Background
Sarcoidosis is a multisystem disorder characterized by an accumulation of activated CD4
+ T cells in the affected organs [
1]. In the majority of cases the lungs are targeted by disease. Many of the patients suffer from respiratory symptoms such as dyspnea, dry cough and chest pain. Bronchoscopy with bronchoalveolar lavage (BAL) is commonly performed to examine patients exhibiting respiratory symptoms and clinical signs of sarcoidosis. BAL can also be used as a research tool because the aspirate includes cells most likely relevant for the pathophysiology [
2]. From studies of BAL cells we know that lung accumulated immune cells are activated in sarcoidosis patients [
3,
4], further, accumulations of CD4
+ cells expressing the T cell receptor (TCR) V gene segment Vα2.3
+ are accumulated in BAL fluid (BALF) of HLA DRB1*03
+ patients, often with Löfgren’s syndrome [
5].
Characterizations of TCR gene usage in lung and blood of patients can provide insights into disease associated processes [
6‐
9]. T cell recognition of a particular antigen presented by an HLA molecule can result in a clonal expansion of T cells bearing identical TCRs. The majority of T cells express the αβ TCR. The variable (V) region of the β-chain is generated by recombination through somatic rearrangement of one V region gene to a D and a J segment selected from a pool of discontinuous gene segments. By RNA splicing the VDJ segment is put together with one of the two C region genes. A similar principle applies to the α-chain, consisting of V and J segments. The TCR V region genes are not randomly used within CD4
+ and CD8
+ T cells. There are several V α and β genes that have a significant skewing to either CD4
+ or CD8
+ cells [
10,
11]. Most likely this is due to interactions of the V region gene products with MHC II molecules (for CD4
+ skewing), or MHC I (for CD8
+), during thymic maturation of the T cells. Hence, we analyzed these cell subsets separately. Previous studies have assessed the TCR α and β chain gene segments in sarcoidosis, mainly by PCR amplification [
5,
12‐
14].
The etiology of sarcoidosis is still not clear. Most evidence supports a genesis including a trigger by one or more still unknown antigens in the lungs and an aberrant immune response in genetically susceptible individuals. We hypothesise that antigenic triggering in the lungs of patients would give rise to T cell clones expressing certain Vβ-segments at higher frequency compared to the corresponding frequency in blood.
In the present study we used flow cytometric analysis of T cells stained with a large panel of 24 TCR Vβ specific antibodies, covering about 70% of the normal TCR Vβ repertoire in both CD4+ and CD8+ T cells. We mapped the Vβ repertoire in BALF cells and peripheral blood mononuclear cells from 15 patients with pulmonary sarcoidosis, six of them with Löfgren’s syndrome.
Discussion
Knowledge about TCR gene segment expression in sarcoidosis can provide insights to the pathogenic process and assist the finding of a potential disease triggering antigen. Previous studies aimed at mapping the TRC Vα and Vβ repertoire has often used PCR technique [
5,
12‐
14]. This technique permit analysis of all Vβ gene segments, however not simultaneously in different populations such as CD4
+ and CD8
+ cells. The PCR technique does not, in contrast to use of monoclonal antibodies and flow cytometry, measure the expression at the protein level. The latter method is the only method to analyze individual cells Vβ usage. Over time the number of available monoclonal antibodies specific for TCR Vβ family members has increased, now allowing approximately 70% of the TCR Vβ repertoire to be mapped. This led us to the use of monoclonal antibodies directed to TCR Vβ-chain proteins and flow cytometry for the present study. Our major finding was that expansions of T cells expressing Vβ5.1, Vβ7.1, Vβ18, Vβ21.3 and Vβ22 were found only in BAL from sarcoidosis patients. Further, Vβ12 and Vβ16 were the Vβ segments most frequently expressed as expansions in present study. Vβ16 TCR expansion has previously been described to appear in sarcoidosis patients [
12].
The patients in our study all had respiratory symptoms, leading to BAL as a part of the clinical examination. This enabled us to study TCR usage in both the lung and blood of the patients. Comparisons between these two compartments in sarcoidosis patients revealed that the expression in the blood, with a few exceptions, exceeded the expression in the lungs. Reference values were provided by the manufacturer of the antibodies. These are based on the Vβ-repertoire in blood lymphocytes from a cohort of healthy subjects, of Mediterranean heritage. In previous studies we have used the definition of a T cell expansion as three times the reference value, or any value above 15% of the gated population. However, since this definition may in fact underestimate T cell expansions we defined a T cell expansion as in a recent study on sporadic inclusion body myositis [
18], which defines an expansion as above the reference value plus two SD. Comparing TCR Vβ repertoires in lung and blood, similarities rather than generalized skewing have previously been described [
19]. Therefore we used the same reference values for BAL as PBMC. When the total repertoire in each patient was added together and compared to the reference values, we did however note a lower expression in the CD4
+ BAL cells than in the CD4
+ PBMCs (data not included). This may indicate a higher normal expression in BAL cells, of certain Vβ segments that is not targeted by the assay in this study.
