Fig 2: aTreg acquired CTL transcriptional signature under the high-inflammatory conditions of GO.(A) Pseudotime analysis of CD4 T cell subpopulations. (B) Dendrogram of CD4 CTL hdWGCNA and representative genes for each module. (C) UMAP plot showed the expression of different module genes in CD4 T cell subpopulations. (D) The expression of module 3 genes in CD4 T cell subpopulations. (E) The expression differences of module 3 genes in Tfh and aTreg between GH and GO groups. Data are represented as the median IQR. ***P < 0.0001 by Mann-Whitney U test. (F) Pseudotime analysis of aTreg, rTreg, and CD4 CTL. Left: Pie chart displayed the proportions of cells in different states over time for Healthy, GH, and GO groups. Right: Distribution of cells in different states along the branching trajectory over time. (G) The scores of CD4 CTL transcriptional feature gene set in aTreg from Healthy, GH, and GO groups; heatmap displayed the expression levels of representative CD4 CTL functional genes in aTreg from 3 groups. Data are represented as the median IQR. ****Padj < 0.00001 by Mann-Whitney U test. (H) Chromatin accessibility plots of KLRC1 and KLRC4 genes in aTreg from 3 groups. (I) Upset plot of aTreg and CD4 effector T cell clonotype sharing. On left, total number of expanded clones for each subpopulation is displayed. On top, the total number of shared clones is displayed. Incidences of clone sharing between aTreg and CD4 CTL are highlighted. (J and K) The expression of KLRC1 (J) and FGFBP2 (K) in peripheral blood CD4+CD25+FOXP3+ Treg from 3 groups; dot plots indicate the target cell proportions (Healthy, n = 9; GH, n = 9; GO, n = 9). Data are represented as the median IQR. *Padj < 0.05, **Padj < 0.001, ***Padj < 0.0001 by Mann-Whitney U test.

Fig 3: Pathogenic role of Treg cytotoxic transition to the orbital lesions of GO.(A) Schematic diagram of the EGFP labeled autoreactive Treg adoptive transfer experiment. (B) The proportion of EGFP+KLRC1+ cells within CD4+ cells in the peripheral blood of Healthy and GO mice after adoptive transfer experiment, representing the cytotoxic transition of Treg in vivo (Healthy, n = 3; GO, n = 9). Data are represented as the median IQR. *P < 0.05 by Mann-Whitney U test. (C) Immunofluorescence staining demonstrated the presence of KLRC1+EGFP+ cells in the orbital tissues of GO model mice. (D and E) The major subtypes of orbital tissues (D) and integrated CD4 T cells from PBMCs and tissues (E) based on scRNA-Seq data of Healthy donors and patients with GO; the upper right UMAP plot illustrates the batch effects. (F) The ligand-receptor interactions involving CD4 CTLs significantly altered in GO among 3 LPF subgroups, MYF, and COF. lipofibroblast, LPF; myofibroblast, MYF; conventional orbital fibroblast, COF. Red represents inflammation-regulating ligand-receptor interactions, while green indicates ligand-receptor interactions related to extracellular matrix remodeling. (G) Experimental model of the pathogenic effect of Treg cytotoxic transformation on localized lesions in the orbit. (H) Immunofluorescence showed the expression of specific proteins of OF cells in the coculture system of healthy control and GO model mouse groups; the histograms represent the quantitative statistics of these proteins. Data are represented as the median IQR. **P < 0.001 by Mann-Whitney U test. (I) qPCR histogram showed the expression of specific genes in cells of the coculture system. Each group consists of 3 independent samples (each n = 6). Data are represented as the median IQR. **P < 0.001 by Mann-Whitney U test. All experiments were repeated 3 times.

Fig 4: NK T cell subsets are altered with MGDep and ABX treatment in BrMet-burdened brains.(A) Dot plot of the relative gene expression level of predicted MHCI receptors across CD3+ T cell SCs. Relative gene expression level is depicted by color scale and percentage by dot size.(B) Feature plots showing cells in the CD3+ T cell subset with co-expression of CD8 (top) and Klrd1 (bottom) with Klrc1.(C–E) Percentages of the indicated (C) NK T cell, (D) CD8 T cell, and (E) NK cell populations in control and MGDep BrMet-burdened brains.(F) Biaxial plot depicting the CD3+ SC2: NK T cell subset with and without Klrc1 and Lamp1 gene expression (left) and stacked bar charts of NK T cell subsets at each time point and condition (right).(G) Violin plot of DEGS on subset NK T cell populations.Plots in (A), (B), (F), and (G) were derived from pooling three biological replicates per time point and condition. Each dot represents 1 mouse in (C)–(E), with data analyzed by two-tailed Student’s t test, error bars representing min and max, and center representing mean. Immune cells in (C)–(E) were enriched from brain tissue and measured by flow cytometry. Data in (G) were analyzed by Wilcoxon rank-sum test. See also Figure S7 and Tables S2 and S6.