Fig 1: PADI2-mediated citrullination and B cell tumor infiltration. (A) Representative IHC sections for PADI2, peptidyl-citrulline (citrulline), B cell markers CD19 and CD20, and the tumor marker PanCK in mammary gland and breast tumors stratified by hormone receptor subtype (original magnification ×200). (B) Immunoprecipitated IgG-bound citrullinome identified by mass spectrometry in plasma from subjects with breast cancer. Distribution of citrullinated proteins normalized against total unique peptides (see the Methods section) in 26 pooled plasma samples of newly diagnosed breast cancer consisting of 156 patients and 12 pooled plasma samples corresponding to 113 cancer-free subjects (see online supplemental table S4). Statistical significance was determined by two-sided Student’s t-test: <0.001. The AUC performance of the same cohort. (C) Autoantibody reactivity against citrullinated VIM in individual patient plasma from 11 stage II TNBC cases and 31 healthy controls. The 11 stage II TNBC patient plasmas were the same used for autoantibody reactivity against unmodified and citrullinated VIM by immunoblotting assay (online supplemental figure S6). Statistical significance was determined by two-sided Wilcoxon rank-sum test. (D) Classifier performance (AUC) of citrullinated VIM for distinguishing TNBC cases (n=11) from healthy controls (n=31). (E) Autoantibody reactivity against citrullinated and unmodified VIM in TNBC case (orange) and healthy control (blue) plasmas. Nodes and connecting lines represent matched samples. AUC, Area under the curve; Statistical significance was determined by two-sided paired t-test. IHC, immunohistochemistry; PADI, protein arginine deiminase; PanCK, pan-cytokeratin; TNBC, Triple Negative Breast Cancer; VIM, vimentin.
Fig 2: CD8+ T cells in ACPA+ RA blood proliferate in response to citrullinated antigens.a, b PBMCs (5 × 105) of ACPA+ RA patients were incubated with anti-CD3/28 antibodies (n = 14), NP (influenza A)/ pp65 (CMV) protein (50 μM of each, n = 14), native vimentin protein (100 μM, n = 5), or citrullinated vimentin (cit-vimentin) protein (100 μM, n = 14) with or without anti-CD8 and/or HLA class I-blocking antibody (anti-CD8 Ab n = 6, anti-HLA class I Ab n = 6 or anti-CD8/ HLA class I Abs n = 10), or no stimulation (n = 15) for 3 days. The percentage of Ki-67-expressing CD8+ (a) or CD4+ (b) T cells in ACPA+ RA PBMCs was measured by flow cytometry. For a, ***P < 0.0001, **P = 0.026, **P = 0.0021, #P = 0.0471, #P = 0.026, ##P = 0.0056 or ##P = 0.0021. For b, ***P < 0.0001, or *P = 0.0383. c, d Native vimentin or cit–vimentin pulsed monocyte-derived dendritic cells (MoDCs) were cocultured with Cell Proliferation Dye eF450-labeled CD3+ T cells isolated from ACPA+ RA PBMCs (n = 9) with or without anti-CD8/HLA class I-blocking antibody for 10 days, followed by flow cytometry analysis. Representative flow cytometry results (c, left) and quantification of the percentage of dyelow proliferating CD8+ T cells (c, right) or CD4+ T cells (d). For c, ***P < 0.0001, **P = 0.0054, **P = 0.0015, #P = 0.0494 or #P = 0.0251. For d, ***P < 0.0001, *P = 0.0171, or #P = 0.0484. e Proliferation capacity of ACPA+ RA CD8+ T cells based on proliferation dye eF450-labeled CD8+ T cells co-cultured with cit-vimentin or native vimentin-loaded MoDCs in the absence or presence of anti-CD8/HLA class I-blocking antibody for 10 days (n = 4). **P = 0.0011, **P = 0.0027, #P = 0.0486, or ###P = 0.0007. Representative histograms (left) and quantification of the proliferating CD8+ T cells (right). Bars represent means ± SEM. *P < 0.05, **P < 0.01, or ***P < 0.001 versus no treatment by unpaired t-test with two-tailed test; and #P < 0.05, ##P < 0.01, or ###P < 0.001 versus cit-vimentin treatment by unpaired t-test with two-tailed test. Source data are provided as a Source Data file. HC healthy control, ACPA anti-citrullinated protein antibodies, RA rheumatoid arthritis, Cit-Vim citrullinated vimentin.
Fig 3: ACPA+ RA blood CD8+ T cells upregulate activation and cytotoxic markers in response to citrullinated antigens.a Stimulation of fresh blood from ACPA+ RA (n = 8, red bars) or HCs (n = 7, blue bars) with anti-CD3/28 Abs, NP/pp65, cit- or native vimentin for 6 h with addition of Golgi inhibitor for the last 5 h. Quantification of CD69+ and GzmB+IFNγ+ CD8+ T cells, and IFNγ+ CD4+ T cells. For CD69+CD8+, *P = 0.049, +P = 0.0397, **P = 0.001, ##P = 0.009, or ###P = 0.0008. For GzmB+IFNγ+CD8+, +P = 0.0175, **P = 0.0071, ##P = 0.0071, or ##P = 0.0037. For IFNγ+CD4+, ++P = 0.0065, +P = 0.0489, *P = 0.0155, #P = 0.0351, or ##P = 0.0038. b PBMCs of ACPA+ RA patients (n = 10) were incubated with anti-CD3/28 Abs, NP/pp65 (50 μM of each), individual citrullinated proteins (100 μM), all citrullinated proteins (20 μM each), or all native proteins (20 μM each) for 16 h. Percentage of IFNγ or Granzyme B (GzmB) expressing CD8+ T cells measured by intracellular staining. c Quantification of IFNγ+ or GzmB+IFNγ+ expressing CD8+ T cells in ACPA+ RA PBMCs treated with cit- or native vimentin in a dose-dependent manner (1, 10 or 100 μM) (n = 7). d Quantitative analysis of IFNγ+ or GzmB+IFNγ+ expressing CD8+ T cells in ACPA+ RA PBMCs stimulated with cit-vimentin in the presence or absence of anti-CD8 and/or HLA class I-blocking antibody (n = 10). Data are presented as means ± SEM. For a, two-tailed unpaired t-test: *P < 0.05 or **P < 0.01 versus no treatment in ACPA+ RA; +P < 0.05, or ++P < 0.01 versus no treatment in HC; and #P < 0.05, ##P < 0.01, or ###P < 0.001 versus cit-vimentin in ACPA+ RA. For b, two-tailed unpaired t-test: *P < 0.05, **P < 0.01, or ***P < 0.001 versus no treatment; #P < 0.05, or ##P < 0.01 versus all native proteins. For c, two-tailed unpaired t-test: *P < 0.05, or **P < 0.01 versus no treatment; #P < 0.05 for cit-vimentin 1 μM versus vimentin 1 μM; $P < 0.05 for cit-vimentin 10 μM versus native vimentin 10 μM by two-tailed unpaired t-test; +P < 0.05 for cit-vimentin 100 μM versus native vimentin 100 μM. For d, by two-tailed unpaired t-test: *P < 0.05, or **P < 0.01 versus no treatment; #P < 0.05, or ##P < 0.01 versus cit-vimentin 100 μM. Source data are provided as a Source Data file. HC healthy control, ACPA anti-citrullinated protein antibodies, RA rheumatoid arthritis, Cit-Vim citrullinated vimentin.
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