462, 2010)     

Aberrations of chromosome 3. A marker of T-cell lymphomas?

Aberrations of chromosome occur in different malignancies, but they are more frequent in lymphoproliferative ones than in the others. In this study here four out of five T-zone lymphomas had abnormalities of chromosome 3. This lead to the question of whether aberrations of chromosome 3 might be a marker of T-cell lymphomas. The conclusion which can be drawn from the cases described in the literature, the own unpublished cases, and these four T-zone lymphomas is that abnormalities of chromosome 3 cannot be regarded as a discriminative marker of T-cell derived malignancies. It is suggested that the relationship between chromosome aberrations and type of disease is not necessarily a direct one. One possible model of the genesis of chromosome aberrations in malignant diseases is presented.     

Rag-dependent and Rag-independent mechanisms of Notch1 rearrangement in thymic lymphomas of Atm−/− and scid mice

The pathways of thymic lymphomagenesis are classified as Rag-dependent or -independent according to their dependence on recombination-activating gene (Rag1/2) proteins. The role of the two-lymphoma pathways in oncogene rearrangements and the connection between lymphoma pathways and rearrangement mechanisms, however, remain obscure. We compared the incidence and latency of thymic lymphomas, and associated rearrangements of the representative oncogene Notch1 among Rag2^?/?, ataxia telangiectasia mutated (Atm)^?/?, and severe combined immune deficiency (scid) mice combined with Rag2 deficiency. Contrary to expectations, Rag2^?/? mice were prone to thymic lymphoma development, suggesting the existence of a Rag2-independent lymphoma pathway in Rag2^?/? mice. The lymphoma incidence in Rag2^?/?Atm^?/? mice was lower than that in Atm^?/? mice, but higher than that in Rag2^?/? mice, indicating that Atm^?/? mice develop lymphomas through both pathways. Scid mice developed lymphomas with an incidence and latency similar to Rag2^?/?scid mice, suggesting that Rag2-mediated V(D)J recombination-driven events are not necessarily required for lymphomagenesis in scid mice. Notch1 rearrangement mechanisms were classified as Rag2-dependent or Rag2-independent based on the presence of recombination signal-like sequences at rearranged sites. In Rag2^?/? lymphomas, Notch1 must be rearranged independently of Rag2 function, implying that Rag2^?/? mice are susceptible to lymphomagenesis due to the presence of other rearrangement mechanisms. The results in Atm^?/? mice suggest that Notch1 was rearranged through both lymphoma pathways. In scid mice, the frequency of Rag2-mediated rearrangements was relatively low compared with that in wild-type mice, suggesting that the Rag2-independent lymphoma pathway prevails in the development of thymic lymphomas in scid mice. Thus, two rearrangement mechanisms underlie the lymphoma pathways and constitute the mechanistic bases for lymphomagenesis, thereby providing the molecular criteria for distinguishing between Rag2-dependent and Rag2-independent lymphoma pathways.     

Are T cells at the origin of B cell lymphomas?

Lymphoma pathogenesis is at least in some cases related to transformed B cells (BCs) arising from germinal centre reactions (GCRs). In this article possible deregulations of GCRs are investigated using in silico simulations. It is found that the final differentiation of BCs as regulated by helper T cells (TCs) is the best candidate mechanism for such a deregulation. This shifts the paradigm of BC lymphoma pathogenesis from BC transformations to an emphasized role of TC-BC interactions.     

