CD8+ bronchoalveolar lavage fluid and peripheral blood T cells, a normal V.2.3 usage was found in all cases, but un-resrtd T-celle s using other TCR V gene.
Proc. Natd. Acad. Sci. USA Vol. 91, pp. 4%5-4969, May 1994 Immunology
T-cell receptor variable region gene usage by CD4+ and CD8+ T cells in bronchoalveolar lavage fluid and peripheral blood of sarcoidosis patients JOHAN GRUNEWALDtI, OLLE OLERUP§, ULLA PERSSON§, MARY BERLIN OHRN1, HANS WIGZELLt, AND ANDERS EKLUND¶ tMicrobiology and Tumorbiology Center, Karolinska Institute; IDepartment of Clinical Immunology, Huddinge Hospital, Karolinska Institute; and rtment of Thoracic Medicine, Karolinska Hospital, Stockholm, Sweden
Communicated by Sune Bergstr6m, January 21, 1994
ABSTRACT Sarcoldosis is a chroniconaing graulomatous disease of unknown etiology. An accumulation of CD4+ T cells in the alveolar space of the lungs is a characteristic feature of the disease. We have in this study analyzed T-cell receptor (TCR) variable region (V) gene usage by CD4+ and CD8+ lung and peripheral blood T cells of 29 sarcoldosis patients and 15 control subjects. In the patient group, we found a 100% postive correlation between TCR Va2.3+ CD4+ lung T-cell exan and the exreion of the HLA-DR3(17),DQ2 haplotype. The remainin TCR Va/Vp gene products analyzed in this study-V.12, Vp2, Vp3, Vp5.1, VpS.2/5.3, VpS.39, Vp6.7, Vp8.1, and Vpl2-were in general normlly exprsd by CD4+ T cells, alogh some of them were used to a nfcnto y higher or lower degree by lung T cells compared to peripheral blood T cells. We also performed repeated TCR V gene analyses on some HLA-DR3+ patients and found an assciation between the ratio bronchoalveolar lavage fluid/periperal blood Va2.3+ CD4+ T cells and dinical signs of disease activity. Finally, when analyzing TCR V gene usage by CD8+ bronchoalveolar lavage fluid and peripheral blood T cells, a normal V.2.3 usage was found in all cases, but un-resrtd T-cell e s using other TCR V gene segment products were Identifed.
at the site of disease-i.e., in the lungs of sarcoidosis patients-and the HLA-DR3(17),DQ2 haplotype. A positive association of the ratio of TCR V.2.3+ lung/peripheral blood CD4+ T cells (PBLs) and clinical signs of disease activity is also shown.
MATERIALS AND METHODS Subjects. Paired samples ofPBLs and lung T cells recruited by BAL were obtained from 13 HLA-DR3Y (values show median age, with minimum and maximum in parentheses) [37 (24-48) years old; 4 women] and 16 HLA-DR3 [38 (27-67) years old; 6 women] Caucasian patients with untreated biopsy-proven sarcoidosis. Six HLA-DR3Y [33 (30-61) years old; 4 women] and 5 HLA-DR3- [37 (22-43) years old; 4 women] healthy volunteers served as controls. Twelve (5 DR3Y/7 DR3) patients were smokers, 14 (5/8) had never smoked and 3 (2/1) were ex-smokers (stopped >5 years). Three of the healthy controls (1 DR3Y and 2 DR3-) were smokers; the other 8 had never smoked. In addition, one individual with healed sarcoidosis, being without any signs of disease activity for >10 years (55-year-old female), and three individuals with acute extrinsic allergic alveolitis (J.G., M.B.O., J.W., H.W., R.L. and A.E., unpublished data) served as DR3+ controls. A minor part of the data from sarcoidosis patients and controls were included in a previous report regarding analyses of only CD4+ T cells (15). Signs of clinically active sarcoidosis and determination of clinical improvement was judged from the results of chest radiography, lung function tests, and the presence of symptoms. In the repeated TCR V gene analyses, all patients had signs of active disease on the first occasion. In all cases except the fourth analysis of patient 2 (see Fig. 4), >6 months passed between the TCR V gene analyses. Chest radiographic stages were as follows: Stage 