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 <i>HLA-DRB1*03</i>, 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 <i>in situ</i> humoral anti-vimentin responses in pulmonary sarcoidosis and their relationship with <i>HLA-DRB1*03</i>. 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 <i>HLA-DRB1*03</i> 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 (Vim_C-term) was enriched in the sarcoid lung. Specifically, HLA-DRB1*03⁺ patient BALF contained higher concentrations of anti-Vim_C-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 <i>in situ</i> 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.