A higher variability in TCR Vβ expression in CD8
+ cells than CD4
+ cells is consistent with previous findings in healthy controls and other diseases [
9]. Somewhat surprisingly, we found no significant difference in number of expansions between CD8
+ cells and CD4
+ cells or between sarcoidosis patients and controls in present study.
In three patients that were positive for the HLA II allele DRB1*04 we identified TCR Vβ 12 expansions in the CD8+ cell population. However, the CD8 molecule is not interacting with HLA II molecules, but with HLA I molecules. To our knowledge there is no association between HLA DRB1*04 and any HLA class I allele. Vβ12 was the segment most frequently found as T cell expansion in present study. However, our control blood donors displayed a significantly higher Vβ12 expression in CD8+ cells than the reference values, suggesting that this may not be a sarcoidosis specific expansion, and may be caused by local deviation from the reference cohort. The reason for this deviation may be the genetic difference between the reference cohort, which is of Mediterranean origin, and our Swedish controls, or due to yet unidentified reasons. The healthy controls in present study were not HLA typed.
Vβ22 expansions were found in CD4
+ T cells of three patients, all being HLA DRB1*03 positive. These expansions moreover, were lung restricted. HLA DRB1*03 and TCR Vβ22 expansion has previously been described in the context of inflammatory disease, albeit at a lower frequency. In a small study, three out of 9 patients, all three HLA DRB1*03 positive, with Idiopathic Inflammatory Myopathies [
6] four Vβ22 T cell expansions were identified. Two of these were located in CD3
+ cells in muscle, one in CD8
+ BAL cells and one in CD4
+ peripheral blood cells.
In a previous report Moller et al. described a biased usage of Vβ8 in sarcoidosis patients’ lung T cells, and to a lesser degree in blood [
20]. Consistent with our previous study [
13] and a study by Forman et al. [
12] in 1994, we noted Vβ8 T cell expansions in BAL in sarcoidosis patients in this study. In the present study we also noted Vβ8 T cell expansions in CD4
+ PBMC of two sarcoidosis patients.
Vβ5.1 and Vβ22 were the only two Vβ segments appearing exclusively as CD4
+ T cell expansion in BAL from more than one sarcoidosis patients. Both these segments have been shown to associate with other diseases, such as oral lichen planus and angioimmunoblastic T cell lymphoma [
21‐
23].
We identified CD8
+ Vβ16 T cell expansions in five sarcoidosis patients, of which three were located in the lung compartment. This TCR expansion has previously been described by Forman
et al. to appear in sarcoidosis patients [
12]. It was not investigated whether these expansions appeared in CD4
+ or CD8
+ cells. However, equally to Vβ12 in present study, our healthy controls had a higher expression of Vβ16 than the reference values.
It is known from our previous studies that T cell accumulations tend to withdraw after spontaneous resolution of clinical and radiographic signs of disease in HLA DRB1*03
+ sarcoidosis patients with Vα2.3 accumulations [
3]. Potentially some clonal expansions may already have resolved. The exact disease onset is not possible to determine, but the BAL was in all cases performed as a part of the diagnostic investigation.
Conclusions
The TCR repertoire is shaped by processes during T cell maturation and exposure to environmental antigens. One pathogen can consist of hundreds of proteins, which each can be broken down to many peptides that are presented in different ways depending on the MHC-molecules on APCs they are presented by. Therefore, even a single specific protein antigen has the potential to give rise to several different Vβ T cell expansions.
Further studies analyzing the T cell responses to candidate antigens and Vβ-repertoire are thus warranted. In the present study we also looked for associations between HLA DRB1 alleles and T cell expansions. However, due to the small sample size only cautious conclusions can be drawn. Further studies could strengthen our conclusion that Vβ8 and Vβ16 T cell expansions are associated to sarcoidosis and Vβ22 associated to the HLA DRB1*03 allele.
Our present results extend the knowledge from the previous studies. This may in the future improve predictions on disease progression and lead to a more individual approach for treatment.
Acknowledgements
The authors thank H. Blomqvist, M. Dahl, B. Dahlberg, and G. de Forest for their excellent technical assistance and Helena Forsslund for valuable input on statistics and linguistics of the manuscript.
This study was supported by The Swedish Heart Lung Foundation, The Swedish Research Council, The Mats Kleberg Foundation, The Stockholm County Council and Karolinska Institutet.
Competing interests
None of the authors has any conflict of interests.
Authors’ contributions
KMA & TR performed the experiments and analysed the results, AE, JW & JG designed the study, KMA wrote the paper. All authors read and approved the final manuscript.