0, normal radiograph; stage I, bilateral hilar lymphadenopathy (BHL); stage II, BHL with parenchymal infiltration; stage III, parenchymal infiltrates without BHL. All 11 healthy volunteers had normal chest x-rays. Lung function tests performed included VC (vital capacity), FVC (forced VC), FEV1 (forced expiratory volume in 1 sec), TLC (total lung capacity), and DLCO (diffusing capacity) and were calculated as percentage of predicted values. All subjects gave their informed consent and the study was approved by the local ethics committee. BAL Procedure and Ha g of Cels. Lavage and handling of cells were performed as described (15). Absolute numbers
Sarcoidosis is characterized by the formation of noncaseating granulomas, a depressed cell-mediated immunity, and an elevated B-lymphocyte immunoglobulin production. Ninety percent of sarcoidosis patients have involvement of the intrathoracic organs (1) in which CD4+ T cells are accumulated (2). Lung T cells are accessible for further analysis through the technique of bronchoalveolar lavage (BAL). Such lung compartmentalized CD4+ T cells are implicated in the pathogenesis of the disease, since they release interleukin 2 (IL-2), proliferate at a high rate (3), and express activation markers (4). T lymphocytes use a receptor (TCR) to specifically recognize antigen. The variable regions of the TCR are constructed through rearrangement of germ-line V (variable), D (diversity), and J (oining) gene segments (5). A particular TCR Va or Vp gene segment can play a dominant role in the reaction against certain peptide-major histocompatibility complexes (6-12). Also, in the recognition of so-called superantigens, T lymphocytes bearing particular TCR Vp gene segment products are of critical importance (reviewed in ref. 13). We have previously described a preliminary correlation between a restricted TCR V gene usage by lung T cells and the HLA haplotype of sarcoidosis patients (14, 15). In this extended study, we definitively establish an association between usage of TCR V,2.3 by CD4+ T cells accumulated
Abbreviations: TCR, T-cell receptor; BAL, bronchoalveolar lavage; IL-2R, interleukin 2 receptor; V, variable region; PBL, peripheral blood lymphocyte; mAb, monoclonal antibody. tTo whom reprint requests should be addressed at: Microbiology and Tumorbiology Center, Karolinska Institute, S-171 77 Stockholm, Sweden.
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of BAL fluid V.2.3+ CD4+ T cells were calculated by using total and differential counts of lung cells and immunofluorescence data showing proportions of CD3Y, CD4+, and V.2.3+ cells. Monoclonal Antibodies (mAbs). Anti-TCR Va2.3-, Vj93-, V195.3-, Vj96.7-, V,98.1-, and V,12V195.1-, mAbs V,95.2/5.3-, were provided by T-Cell Sciences (Cambridge, specific MA). mAb 6D6 (Va12.1) was a kind gift of H. DerSimonian and M. Brenner (16). The V,2-specific mAb was purchased from Immunotech (Luminy, France). Phycoerythrin (PE)-conjugated leu-2 (CD8), leu-3a (CD4), IL-2 receptor (IL-2R) (CD25), or HLA-DR mAb was obtained from Becton Dickinson. F(ab')2 fragments of rabbit anti-mouse immunoglobulin, used as secondary antibodies, were conjugated with either fluorescein isothiocyanate (FITC) or PE (Dakopatts, Glostrup, Denmark). Normal mouse serum (NMS), from BALB/c mice, was used for a negative control at a dilution of 1:500 in PBS. The OKT3 (CD3) hybridoma, used for positive controls, was acquired from American Type Culture Collection. Immunofluorescence and Flow Cytometry. Briefly, cells were incubated with unlabeled TCR V-specific mAb and washed twice; FITC-conjugated F(ab')2 fragments of rabbit anti-mouse immunoglobulin were added for detection of bound antibodies. NMS, diluted 1:500, was used to block rabbit anti-mouse immunoglobulin before adding re