对病原体和候选疫苗的免疫应答的研究的进展受到研究响应于由抗原呈递细胞(APC)加工和呈递的抗原而增殖的T细胞亚群的功能活性的方法的限制的阻碍。如这篇评论所述,在我们研究牛副结核分枝杆菌(Map)和牛分枝杆菌(BCG)中对基于候选肽的疫苗和候选rel缺失突变体的免疫反应期间,我们开发了使用离体平台研究原发性和回忆性CD4和CD8T细胞反应的方法.开发了一种测定法来研究由CD8T细胞介导的细菌的细胞内杀伤,使用定量PCR来区分活细菌和死细菌的混合群体中的死细菌。通过使用这些化验,我们能够证明使用活relMap和BCG缺失突变体进行疫苗接种,并且基于Map肽的疫苗可引发CD8细胞毒性T细胞的发展,并能够使用穿孔素-颗粒酶B途径杀死细胞内细菌.我们还证明了CD4和CD8T细胞与抗原引发的APC之间的三向信号传导对于引发CD8细胞毒性T细胞是必不可少的。在这里,我们描述了这些检测方法的发展,并回顾了它们在分枝杆菌病原体和候选疫苗免疫反应研究中的应用进展。该方法避免了在表征对病原体的细胞介导的免疫应答以及减毒和基于肽的疫苗的开发中遇到的一些主要困难。
Progress in the
study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to
study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to
study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to
study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the
study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.