Sarcoidosis

In collaboration with epidemiologists (E Arkema et al) we showed that in Sweden there are geographical differences in disease prevalence, and a substantial genetic influence to get the disease. Thus, there is a 3.7-fold increased risk among siblings, and the heritability is estimated to be 39%.

Patients are routinely investigated with bronchoscopy and lavage (BAL), to diagnose and evaluate the local inflammatory process in the lungs. Patients are sub-grouped into Löfgren´s syndrome (LS) respectively non-LS and genetically characterized. LS patients have an acute onset usually with fever, bilateral ankle arthritis and/or erythema nodosum, bilateral hilar lymph adenopathy and mostly a good prognosis.

It is generally believed that sarcoidosis is caused by exposure to specific antigens in genetically predisposed individuals. Our hypothesis is that such antigens are possible to identify using sophisticated methods that we are adopting. Patients with LS have lung accumulated T cells expressing specific T cell receptors (TCRs). Around 30 years ago, we and others reported that lung accumulated Th1 cells expressed a biased TCR repertoire, suggesting antigen recognition. Moreover, lung accumulated Va2.3+ T cells associated with HLA-DR3, with the clinical course, the prognosis, and diagnose of the disease. These T cells also expressed identical sequences of TCR Va2+ alpha and Vb22 chains within and between individual patients. The pronounced lung accumulated Th1 cells, the strong association with DR3, and the granuloma formation per se strongly favour immune reactivity against a particular antigen in the lungs of these patients. We hypothesize that such specific sarcoidosis-antigen(s) can be effectively eliminated by the TCR Va2.3+ T cells and thus explain why LS patients usually have a favourable outcome. We investigated the proportion of Va2.3pos BALF T cells and found a strong association with the diagnose. A proportion of CD4+ Vα2.3+ T-cells in BALF > 10.5% is highly specific for sarcoidosis, with a specificity of 97% and a sensitivity of 36%. We now will to extend these observations to include also T cells expressing simultaneously TCR Va2.3and Vb22 genes, to investigate if they in any way associate with the clinical features. We plan to stimulate blood cells with autologous and irradiated BAL cells, to investigate whether BAL cells have the capacity to trigger blood T-cells to proliferate. Both JAK-STAT and metabolic checkpoint kinase mTORC1 may influence granuloma formation and maintenance. We will evaluate whether addition or blocking of JAK-STAT or mTORC1 could influence the stimulation of lung-, blood- or T cell hybridomas, using Elispot as a readout. We also plan to see if blocking CD44 could influence the T cell stimulation.

Our search for sarcoidosis-specific antigens (candidate antigens) involves several different techniques; one has been to reveal peptides presented by HLA-molecules. Here, we identified a number of candidate antigen-peptides such as e.g. ATP synthase, lysyl-tRNA synthetase and vimentin (see below). We use our own modified technique to identify peptides presented by HLA molecules on BALF cells.

With collaborators in Denver (A Fontenot et al) hybridomas expressing TCRs of interest were generated and screened in peptide libraries consisting of synthetic deca-peptides to identify candidate antigens capable of stimulating the hybridomas. We recently identified an interesting peptide derived from an enzyme in Aspergillus Nidulans, ie. a fungus possibly involved in the inflammatory process. Thus, we generated T cell hybridomas expressing “public” TCR sequences that were previously found repeatedly on BALF T cells from different individual patients. We mixed these cells with a DR3+ fibroblast cell line as APC and added decapeptides in a high through-put fashion to identify peptides capable of stimulating such T cell hybridomas. The detection of such synthetic peptides ("mimotopes") may lead to the identification of naturally occurring peptides using viral, bacterial, fungal and human protein databases.

A Nidulans will, together with other candidate antigens, be scrutinized for any functional relevance.  Such “candidate antigens” will be identified when they are capable of stimulating T cell hybridomas that express previously identified repeated TCRs from different individual patients. We also apply techniques apart from the T-cell hybridomas assay; mass cytometry, microbead arrays, proteomics (Silva), techniques to select proteins using IgG, and genetics.

All candidate antigens including the A Nidulans peptide will be scrutinized for their expression in relevant tissues, and their functional capacity e.g. to stimulate T cells in proliferation- and cytokine- production assays, using FACS, Elispot and Fluorospot. For A Nidulans, we plan to investigate the entire fungus, to detect any other antigen-epitopes. If we could create a kit including several antigenic epitopes of the A Nidulans, that may become a valuable diagnostic test for all patients, similar to the M Tb kit (Quantipheron test). We are in the process of validating a tetramer (produced by NIH) that will stain T cells specific for DR3+/A Nidulans peptide and allow us to identify and follow these T cells geographically and repeatedly.

