Disruption of Lymphocyte Function and Signaling in CD45–associated Protein–null Mice

CD45-AP specifically associates with CD45, a protein tyrosine phosphatase essential for lymphocyte differentiation and antigen receptor–mediated signal transduction. CD45 is thought to mediate antigen receptor signaling by dephosphorylating regulatory tyrosine residues on Src family protein tyrosine kinases such as Lck. However, the mechanism for regulating CD45 protein tyrosine phosphatase activity remains unclear. CD45-AP–null mice were created to examine the role of CD45-AP in CD45-mediated signal transduction. T and B lymphocytes showed reduced proliferation in response to antigen receptor stimulation. Both mixed leukocyte reaction and cytotoxic T lymphocyte functions of T cells were also markedly decreased in CD45-AP–null mice. Interestingly, the interaction between CD45 and Lck was significantly reduced in CD45-AP–null T cells, indicating that CD45-AP directly or indirectly mediates the interaction of CD45 with Lck. Our data indicate that CD45-AP is required for normal antigen receptor signaling and function in lymphocytes.     

The Functional Paradox of CD43 in Leukocyte Recruitment: A Study Using CD43-deficient Mice

Although there is considerable evidence implicating a role for CD43 (leukosialin) in leukocyte cell–cell interactions, its precise function remains uncertain. Using CD43-deficient mice (CD43^−/−) and intravital microscopy to directly visualize leukocyte interactions in vivo, we investigated the role of CD43 in leukocyte–endothelial cell interactions within the cremasteric microcirculation under flow conditions. Our studies demonstrated significantly enhanced leukocyte rolling and adhesion after chemotactic stimuli in CD43^−/− mice compared with wild type mice. Using an in vitro flow chamber, we established that the enhanced rolling interactions of CD43^−/− leukocytes, primarily neutrophils, were also observed using immobilized E-selectin as a substrate, suggesting that passive processes related to steric hindrance or charge repulsion were likely mechanisms. Despite increased adhesion and rolling interactions by CD43^−/− leukocytes, we uncovered a previously unrecognized impairment of CD43^−/− leukocytes to infiltrate tissues. Oyster glycogen–induced neutrophil and monocyte infiltration into the peritoneum was significantly reduced in CD43^−/− mice. In response to platelet activating factor, CD43^−/− leukocytes were impaired in their ability to emigrate out of the vasculature. These results suggest that leukocyte CD43 has a dual function in leukocyte–endothelial interactions. In addition to its role as a passive nonspecific functional barrier, CD43 also facilitates emigration of leukocytes into tissues.     

Loss of Shp2 Rescues BDNF/TrkB Signaling and Contributes to Improved Retinal Ganglion Cell Neuroprotection

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玫瑰糠疹患者皮损中CD45RA与CD45RO的表达

目的：观察玫瑰糠疹患者皮损中CD45RA与CD45RO的表达情况，探讨玫瑰糠疹的发病机制。方法：免疫组化SP法检测30例玫瑰糠疹患者皮损中CD45RA与CDRTRO的表达，取10例正常人躯干部皮肤作对照。结果：玫瑰糠疹患者皮损中CD45RO的表达明显高于正常对照组（P〈0．01），CD45RA的表达和正常对照组比较差异无统计学意义（P〉0．05）。结论：玫瑰糠疹患者皮损真皮中浸润的淋巴细胞以记忆性T淋巴细胞为主，推测细胞免疫在玫瑰糠疹的发病中起主要作用。     

Single and Dual Vector Gene Therapy with AAV9-PHP.B Rescues Hearing in Tmc1 Mutant Mice

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Neutrophil Emigration in the Skin, Lungs, and Peritoneum: Different Requirements for CD11/CD18 Revealed by CD18-deficient Mice

To determine the role of CD11/CD18 complexes in neutrophil emigration, inflammation was induced in the skin, lungs, or peritoneum of mutant mice deficient in CD18 (CD18^−/− mutants). Peripheral blood of CD18^−/− mutants contained 11-fold more neutrophils than did blood of wild-type (WT) mice. During irritant dermatitis induced by topical application of croton oil, the number of emigrated neutrophils in histological sections of dermis was 98% less in CD18^−/− mutants than in WT mice. During Streptococcus pneumoniae pneumonia, neutrophil emigration in CD18^−/− mutants was not reduced. These data are consistent with expectations based on studies using blocking antibodies to inhibit CD11/CD18 complexes, and on observations of humans lacking CD11/CD18 complexes. The number of emigrated neutrophils in lung sections during Escherichia coli pneumonia, or in peritoneal lavage fluid after 4 h of S. pneumoniae peritonitis, was not reduced in CD18^−/− mutants, but rather was greater than the WT values (240 ± 30 and 220 ± 30% WT, respectively). Also, there was no inhibition of neutrophil emigration during sterile peritonitis induced by intraperitoneal injection of thioglycollate (90 ± 20% WT). These data contrast with expectations. Whereas CD11/CD18 complexes are essential to the dermal emigration of neutrophils during acute dermatitis, CD18^−/− mutant mice demonstrate surprising alternative pathways for neutrophil emigration during pneumonia or peritonitis.     

