Cellular & Molecular Immunology www.nature.com/cmi ARTICLE OPEN A novel CD4+ CTL subtype characterized by chemotaxis and inflammation is involved in the pathogenesis of Graves’ orbitopathy Yue Wang1,2,3,4, Ziyi Chen 1, Tingjie Wang1,2, Hui Guo1, Yufeng Liu2,3,5, Ningxin Dang3, Shiqian Hu1, Liping Wu1, Chengsheng Zhang4,6,KaiYe2,3,7 and Bingyin Shi1 Graves’ orbitopathy (GO), the most severe manifestation of Graves’ hyperthyroidism (GH), is an autoimmune-mediated inflammatory disorder, and treatments often exhibit a low efficacy. CD4+ T cells have been reported to play vital roles in GO progression. To explore the pathogenic CD4+ T cell types that drive GO progression, we applied single-cell RNA sequencing (scRNA-Seq), T cell receptor sequencing (TCR-Seq), flow cytometry, immunofluorescence and mixed lymphocyte reaction (MLR) assays to evaluate CD4+ T cells from GO and GH patients. scRNA-Seq revealed the novel GO-specific cell type CD4+ cytotoxic T lymphocytes (CTLs), which are characterized by chemotactic and inflammatory features. The clonal expansion of this CD4+ CTL population, as demonstrated by TCR-Seq, along with their strong cytotoxic response to autoantigens, localization in orbital sites, and potential relationship with disease relapse provide strong evidence for the pathogenic roles of GZMB and IFN-γ-secreting CD4+ CTLs in GO. Therefore, cytotoxic pathways may become potential therapeutic targets for GO. 1234567890();,: Keywords: Graves’ orbitopathy; single-cell RNA sequencing; CD4+ cytotoxic T lymphocytes Cellular & Molecular Immunology (2021) 18:735–745; https://doi.org/10.1038/s41423-020-00615-2 INTRODUCTION stimulated by T cells expressing cytokines and chemokines, such Graves’ hyperthyroidism (GH) is an organ-specific autoimmune as interferon-γ (IFN-γ), tumor necrosis factor and C–X–C motif disease with a lifetime risk of 3% in women and 0.5% in men.1 ligands.14 Furthermore, upon exposure to their cognate antigen, Graves’ orbitopathy (GO), which is characterized by disfiguring CD4+ T cells undergo differentiation and can be classified as T and dysfunctional manifestations of eyelid retraction, proptosis or helper (Th)1 cells, Th2 cells, regulatory T cells, Th17 cells and newly optic neuropathy, occurs in 20–50% of GH patients.2 GO involves reported CD4+ cytotoxic T lymphocytes (CTLs) on the basis of autoimmune-mediated inflammation of the orbit characterized by functional properties.15,16 Previous studies have shown that the fibroblast activation, adipogenesis and enlargement of the balance between Th1 and Th2 cells is involved in the progression extraocular muscles. However, the precise pathogenesis of GO of GO, although some findings have contradicted these remains poorly understood.3 Approximately 20–30% of patients results.17,18 The pathogenicity of Th17 cells in the initiation and have no response to glucocorticoids, the mainstay treatment for progression of GO was also confirmed according to antigen- GO, and 10–20% relapse after treatment withdrawal.2 Based on presenting functions and proinflammatory cytokines.19,20 A recent current knowledge of GO pathogenesis, novel clinical trials for GO study indicated that the immune microenvironment in the various treatments, including mycophenolate4,5 and azathioprine6 orbit is composed of natural killer (NK) cells, IFN-γ-producing and targeting the overactivation and proliferation of lymphocytes RORγt+TBX21+ T cells and CD34+ orbital fibroblasts.21 and the IGF-IR inhibitors teprotumumab,7,8 tocilizumab9 and CD4+ CTLs were initially identified in chronic viral infections or selenium,10 have been conducted. Therefore, addressing the vaccinations against certain viral infections.22–24 They have also pathogenesis of GO has always been recognized as imperative for been reported to undergo expansion in the peripheral blood or the identification of new therapeutic targets. diseased tissue under inflammatory conditions, such as antitumor CD4+ T cells are the most abundant lymphocytes infiltrating immune responses or autoimmune disorders.25–29 Systemic GO orbital tissues and have been reported to initiate and sclerosis, an autoimmune fibrotic disease, was reported to involve perpetuate orbital inflammation in GO.11,12 Orbital fibroblasts, as CD4+ CTL-induced apoptotic death of endothelial and other the target cells in GO, are specifically activated by the T cell cells.27 Mass cytometry and flow cytometry analyses of rheuma- receptor (TCR) on antigen-specific CD4+ T cells13 and perpetually toid arthritis patients revealed an expanded population of 1Department of Endocrinology, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; 2MOE Key Lab for Intelligent Networks & Networks Security, School of Electronic and Information