GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against <i>Cryptococcus</i> spp. And had difference in the strength of tonic signaling

<i>Cryptococcus</i> spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target <i>Cryptococcus</i> spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3ζ. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to <i>Cryptococcus</i> spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of <i>Cryptococcus</i> spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling. 2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface;2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands;18B7-GXMR-CAR provided highest levels of tonic signaling;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR; 2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface; 2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands; 18B7-GXMR-CAR provided highest levels of tonic signaling; Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR; Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR;

隐球菌属（Cryptococcus）菌株拥有由葡萄糖醛酸木糖甘露聚糖（glucuronoxylomannan, GXM）构成的多糖荚膜，GXM作为其主要毒力因子，可阻碍免疫细胞对真菌的识别。嵌合抗原受体（Chimeric Antigen Receptor, CAR）可重定向T细胞靶向隐球菌属菌株，此前已有靶向GXM的CAR——GXMR-CAR的相关研究报道。本研究评估了由单链可变片段（single-chain variable fragment, scFv）来源的特异性抗原结合结构域构成的GXMR-CAR所触发的信号转导强度。本研究分别以单克隆抗体2H1和18B7来源的GXM特异性scFv构建了2H1-GXMR-CAR与18B7-GXMR-CAR，二者均设计为以CD8分子作为铰链/跨膜结构域，并偶联CD3ζ链的共刺激分子CD137（4-1BB）。表达2H1-GXMR-CAR或18B7-GXMR-CAR的Jurkat细胞可识别来自加蒂隐球菌（C. gattii）和新生隐球菌（C. neoformans）的可溶性GXM；经隐球菌属菌株刺激后，两种GXMR-CAR载体修饰的细胞所释放的白细胞介素-2（IL-2）水平无显著差异。在修饰后的Jurkat细胞中，18B7-GXMR-CAR触发的基础信号（tonic signaling）更为显著；Src家族蛋白激酶抑制剂达沙替尼（dasatinib）可显著降低GXMR-CAR的基础信号，并抑制细胞对配体的活化反应。18B7 scFv的重链可变区（VH）存在结构修饰，这阐明了2H1-GXMR-CAR与18B7-GXMR-CAR之间基础信号强度及细胞活化水平的差异。GXMR-CAR载体可诱导T细胞针对隐球菌属临床分离株的活化；隐球菌病患者血清可诱导高水平的IL-2释放，尤以18B7-GXMR-CAR修饰的细胞最为显著。综上，2H1-GXMR-CAR与18B7-GXMR-CAR均可介导T细胞针对隐球菌属菌株的活化，且2H1与18B7 scFv会影响基础信号的强度：其一，2H1-GXMR-CAR与18B7-GXMR-CAR可在细胞表面高效表达；其二，二者均可重定向T细胞靶向相关配体；其三，18B7-GXMR-CAR可触发最强的基础信号；其四，18B7 scFv的结合口袋可促进GXMR-CAR所触发的基础信号。

搜集汇总

背景与挑战

背景概述

该数据集聚焦于GXMR-CAR，这是一种针对隐球菌属的嵌合抗原受体，使用两种不同的单链可变片段（scFv）——2H1和18B7——来介导T细胞激活。数据集包括实验数据文件，显示两种CAR变体均能有效识别隐球菌多糖并触发细胞反应，但18B7-GXMR-CAR产生更强的基础信号，这与其scFv的结构差异有关。