COBRE: DMS: CD1 ACQUISITION OF BCR & FC RECEPTOR TARGETED LIGAND

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。结核分枝杆菌(MTB)细胞壁糖脂(磷脂酰肌醇甘露糖苷，PIM)和脂多糖(脂阿拉伯甘露聚糖，LAM)是强大的毒力因子，通过抑制天然和获得性免疫反应，促进MTB在宿主巨噬细胞中的存活。专用于PIM和LAM的ABS可提供对MTB的保护。PIM和LAM是特异性结合CD1d和CD1b的AGs，以激活CD1d或CD1b限制的T细胞。CD1d限制性T细胞(Natural Killer T cell，NKT)为抗体的产生提供CD1d依赖的帮助。这表明B细胞抗原受体(BCR)可以捕获和内化PIM，以促进CD1d依赖的NKT的激活，以及随后的抗体产生。特定目的1：BCR是否促进CD1对糖脂抗原的提呈？实验进展：我们测试了BCR靶向生物素修饰的CD1d结合的Agα-半乳糖基神经酰胺(生物素-α-GalCer，Ag)与非靶向Ag相比导致CD1d呈递增强的假设。这种新型的银是与英国伯明翰大学的贝斯拉博士合作合成的。与非靶向抗原相比，BCR靶向抗原对NKT细胞的呈递能力提高了100倍。在4小时的银脉冲后观察到BCR靶向抗原的CD1d呈现，这与BCR/Ag的内体转运要求是一致的。在Ag脉冲前固定APC可防止BCR靶向而不是非靶向Ag的出现。用Syk激酶抑制剂Piceatanyl阻断BCR信号，可抑制BCR靶向抗原的呈递，但不抑制非靶向抗原的呈递。Piceat注l阻断了BCR向含CD1d的囊泡的转运，表明BCR靶向的Ag与细胞内含CD1d的囊泡的交叉点是Ag提呈所必需的。我们的数据表明，BCR有助于捕获少量的糖脂抗原，以增强CD1d依赖的NKT的激活。在与Reinhold博士的合作下，我们对含有CD1d的内体进行了质谱分析，脉冲作用于bcr靶向或非靶向的Ag。令人惊讶的是，没有检测到Ag，这表明如此低水平的糖脂Ag刺激NKT激活，以至于无法用质谱仪检测到。我们正在继续尝试用质谱仪在细胞内检测这些抗原。生产力：我们已经完成了BCR介导的抗原呈递的实验，并在《国际免疫学》上发表了这一发现(在印刷中)。特异性目的2：CD1抗原递呈是否代表抗体产生的新途径？实验进展：我们测试了在没有第二类/TCR同源相互作用的情况下，α-GalCer在体内刺激抗体产生的假设。我们免疫II-/-类小鼠S.C.使用OVA、α-GalCer或α-GalCer+OVA，没有观察到OVA特异性血清抗体效价的任何增加。当抗CD40激动型单抗在Ag(OVA+α-GalCer)免疫后24小时注射时，我们观察到OVA特异性抗体滴度增加，但HEL特异性抗体效价没有增加，这表明抗体反应也是抗原特异性的。分别用OVA和α-GalCer免疫(进入相对侧翼)不会导致抗体的产生，这表明预先混合的OVA和α-GalCer的物理结合以及OVA/α-GalCer复合体的BCR内化。初步数据还表明，用PIM免疫，然后再用抗CD40单抗免疫，会在II-/-小鼠中产生抗体。我们的研究结果表明，NKT细胞在体内有助于产生对蛋白质和糖脂AGS有反应的抗体。生产力：这些研究构成了新手稿的基础。为了完成我们的研究，我们需要CD1d/ClassII-/-双重敲除来确定在没有CD1d和Class II的情况下，抗体的产生是否有缺陷。我们已经获得了CD1-/-小鼠(Exley博士，哈佛医学院)，而Cobre Core C正在产生必要的？双重敲除？控制。当前/迫在眉睫的方向：我们由Cobre资助的实验表明，NKT细胞为抗体的产生提供了帮助，并表明抗体的产生需要糖脂特异性的BCR捕获抗原，并随后将CD1d呈递给NKT细胞。我们的发现被用于与Erickson博士(项目4负责人)合作开发NIH R21拨款提案(提交于2005年2月)。在这一应用中，我们提出了一个假设，即表达CD1d的边缘区B细胞对MTB来源的PIM的BCR捕获导致CD1d/NKT依赖的抗PIM抗体的产生。我目前正在使用PIM获取新的数据，以便纳入NIH的拨款提案(2005年6月截止日期的RO1)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. M. tuberculosis (MTB) cell wall glycolipids (phosphatidylinositol mannoside, PIM) and lipoglycans (lipoarabinomannan, LAM) are potent virulence factors and contribute to MTB survival in host macrophages, by inhibiting innate and adaptive immune responses. Abs specific for PIM and LAM confer protection against MTB. PIM and LAM are Ags that specifically bind CD1d and CD1b, to activate CD1d- or CD1b-restricted T cells. CD1d-restricted T cells (Natural Killer T cells, NKT) provide CD1d-dependent help for Ab production. This suggests that the B cell antigen receptor (BCR) may capture and internalize PIM, to facilitate CD1d-dependent NKT activation, and subsequent Ab production. SPECIFIC AIM 1: Does the BCR facilitate enhanced presentation of glycolipid Ag by CD1? Experimental Progress: We tested the hypothesis that BCR-targeting of a biotin-modified CD1d-binding Ag alpha-Galactosylceramide (biotin-alpha-GalCer, Ag) led to in enhanced CD1d presentation as compared to presentation of non-targeted Ag. This novel Ag was synthesized in collaboration with Dr. Besra (University of Birmingham, U.K.). Presentation of BCR-targeted Ag to NKT cells was enhanced 100-fold compared to non-targeted Ag. CD1d presentation of BCR-targeted Ag was observed after 4 hours Ag pulse, consistent with a requirement for endosomal trafficking of BCR/Ag. Fixing APCs before Ag pulse