BAY 41-2272防治大鼠Thy-1肾炎的实验研究

目的:观察比较BAY41-2272和己酮可可碱(PTX)这两种增强环磷酸鸟苷(cGMP)活性的物质对大鼠Thy-1诱导的进展性肾病模型的疗效,探讨肾纤维化治疗的新途径。方法:成功诱导Thy-1肾炎后,将大鼠分为四组:即肾纤维化组(RF组)、BAY 41-2272治疗组、PTX治疗组和对照组。治疗15周后,分别检测各组大鼠血清肌酐和尿蛋白排泄量,肾组织病理和免疫组化方法检测肾小球/小管间质基质堆积程度和ED1/PCNA阳性细胞数,检测肾小球和皮质转化生长因子TGF-β1、纤粘蛋白(fi- bronectin)、基质金属蛋白酶抑制物(TIMP-1)等蛋白能表达水平,ELISA方法检测肾小球/小管间质细胞的基础、刺激后cGMP水平。RT-PCR方法检测内皮细胞一氧化氮合成酶、α1/β1可溶性鸟苷酸环化酶mRNA水平。结果:观察结束时,BAY41-2272显著降低大鼠尿蛋白排泄和血肌酐水平,显著减轻肾小球/小管间质基质堆积程度,降低巨噬细胞浸润数目和TGF-β1、fibronectin表达水平,而PTX治疗仅产生轻微的变化。肾纤维化组大鼠的小管间质sGC mRNA、NO刺激的cGMP水平显著高于对照组,而肾小球的则较对照组降低;BAY41-2272治疗后肾小球/小管间质sGC mRNA、刺激后cGMP水平可显著提高,而PTX治疗的只有轻度提高而无统计学意义。结论:BAY41-2272治疗可显著改善肾组织NO-cGMP信号通路转导和cGMP的活性,可有效的延缓肾纤维化的进展,而PTX疗效则相对很弱。采用BAY41-2272提高cGMP活性的方案可能为肾纤维化提供新颖、极具前景的治疗策略。     

Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, α(V)β(3)-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.     

Gene expression profile and overexpression of apoptosis-related genes (NGFI-B and Gadd 45 γ) in early phase of Thy-1 nephritis model

Mesangioproliferative glomerulonephritis (MPGN) is a disease of high incidence in humans. Rat Thy-1 nephritis (Thy-1 N), namely, anti-thymocyte serum (ATS)-induced nephritis, is considered to be an animal model for studying MPGN. Although previous studies have demonstrated that glomerular mesangial cell (GMCs) injury might be a feature of Thy-1 N, the mechanism of the disease (i.e., GMC apoptosis) remains unclear. We have examined the pathologic changes of GMCs and the gene expression profile of renal tissues in Thy-1 N. The pathologic changes of Thy-1 N include three phages: GMC apoptosis (40 min), necrosis (2 h), and proliferation (5 days). Many TUNEL-positive cells are found 40 min after administration of ATS. Concomitantly, 341 genes are up-regulated, whereas 392 genes are down-regulated, as shown by microarrays analysis. The mRNA and protein of two of the up-regulated genes (nerve growth factor induced protein I-B, NGFI-B; growth arrest- and DNA-damage-inducible protein 45 gamma, Gadd 45 γ) in the GMC apoptotic phase of Thy-1 N are markedly elevated, as observed by real-time polymerase chain reaction and immunohistochemistry. Our data indicate that pathologic changes of Thy-1 N are involved in the abnormal gene profile. The overexpression of the NGFI-B and Gadd 45 γ genes may be associated with GMC apoptosis of Thy-1 N.This work was supported by grants (no. 30571728, 30471615, and no. 03KJA310074) from the National Natural Science Foundations of China and Jiangsu Province.     

Evidence for a genetic basis for the class A Thy-1− defect

When Thy-1^– cell lines derived from different Thy-1⁺ murine thymic lymphomas are analyzed by complementation analysis, most fall into the A complementation class. A possible explanation for this result is that the Class A phenotype is due to a mutation in a gene on the X chromosome. To test this idea, selection for 6-thioguanine resistance was carried out on Thy-1⁺ hybrid cell lines between complementary Class A and Class C Thy-1^– mutant cell lines. In some hybrid clones, there was complete concordance between 6-thioguanine resistance and a change of the phenotype of the hybrid from Thy-1⁺ to Thy-1^–. Detailed study of one of these hybrid clones showed that 6-thioguanine resistance was accompanied by loss of hypoxanthine guanine phosphoribosyltransferase activity and that the Thy-1^– phenotype was attributable to loss of the gene complementing the Class A Thy-1^– mutation.Other hybrid clones, however, had some thioguanine resistant lines which remained Thy-1⁺. The degree of concordance was a characteristic of the particular hybrid clone examined and subclones which showed complete concordance could be derived from clones showing incomplete concordance. The variability in the degree of concordance between 6-thioguanine resistance and the Thy-1^– phenotype in different hybrid cell lines was also seen among individual hybrid clones isolated from a fusion between a Class A mutant and normal spleen cell blasts.We conclude from these results that the basis of the Class A Thy-1^– phenotype is genetic, but given the variability in the degree of linkage observed, we cannot determine whether the gene determining the Class A mutant phenotype is X-linked in the normal situation.     

