Abstract：
For the rational design of epitope-specific vaccines, identifying epitopes that can be processed and presented is essential. As algorithm-based epitope prediction is frequently discordant with actually recognized CD8+ T-cell epitopes, we developed an in vitro CD8 T-cell priming protocol to enable the identification of truly and functionally expressed HLA class I epitopes. The assay was established and validated to identify epitopes presented by hepatitis C virus (HCV)-infected cells. In vitro priming of naïve CD8 T cells was achieved by culturing unfractionated PBMCs in the presence of a specific cocktail of growth factors and cytokines, and next exposing the cells to hepatic cells expressing the NS3 protein of HCV. After a 10-day co-culture, HCV-specific T-cell responses were identified based on IFN-γ ELISpot analysis. For this, the T cells were restimulated with long synthetic peptides (SLPs) spanning the whole NS3 protein sequence allowing the identification of HCV-specificity. We demonstrated that this protocol resulted in the in vitro priming of naïve precursors to antigen-experienced T-cells specific for 11 out of 98 SLPs tested. These 11 SLPs contain 12 different HLA-A*02:01-restricted epitopes, as predicted by a combination of three epitope prediction algorithms. Furthermore, we identified responses against 3 peptides that were not predicted to contain any immunogenic HLA class I epitopes, yet showed HCV-specific responses in vitro. Separation of CD8+ and CD8- T cells from PBMCs primed in vitro showed responses only upon restimulation with short peptides. We established an in vitro method that enables the identification of HLA class I epitopes resulting from cross-presented antigens and that can cross-prime T cells and allows the effective selection of functional immunogenic epitopes, but also less immunogenic ones, for the design of tailored therapeutic vaccines against persistent viral infections and tumor antigens.
摘要：
为了合理设计表位特异性疫苗，鉴定可以加工和呈递的表位是必不可少的。由于基于算法的表位预测经常与实际识别的CD8+T细胞表位不一致，我们开发了一种体外CD8T细胞引发方案，以鉴定真正和功能表达的HLAI类表位。建立并验证该测定以鉴定由丙型肝炎病毒（HCV）感染的细胞呈递的表位。通过在生长因子和细胞因子的特定混合物存在下培养未分级的PBMC，可以实现初始CD8T细胞的体外引发。然后将细胞暴露于表达HCV的NS3蛋白的肝细胞。经过10天的共同培养，基于IFN-γELISpot分析鉴定HCV特异性T细胞应答。为此，用跨越整个NS3蛋白序列的长合成肽(SLP)再刺激T细胞,从而鉴定HCV特异性.我们证明了该方案导致初始前体对所测试的98个SLP中的11个具有特异性的抗原经历T细胞的体外引发。这11个SLP包含12个不同的HLA-A*02:01限制性表位，通过三种表位预测算法的组合预测。此外，我们确定了针对3种预测不包含任何免疫原性HLAI类表位的肽的反应，但在体外显示HCV特异性应答。从体外引发的PBMC中分离CD8+和CD8-T细胞仅在用短肽再刺激时显示出应答。我们建立了一种体外方法，该方法能够鉴定由交叉呈递抗原产生的HLAI类表位，并且可以交叉引发T细胞，并允许有效选择功能性免疫原性表位。但免疫原性也较低，用于设计针对持续性病毒感染和肿瘤抗原的定制治疗性疫苗。
