背景：化脓性汗腺炎（Hidradenitis suppurativa，HS）是一种发生在皮肤皱褶部位、反复发作的慢性炎症性疾病，已知γ-分泌酶（γ-secretase complex，GSC）突变与家族性HS相关（FHS），突变亚基主要为呆蛋白（Nicastrin，NCT/NCSTN）。研究证实GSC作为一种跨膜多亚基酶复合物，参与Notch、CD44、钙粘蛋白等多种I型跨膜蛋白的切割，调控细胞增殖分化、炎症反应及免疫代谢。Notch是GSC水解的重要底物之一，NCSTN表达下调引起Notch通路受损造成毛囊口上皮细胞角化过度，部分阐释了GSC突变引起HS的机理。而HS发病过程中的核心环节是炎症反应，在HS患者皮损及血液中均有研究检测到CD4⁺ T细胞分泌细胞因子谱变化及CD4⁺ T细胞亚群失衡，然而GSC基因突变是否通过影响CD4⁺ T细胞功能参与HS发病的机制尚不清楚。

目的：

1. 筛查并验证FHS患者中GSC关键亚基的新发致病性突变位点；

2. 探究GSC突变后外周血单个核细胞在单细胞分辨率下的免疫图谱变化；

3. 探讨GSC突变对外周血CD4⁺ T细胞功能及其亚群的影响。

方法：

1. 从44例HS患者中采集FHS患者及其家属的血液样本进行全外显子组测序（whole exome sequencing，WES），筛选可能致病的GSC突变位点，使用Sanger及qPCR测序进行验证。通过PyMOL软件构建突变蛋白三维结构，预测其功能影响。

2. 采集已验证的FHS患者、散发HS患者及健康对照（Healthy Control，HC）的外周血，收集9例外周血单个核细胞（Peripheral blood mononuclear cell，PBMC）进行单细胞转录组测序，重点分析其中CD4⁺ T细胞变化。

3. 对PBMC进行磁珠分选（magnetic activated cell sorting，MACS）后得到CD4⁺ T细胞，体外采用CD3/28进行刺激后扩增培养。以DLL4激活Notch通路，干扰素-α（Interferon-α，IFN-α）/白介素-6（Interleukin-6）IL-6/IL-21激活STAT3通路，分别于D3、D0、D5进行流式细胞术检测CD3、CD4、CD8、IL-17A、IL-10、IL-4、IL-22、TNF-α、IFN-γ的表达情况。Bulk RNA测序检测DLL4刺激组、非刺激组FHS患者与HC的CD4⁺ T细胞的转录组变化，筛序差异表达基因并进行相关通路富集分析。

结果：

1. 发现3个未被报道过的新发致病突变，包括PSENEN c.134del、NCSTN c.C1935A和NCSTN EX2 Del，GSC基因突变均为杂合突变，符合遗传共分离定律。经分子结构模拟后发现GSC突变对其结构影响较大，部分功能性结构域缺失可能会影响其对Notch等一系列底物发挥剪切作用。

2. 单细胞测序中3组细胞分群类似，未发现特殊细胞群，突变组中CD4⁺ T细胞明显增多，中央记忆型CD4⁺ T细胞在2个患者组均明显较高，Notch激活的CD4⁺ T细胞在突变组较低，Treg、Th1细胞及耗竭型CD4⁺ T细胞则在3组细胞中占比均较少。2个患者组CD4⁺ T细胞中Notch2、RBPJ、CD44、IL-10RA、IL-10RB表达较HC组降低，突变组中JAK1、STAT3升高，此外还富集到了T细胞调控及分化等相关通路。

3. FHS患者非刺激组中IL-17A⁺CD4⁺ T细胞比例显著升高，此外TNF-α⁺CD4⁺ T细胞及IFN-γ⁺CD4⁺ T细胞亦有明显升高。培养5天后刺激组的IL-10⁺CD4⁺ T细胞比例较少，FHS及SHS患者外周血的CD4⁺ T细胞分泌IL-10抑炎因子不足。FHS患者组CD4⁺ T细胞与正常人相比，IL-4及IL-22未检测到明显升高。Bulk RNA测序显示FHS患者外周血CD4⁺ T细胞中RORC、IL-17A/F等Th17相关基因显著上调。富集分析提示JAK-STAT及细胞因子-受体相互作用等相关免疫通路参与HS发病。

