Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab′ antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab′-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab′-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab′-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

在生理条件下，肽主要组织相容性复合物 (pMHC) 分子可以触发作为单价配体的 T 细胞受体 (TCR)，这些配体稀疏地分布在抗原呈递细胞的质膜上。TCR 也可以通过人工聚类触发，例如 pMHC 四聚体或抗体；然而，这些策略绕过了 T 细胞的许多天然配体识别机制，并且可以引发非生理信号传导活动。我们最近推出了一种由抗 TCR β组成的合成 TCR 激动剂Fab' 抗体片段与 DNA 寡核苷酸共价结合，充当膜锚。这种 Fab'-DNA 配体在膜结合时有效地触发 TCR 作为单体，并表现出类似于激动剂 pMHC 的效力和激活曲线。在本报告中，我们探讨了 Fab'-DNA 配体有效触发 TCR 和细胞激活的几何要求。我们发现 T 细胞对 TCR 复合体上的配体结合表位不敏感，但 DNA 系链的长度很重要。增加，Fab'-DNA:TCR 复合物跨越的膜间距离降低了 TCR 触发效率和 T 细胞活化效力，这与 TCR 触发的动力学分离模型一致。