Abstract：
BACKGROUND: The invariant TCR ζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its 3 immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects.
OBJECTIVE: We sought to analyze and model the potential impact on T-cell receptor (TCR) expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity.
METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with WT CD247 or mutations ablating 1 (Q142X), 2 (Q101X), or 3 (Q70X) ITAMs.
RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2 and ITAM-3, respectively. Both mutations were associated with low surface CD3 expression and normal intracellular CD247 levels using a transmembrane-specific antibody, but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO cells could not restore normal surface CD3 expression (only 60%, 22%, and 10%, respectively), whereas in WT cells, normal surface CD3 expression was reduced (to 39%, 19%, and 9% of normal levels), and both effects were dependent on ITAM number. All 6 transfectants showed reduced CD69 induction (25% to 50%), indicating that they were unable to signal downstream properly, neither isolated nor associated with WT CD247.
CONCLUSIONS: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.
OBJECTIVE: We sought to analyze and model the potential impact on T-cell receptor (TCR) expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity.
METHODS: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with WT CD247 or mutations ablating 1 (Q142X), 2 (Q101X), or 3 (Q70X) ITAMs.
RESULTS: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2 and ITAM-3, respectively. Both mutations were associated with low surface CD3 expression and normal intracellular CD247 levels using a transmembrane-specific antibody, but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO cells could not restore normal surface CD3 expression (only 60%, 22%, and 10%, respectively), whereas in WT cells, normal surface CD3 expression was reduced (to 39%, 19%, and 9% of normal levels), and both effects were dependent on ITAM number. All 6 transfectants showed reduced CD69 induction (25% to 50%), indicating that they were unable to signal downstream properly, neither isolated nor associated with WT CD247.
CONCLUSIONS: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.
摘要：
背景：恒定TCRζ/CD247同源二聚体对于TCR/CD3表达和通过其三个基于酪氨酸的免疫受体激活基序（ITAM）的信号传导至关重要。CD247中的纯合无效突变导致免疫缺陷，而载体表现出50%的表面CD3减少。目前尚不清楚其他CD247变体的携带者是否显示显性负面影响。
目的：分析和模拟在有免疫缺陷或自身免疫体征的患者中发现的杂合无义CD247突变对TCR表达和功能的潜在影响。
方法：JurkatT细胞，野生型（WT）或CRISPR/Cas9编辑的CD247缺陷型（ZKO），用野生型CD247或突变消除一个慢病毒转导（Q142X），两个（Q101X），或三个（Q70X）ITAM。
结果：研究了三名来自无关家庭的患者。鉴定出两个杂合无义CD247突变（p。Y152Xandp.Q101X),分别影响ITAM-3和ITAM-2+3。两种突变都与低表面CD3表达有关，使用跨膜特异性抗体的正常细胞内CD247水平，但使用ITAM-3特异性抗体的细胞内CD247水平非常低，提示T细胞中存在截短的变体。将缺乏1、2或3个ITAMs的突变导入ZKO无法恢复正常的表面CD3表达（仅60％，22%和10%，分别)，而在WT，他们将其降低(至39%，正常水平的19%和9%)，两种效应均与ITAM数量相关。所有六个转染子显示减少的CD69诱导（25-50％），表明它们无法正确地向下游发出信号，既不分离也不与野生型CD247相关。
结论：我们的结果表明，CD247变体由于无义而缺乏ITAMs，但不是null，突变对于正常的TCR组装是有缺陷的,并且在体外对TCR表达和信号传导发挥显性负效应。这个,反过来,可能与体内临床特征相关。
目的：分析和模拟在有免疫缺陷或自身免疫体征的患者中发现的杂合无义CD247突变对TCR表达和功能的潜在影响。
方法：JurkatT细胞，野生型（WT）或CRISPR/Cas9编辑的CD247缺陷型（ZKO），用野生型CD247或突变消除一个慢病毒转导（Q142X），两个（Q101X），或三个（Q70X）ITAM。
结果：研究了三名来自无关家庭的患者。鉴定出两个杂合无义CD247突变（p。Y152Xandp.Q101X),分别影响ITAM-3和ITAM-2+3。两种突变都与低表面CD3表达有关，使用跨膜特异性抗体的正常细胞内CD247水平，但使用ITAM-3特异性抗体的细胞内CD247水平非常低，提示T细胞中存在截短的变体。将缺乏1、2或3个ITAMs的突变导入ZKO无法恢复正常的表面CD3表达（仅60％，22%和10%，分别)，而在WT，他们将其降低(至39%，正常水平的19%和9%)，两种效应均与ITAM数量相关。所有六个转染子显示减少的CD69诱导（25-50％），表明它们无法正确地向下游发出信号，既不分离也不与野生型CD247相关。
结论：我们的结果表明，CD247变体由于无义而缺乏ITAMs，但不是null，突变对于正常的TCR组装是有缺陷的,并且在体外对TCR表达和信号传导发挥显性负效应。这个,反过来,可能与体内临床特征相关。