结论：

1. 本研究报道3个GSC新发致病性突变，扩展了与HS相关的GSC基因突变谱。

2. 单细胞测序显示FHS患者外周血CD4⁺ Th细胞升高，Notch相关通路表达下调。

3. FHS患者外周血CD4⁺ T细胞依赖Notch/STAT3产生IL-10不足，无法限制IL-17A升高。Bulk RNA-seq发现其CD4⁺ T细胞中IL-17A/F、RORC表达升高，同时富集到免疫相关通路参与FHS发病。

Background: Hidradenitis suppurativa (HS) is a chronic, recurrent inflammatory skin disorder predominantly affecting intertriginous areas. Germline mutations in the γ-secretase complex (GSC) have been associated with familial HS (FHS), with nicastrin (NCT/NCSTN) being the most frequently implicated subunit. The GSC is a multi-subunit transmembrane protease complex responsible for cleaving various type I transmembrane proteins, including Notch, CD44, and cadherins, thereby regulating cellular proliferation, differentiation, inflammation, and immune metabolism. Among these substrates, Notch is a key target, and downregulation of NCSTN has been shown to impair Notch signaling, contributing to follicular hyperkeratosis—a known pathophysiological feature of HS. Increasing evidence also supports a central role for inflammation in HS pathogenesis. Altered cytokine profiles and imbalanced CD4⁺ T cell subsets have been observed in both HS skin lesions and peripheral blood. However, whether GSC mutations contribute to HS by disrupting CD4⁺ T cell function remains unclear.

Objectives:

1. To identify and validate new pathogenic mutations in key GSC subunits in FHS patients.

2. To characterize the immune landscape of peripheral blood mononuclear cells (PBMCs) at single-cell resolution in individuals harboring GSC mutations.

3. To assess the impact of GSC mutations on the function and subset distribution of peripheral CD4⁺ T cells.

Methods:

1. Blood samples were collected from 44 HS patients, and whole exome sequencing (WES) was performed in FHS patients and their family members to screen for pathogenic mutations in GSC genes. Putative variants were verified by Sanger sequencing and quantitative PCR (qPCR). PyMOL was used to construct 3D protein models to predict the structural consequences of mutations.

2. PBMCs were collected from three validated FHS patients, three sporadic HS (SHS) patients, and three healthy controls (HC) for 10× Genomics single-cell RNA sequencing (scRNA-seq). A particular focus was placed on the analysis of CD4⁺ T cells.

3. CD4⁺ T cells were isolated from PBMCs via magnetic-activated cell sorting (MACS), stimulated with anti-CD3/CD28 in vitro, and expanded. Notch signaling was activated using DLL4, while the STAT3 pathway was induced using interferon-α (IFN-α), interleukin-6 (IL-6), and IL-21. Cells were collected at days 0, 3, and 5 for flow cytometry analysis of CD3, CD4, CD8, IL-17A, IL-10, IL-4, IL-22, TNF-α, and IFN-γ expression. Bulk RNA-seq was performed on CD4⁺ T cells from FHS patients (stimulated vs unstimulated) and HCs, followed by differential gene expression and pathway enrichment analyses.

Results:

1. Three novel pathogenic heterozygous mutations were identified in GSC subunits: PSENEN c.134del, NCSTN c.C1935A, and NCSTN exon 2 deletion. All mutations conformed to Mendelian segregation. Structural modeling revealed substantial disruption in GSC architecture, potentially impairing substrate cleavage such as Notch signaling.

2. scRNA-seq revealed no unique cell clusters across the three groups. However, CD4⁺ T cell frequencies were significantly increased in the mutation group. Central memory CD4⁺ T cells were elevated in both HS and FHS groups, while Notch-active CD4⁺ T cells were reduced in the mutation group. Regulatory T cells (Tregs), Th1 cells, and exhausted CD4⁺ T cells remained scarce in all groups. Notably, Notch2, RBPJ, CD44, IL-10RA, and IL-10RB were downregulated in patient groups, while JAK1 and STAT3 were elevated in FHS samples. T cell regulatory and differentiation pathways were enriched.

3. In unstimulated FHS patients, IL-17A⁺ CD4⁺ T cells were significantly elevated, along with TNF-α⁺ and IFN-γ⁺ CD4⁺ T cells. After 5 days of stimulation, IL-10⁺ CD4⁺ T cells remained low in both FHS and SHS patients, indicating deficient anti-inflammatory responses. No significant elevation of IL-4 or IL-22 was observed in patient CD4⁺ T cells compared to HCs. Bulk RNA-seq confirmed upregulation of Th17-associated genes such as RORC and IL-17A/F. Enrichment analysis indicated that JAK-STAT and cytokine–receptor interaction pathways were involved in HS pathogenesis.

Conclusions:

1. This study identified three new GSC pathogenic mutations, expanding the mutational spectrum of HS-associated γ-secretase components.

2. Single-cell analysis revealed an increased proportion of CD4⁺ T helper cells and reduced expression of Notch pathway components in the peripheral blood of FHS patients.

3. CD4⁺ T cells from FHS patients displayed impaired IL-10 production via Notch/STAT3 signaling, leading to unchecked IL-17A elevation. Bulk RNA-seq further demonstrated upregulation of IL-17A/F and RORC and enrichment of immune-related pathways implicated in FHS.