Mixed-effects association of single cells identifies an expanded effector CD4⁺T cell subset in rheumatoid arthritis

Abstract

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 due to technical and inter-individual 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 (OR = 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 ~5-fold 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 Th1- and cytotoxicity-associated features, and produced abundant 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.

Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

“…This suggest these cells are not from the classical TFH cell population 18,[22][23][24][25] . We observed some differences between our study on AIH patients and previous studies on gluten-specific CD4 T cells 18 , autoreactive CD4 T cell described in infants with autoimmune diabetes 20 and expanded effector CD4 T cells in rheumatoid arthritis 47 . For instance, the PD-1 + CXCR5memory CD4 T cell population identified in our study expressed CD27 but did not express the chemokine receptor CCR6.…”

Multimodal memory T cell profiling identifies a reduction in a polyfunctional Th17 state associated with tuberculosis progression

“…Because T cell phenotypes can be characterized by both transcriptional and surface protein markers [27][28][29][30][31][32][33] , we profiled single cells with Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), a multimodal method that combines unbiased single-cell RNA-seq with surface marker profiles obtained via oligonucleotide-tagged antibodies 34,35 . We optimized a panel of 31 surface markers ( Supplementary Table 3), including markers of lineage (e.g., CD4, CD8), activation (e.g., CD25, HLA-DR), migration (e.g., CCR6, CXCR3), and other functions, and mouse immunoglobulin G (IgG) as a control.…”

Advances in genetics toward identifying pathogenic cell states of rheumatoid arthritis

“…One such approach called MASC (Mixed effects association of single cells) uses patient sample data rather than genetic association data to identify which cell types are expanded among cases vs the controls, while carefully accounting for inter‐individual variation . In one application, our group examined blood from RA cases and controls and applied mass cytometry.…”

“…One such approach called MASC (Mixed effects association of single cells) uses patient sample data rather than genetic association data to identify which cell types are expanded among cases vs the controls, while carefully accounting for inter-individual variation. 78 In one application, our group examined blood from RA cases and controls and applied mass cytometry. Then using MASC to explicitly model case-control status, we identified a novel cellular subset CD27 − HLA-DR+ effector memory CD4 T cells in RA with an odds ratio of 1.7 in blood compared to controls.…”