Tissue-associated self-antigens containing exosomes: Role in allograft rejection

Exosomes are extracellular vesicles that express self-antigens (SAgs) and donor human leukocyte antigens. Tissue-specific exosomes can be detected in the circulation following lung, heart, kidney and islet cell transplantations. We collected serum samples from patients who had undergone lung (n?=?30), heart (n?=?8), or kidney (n?=?15) transplantations to isolate circulating exosomes. Exosome purity was analyzed by Western blot, using CD9 exosome-specific markers. Tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV), were identified using western blot. Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2-fold) and Kα1T (37.1-fold) than stable. Exosomes isolated from heart transplant recipients diagnosed with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Further, Kidney transplant recipients diagnosed with transplant glomerulopathy had circulating exosomes with a 2-fold increased expression of fibronectin and 2.5-fold increase in Col-IV compared with stable. We conclude that circulating exosomes with tissue associated SAgs have the potential to be a noninvasive biomarker for allograft rejection.     

Xenogeneic cells and superantigen induce human T-cell activation in the absence of T-cell recognition of xenoantigen

The extent of interaction between human T-lymphocytes and xenogeneic antigen-presenting cells (APCs) is species-dependent. A successful interaction requires high-affinity receptor-ligand pairing across species and recognition of antigens presented by foreign major histocompatibility complex (MHC). A deficient human T-lymphocyte response to xenogeneic cells is likely the result of a defect in these interactions. However, the requirements for a T-cell response to superantigen (SAg) may differ from those of other T-cell responses. Using irradiated murine splenocytes, which are believed to be incapable of eliciting human T-cell responses, and porcine aortic endothelial cells (PAECs) as the APC populations, we studied the human T-lymphocyte response to antigens presented by these cells. Direct proliferation of human T-lymphocytes to SAg presented by murine APCs was demonstrated; it was blocked by anti-human LFA-1 and anti-murine MHC class II but not by anti-human MHC class II. PAECs also presented SAg to human T-cells, generating a proliferative response greater than the primary response to porcine xenoantigen. Culture of human T-cells with murine splenocytes or PAECs and SAg Staphylococcus enterotoxin A (SEA) for 7 days primed human T-cells to proliferate in a secondary culture in response to autologous APCs. This autologous secondary response was human MHC class II-dependent and was inhibited by anti-human LFA-1, anti-human CD2, and anti-human CD98. Surprisingly, both of these responses were also blocked by anti-SEA, suggesting that despite vigorous washing, a small amount of functionally important SAg was carried over from primary to secondary culture, probably bound to the surface of T-cells. Xenogeneic APCs, even those that fail to stimulate human T-cells directly, can serve as APCs for primary human T-cell responses. After such interactions T-cells can develop secondary responses in autologous interactions based on retention of minute amounts of SAg. Such interactions may have important implications for xenotransplantation.     

SEA基因与GPI信号肽序列融合基因的真核表达载体的构建和序列分析

目的：将超抗原金黄色葡萄球菌肠毒素A（staphylococcus enterotoxinA,SEA)基因的人胎盘碱性磷酸酶（hPLAP-1)C末端信号肽序列，即糖基化磷脂酰肌醇（glycosyl-phosphatidyl inositol,GPI)附着信号序列，拼接成融合基因，并构建该融合基因的真核表达载体。方法：利用基因SOEing法，分别扩增hPLAP-1 C末端信号肽序列和SEA基因，然后将二段基因拼接起来，拼接后与pGEM-T克隆载体连接，再亚克隆到真核表达载体pcDNA3.1( )中，测序鉴定。结果：分别成功扩增出hPLAP-1 C末端信号肽序列131bp和SEA基因789bp，并将二段基因序列成功拼接（901bp），且成功构建了真核表达载体pcDNA3.1( )/SEA-GPI，经测序是正确的。结论；真核表达载体pcDNA3.1( )/SEA-GPI的构建，为研究SEA－GPI抗肿瘤作用奠定了基础。     

抗体——超抗原融合蛋白及其应用的研究进展

超抗原(Superantigen,SAg)是一类具有多种免疫活性的蛋白分子,在一定条件下能比普通抗原十至数千倍激活T细胞和抗原呈递细胞。超抗原能引导T细胞产生细胞毒作用(SDCC)和诱导产生一些细胞因子来杀伤肿瘤细胞。然而单用SAg效果差,毒副作用强,其作用受到限制。研究表明使用肿瘤特异性的单抗Fab-SAg融合蛋白,能比单用SAg取得更稳定、更明显的效果。该融合蛋白对肿瘤部位的靶向性强、亲和力高,并能很好地减少系统的免疫原性。另外发现使用突变的Fab-SAg融合蛋白,能进一步增加动物的耐受性、降低毒副作用。目前已有多个用单抗Fab-SAg融合蛋白药物进入临床研究,该药物将会为治疗人类恶性肿瘤带来希望。