The role of juxtaposition of activating and inhibitory receptors in signal inhibition of cytotoxic lymphocytes remains strongly debated. The challenge lies in the lack of tools that allow simultaneous spatial manipulation of signaling molecules. To circumvent this, we produced a nanoengineered multifunctional platform with molecular-scale spatial control of ligands, which was applied to elucidate KIR2DL1-mediated inhibition of NKG2D signaling—receptors of natural killer cells. This platform was conceived by bimetallic nanodot patterning with molecular-scale registry, followed by a ternary functionalization with distinct moieties. We found that a 40-nm gap between activating and inhibitory ligands provided optimal inhibitory conditions. Supported by theoretical modeling, we interpret these findings as a consequence of the size mismatch and conformational flexibility of ligands in their spatial interaction. This highly versatile approach provides an important insight into the spatial mechanism of inhibitory immune checkpoints, which is essential for the rational design of future immunotherapies.

激活和抑制受体并置在细胞毒性淋巴细胞信号抑制中的作用仍然存在激烈争论。挑战在于缺乏允许同时空间操纵信号分子的工具。为了避免这种情况，我们制造了一个具有分子尺度空间控制配体的纳米工程多功能平台，该平台用于阐明 KIR2DL1 介导的 NKG2D 信号传导抑制——自然杀伤细胞的受体。该平台是通过具有分子级注册的双金属纳米点图案构想的，然后是具有不同部分的三元功能化。我们发现激活配体和抑制配体之间的 40 nm 间隙提供了最佳的抑制条件。在理论模型的支持下，我们将这些发现解释为配体在空间相互作用中的大小不匹配和构象灵活性的结果。这种高度通用的方法提供了对抑制性免疫检查点空间机制的重要洞察，这对于未来免疫疗法的合理设计至关重要。