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Restricted Usage of T Cell Receptor Vα/Jα Gene Segments with Different Nucleotide but Identical Amino Acid Sequences in HLA-DR3+ Sarcoidosis Patients
Molecular Medicine volume 1, pages287–296(1995)
Sarcoidosis is a granulomatous disease characterized by the accumulation of activated T cells in the lungs. We previously showed that sarcoidosis patients expressing the HLA haplotype DR3(17), DQ2 had increased numbers of lung CD4+ T cells using the T cell receptor (TCR) variable region (V) α 2.3 gene segment product. In the present study, the composition of both the TCR α- and β-chains of the expanded CD4+ lung T cells from four DR3(17), DQ2+ sarcoidosis patients was examined.
Materials and Methods
TCR α-chains were analyzed by cDNA cloning and nucleotide sequencing. TCR β-chains were analyzed for Vβ usage by flow cytometry using TCR V-specific monoclonal antibodies or by the polymerase chain reaction (PCR) using Vβ- and Cβ-specific primers. Jβ usage was analyzed by Southern blotting of PCR products and subsequent hybridization with radiolabeled Jβ-specific probes.
Evidence of biased Jα gene segment usage by the α-chains of Vα 2.3+ CD4+ lung T cells was found in four out of four patients. Both different α-chain nucleotide sequences coding for identical amino acid sequences and a number of identically repeated α-chain sequences were identified. In contrast, the TCR β-chains of FACS-sorted Vα 2.3+ CD4+ lung T cells were found, with one exception, to have a nonrestricted TCR Vβ usage.
The finding of Vα 2.3+ CD4+ lung T cells with identical TCR α-chain amino acid sequences but with different nucleotide sequences strongly suggests that different T cell clones have been selected to interact with a specific sarcoidosis associated antigen(s). The identification of T cells with restricted TCR usage, which may play an important role in the development of sarcoidosis, and the possibility of selectively manipulating these cells should have important implications for the treatment of the disease.
Sarcoidosis is characterized by the formation of non-caseating granulomas in affected organs, depressed cell-mediated immunity and elevated B lymphocyte activity. Ninety percent of patients with sarcoidosis have involvement of the intrathoracic organs (1). The CD4+ T cells, which accumulate in lungs and can be analyzed through bronchoalveolar lavage (BAL), show signs of being activated and are implicated in the pathogenesis of sarcoidosis (2,3). In order to specifically identify an antigen, T cells need to have the antigen presented as peptides in the context of the major histocompatibility complex (MHC) (in humans called HLA) molecules (4). Most T lymphocytes use αβ T cell receptor (TCR), consisting of α and β polypeptide chains for antigen recognition. Each chain is composed of a constant and a variable part, the latter made up of random combinations of V (variable), D (diversity; only the β-chain) and J (joining) gene segments (5). T cells with preferential use of particular TCR V genes have been described in several human diseases, such as multiple sclerosis (6), rheumatoid arthritis (7,8) and inflammatory vascular diseases (9,10). If T cells with restricted TCR V gene usage prove to be important for the development of the disease, a highly selective type of immune intervention may be undertaken (6). In animal models, it has been possible to selectively manipulate T cells with a restricted TCR V gene usage to succesfully prevent and modulate disease (11,12).
In sarcoidosis, several groups have shown a preferential TCR V gene usage by lung T cells and have suggested that there is a specific sarcoidosis-associated antigen in the lungs of these patients (13–21). We showed previously that CD4+ cells bearing the Vα 2.3 gene segment on the α-chain of the TCR specifically accumulate in the lungs of sarcoidosis patients of HLA type DR3(17), DQ2, suggesting that, in this subgroup of patients, T cells expressing this particular V gene element may have a role in the development of the disease (19,20). In the present study, a more extensive analysis of the TCR expressed by these CD4+ T cell was undertaken. In particular, the TCR α-chains and the Vα 2.3-associated Vβ- and Jβ-gene segments of the β chains have been analyzed using different approaches: cloning and nucleotide sequencing of the rest of the TCR α-chains; PCR amplifications using Vβ- and Cβ-specific primers to identify the Vα 2.3-associated Vβ gene segments; a recently developed PCR based technique using radiolabeled Jβ-specific probes to determine the usage of Vβ-associated Jβ gene segments (22); and flow cytometric triple staining techniques.