Thy-1 Attenuates TNF-α-Activated Gene Expression in Mouse Embryonic Fibroblasts via Src Family Kinase

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-α. Our study demonstrates distinct profiles of TNF-α-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1−) subsets of mouse embryonic fibroblasts (MEF). TNF-α induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1− MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1− MEFs significantly attenuated TNF-α-activated gene expression. Mechanistically, TNF-α activated Src family kinase (SFK) only in Thy-1− MEFs. Blockade of SFK activation abrogated TNF-α-activated gene expression in Thy-1− MEFs, whereas restoration of SFK activation rescued the TNF-α response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-α-activated gene expression via interfering with SFK- and NF-κB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.     

Rap GTPase-mediated adhesion and migration: A target for limiting the dissemination of B-cell lymphomas?

B-cell lymphomas, which arise in lymphoid organs, can spread rapidly via the circulatory system and form solid tumors within multiple organs. Rate-limiting steps in this metastatic process may be the adhesion of lymphoma cells to vascular endothelial cells, their exit from the vasculature and their migration to tissue sites that will support tumor growth. Thus proteins that control B-cell adhesion and migration are likely to be key factors in lymphoma dissemination, and hence potential targets for therapeutic intervention. The Rap GTPases are master regulators of integrin activation, cell motility and the underlying cytoskeletal, adhesion and membrane dynamics. We have recently shown that Rap activation is critical for B-lymphoma cells to undergo transendothelial migration in vitro and in vivo. As a consequence, suppressing Rap activation impairs the ability of intravenously injected B-lymphoma cells to form solid tumors in the liver and other organs. We discuss this work in the context of targeting Rap, its downstream effectors, or other regulators of B-cell adhesion and migration as an approach for limiting the dissemination of B-lymphoma cells and the development of secondary tumors.Key words: B-cell lymphomas, Rap GTPases, extravasation, chemokines, integrins, metastasisB-cell lymphomas are frequently occurring malignancies that are often aggressive and difficult to treat. Abnormally proliferating B cells that acquire survival-promoting mutations originate within the bone marrow or the lymphoid organs but can traffic via the blood and lymphatic systems to other organs, where they can form solid tumors. A consequence of the genetic mechanisms that generate a large repertoire of antigen-detecting B-cell receptors (BCR) and antibodies is an increased frequency of chromosomal translocations and mutations that can lead to oncogenic transformation.¹ During B-cell development in the bone marrow, the vast diversity of the BCR repertoire within an individual is generated by the random rearrangement of the VDJ gene segments that encode the BCR. Subsequent to antigen binding, highly proliferating B cells within the germinal centers of secondary lymphoid organs undergo somatic hypermutation of the genes encoding the immunoglobulin portion of the BCR in order to generate antibodies of higher affinity (“affinity maturation”). These cells can also undergo a second DNA rearrangement event associated with immunoglobulin class switching. Aberrant DNA rearrangements or somatic hypermutation can lead to oncogenic transformation. As examples, translocation of the c-myc gene into the IgH locus is characteristic of Burkitt''s lymphoma whereas somatic hypermutation of genes that encode prosurvival proteins (e.g., pim-1) is associated with diffuse large B-cell lymphomas,² the most common type of non-Hodgkin lymphoma.The ability of B-cell lymphomas to spread to multiple organs reflects the migratory capacity of their normal counterparts. B cells circulate continuously throughout the body via the blood and lymphatic systems. The extravasation of B cells out of the blood and into tissues is a multi-step process that requires selectin-mediated rolling on the surface of vascular endothelial cells, intergin-mediated firm adhesion to the endothelial cells, and migration across the endothelial cell monolayer that makes up the vessel wall (Fig. 1).^3–6 These steps are orchestrated by chemokines and adhesion molecules that are displayed on the surface of the vascular endothelial cells. Chemokines initiate signaling within the B cell that results in integrin activation. The collaboration between chemokine receptor signaling and outside-in integrin signaling causes B cells to reorganize their cytoskeleton. This cytoskeletal reorganization allows B cells to spread on the surface of the vascular endothelial cells, migrate to sites suitable for extravasation (e.g., junctions between endothelial cells) and then deform themselves in order to move across the endothelial cell layer.⁷ The ability of B-cell lymphomas to follow these constitutive organ-homing cues allows them to spread to multiple organs throughout the body, making them difficult to combat. Diffuse large B-cell lymphomas are highly aggressive precisely for this reason and readily spread to the liver, kidneys and lungs.⁸ Thus, identifying key proteins that regulate the extravasation of B-cell lymphomas could suggest new therapeutic strategies for treating these malignancies.