Howard Hughes Medical Institute, Department of Chemistry, and Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2011 May 31;108(22):9089-94. doi: 10.1073/pnas.1018771108. Epub 2011 May 16.
T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.
T 细胞对极少量的激活激动肽作出反应。T 细胞受体 (TCR) 和它们的肽-主要组织相容性复合物 (pMHC) 配体的空间组织成微簇与 T 细胞激活相关。在这里，我们设计了一种实验策略，能够控制每个 TCR 簇中的激动肽数量，而不改变细胞所涉及的总数量。支持膜，用障碍物划分成格子以限制横向流动性，为限制单个 TCR 簇中可能组装的 pMHC 配体的总数提供了一种有效方法。观察结果直接表明，限制单个 TCR 簇内的 pMHC 含量可以降低 T 细胞在固定的总 pMHC 密度下引发初始钙流的敏感性。进一步的分析表明，触发阈值由单个 TCR 簇中可用的激活配体数量决定，而不是由细胞遇到的总数量决定。在原代 T 细胞中独立改变总体激动剂密度和每个 TCR 簇中最大激动剂数量的一系列实验的结果表明，对于该系统，钙信号传导的最可能最小触发单位至少为单个簇中的四个 pMHC。包含共激动剂 pMHC 不会改变这个阈值，但 CD80 对 CD28 的共刺激可以将阈值调低。