Engineering, Xi’an Jiaotong University, Xi’an, China; 3Genome Institute, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; 4Precision Medicine Center, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; 5BioBank, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China; 6The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA and 7The School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, China Correspondence: Kai Ye ([email protected]) or Bingyin Shi ([email protected]) These authors contributed equally: Yue Wang, Ziyi Chen and Tingjie Wang Received: 16 July 2020 Revised: 2 December 2020 Accepted: 3 December 2020 Published online: 29 January 2021 © The Author(s) 2021 A novel CD4+ CTL subtype characterized by chemotaxis andy Y Wang et al. 736 CD27-HLA-DR+ cells that expressed Th1- and cytotoxicity- were read on a Canto II instrument (BD Biosciences) and analyzed associated features and produced abundant IFN-γ and granzyme using FlowJo software version 10.0.7 (Tree Star). The following A proteins.28 Studies of IgG4-related disease have also shown that monoclonal antibodies were used: anti-CD4 (clone RPA-T4), anti- CD4+ effector/memory T cells with a cytotoxic phenotype are CCR6 (clone 11A9), anti-CCR4 (clone 1G1), anti-CXCR3 (clone 1C6), expanded in inflamed tissue sites and secrete the profibrotic anti-CD45RA (clone Hi100), anti-CCR7 (clone 150503), anti-ICOS cytokines IFN-γ, IL-1β and TGF-β1.30,31 Granzymes stored in CTLs (clone DX29), and anti-PD-1 (clone EH12.1) from BD Biosciences; directly influence inflammation by releasing proinflammatory anti-CD25 (clone BC96), anti-CXCR5 (clone MU5UBEE), anti-Foxp3 cytokines, including IL-6, tumor necrosis factor (TNF) α and IL-1β, (clone PCH101), and anti-CD45RO (clone UCHL1) from eBioscience; which are affected by GZMA, GZMB, GZMK and GZMM.32–34 and anti-granzyme A (clone CB9), anti-granzyme B (clone Granzymes can also degrade several extracellular matrix compo- QA16A02), anti-killer cell lectin-like G1 (KLRG1; clone SA231A2), nents, contributing to inflammation and mediating tissue anti-granulysin (clone DH2), anti-FGFBP2 (clone TDA3), and anti- remodeling.35,36 Thus, further characterization of CD4+ T cell Perforin 1 (clone B-D48) from BioLegend. subsets may better decipher the immune mechanisms of GO For cell sorting, PBMCs were prepared as described above. After progression. labeling with anti-CD3 and anti-CD4 antibodies (BD Biosciences, Given the importance of CD4+ T cells in the pathogenesis of San Jose, CA, USA) for 30 min at 4 °C in FACS buffer (2% FBS in GO, we undertook unbiased single-cell RNA sequencing (scRNA- Dulbecco’s PBS), viable CD3+ CD4+ T cells were sorted into PBS+ Seq) and identified a novel CD4+ CTL subtype with chemotactic 0.04% BSA by fluorescence-activated cell sorting (FACS; FACSAria, and inflammatory properties; the subset comprised GO-specific, BD) and retained on ice. Sorted cells were confirmed to be clonally expanded, granzyme-releasing cells and was present as a >90–95% pure before RNA extraction or scRNA-Seq. A MACS bead large fraction of the total CD4+ T cell population within orbital system (Miltenyi, Bergisch, Germany) was used to sort CD4+ tissues. Moreover, increased levels of cytotoxicity and chemotaxis KLRG1+ and naïve CD4+ T cells according to the manufacturer’s in CD4+ CTLs were probably correlated with GO relapse. Our guidelines. Sorted cells were confirmed to be >85–95% pure findings suggest that this novel proinflammatory CD4+ CTL before RNA extraction. subtype contributes to the pathogenesis of GO and is worth exploring as a new treatment target in the future. scRNA-Seq FACS-sorted CD4+ T cells were suspended in ice-cold PBS+ 0.04% BSA and then counted and assessed for viability with Trypan blue MATERIALS AND METHODS staining. Cells were resuspended at a concentration of 5 × 105–1× Study design 106 cells/ml with a final viability >80%. A single-cell library was This study was approved by the Ethics Committee of the First prepared following the protocol of the v2 Reagent Kit from 10× Affiliated Hospital of Xi’an Jiaotong University (XJTU1AF2016LSK- Genomics37 (10× Genomics, CA, USA) aiming for an estimated 35), and the clinical trial registration number is ChiCTR-IPR- 8000 cells per library. Briefly, cell suspensions were processed in 16009305. Informed consent was acquired from all patients before the chip (10× Genomics) along with reverse transcription (RT) they were screened or included in the study. No statistical master mix and single-cell 3’ gel beads. After RT, barcoded cDNA methods were used to predetermine the sample size. was purified, followed by PCR amplification. Then, the cDNA was To evaluate pathogenic CD4+ T cell subsets and explore the fragmented and double size selected