⁹Open in a separate windowFigure 1Rap activation is required for multiple steps in lymphoma dissemination. B-cell lymphomas exit the vasculature using the same mechanisms as normal B cells. Once B cells are tethered via selectin-mediated rolling, chemokines immobilized on the surface of vascular endothelial cells convert integrins to a high affinity state via a mechanism that involves activation of the Rap GTPases. This permits firm adhesion. Adhered B cells migrate across the endothelium and then send out actin-rich protrusions, which penetrate the endothelial barrier to reach the subendothelial matrix. The formation of these membrane processes, and the subsequent movement of the cells through the junctions, requires activation of the Rap, Rho and Rac GTPases. Once in the tissue, B-lymphoma cells assume a polarized morphology and can migrate towards optimal growth niches.The ubiquitously-expressed Rap GTPases are master regulators of cell adhesion, cell polarity, cytoskeletal dynamics and cell motility.¹⁰ Receptor-induced conversion of the Rap GTPases to their active GTP-bound state (Rap-GTP) allows them to bind multiple effector proteins and thereby orchestrate their localization and function. These downstream effectors of Rap-GTP control integrin activation, actin polymerization and dynamics and the formation of protrusive leading edges in migrating cells (see below and Fig. 2). In both normal B cells and B-lymphoma cell lines, signaling via chemoattractant receptors, the BCR and integrins all activate Rap.^11–13 Moreover, we have shown that chemokine-induced Rap activation is essential for the chemoattractants CXCL12 (SDF-1), CXCL13 and sphingosine-1-phosphate (S1P) receptors to stimulate B-cell migration and adhesion.^12,14 Rap activation is also important for receptor-induced actin polymerization, cell spreading and cytoskeletal reorganization in both primary B cells and B-lymphoma cells.¹⁵ These findings suggested that Rap activation might be essential for the in vivo metastatic spread of B-cell lymphomas.Open in a separate windowFigure 2The Rap GTPases are master regulators of actin dynamics, cell morphology, cell polarity and integrin-mediated adhesion. The Rap GTPases are activated subsequent to the binding of chemokines to their receptors or activated integrins to their ligands. The active GTP-bound form of Rap binds effector proteins that promote integrin activation, actin polymerization and membrane protrusion, as well as activation of the Pyk2 and FAK tyrosine kinases, which modulate cell spreading, adhesion and migration. Rap-GTP also plays a key role in establishing cell polarity and may direct membrane vesicles to the leading edge of the cell. See text for details. MTOC, microtubule-organizing center.To test this hypothesis, we suppressed Rap activation in A20 murine B-lymphoma cells, a cell line derived from an aggressive diffuse large B-cell lymphoma. We blocked Rap activation in these cells by expressing a Rap-specific GTPase-activating protein (GAP), RapGAPII, which enzymatically converts Rap1 and Rap2 proteins to their inactive GDP-bound states. Injecting stable A20/RapGAPII and A20/empty vector transfectants intravenously into mice showed that Rap activation was required for these cells to form solid lymphomas within organs such as the liver.¹⁶ Solid tumor formation was delayed and reduced when A20/RapGAPII cells were injected instead of A20/control cells. Strikingly, the lymphoma cells isolated from the tumors that developed in mice injected with A20/RapGAPII cells had downregulated RapGAPII expression and regained the ability to activate Rap. Thus tumor formation reflected a strong in vivo selection for lymphoma cells capable of activating Rap. This indicates that Rap-dependent signaling is critical for the metastatic spread of B-cell lymphomas.The ability of B-lymphoma cells to exit the vasculature and migrate into the underlying tissue is likely to be a rate-limiting step in the metastasis of B-cell lymphomas. We showed that this extravasation step is a Rap-dependent process for B-cell lymphomas. To do this, we performed competitive in vivo homing assays in which differentially-labeled A20/vector and A20/RapGAPII cells were co-injected into the tail veins of mice.¹⁶ Analyses performed 1–3 days after injecting the cells showed that A20/RapGAPII cells exhibited a greatly reduced ability to arrest and lodge in the liver, compared to control cells. The liver produces large amounts of the chemokine CXCL12 and is a major site of lymphoma homing and tumor formation. More detailed studies revealed that the control A20 cells that lodged in the liver had entered the liver parenchyma and had an elongated morphology, as expected for cells that are migrating within the tissue and interacting with resident cells. In contrast, a larger fraction of the A20/RapGAPII cells were round and appeared to still be within the vasculature. These findings suggest that Rap activation is required for efficient extravasation of lymphoma cells in vivo, as had previously been shown for in T cells in vitro.¹⁷Leukocyte extravasation is a multi-step process that requires initial adhesion to the vascular endothelium followed by crawling on the luminal surface of the endothelial cells until a suitable site for migration through the endothelial barrier is located. We found that Rap activation was required for the initial adhesion of A20 cells to vascular endothelial cells in vitro.