Spontaneous Skin Ulceration and Defective T Cell Function in CD18 Null Mice

A null mutation was prepared in the mouse for CD18, the β₂ subunit of leukocyte integrins. Homozygous CD18 null mice develop chronic dermatitis with extensive facial and submandibular erosions. The phenotype includes elevated neutrophil counts, increased immunoglobulin levels, lymphadenopathy, splenomegaly, and abundant plasma cells in skin, lymph nodes, gut, and kidney. Very few neutrophils were found in spontaneously occurring skin lesions or with an induced toxic dermatitis. Intravital microscopy in CD18 null mice revealed a lack of firm neutrophil attachment to venules in the cremaster muscle in response to N-formyl- methionyl-leucyl-phenylalanine. A severe defect in T cell proliferation was found in the CD18 null mice when T cell receptors were stimulated either by staphylococcal enterotoxin A or by major histocompatibility complex alloantigens demonstrating a greater role of CD11/CD18 integrins in T cell responses than previously documented. The null mice are useful for delineating the functions of CD18 in vivo.     

CD45RA和CD45RO抗原在白血病细胞上的表达及意义

探讨ＣＤ４５ＲＡ和ＣＤ４５ＲＯ抗原在白血病及其亚型中的表达和分布及其在白血病鉴别诊断中的应用价值。     

Indirect Recruitment of a CD40 Signaling Pathway in Dendritic Cells by B7-DC Cross-Linking Antibody Modulates T Cell Functions

The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40^−/− DCs treated with B7-DC XAb (DC^XAb) activated DAP12, but failed to activate NFκB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D₃. CD40^−/− DC^XAb also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DC^XAb to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant.     

自身免疫病患者自体造血干细胞移植后CD4+CD45RA+及CD4+CD45RO+T辅助淋巴细胞亚群的改变

目的检测严重自体免疫病(SA ID)患者自身造血干细胞移植(AHSCT)前后外周血中CD 4 CD 45RA (na ive T h细胞)及CD 4 CD 45RO (记忆T h细胞)T辅助淋巴细胞亚群的变化,探讨na ive T h细胞和记忆T h细胞在AHSCT诱导自身免疫病缓解中的作用。方法使用三色流式细胞术检测SA ID患者AHSCT前1 w及移植后1~3 m o外周血中CD 4 CD 45RA 及CD 4 CD 45RO T辅助淋巴细胞亚群的改变,na ive T h细胞表型为CD 4 CD 45RA ,记忆T h细胞表型为CD 4 CD 45RO 。分析各细胞亚群占CD 4 T辅助淋巴细胞的百分比,并比较移植前后na ive T h、记忆T h细胞的变化。结果AHSCT后,患者外周血中记忆T h细胞显著增高,P<0.05;na ive T h细胞和CD 45RO CD 45RA 双阳性细胞均显著下降,P<0.05。结论AHSCT治疗SA ID患者,患者体内na ive T h细胞和CD 45RO CD 45RA 双阳性T h细胞百分率显著减少,这是AHSCT诱导SA ID缓解的机制之一。     

CD19-Regulated Signaling Thresholds Control Peripheral Tolerance and Autoantibody Production in B Lymphocytes

The CD19 cell surface molecule regulates signal transduction events critical for B lymphocyte development and humoral immunity. Increasing the density of CD19 expression renders B lymphocytes hyper-responsive to transmembrane signals, and transgenic mice that overexpress CD19 have increased levels of autoantibodies. The role of CD19 in tolerance regulation and autoantibody generation was therefore examined by crossing mice that overexpress a human CD19 transgene with transgenic mice expressing a model autoantigen (soluble hen egg lysozyme, sHEL) and high-affinity HEL-specific IgM^a and IgD^a (Ig^HEL) antigen receptors. In this model of peripheral tolerance, B cells in sHEL/Ig^HEL double-transgenic mice are functionally anergic and do not produce autoantibodies. However, it was found that overexpression of CD19 in sHEL/Ig^HEL double-transgenic mice resulted in a breakdown of peripheral tolerance and the production of anti-HEL antibodies at levels similar to those observed in Ig^HEL mice lacking the sHEL autoantigen. Therefore, altered signaling thresholds due to CD19 overexpression resulted in the breakdown of peripheral tolerance. Thus, CD19 overexpression shifts the balance between tolerance and immunity to autoimmunity by augmenting antigen receptor signaling.     

Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.     

CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and B cell maturation

The CD45 transmembrane glycoprotein has been shown to be a protein phosphotyrosine phosphatase and to be important in signal transduction in T and B lymphocytes. We have employed gene targeting to create a strain of transgenic mice that completely lacks expression of all isoforms of CD45. The spleens from CD45-null mice contain approximately twice the number of B cells and one fifth the number of T cells found in normal controls. The increase in B cell numbers is due to the specific expansion of two B cell subpopulations that express high levels of immunoglobulin (IgM) staining. T cell development is significantly inhibited in CD45-null animals at two distinct stages. The efficiency of the development of CD4-CD8- thymocytes into CD4+ CD8+ thymocytes is reduced by twofold, subsequently the frequency of successful maturation of the double positive population into mature, single positive thymocytes is reduced by a further four- to fivefold. In addition, we demonstrate that CD45-null thymocytes are severely impaired in their apoptotic response to cross-linking signals via T cell receptor (TCR) in fetal thymic organ culture. In contrast, apoptosis can be induced normally in CD45-null thymocytes by non-TCR- mediated signals. Since both positive and negative selection require signals through the TCR complex, these findings suggest that CD45 is an important regulator of signal transduction via the TCR complex at multiple stages of T cell development. CD45 is absolutely required for the transmission of mitogenic signals via IgM and IgD. By contrast, CD45-null B cells proliferate as well as wild-type cells to CD40- mediated signals. The proliferation of B cells in response to CD38 cross-linking is significantly reduced but not abolished by the CD45- null mutation. We conclude that CD45 is not required at any stage during the generation of mature peripheral B cells, however its loss reveals a previously unrecognized role for CD45 in the regulation of certain subpopulations of B cells.     

Induction of unresponsiveness and impaired T cell expansion by staphylococcal enterotoxin B in CD28-deficient mice

We used CD28-deficient mice to analyze the importance of CD28 costimulation for the response against Staphylococcal enterotoxin B (SEB) in vivo. CD28 was necessary for the strong expansion of V beta 8+ T cells, but not for deletion. The lack of expansion was not due to a failure of SEB to activate V beta 8+ T cells, as V beta 8+ T cells from both CD28-/- and CD28+/+ mice showed similar phenotypic changes within the first 24 h after SEB injection and cell cycle analysis showed that an equal percentage of V beta 8+ T cells started to proliferate. However, the phenotype and the state of proliferation of V beta 8+ T cells was different at later time points. Furthermore, in CD28-/- mice injection with SEB led to rapid induction of unresponsiveness in SEB responsive T cells, indicated by a drastic reduction of proliferation after secondary SEB stimulation in vitro. Unresponsiveness could also be demonstrated in vivo, as CD28-/- mice produced only marginal amounts of TNF alpha after rechallenge with SEB. In addition CD28-/- mice were protected against a lethal toxic shock induced by a second injection with SEB. Our results indicate that CD28 costimulation is crucial for the T cell-mediated toxicity of SEB and demonstrate that T cell stimulation in the absence of CD28 costimulation induces unresponsiveness in vivo.     

系统性红斑狼疮患者CD4+T淋巴细胞CD45RO/CD45RA的表达

目的：检测系统性红斑狼疮患者CD4^ T辅助淋巴细胞CD45RO和CD45RA亚群的变化及临床意义。方法：用流式细胞术检测了8例活动期SLE患者CD4^ T辅助细胞CD45RO、CD45RA的表达率，并与对照组进行了比较。结果：SLE患者CD4^ T辅助淋巴细胞CD45RO的表达率与对照组无显著性差异（59．30％vs.59.74%),CD45RA下降（21．40％vs.31.64%),CD45RD、CD45RA双阳性T辅助淋巴细胞显著增高（18．57％vs.7.92%)。结论：SLE患者体内CD45RA^ T辅助淋巴细胞被激活，向CD45RO^ 细胞转化。CD45RA^ T辅助细胞的减少是导致机体免疫功能失去平衡的重要原因。     

Regulation of B Cell Development by Variable Gene Complexity in Mice Reconstituted with Human Immunoglobulin Yeast Artificial Chromosomes

The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and κ light (L) chain loci. Each of the two H and the two κ chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique V_H (5 versus 66) or Vκ genes (3 versus 32). Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by V_H and Vκ gene complexity: (a) production of functional μ chains at the transition from the pre B-I to the pre B-II stage; (b) productive VκJκ recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of μ chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.     

Development of spontaneous autoimmune peripheral polyneuropathy in B7-2-deficient NOD mice

An increasing number of studies have documented the central role of T cell costimulation in autoimmunity. Here we show that the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain, deficient in B7-2 costimulation, is protected from diabetes but develops a spontaneous autoimmune peripheral polyneuropathy. All the female and one third of the male mice exhibited limb paralysis with histologic and electrophysiologic evidence of severe demyelination in the peripheral nerves beginning at 20 wk of age. No central nervous system lesions were apparent. The peripheral nerve tissue was infiltrated with dendritic cells, CD4(+), and CD8(+) T cells. Finally, CD4(+) T cells isolated from affected animals induced the disease in NOD.SCID mice. Thus, the B7-2-deficient NOD mouse constitutes the first model of a spontaneous autoimmune disease of the peripheral nervous system, which has many similarities to the human disease, chronic inflammatory demyelinating polyneuropathy (CIDP). This model demonstrates that NOD mice have "cryptic" autoimmune defects that can polarize toward the nervous tissue after the selective disruption of CD28/B7-2 costimulatory pathway.