CD62-L down-regulation after L18-MDP stimulation as a complementary flow cytometry functional assay for the diagnosis of XIAP deficiency

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS
Agustín D. Rizzo, Marianela Sanz, Georgina Roffe, Elisa O. Sajaroff, Damian A. Prado, Emma Prieto, Verónica Goris, Jorge G. Rossi, Andrea R. Bernasconi
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Abstract

X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.

将 L18-MDP 刺激后的 CD62-L 下调作为诊断 XIAP 缺乏症的辅助流式细胞术功能测定。
X连锁细胞凋亡抑制因子(XIAP)缺乏症是由 XIAP 基因突变引起的一种不常见的先天性免疫错误。大多数病例表现为 XIAP 蛋白缺失,可通过流式细胞术(FC)检测,是一种快速诊断方法。然而,由于某些基因缺陷可能并不排除蛋白的表达,因此在这些患者的实验室检查中加入辅助功能检测非常重要。L-选择素(CD62-L)是一种在不同受体(如类收费受体(TLR)和核苷酸结合寡聚化结构域样受体(NLR),包括 NOD2)刺激下从白细胞表面膜上裂解的分子。考虑到 XIAP 缺乏会损害 NOD2 信号传导,我们决定用 NOD2 特异性激动剂 L18-氨酰双肽(L18-MDP)刺激中性粒细胞和单核细胞后,通过 FC 评估 CD62-L 的下调情况,从而开发出一种新型检测方法,用于对怀疑存在 XIAP 缺陷的患者进行功能评估。用 200 纳克/毫升的 L18-MDP 刺激 20 名健康对照组(HC)和 4 名经分子诊断确诊为 XIAP 缺乏症的患者的全血样本 2 小时,同时用 100 纳克/毫升的脂多糖(LPS)刺激,作为 CD62-L 脱落的阳性对照。使用抗 CD62-L- 抗体通过 FC 评估 CD62-L 的表达,并通过计算刺激前后 CD62-L 表达的差异(CD62-L 表达细胞的百分比(Δ%CD62-L)和荧光强度中值(ΔMFI%))评估下调情况。与 HC 相比,XIAP 缺陷患者的中性粒细胞和单核细胞对 L18-MDP 刺激的反应明显减弱(p
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来源期刊
ACS Applied Bio Materials
ACS Applied Bio Materials Chemistry-Chemistry (all)
CiteScore
9.40
自引率
2.10%
发文量
464
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