¹⁶ Whether integrin-mediated adhesion is an absolute requirement for tumor cells to arrest within organ vasculature remains an open question as tumor cells can be physically trapped in small vessels in a manner that is independent of integrins or other adhesion molecules (Freeman SA, unpublished data). In contrast, the ability of lymphoma cells to generate polarized membrane protrusions that invade junctions between vascular endothelial cells and then move through the junctions is likely to have a strong dependence on Rap-mediated integrin activation and Rap-mediated cell polarization and cytoskeletal reorganization. Indeed, we found that Rap activation was required for A20 B-lymphoma cells to form membrane projections that penetrated endothelial junctions in vitro, and for the subsequent transendothelial migration of A20 cells.¹⁶In addition to this well-characterized paracellular mode of extravasation in which leukocytes crawl across endothelial cells until they arrive at cell-cell junctions and then migrate across the endothelial cell layer, leukocytes can also extravasate via a transcellular route.¹⁸ T cells can send invadopodia through endothelial cells, which upon contacting the subendothelial matrix pull the cell through and across the endothelial cell. The paracellular and transcellular routes of leukocyte extravasation may involve distinct modes of leukocyte motility and cytoskeletal reorganization. For example, activation of WASp and Src is required for transcellular extravasation of T cells, but dispensable for paracellular extravasation.¹⁸ Our data suggest that Rap activation is involved in the paracellular extravasation of B-cell lymphomas. It is not known if lymphoma cells, which are considerably larger than normal leukocytes, can undergo transcellular extravasation, and if so, whether Rap-dependent signaling is required. Determining the relative contributions of these two modes of extravasation, as well as their underlying molecular mechanisms, could facilitate the development of therapeutic approaches for reducing lymphoma cell extravasation and dissemination.Rap GTPases are ubiquitously expressed and are involved in critical processes such as the formation of tight junctions between vascular endothelial cells.¹⁹ Therefore, targeting downstream effectors of Rap that mediate specific aspects of adhesion and migration may be a more reasonable way to limit lymphoma dissemination than targeting Rap activation. As shown in Figure 2 and reviewed by Bos,¹⁰ the effector proteins that are regulated either directly or indirectly by Rap-GTP control several modules that are critical for cell adhesion and migration.Activated Rap is an essential component of the inside-out signaling pathway by which chemokine receptors activate integrins. Rap-GTP recruits the adaptor protein RapL as well as RIAM/talin complexes to the cytoplasmic domains of integrins.^20,21 This results in conformational changes in the integrin extracellular domains that increase their affinity for adhesion molecules, such as those present on the surface of vascular endothelial cells. Actin-dependent intracellular forces exerted by talin on the integrin cytoplasmic domains also increase integrin affinity²² and may be regulated by Rap-GTP, which promotes actin polymerization (see below).Effector proteins that bind Rap-GTP include upstream activators of Rac and Cdc42,^23,24 GTPases that promote dynamic actin polymerization at the leading edge of migrating cells and at the growing ends of membrane protrusions. Activated Rac and Cdc42 act via the WASp and WAVE proteins to induce branching actin polymerization that drives membrane protrusion and the formation of lamellipodia and filopodia. Other Rap effectors, the RIAM²⁵ and AF-6 adaptor proteins,²⁶ allow Rap-GTP to recruit Ena/Vasp and profilin, proteins that prime actin monomers for incorporation into actin filaments, a rate-limiting step in actin filament assembly.The Pyk2 and FAK tyrosine kinases are key regulators of cell adhesion, cell migration and cell morphology, and we have shown that they are also downstream targets of Rap-GTP signaling.²⁷ Rap-dependent actin dynamics is critical for the activation of Pyk2 and FAK in B-lymphoma cells. Moreover the kinase activities of Pyk2 and FAK are required for B cell spreading, a key aspect of cell adhesion and motility.²⁷ The importance of this Rap/Pyk2 signaling module is supported by the observation that B cells from Pyk2-deficient mice have a severe defect in chemokine-induced migration.²⁸Rap effectors also promote the establishment of cell polarity, another key aspect of cell motility. Rap-GTP binds the evolutionarily-conserved Par3/6 polarity complex²⁹ and promotes the microtubule-dependent transport of vesicles containing integrins to the leading edge of migrating cells and to cell-cell contact sites such as immune synapses.^30,31A key question is whether modulating the expression or activity of individual targets of Rap signaling can effectively limit the dissemination of B-cell lymphomas. An exciting recent paper supports the idea that targeting proteins involved in cell motility may be an effective way to limit the spread and growth of B-cell lymphomas.