QRICH1 Mediates an Intracellular Checkpoint for CD8+ T Cell Activation via the CARD11 Signalosome

Antigen receptor signaling pathways that control lymphocyte activation depend upon signaling hubs and negative regulatory proteins to fine-tune signaling output to ensure host defense and avoid potentially pathogenic hyperresponses. CARD11 is a critical signaling scaffold that translates T Cell Receptor triggering into the activation of branching NF-kB, JNK, mTOR and Akt pathways. Here we identify QRICH1 as a regulator of CARD11 signaling that mediates a previously undiscovered intracellular checkpoint for CD8+ T cell activation. QRICH1 inducibly associates with CARD11 following TCR engagement and negatively regulates CARD11 signaling to the IKK complex and NF-kB. QRICH1 binding to CARD11 occurs after TCR-induced CARD11 opening and is controlled by an autoregulatory intramolecular interaction between QRICH1 protein domains of previously uncharacterized function. Using mice deficient in QRICH1 in the T cell lineage, we show that QRICH1 controls the antigen-induced activation, proliferation, and effector status of CD8+ T cells by regulating numerous gene targets critical for CD8+ T cell function. Our results define a previously unappreciated component of antigen receptor signaling circuitry that fine-tunes effector output in response to antigen recognition. Overall design: To analyze differentially expressed genes between QRICH1-KO and control CD8+ T cells in response to antigen-specific stimulation, OT-1 CD8+ T cells were harvested from spleens of QRICH1-KO (QRICH1-fl/fl, OT-1/CD4-Cre) or control (QRICH1-fl/fl, OT-1/+) mice and stimulated under the following conditions: unstim; 1nM MHC Class I N4 Ova tetramer (4hrs); 1nM MHC Class I N4 Ova tetramer + 2µg/mL anti-mouse CD28 (4hrs); 1nM MHC Class I N4 Ova tetramer (24hrs) or 1nM MHC Class I N4 Ova tetramer + 2µg/mL anti-mouse CD28 (24hrs). Three replicates per stimulation condition per genotype were included.

调控淋巴细胞活化的抗原受体信号通路依赖于信号枢纽与负调控蛋白，以精细调节信号输出，保障宿主防御并避免潜在的病理性过度应答。CARD11是关键的信号支架蛋白，可将T细胞受体（T Cell Receptor）触发的信号转化为NF-κB、JNK、mTOR及Akt分支信号通路的活化。本研究鉴定QRICH1为CARD11信号的调控因子，其介导了此前未被发现的CD8阳性T细胞（CD8+ T cell）活化的细胞内检查点。在T细胞受体接合后，QRICH1可诱导性地与CARD11结合，并负向调控CARD11向IκB激酶（IKK）复合物及NF-κB的信号通路。QRICH1与CARD11的结合发生于T细胞受体诱导的CARD11构象开放之后，且受QRICH1蛋白结构域间此前功能未被阐明的自身调控分子内相互作用所控制。利用T细胞谱系中QRICH1缺陷的小鼠，我们证实QRICH1通过调控诸多对CD8阳性T细胞功能至关重要的基因靶点，控制抗原诱导的CD8阳性T细胞活化、增殖及效应状态。本研究结果定义了抗原受体信号通路网络中此前未被重视的组分，该组分可响应抗原识别精细调节效应分子的输出。总体实验设计：为分析抗原特异性刺激下QRICH1敲除（QRICH1-KO）与对照CD8阳性T细胞间的差异表达基因，我们从QRICH1敲除（QRICH1-fl/fl, OT-1/CD4-Cre）或对照（QRICH1-fl/fl, OT-1/+）小鼠的脾脏中分离OT-1 CD8阳性T细胞，并在以下条件下进行刺激：未刺激组；1nM MHC I类（MHC Class I）N4卵清蛋白四聚体刺激4小时；1nM MHC I类N4卵清蛋白四聚体+2 μg/mL抗小鼠CD28抗体刺激4小时；1nM MHC I类N4卵清蛋白四聚体刺激24小时；或1nM MHC I类N4卵清蛋白四聚体+2 μg/mL抗小鼠CD28抗体刺激24小时。每种基因型的每种刺激条件均设置3个生物学重复。