Nanobody-based in vivo blockade of the P2X7 ion channel uncovers a hitherto unknown large subpopulation of parenchymal kidney TRM and NKT cells. Nanobody-based in vivo blockade of the P2X7 ion channel uncovers a hitherto unknown large subpopulation of parenchymal kidney TRM and NKT cells

The P2X7 ion channel, a key sensor of sterile inflammation, has been implicated as a therapeutic target in glomerulonephritis and P2X7-antagonistic nanobodies can attenuate experimental glomerulonephritis. However, little is known about the expression of P2X7 on renal immune cells. We used i.p. injection of nanobodies in mice followed by flow cytometry analysis of parenchymal T cells and RNA-sequencing to elucidate the expression and function of P2X7 on parenchymal and vascular immune cells in the mouse kidney. We show that parenchymal T cells, including a large subset NKT cells and tissue-resident memory T cells, display much higher cell surface levels of P2X7 than vascular T cells. These parenchymal T cells are highly sensitive to NAD+-induced cell death unless injection of P2X7-antagonistic nanobodies was performed before cell preparation. Remarkably, within 30 min after a single intraperitoneal injection of P2X7-blocking nanobodies, P2X7 is fully occupied by the injected nanobodies on parenchymal T cells in the kidney. This results in an effective protection of these cells from NAD+-induced cell death. Conversely, systemic injection of NAD+ that mimics sterile inflammation results in the selective depletion of P2X7hiCD69hi T cells from the kidney parenchyma. In summary, our study uncovers a novel purinergic regulatory mechanism affecting kidney resident T cell populations. Overall design: T cell subpopulations (vascular CD4+, parenchymal NKT CD1dtet+/CD69+, TRM CD1dtet-/CD4+/CD69+ and non TRM CD1dtet-/CD69-) were sorted by FACS and total RNA was extracted using the Qiagen RNeasy Micro Kit. RNA sequencing was performed by the core facility of the Leibniz Institute of Virology,

P2X7离子通道是无菌炎症(sterile inflammation)的关键感受器，已被认定为肾小球肾炎(glomerulonephritis)的治疗靶点，而P2X7拮抗纳米抗体(nanobodies)可减轻实验性肾小球肾炎。但目前对于P2X7在肾脏免疫细胞上的表达情况仍知之甚少。我们通过对小鼠腹腔注射（intraperitoneal, i.p.）纳米抗体，随后开展肾实质T细胞的流式细胞术(flow cytometry)分析与RNA测序(RNA-sequencing)，以阐明P2X7在小鼠肾脏实质与血管免疫细胞上的表达模式与功能。研究结果显示，包括大量NKT细胞亚群与组织驻留记忆T细胞(tissue-resident memory T cells, TRM)在内的肾实质T细胞，其细胞表面P2X7的表达水平显著高于血管T细胞。除非在细胞制备前注射P2X7拮抗纳米抗体，否则这类肾实质T细胞会对烟酰胺腺嘌呤二核苷酸(NAD+)诱导的细胞死亡表现出高度敏感性。值得注意的是，单次腹腔注射P2X7阻断纳米抗体后的30分钟内，肾脏实质T细胞表面的P2X7即可被注射的纳米抗体完全占据，从而有效保护这些细胞免受NAD+诱导的细胞死亡。相反，模拟无菌炎症的系统性烟酰胺腺嘌呤二核苷酸(NAD+)注射，会导致肾脏实质内P2X7高表达且CD69高表达的T细胞发生选择性耗竭。综上，本研究揭示了一种全新的嘌呤能调控机制，可影响肾脏驻留T细胞群体。整体实验设计：通过荧光激活细胞分选(Fluorescence-Activated Cell Sorting, FACS)分选出T细胞亚群（血管性CD4+ T细胞、肾实质NKT细胞CD1d四聚体+/CD69+、TRM CD1d四聚体-/CD4+/CD69+以及非驻留记忆T细胞CD1d四聚体-/CD69-），并使用Qiagen RNeasy微量试剂盒提取总RNA。RNA测序由莱布尼茨病毒学研究所核心实验室完成。