⁹ Using a library of short hairpin RNAs (shRNAs) directed against 1,000 genes thought to be involved in lymphoma progression, Meachem et al. found that two regulators of the actin cytoskeleton, Rac2 and twinfilin (Twf1), were key determinants of lymphoma motility, invasiveness and progression. shRNA-mediated knockdown of either Rac2 or Twf1 expression dramatically inhibited the growth of Eµ-myc B-cell lymphomas in mice, a model for the development of human Burkitt lymphomas. The decreased lymphoma tumorgenicity, as well as the decreased ability of the lymphoma cells to engraft in the spleen and bone marrow and then metastasize to secondary sites such as the liver was associated with the cells'' inability to migrate and crawl in vitro. This is consistent with our finding that inhibiting the in vitro migration and adhesion of B-lymphoma cells by suppressing Rap activation correlated with reduced extravasation and tumor formation in vivo.The involvement of both Rap and Rac2 in lymphoma motility and dissemination may reflect the fact that these two GTPases lie in the same pathway. Rap-GTP has been shown to bind the Rac activator Vav2 and promote Rac activation.²³ Conversely, Batista and colleagues showed that Rac2 acts upstream of Rap to promote Rap activation and modulate B-cell adhesion and immune synapse formation.³² Although the interrelationship of Rap and Rac2 in B-cell lymphomas remains to be clarified, the Rac2/Rap signaling module is a potential target for limiting the spread of B-cell lymphomas. Inhibiting this Rac2/Rap module that controls B-cell motility and adhesion may reduce both the extravasation of lymphoma cells into organs as well as the ability of B-lymphoma cells to crawl to sites within the organ where they can establish a suitable metastatic niche. Migration through the subendothelial stroma to find optimal growth niches is a rate-limiting step in the dissemination of many types of tumors.³³ Blocking Rap-dependent adhesion may also prevent B-lymphoma cells from forming critical adhesive interactions with tissue-resident stromal cells. In vitro, the survival of many B-cell lymphomas depends on integrin engagement^34,35 and the subsequent activation of pro-survival signaling pathways (e.g., the PI 3-kinase/Akt pathway) by integrin signaling.³⁶ It is not known whether Rap-dependent adhesion and the ensuing integrin-mediated survival signaling are required for B-cell lymphomas to form solid tumors at secondary sites in vivo.A series of recent papers has identified the hematopoietic lineage-restricted adaptor protein kindlin-3 as a key regulator of integrin activation in leukocytes. Kindlin-3 is required for leukocyte adhesion in vitro and for in vivo extravasation,^37–39 making it a potential target for limiting the spread of B-cell lymphomas. Kindlin-3 binds to the cytoplasmic domain of several integrin beta subunits but the mechanism by which it promotes integrin activation is not known. An interesting question is whether Rap-GTP, or the RapL/RIAM/talin complexes that are recruited to integrins by Rap-GTP, regulate the localization or function of kindlin-3. Whether or not Rap and kindlin-3 act in the same pathway, it would be interesting to test whether knocking down the expression of kindlin-3 reduces the dissemination of B-cell lymphomas in either the A20 cell model we have used or the Eµ-myc B-cell lymphoma model used by Meachem et al.⁹Although we have thus far referred to the Rap GTPases collectively as “Rap,” there are five Rap GTPases in humans and mice, Rap1a, Rap1b, Rap2a, Rap2b and Rap2c, each encoded by a separate gene. Several reports have suggested distinct functions for Rap1 versus Rap2,^14,40 but it is not known to what extent the functions of the five Rap proteins are redundant or unique. Although many studies have not assessed Rap2 activation, loss-of-function approaches such as overexpressing Rap-specific GAPs or expressing the dominant-negative Rap1N17 protein may suppress the activation of all Rap proteins. Nevertheless, the possibility that different Rap proteins have distinct functions, coupled with cell type-specific differences in the expression of the Rap proteins, may present additional opportunities for targeting Rap signaling in tumor cells. Rap1b is much more abundant than Rap1a in B cells and recent work has shown that Rap1b-deficient murine B cells exhibit impaired migration and adhesion in vitro, as well as impaired in vivo homing.^41,42 If B-lymphoma cells also express much more Rap1b than Rap1a, then Rap1b could be a target for limiting the spread of these malignant B cells. An important caveat is that Rap1b is also the most abundant Rap1 isoform in platelets and plays a critical role in platelet aggregation and clotting.^43,44As master regulators of cell adhesion and migration, the Rap GTPases and the signaling pathways they control are obvious therapeutic targets for limiting the spread of B-cell lymphomas. Other signaling pathways that impact B-cell migration and adhesion, perhaps independently of Rap, are also attractive targets. Our in vivo experiments and those of Meachem et al.⁹ provide direct evidence that interfering with key regulators of adhesion and migration can dramatically limit the dissemination of B-cell lymphomas and the development of secondary tumors in critical organs. Further studies are needed to determine if this approach would be a useful therapeutic strategy for patients with B-cell lymphoma.Finally, it will be of interest to determine whether gain-of-function mutations that increase Rap signaling, or activate other pathways that promote B cell migration and adhesion, contribute to the aggressiveness of certain types of B-cell lymphomas. Increased Rap activation is associated with enhanced invasiveness in several types of tumors.^45,46 If this were true for B-cell lymphomas, then Rap-GTP levels could be a useful prognostic marker for aggressive lymphomas, in addition to being a potential therapeutic target.     

