table_1_Determination of a Predictive Cleavage Motif for Eluted Major Histocompatibility Complex Class II Ligands.xlsx

CD4+ T cells have a major role in regulating immune responses. They are activated by recognition of peptides mostly generated from exogenous antigens through the major histocompatibility complex (MHC) class II pathway. Identification of epitopes is important and computational prediction of epitopes is used widely to save time and resources. Although there are algorithms to predict binding affinity of peptides to MHC II molecules, no accurate methods exist to predict which ligands are generated as a result of natural antigen processing. We utilized a dataset of around 14,000 naturally processed ligands identified by mass spectrometry of peptides eluted from MHC class II expressing cells to investigate the existence of sequence signatures potentially related to the cleavage mechanisms that liberate the presented peptides from their source antigens. This analysis revealed preferred amino acids surrounding both N- and C-terminuses of ligands, indicating sequence-specific cleavage preferences. We used these cleavage motifs to develop a method for predicting naturally processed MHC II ligands, and validated that it had predictive power to identify ligands from independent studies. We further confirmed that prediction of ligands based on cleavage motifs could be combined with predictions of MHC binding, and that the combined prediction had superior performance. However, when attempting to predict CD4+ T cell epitopes, either alone or in combination with MHC binding predictions, predictions based on the cleavage motifs did not show predictive power. Given that peptides identified as epitopes based on CD4+ T cell reactivity typically do not have well-defined termini, it is possible that motifs are present but outside of the mapped epitope. Our attempts to take that into account computationally did not show any sign of an increased presence of cleavage motifs around well-characterized CD4+ T cell epitopes. While it is possible that our attempts to translate the cleavage motifs in MHC II ligand elution data into T cell epitope predictions were suboptimal, other possible explanations are that the cleavage signal is too diluted to be detected, or that elution data are enriched for ligands generated through an antigen processing and presentation pathway that is less frequently utilized for T cell epitopes.

CD4+ T细胞在调节免疫反应中发挥着重要作用。它们通过主要组织相容性复合物（MHC）II类途径识别主要由外源性抗原生成的肽段而被激活。表位的识别至关重要，而表位的计算预测被广泛用于节省时间和资源。尽管存在预测肽段与MHC II分子结合亲和力的算法，但尚无准确的方法可预测由天然抗原处理产生的配体。我们利用了一个由约14,000个通过从表达MHC II类的细胞中洗脱的肽段的质谱鉴定出的天然处理配体数据集，以研究可能与从其来源抗原释放所呈现肽段的切割机制相关的序列特征的存在。该分析揭示了围绕配体N端和C端的优先氨基酸，表明了序列特异性的切割偏好。我们利用这些切割模式开发了一种预测天然处理MHC II配体的方法，并验证了其具有从独立研究中识别配体的预测能力。我们进一步证实，基于切割模式的配体预测可以与MHC结合预测相结合，并且联合预测的性能更优。然而，当试图单独或与MHC结合预测相结合预测CD4+ T细胞表位时，基于切割模式的预测并未显示出预测能力。鉴于基于CD4+ T细胞反应性识别的肽段通常没有明确的末端，可能存在但位于映射表位之外的模式。我们尝试在计算中考虑这一点并未显示出在已表征的CD4+ T细胞表位周围切割模式的增加迹象。虽然我们尝试将MHC II配体洗脱数据中的切割模式转换为T细胞表位预测可能是次优的，但其他可能的解释是切割信号过于稀释而无法检测，或者洗脱数据富含通过较少用于T细胞表位的抗原处理和呈递途径产生的配体。