Mass cytometry data support two divergent immune reactions; non-LS patients more often had clusters of markers associated with prolonged inflammation. Based on our TCR sequencing data, a molecular 3D model of the trimolecular TCR Vα2.3/Vβ22-HLA-DR3 complex was generated in collaboration with Prof A Achour, KI. This model showed a perfect fit for a peptide derived from the C-terminal end of cytoskeletal protein vimentin into the peptide-binding cleft, implicating vimentin as a potential autoantigen in sarcoidosis. In addition, the same vimentin peptide was in separate experiments eluted from HLA-DR3 molecules of BAL cells, and stimulated T cells could produce IFNγ in HLA-DR3+ patients. Eberhardt and colleagues identified vimentin in “Kveim-Silzbach (KS) reagent”, an insoluble homogenate of sarcoidosis lymph node tissue previously used for diagnosing sarcoidosis. KS derived vimentin also promoted IFNγ production by T cells of HLA-DR3+ patients and was suggested to act as an autoantigen in SLE. We also revealed a co-localization of B- and T-cells and vimentin in lung tissues´ granulomas, suggesting B-cells to act as APC and triggering T-cells in situ.

We developed a microbead arrays system together with Prof P Nilsson (SciLife), and revealed the autoantigenic enzymes NCOA2, ZNF688, MRPL43 and ARFGAP1. Their capacity to induce proliferation (CFSE), or cytokine-production (Elispot) will be investigated. Our technique to elute and identify (through mass spectrometry, MS) peptides derived from HLA molecule has been improved and will disclose peptides of interest, as they are presented by HLA molecules at the place for the local inflammation. We have collected BAL cells from HLA-homozygous patients to avoid “contaminating” the peptide pool with peptides presented by other HLA molecules. This technique could be used for diagnostic or prognostic purposes, by expressing disease-associated “peptidomes”.

My research focus at KS/KI during the last four decades has been to explore inflammatory and immunological responses in the airways and lung tissue to a variety of stimuli originating from within or outside the body. This has been an interesting field of research as the ways of characterizing different pheno- and genotypes have greatly improved over the years as well as the techniques to get an impressive amount of information even from small retrieved samples from the lungs. At the same time handling of big data became possible. My group focus was initially on interstitial lung diseases (ILD) sometimes ending-up in non-reversible fibrotic lesions. Specific interest has for my part been in sarcoidosis and our epidemiological findings and the findings on the relationship between different geno- and phenotypes have been internationally very well received. Also, the research has given rise to potentially causing agents and new treatment options. As the research group grew larger in a constructive way subgroups with separate leaders developed focusing more specifically on COPD, IPF, sarcoidosis and lately Covid. Our colleagues at the Department of Medicine at KI, Huddinge have created another lung related profile, mainly on asthma.

My own contribution to the team is nowadays more of an advising character based on many years of research and several years in a central position in the administration of the Department of Medicine at KI, Solna.

Having a background in preclinical research and now working in the clinic, I have a great interest in, and am highly devoted to translational research. My clinical work is mainly at the Sarcoidosis Center at the Department of Respiratory Medicine, where about 1000 sarcoidosis patients are examined every year. We are a referral center for patients with severe sarcoidosis and accept patients from all Sweden. My qualifications to decide what important research from the patient´s perspective is therefore excellent. In my research, I focus mainly on immunological and genetic differences responsible for differences in disease course and response to given therapies. The patients are clinically phenotyped, and in cooperation with researchers in immunology and genetics also characterized immunologically and genetically. The aim is to better understand why some patients resolve spontaneously, and why some experience a progressive disease course despite treatment with immunosuppressive drugs. My long-term goal is to be able to tailor therapy for each individual patient based on immunological, genetic and phenotypic data.

Pernilla Darlington defended her thesis on “Studies of Different Clinical Manifestations of Sarcoidosis and the Role of Genetic Factors” in 2013. She works clinically as a physician and conducts studies on sarcoidosis. The research aims to study links between genetics and different clinical manifestations of the disease as well as their role for the prognosis. The project is mainly based on a sarcoidosis register that was started in the 1990s at the Lung Research laboratory, Solna. She also pays interest in the genetic influences of the different immunological reactions and mechanisms in the disorder. In this context various biomarkers are observed and related to the outcome.