CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum.
CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum.
复制标题
DOI：
10.1128/mcb.00383-18
复制
发表时间：
2018-11-01
影响因子：
5.3
通讯作者：
Thomas-Tikhonenko A
中科院分区：
文献类型：
--
作者：
Bagashev A;Sotillo E;Tang CH;Black KL;Perazzelli J;Seeholzer SH;Argon Y;Barrett DM;Grupp SA;Hu CC;Thomas-Tikhonenko A
我们先前描述了B细胞急性淋巴细胞白血病对CD19指导的嵌合抗原受体T细胞(CART)免疫治疗获得性耐药的机制。它是基于CD19外显子2的框内插入或跳过。我们先前描述了B细胞急性淋巴细胞白血病对CD19指导的嵌合抗原受体T细胞(CART)免疫治疗获得性耐药的机制。它是基于CD19外显子2的框内插入或跳过。为了区分表位缺失和表面定位缺陷，我们利用逆转录病毒转导和基因组编辑建立了带有水泡性口炎病毒G蛋白(VSVg)标签的表达CD19外显子2变异体(CD19ex2vs)的细胞系。经活细胞流式细胞术检测，抗VSVg抗体呈阴性，且对VSVg导向的抗体-药物结合物(ADCs)具有杀伤作用，提示细胞表面定位存在缺陷。实际上，脉冲追逐和α-甘露糖苷酶抑制剂分析表明，所有CD19ex2v都获得了内质网(ER)特异性的高甘露糖型糖，但不是高尔基体合成的复合型多糖。当CD19ex2v与绿色荧光蛋白(GFP)融合时，CD19ex2v(包括一个缺失相应二硫键的突变体)与ER标记显示共定位，表明蛋白质错误折叠。对CD19相互作用蛋白的质谱学分析表明，CD19ex2v不能与关键的Tetraspanin CD81结合，而是与内质网驻留的伴侣蛋白(如Calnexin)和参与抗原提呈的ER转运体相互作用。因此，即使是CD19ex2v的完整结构域也不能很容易地被ADC或当前的CD19购物车靶向，但可以作为主要组织相容性复合体(MHC)限制性递呈和T细胞受体(TCR)介导的杀伤的多肽来源。
We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.