prevented presentation of BCR-targeted but not non-targeted Ag. Blocking BCR signaling with the Syk kinase inhibitor, piceatannol, inhibited presentation of BCR-targeted Ag but not non-targeted Ag. Piceatannol blocked BCR transport to CD1d-containing vesicles, showing that intersection of BCR-targeted Ag with intracellular CD1d-containing vesicles is required for Ag presentation. Our data suggest that the BCR facilitates capture of low quantities of glycolipid Ag to enhance CD1d-dependent NKT activation. In collaboration with Dr. Reinhold (Consortium PI, UNH), we have performed mass spectrometry analysis of CD1d-containing endosomes following a pulse with BCR-targeted or non-targeted Ag. Surprisingly, the Ag was not detected, suggesting that such low levels of glycolipid Ag stimulate NKT activation, as to be undetectable by mass spectrometry. We are continuing to attempt detection of these antigens intracellularly by mass spectrometry. Productivity: We have completed our experiments on BCR-mediated antigen presentation and published the findings in International Immunology (in press). SPECIFIC AIM 2: Does CD1 Ag presentation represent a novel pathway for Ab production? Experimental Progress: We tested the hypothesis that alpha-GalCer stimulates Ab production in vivo in the absence of class II/TCR cognate interactions. We immunized class II-/- mice s.c. with OVA, alpha-GalCer or alpha-GalCer plus OVA, and did not observe any increase in OVA-specific serum Ab titers. When anti-CD40 agonistic mAbs were injected 24 hours following Ag (OVA plus alpha-GalCer) immunization, we observed increased OVA- but not HEL-specific Ab titers, indicating that Ab responses were also Ag specific. Immunization with OVA and alpha-GalCer separately (into opposing flanks), did not lead to Ab production, indicating physical association of pre-mixed OVA and alpha-GalCer and BCR internalization of OVA/alpha-GalCer complexes. Preliminary data also shows that immunization with PIM, followed by anti-CD40 mAb results in Ab production in class II-/- mice. Our findings show that NKT cells contribute to production of Ab reactive to protein and glycolipid Ags in vivo. Productivity: These studies form the basis for a new manuscript. To complete our studies, we require a CD1d/classII-/- double knockout to determine if Ab production is defective in the absence of CD1d and class II. We have obtained CD1-/- mice (Dr. Exley, Harvard Medical School) and COBRE Core C is generating the necessary ?double knockout? controls. CURRENT/IMMINENT DIRECTIONS: Our COBRE-funded experiments show that NKT cells provide help for Ab production, and suggest that Ab production requires glycolipid-specific BCR capture of Ag and subsequent CD1d presentation to NKT cells. Our findings were used to develop an NIH R21 grant proposal (submitted Feb 2005) in collaboration with Dr. Erickson (Project 4 leader). In that application, we proposed the hypothesis that BCR capture of MTB-derived PIM by CD1d-expressing marginal zone B cells results in CD1d/NKT-dependent anti-PIM Ab production. I am currently working to obtain new data using PIM to incorporate into an NIH grant proposal (RO1 for June 2005 deadline).