In Situ Humoral Immunity to Vimentin in HLA-DRB1*03+ Patients With Pulmonary Sarcoidosis

Abstract

Vimentin has been implicated in pulmonary sarcoidosis as a T-cell autoantigen, particularly in the context of HLA-DRB1*03, the Vα2.3/Vβ22 T-cell receptor (TCR), and Löfgren’s syndrome. As vimentin is a known antigenic target in B-cell-mediated autoimmunity, we investigated in situ humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with HLA-DRB1*03. Sarcoid and healthy control (HC) lung biopsies were analyzed by multi-color confocal microscopy for B-cells, T-cells, proliferation, and vimentin, and compared to tonsillectomy tissue. Bronchoalveolar lavage fluid (BALF) and serum from 48 sarcoidosis patients and 15 healthy volunteers were typed for HLA-DRB1*03 and titrated for antibodies to full-length vimentin, vimentin truncations, and total IgG and IgA by ELISA. Presence of extracellular vimentin in BALF was determined by mass spectrometry and T-cell populations measured by flow cytometry. Sarcoid lung samples, especially from HLA-DRB1*03+ patients, contained vimentin-rich tertiary lymphoid structures and corresponding BALF was highly enriched for both IgG and IgA anti-vimentin antibody (AVA) titers. Furthermore, sarcoidosis patient BALF AVA concentrations (expressed as arbitrary units per milligram of total immunoglobulin isotype) correlated with the percentage of CD4+ T-cells expressing the Vα2.3/Vβ22 TCR. BALF antibody reactivity to the vimentin N-terminus was most prominent in HCs, whereas reactivity to the C-terminus (VimC-term) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03+ patient BALF contained higher concentrations of anti-VimC-term antibodies than BALF from both HCs and HLA-DRB1*03− patients. Consistent with the lung as a site of AVA production, the concentration of AVAs in BALF was dramatically higher than in matched serum samples. Overall, there was a poor correlation between BALF and serum AVA concentrations. Together, these studies reveal the presence of linked in situ recognition of vimentin by both T- and B-cells in HLA-DRB1*03+ sarcoidosis patients, associated with a selective humoral immune response to the vimentin C-terminus.

Proinflammatory Histidyl–Transfer RNA Synthetase–Specific CD4+ T Cells in the Blood and Lungs of Patients With Idiopathic Inflammatory Myopathies

“…In our case, the sequence of the first 60 amino acids of HisRS (UniProtKB identifier P12081) was examined to predict peptides that can bind DRB1*03:01; residues 11-23 (HisRS 11-23 ) contained 2 such motifs and therefore had a high probability of containing a bona fide DRB1*03:01 epitope. Thus, HisRS [11][12][13][14][15][16][17][18][19][20][21][22][23] peptide was selected for further functional assays ( Figure 1A). Immunohistochemistry and histopathology.…”

“…To identify the potential presence of antigen-specific T cells against HisRS in the blood of patients with IIM/antisynthetase syndrome, we assessed the up-regulation of CD40L in CD4+ T cells following stimulation with HisRS protein or HisRS [11][12][13][14][15][16][17][18][19][20][21][22][23] After stimulation of PBMCs with HisRS 11-23 , we detected up-regulation of CD40L on CD4+ T cells in 11 of 14 patients with IIM/antisynthetase syndrome, with a median fold change of 3.4 (IQR 1.87-10.9) relative to unstimulated cells (P = 0.0012). The highest response toward HisRS 11-23 (>10-fold change versus unstimulated cells) was observed in 3 of 11 patients with IIM/ antisynthetase syndrome (2 anti-Jo-1+ and 1 anti-Jo-1−), with a median fold change of 12.75 (IQR 10.9-37) ( Figures 1B and C).…”

“…A, A 13-mer peptide within the first 60 amino acids of HisRS was predicted to represent a potential T cell epitope, HisRS [11][12][13][14][15][16][17][18][19][20][21][22][23] . B, Representative plots from fluorescence-activated cell sorter analysis (gated on lymphocytes and CD4+CD3+ T cells, with exclusion of dead cells and monocytes) show up-regulation of CD40L in CD4+ T cells as well as production of interferon-γ (IFNγ), interleukin-2 (IL-2), and IL-17 on peripheral blood mononuclear cells (PBMCs) from an anti-Jo-1+ patient with IIM/antisynthetase syndrome, in unstimulated (un-stim) conditions or after stimulation with full-length HisRS protein or HisRS [11][12][13][14][15][16][17][18][19][20][21][22][23] . C-E, Up-regulation of CD40L expression was examined on CD4+ T cells in PBMCs from anti-Jo-1+ patients with IIM/antisynthetase syndrome (n = 18) (patients P1-P18 in Table 1) (C), patients with sarcoidosis (n = 6) (D), and HLA-DRB1*03-positive healthy controls (HCs) (n = 12) (E).…”

“…C-E, Up-regulation of CD40L expression was examined on CD4+ T cells in PBMCs from anti-Jo-1+ patients with IIM/antisynthetase syndrome (n = 18) (patients P1-P18 in Table 1) (C), patients with sarcoidosis (n = 6) (D), and HLA-DRB1*03-positive healthy controls (HCs) (n = 12) (E). Solid symbols in C-E represent the median fold change in PBMCs from these groups, while gray-shaded symbols represent the median fold change in PBMCs from anti-Jo-1− patients (C), patients with sardoidosis who showed reactivity toward HisRS protein and HisRS [11][12][13][14][15][16][17][18][19][20][21][22][23] Figure 1D). The HisRS-reactive patients with sarcoidosis were positive for either HLA-DRB1*04/*15 or HLA-DRB1*01/*03 and were former smokers (see Supplementary Table 3 [http://onlin e libr ary.wiley.com/doi/10.1002/art.41075/ abstract]).…”

“…To confirm an HLA-DR dependency of the observed T cell responses, PBMCs from 2 anti-Jo-1+ patients were stimulated with HisRS [11][12][13][14][15][16][17][18][19][20][21][22][23] in the presence of anti-HLA-DR or anti-HLA-DQ blocking antibodies. Anti-HLA-DR blocking efficiently abrogated the up-regulation of CD40L upon stimulation with HisRS 11-23 , while anti-HLA-DQ blocking did not (Figures 2A and B), thereby indicating that the HisRS 11-23 epitope is presented by HLA-DR.…”