Materials and Methods
Paired samples of PBL and lung T cells recruited by BAL were obtained from 5 HLA-DR3(17), DQ2+ Caucasian patients (three males and two females; ages 24–42 years, median 37 years) with biopsy-proven sarcoidosis. Samples from Patient 3, with persistent clinical disease activity over a period of 1 year, were obtained by BAL on two separate occasions, when the patient received no medication and 30 mg prednisolone/every other day, respectively. BAL samples showed signs of lung restricted Vα 2.3+ CD4+ T cell expansions at both times. The other four patients received no medication. Three patients were never smokers and two exsmokers (quit >5 years ago). These sarcoidosis patients were included in a previous report (20), where more detailed clinical information, including bronchoalveolar lavage procedures, is given.
Anti-TCR Vα 2.3-, Vβ 3-, Vβ 5.1-, Vβ 5.2/5.3-, Vβ 5.3, Vβ 6.7, Vβ 8.1-, and Vβ 12-specific MAb were provided by T Cell Sciences, Inc. (Cambridge, MA, U.S.A.). MAb 6D6 (Vα 12.1) was a kind gift of Dr. H. DerSimonian and Dr. M. Brenner (23). The Vβ 2-, Vβ 13-, and Vβ 17-specific MAb were purchased from Immunotech (Luminy, France).
PE or PerCp-conjugated leu-3a (CD4) Mab were obtained from Becton-Dickinson (Mountain View, CA, U.S.A.). F(ab′)2 fragments of rabbit anti-mouse Ig, used as secondary antibodies, were conjugated with either FITC or PE (Dakopatts A/S, Glostrup, Denmark). Normal mouse serum (NMS) from BALB/c mice was used for negative control (in all cases ≤0.5%) at a dilution of 1:500 in PBS.
Immunofluorescence and Flow Cytometry (Fluorescence-Activated Cell Sorting)
Cells were first stained with the Vα 2.3-specific Mab (F1), detected with FITC-conjugated F(ab′)2 fragments of rabbit anti-mouse Ig, and, after a blocking step using NMS, PE-conjugated anti-CD4 Mab was added (described in detail in Ref. 19). To analyze TCR Vα 2.3-associated Vβ segments, cells from Patients 3 and 5 were incubated with the respective anti-TCR Vβ segment-specific MAb for 30 min, washed twice, and PE-conjugated F(ab′)2 fragments of rabbit anti-mouse Ig were added to detect bound antibodies. After three washes, NMS (diluted 1:500) was used to block remaining rabbit anti-mouse Ig before adding the FITC-conjugated F1 (anti-Vα 2.3), as well as PerCp-conjugated (anti-CD4) MAb. For positive separation of Vα 2.3+ CD4+ BAL T cells, a FACStar PLUS (Becton Dickinson), with standard optical equipment and a 90-µm nozzle diameter (24) was used, giving a sorting purity of >98%.
Sequencing of TCR α-Chains
Total RNA was extracted from 0.5 × 106 FACS sorted Vα 2.3+ T cells, using a RNA isolation kit (Stratagene, La Jolla, CA, U.S.A.), and cDNA synthesized using random hexamer primers, by the reverse transcriptase (RT) reaction according to the manufacturer’s recommendation (BRL, Gaithersburg, MD, U.S.A.). Amplification of cDNA was performed as described previously (25), using a Vα 2.3 sense primer (5′-AAGCATGCTCCTGGACCCTTCAATGTTCC-3′), including a Sph I cleavage site at the 5′ end (underlined), in combination with a Cα-antisense primer (5′-GGGTCGACTAGGCAGACAGACTTGTCACT-3′) with a Sal I cleavage site (underlined). The PCR profile used was the following: 94°C (60 sec), 55°C (60 sec) and 72°C (60 sec) for 38 cycles, with a final extension of 5 min. After PCR amplification, PCR products were cleaved with Sal I/Sph I and loaded on a low melting agarose gel. DNA bands corresponding to 500 bp were subsequently cut out from the gel, purified using Geneclean kit (Bio 101, La Jolla, CA, U.S.A.) and ligated into a Sal I/Sph I-cleaved pUC18 plasmid. Alternatively, PCR fragments amplified using primers without restriction enzyme cleavage sites were inserted blunt ended into a linearized pUC18 plasmid. The vector was inserted into competent Escherichia coli (JM 105) and grown on ampicillin/streptomycin plates. Positive colonies were picked using blue/white selection, and further PCR amplified and biotin labeled using pUC18 primers as described (26).
Amplified biotin-labeled clones were subjected to solid-phase sequencing as described (27,28). Briefly, the PCR mixture was immobilized on streptavidin coupled magnetic beads (Dynabeads; Dynal, Oslo, Norway). After washing and denaturation, single-stranded DNA was eluted and subsequently neutralized. Sequencing reactions were performed using AutoRed T7 sequencing kit (Pharmacia, Uppsala, Sweden) in a robot workstation (Biomek-1000; Beckman, Palo Alto, CA, U.S.A.). Samples were loaded on a sequencer (Pharmacia) with detection of fluorescent bands during electrophoresis. A total of 118 clones were sequenced (31 from Patient 1, 51 from Patient 2, 18 from Patient 3, and 18 from Patient 4).