Molecular pathogenesis of lymphomas of mucosa-associated lymphoid tissue—from(auto)antigen driven selection to the activation of NF-?B signaling

Lymphomas of mucosa-associated lymphoid tissue(MALT) are typically present at sites such as the stomach, lung or urinary tract, where lymphoid tissues scatter in mucosa lamina propria, intra- or sub-epithelial cells. The infection of certain pathogens, such as Helicobacter pylori, Chlamydophila psittaci, Borrelia burgdorferi, hepatitis C virus, or certain autoantigens cause these sites to generate a germinal center called the "acquired lymphoid tissue". The molecular pathogenesis of MALT lymphoma is a multi-step process. Receptor signaling, such as the contact stimulation of B cell receptors and CD4 positive T cells mediated by CD40/CD40-ligand and T helper cell type 2 cytokines like interleukin-4, contributes to tumor cell proliferation. A number of genetic alterations have been identified in MALT lymphoma, and among them are important translocations, such as t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21) and t(3;14)(p13;q32). Fusion proteins generated by these translocations share the same NF-?B signaling pathway, which is activated by the caspase activation and recruitment domain containing molecules of the membrane associated guanylate kinase family, B cell lymphoma-10 and MALT1(CBM) protein complex. They act downstream of cell surface receptors, such as B cell receptors, T cell receptors, B cell activating factors and Toll-like receptors, and participate in the biological process of MALT lymphoma. The discovery of therapeutic drugs that exclusively inhibit the antigen receptor signaling pathway will be beneficial for the treatment of B cell lymphomas in the future.     