ini the PE-conjugated mAb. Cells were analyzed in a FACScan flow cytometer (Becton Dickinson) and a Hewlett-Packard 300 computer. Lymphocytes were gated out by forward and side scatter and dead cells were identified by staining with propidium iodide. NMS was used as a negative control (in all cases c0.5%). (See ref. 15 for additional details.) Sttscal Analyses. X-ray statistics were calculated by converting the chest radiographic stages into values as follows: 1, stage 0; 2, stage I; 3, stage II; 4, stage III. These values were compared in the two groups of patients. The nonparametric Mann-Whitney two-tailed U test was used for calculation of statistical significance (performed in groups of 17). All results are presented as median values; minimum and maximum values indicate the range. HLA Typing. HLA class I (A, B, and C) were determined by the microlymphocytotoxicity technique. HLA-DR and HLA-DQ alleles were in most cases determined by Taq I DRB-DQA-DQB restriction fragment length polymorphism analysis or PCR amplification with sequence-specific primers. Eight of the patients and one of the controls were DR typed by serology. RESULTS HILA Typing. Because of our previous finding that sarcoidosis patients expressing HLA-DR3(17),DQ2 had expansions of lung CD4+ T cells using TCR Va2.3 (15), we divided our sarcoidosis patients into two groups: those carrying DR3 (DR3+) (n = 13) and those who were DR3 negative (DR3) (n = 16). All 13 DR3+ sarcoidosis patients and 9/10 of the DR3+ controls were HLA class II typed genomically and found to carry the HLA-DR3(17),DQ2 haplotype (or with the genomic nomenclature DRB1*0301,DRB3*0101, DQA1*0501,DQBI*0201). General Characteristics. There were no differences between the two patient groups regarding signs of clinical symptoms or general characteristics of BAL cells. In both groups of patients, an accumulation in the lungs of CD4+ T lymphocytes expressing enhanced levels of HLA-DR, but not IL-2R, was found (Table 1). In contrast, chest radiographs differed significantly (P < 0.01) between the two groups of patients. For DR3+ patients, two were classified into stage 0, eight into stage I, and three into stage II, while for DR3- patients, one was classified into
Proc. Natl. Acad. Sci. USA 91
(1994)
Table 1. General characteristics of BAL T cells of DR3+ (n = 13) and DR3 (n = 16) sarcoidosis patients DR3- patients DR3+ patients Analysis 196 (30-449) 155 (60-349) TCC 18.4 (2.3-40.4) 25.4 (7.9-65.8) % lymphocytes 84 (38-97) 88 (50-99) % CD3 77 (49-89) 88 (58-96) % CD4/CD3 4.0 (2.4-6.5)t 3.6 (1.1-6.2)* % IL-2R/CD3 45 (20.4-64.0)t 49 (8.5-84)* % HLA-DR/CD3 Boldface numbers show median values. Numbers in parentheses show minimum and maximum values. TCC, total cell concentration (cells per A4 of BAL fluid). *n= 5. tn = 9.
stage 0, three into stage I, nine into stage II, and three into stage III. Moreover, lung function tests revealed a significant difference between the patient groups regarding diffusing capacity, as the DLCO median value was 74% (range 2495%) predicted in the DR3- patient group, compared to 92% (range, 64-112%) predicted in DR3Y patients (P < 0.05). TCR V Gene Usage In Sarcoidouis Patients. In Table 2, the median and range values for TCR V gene usage in CD4+ T cells of peripheral blood and BAL of DR3Y and DR3patients are shown. In the DR3Y patients, the TCR Vc2.3 gene segment was used significantly (P < 0.001) more by CD4+ BAL T cells (21.7%), compared to paired CD4+ PBLs (4.3%). In fact, every single HLA-DR3Y sarcoidosis patient had a more or less dramatic lung restricted Va2.3+ CD4+ T-cell expansion (Fig. 1A). When considering only DR3Y patients not included in a previous report (Fig. 1A, nos. 5-13) (15), the corresponding values were 22.5% and 4.3%, respectively, and the P value was
