Zarei Shohreh, Leuba Florence, Arrighi Jean-François, Hauser Conrad, Piguet Vincent
Division of Allergy and Immunology and the Division of Hematology, Department of Dermatology, University Hospital of Geneva, Switzerland.
J Allergy Clin Immunol. 2002 Jun;109(6):988-94. doi: 10.1067/mai.2002.124663.
Because antigen-presenting dendritic cells (DCs) play a major role in the polarization of T cells, including T(H)2 cells involved in allergy, strategies to modify DCs genetically are required.
The purpose of this investigation was to transduce murine bone marrow-derived DCs with lentiviral vectors encoding antigen to demonstrate antigen processing and MHC class I-dependent presentation.
Bone marrow leukocytes were incubated with antigen-encoding lentiviral constructs and cultured with GM-CSF, IL-4, and Flt-3 ligand. The capacity of the resulting DCs to express, process, and present antigen was tested in vitro.
An average of 40% of DCs expressed antigen after 1 week of culture when antigen encoded by the lentiviral vector construct was green fluorescent protein. To demonstrate that transduced antigen can be presented by DCs on MHC class I, we chose the lymphocytic choriomeningitis virus glycoprotein (gp) as a model antigen, inasmuch as it is recognized by CD8 T cells from transgenic mice expressing an MHC class I-restricted T-cell receptor specific for the epitope of positions 33 through 41 of gp. DCs transduced with lentiviral construct encoding gp and matured with LPS activated transgenic T cells in an antigen-specific fashion. Using transporter associated with antigen presentation (TAP)-deficient mice, we show that presentation of the gp33-41 epitope is TAP-dependent, confirming processing of gp by the endogenous pathway.
These results demonstrate that CD8 T cells can recognize MHC class I epitopes processed from antigen in DCs transduced with lentiviral vectors. Lentiviral transduction of DCs and antigen presentation to CD8 T cells could be exploited for immunotherapy, because allergen-specific CD8 T cells have been shown to be suppressive in IgE-dependent allergy models.
由于抗原呈递树突状细胞（DCs）在T细胞极化中起主要作用，包括参与过敏反应的辅助性T细胞2（TH2），因此需要采用基因改造DCs的策略。
本研究旨在用编码抗原的慢病毒载体转导小鼠骨髓来源的DCs，以证明抗原加工及MHC I类分子依赖性呈递。
将骨髓白细胞与编码抗原的慢病毒构建体一起孵育，并用粒细胞巨噬细胞集落刺激因子（GM-CSF）、白细胞介素4（IL-4）和Flt-3配体进行培养。在体外测试所得DCs表达、加工和呈递抗原的能力。
当慢病毒载体构建体编码的抗原为绿色荧光蛋白时，培养1周后平均40%的DCs表达抗原。为证明转导的抗原可由DCs在MHC I类分子上呈递，我们选择淋巴细胞性脉络丛脑膜炎病毒糖蛋白（gp）作为模型抗原，因为它可被表达针对gp第33至41位表位的MHC I类限制性T细胞受体的转基因小鼠的CD8 T细胞识别。用编码gp的慢病毒构建体转导并用脂多糖（LPS）成熟的DCs以抗原特异性方式激活转基因T细胞。利用与抗原呈递相关的转运体（TAP）缺陷小鼠，我们发现gp33-41表位的呈递依赖于TAP，证实了通过内源性途径对gp的加工。
这些结果表明，CD8 T细胞可识别由慢病毒载体转导的DCs中抗原加工产生的MHC I类表位。DCs的慢病毒转导及向CD8 T细胞的抗原呈递可用于免疫治疗，因为在IgE依赖性过敏模型中，过敏原特异性CD8 T细胞已被证明具有抑制作用。
