The blind spot : inflammatory pathways and specific antigens in clinical phenotypes of pulmonary sarcoidosis

Abstract

Sarcoidosis is a granulomatous multisystem disorder of unknown aetiology, primarily manifesting in the lungs. The disease appears in two forms, which are mainly distinguished based on clinical criteria; Löfgren's syndrome (LS) presents with acute onset, usually fever, active inflammation and characteristic symptoms such as bilateral hilar lymphadenopathy (BHL), erythema nodosum and/or ankle arthritis, but is also associated with spontaneous resolution and a good prognosis. In contrast, "non-LS" comprises a more heterogeneous patient group, with common characteristics being an insidious onset, slower disease progression and a higher risk of developing chronic disease, extrapulmonary manifestations and eventually pulmonary fibrosis. Despite advances in genetics, epidemiology, immunology and therapeutics during the past decades, the disease-triggering antigen(s) and the immunological mechanisms underlying different clinical phenotypes remain a "blind spot" - something that is known to exist but that the eye is, as yet, unable to perceive. Previous studies have shown the human leukocyte antigen (HLA)-DR allele HLA-DRB1*03 to associate closely with LS and a favourable clinical outcome. In these patients, local expansion of CD4+ T cells carrying the specific T cell receptor (TCR) variable (V) segment Vα2.3 in the lungs suggests specific antigen recognition, making these cells ideal tools in the quest for disease-triggering factors in sarcoidosis. The aims of this thesis were therefore to investigate on one hand the phenotype, functionality and ultimately antigen-specificity of TCR-specific CD4+ T cells, and on the other to improve understanding of immunological mechanisms underlying distinct clinical phenotypes of sarcoidosis.

In HLA-DRB1*03+ patients with sarcoidosis, Vα2.3 was found to preferentially pair with the Vβ22 TCR segment on CD4+ T cells in the lung. Expression of activation markers on these cells indicated an advanced state of differentiation, consistent with prolonged antigen exposure, and a high degree of clonality, marked by appearance of identical and near-identical TCR α and β CDR3 sequences between patients. In addition, molecular modelling of the TCR Vα2.3/Vβ22-HLA-DRB1*03 complex revealed an ideal fit into the peptide-binding cleft of a peptide derived from the carboxyl (C)-terminal of cytoskeletal protein vimentin, implicating vimentin as a potential (auto)antigen in sarcoidosis and suggesting especially LS to harbour traits of autoimmunity. Accordingly, anti-vimentin IgG and IgA antibodies were detected in the lungs of sarcoidosis patients to a higher degree than in healthy individuals, and correlated with expression of the Vα2.3/Vβ22 TCR. HLA-DRB1*03+ patients also demonstrated lower total Ig counts, but stronger reactivity towards the vimentin C-terminal, ultimately suggesting a more antigen-driven, but less aggressive, cooperative T-B cell response in HLADRB1* 03-mediated disease.

Particularly in LS patients, CD4+ T cells in the lung demonstrated simultaneous expression of T helper (TH) 1 transcriptional regulator T-bet and TH17 counterpart RORγT, which intriguingly correlated with non-chronic disease, and responded to stimuli with a broad array of cytokines, including interleukin (IL)-10, IL-2, IL-17A and IL-22, in addition to interferon (IFN)-γ, the main cytokine produced in non-LS patients. Moreover, expression of inhibitory receptors CTLA-4 and PD-1 indicated a higher degree of regulatory capacity compared to non-LS CD4+ T cells, where elevated expression of HLA-DR, CD127 and CD39 pointed towards a more pronounced effector profile. The discovery of marked CD8+ T cell proliferation in both patient groups provides further evidence for a hitherto unappreciated role of CD4+ T cells in restriction of cytotoxic T cell activity to prevent tissue damage in sarcoidosis, with loss of CTLA-4- and PD-1-mediated inhibition in non-LS patients exacerbating the risk of developing chronic disease and permanent scarring. Combined with reduced expression of adhesion marker CD44 in LS, this detailed characterisation outlines a possible mechanism for spontaneous resolution of granulomas.

The collected findings of this thesis suggest fundamental differences in CD4+ T cell biology that may be of key importance for disease resolution and progression, respectively, in clinical phenotypes of pulmonary sarcoidosis. Moreover, the results presented constitute an important step forward in the search for diseasespecific antigens, and provide incentive for further exploration of sarcoidosis, and LS in particular, as an autoimmune condition, at least partly driven by cognate T and B cell reactivity to vimentin. Hopefully, these discoveries may shed further light upon the still-elusive enigma of sarcoidosis, act as a foundation for continued investigation of disease-instigating factors, and ultimately contribute to reconsideration of current disease classification.