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin α_vβ₅-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin α_vβ₅, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin α_vβ₅ interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin α_vβ₅-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-α_vβ₅ interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin α_vβ₅ interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin α_vβ₅. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.     

Stromal interferon-γ signaling and cross-presentation are required to eliminate antigen-loss variants of B cell lymphomas in mice

To study mechanisms of T cell-mediated rejection of B cell lymphomas, we developed a murine lymphoma model wherein three potential rejection antigens, human c-MYC, chicken ovalbumin (OVA), and GFP are expressed. After transfer into wild-type mice 60-70% of systemically growing lymphomas expressing all three antigens were rejected; lymphomas expressing only human c-MYC protein were not rejected. OVA expressing lymphomas were infiltrated by T cells, showed MHC class I and II upregulation, and lost antigen expression, indicating immune escape. In contrast to wild-type recipients, 80-100% of STAT1-, IFN-γ-, or IFN-γ receptor-deficient recipients died of lymphoma, indicating that host IFN-γ signaling is critical for rejection. Lymphomas arising in IFN-γ- and IFN-γ-receptor-deficient mice had invariably lost antigen expression, suggesting that poor overall survival of these recipients was due to inefficient elimination of antigen-negative lymphoma variants. Antigen-dependent eradication of lymphoma cells in wild-type animals was dependent on cross-presentation of antigen by cells of the tumor stroma. These findings provide first evidence for an important role of the tumor stroma in T cell-mediated control of hematologic neoplasias and highlight the importance of incorporating stroma-targeting strategies into future immunotherapeutic approaches.     

Genetic basis for Ly-6− defect: Complementation between Ly-6− and Thy-1− mutant cell lines

Selection of antigen loss variants from the BW5147 cell line after chemical mutagenesis, using antibodies to Thy-1.1, Ly-6.2 and H9/25 allospecificities, produced cell lines with a pleiotropic defect in cell surface antigen expression; selection against any antigen led to the loss of all three determinants. The genetic basis for the mutant phenotype has been analyzed by gene complementation by somatic cell hybridization between Ly-6 ^– or H9/25 ^– and Thy-1 ^– mutants, which comprise different Thy-1 gene complementation classes. The Ly-6 ^– and H9/25 ^– mutants can be classified as belonging to either the A or E Thy-1^– mutant class.