High-dimensional single-cell analyses have improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging because of technical and interindividual variation. Here, we present mixed-effects modeling of associations of single cells (MASC), a reverse single-cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounders and biological variation. Applying MASC to mass cytometry analyses of CD4⁺ T cells from the blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4⁺ T cells, identified as CD27⁻ HLA-DR⁺ effector memory cells, in RA patients (odds ratio, 1.7; P = 1.1 × 10⁻³). The frequency of CD27⁻ HLA-DR⁺ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27⁻ HLA-DR⁺ cell frequency decreased in RA patients who responded to immunosuppressive therapy. Mass cytometry and flow cytometry analyses indicated that CD27⁻ HLA-DR⁺ cells were associated with RA (meta-analysis P = 2.3 × 10⁻⁴). Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained about fivefold higher frequencies of CD27⁻ HLA-DR⁺ cells, which comprised ~10% of synovial CD4⁺ T cells. CD27⁻ HLA-DR⁺ cells expressed a distinctive effector memory transcriptomic program with T helper 1 (T_H1)– and cytotoxicity-associated features and produced abundant interferon-γ (IFN-γ) and granzyme A protein upon stimulation. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single-cell data.

高维单细胞分析提高了从人类疾病样本中分辨复杂细胞混合物的能力；然而，由于技术和个体间的差异，识别患者样本中与疾病相关的细胞类型或细胞状态仍然具有挑战性。在这里，我们提出了单细胞关联（MASC）的混合效应模型，这是一种反向单细胞关联策略，用于测试病例对照状态是否影响多个细胞亚群中单细胞的成员资格，同时考虑技术混杂因素和生物学因素。变化。应用 MASC 对来自类风湿性关节炎 (RA) 患者和对照者血液的CD4 ⁺ T 细胞进行大规模细胞计数分析，结果显示 CD4 ^{+ T 细胞群显着增加}RA患者中的T细胞，被鉴定为CD27 ^- HLA-DR ⁺效应记忆细胞（优势比，1.7；P = 1.1 × 10 ^-3）。在来自第二个 RA 患者队列的血液样本中，CD27 - HLA-DR + 细胞的频率同样升高，^而对免疫^{抑制治疗有反应的 RA 患者的 CD27 - HLA-DR +}细胞频率降低。质谱流式细胞术和流式细胞术分析表明 CD27 ^- HLA-DR ⁺细胞与 RA 相关（荟萃分析P = 2.3 × 10 ^-4）。与外周血相比，来自 RA 患者的滑液和滑膜组织样本含有大约 5 倍频率的 CD27 ^- HLA-DR ⁺细胞，其中包括约 10% 的滑膜 CD4 ⁺ T 细胞。CD27 ^- HLA-DR ⁺细胞表达具有 T 辅助 1 (T _H 1) 和细胞毒性相关特征的独特效应记忆转录组程序，并在刺激后产生丰富的干扰素-γ (IFN-γ) 和颗粒酶 A 蛋白。我们建议 MASC 是一种广泛适用的方法，可在高维单细胞数据中识别疾病相关细胞群。