PCR Amplification of Vα 2.3-Associated TCR β-Chains
Total RNA and cDNA was prepared as described above, and amplification of cDNA was performed using TCR Vβ- and Cβ-specific primers (for detailed information, see Ref. 25), and a PCR profile as above. The amplified products were separated on an ethidium bromide stained 2.0% agarose gel to show the expected size (270 bp), and subsequently blotted over night (Southern blot) onto Hybond-N nylon filters (Amersham, United Kingdom). After prehybridization of filters, hybridizations were performed over night using a 5′ end-labeled (using 5′-[γ-32P]-ATP; Amersham) Cβ-specific oligonucleotide probe (25). After washing the filters, they were exposed to Hyperfilm MP (Amersham) overnight at −70°C. Gel scanning technique (2400 GelScan XL; Pharmacia-LKB) was used for quantification of exposed films.
Analysis of Jβ Gene Segment Usage
Vβ 7-, Vβ 12-, Vβ 13.1-, and Vβ 18-amplified PCR products of Patient 2 were transferred to Hybond-N nylon filters, using a Bio-Dot SF micro-filtration unit (Bio-Rad, Hercules, CA, U.S.A.). Filters were then treated as above, and hybridizations were performed overnight using 5′-(γ-32P)-ATP end-labeled Jβ-specific oligonucleotide probes. Washing, film-exposure, and gel scanning (for quantification) were performed as described above and in detail in Ref. 22.
Flow Cytometric Analyses
TCR Vα 2.3 was used by 31.3, 12.8, 21.8 (mean value), 15.4, and 27.6% of CD4+ T cells obtained by bronchoalveolar lavage (BAL T cells) from Patients 1–5, respectively. The cells were analyzed at the time disease was discovered, except in Patient 3, in whom BAL T cells were obtained at different times for PCR analyses and for flourescence-activated cell sorting (FACS) analyses.
TCR Jα Usage
TCR Jα usage by Vα 2.3+ TCR α-chains, obtained from Vα 2.3+ CD4+ BAL T cells of Patients 1–4, was found to be restricted. In Patient 1, Jα family 17 gene segments were used by about 50% of the TCR α-chains. In Patients 3 and 4, a restricted usage of Jα family 1 gene segments was noted in particular, with frequencies of about 35 and 50%, respectively.
In addition, a preference for certain individual Jα gene segments by Vα 2.3+ TCR α-chains was noted (Table 1). In Patient 1, 26% of the TCR α-chains used the Jα 17.3 gene segment and, within this group, two distinct nucleotide sequences were found twice (Table 1). In comparison, the Jα 17.3 gene segment has a normal usage of 3% in PBLs (29). The reported normal usage in PBL for the Jα 9.11, 15.2, and 16.6 gene segments is 1.3, 1, and 4%, respectively (29). These Jα gene segments were preferentially used in Patient 2, in whom 9.11 was used by 18%, Jα 15.2 by 14%, and Jα 16.6 by 14% of the α-sequences (Table 1). Interestingly, identical Jα 9.11 nucleotide sequences were found in Patients 1 and 2. Moreover, several different nucleotide sequences were found to code for identical amino acid sequences (Table 2). In two different cloning experiments, 6/7 Jα 15.2 as well as 6/7 Jα 16.6 nucleotide sequences were found to be identical.
In Patient 3, the Jα 1.8 (17%) and Jα 9.7 (17%) gene segments were found to be preferentially used. The Jα 9.7 gene segment (normally used at a frequency of 3% in PBLs ), included 3/3 identically repeated nucleotide sequences (Table 1). Similarily, in Patient 4 a high number of Jα 1.8 gene segments (39%) was identified. Here, 5/7 Jα 1.8+ clones with different nucleotide sequences had identical amino acid sequences, while a sixth clone coded for an amino acid sequence with matching properties (Tables 1 and 3). Amino acid sequences with similar properties were also seen when comparing Jα 1.4+ clones from Patients 1 and 4, and Jα 15.3+ clones from Patient 2. In both cases, an arginine was found to be substituted with a lysine (both positively charged).
TCR Vβ and Jβ Usage
TCR Vβ usage by Vα 2.3+ CD4+ BAL T cells was investigated using two different approaches. First, for Patients 3 and 5 triple staining techniques and flow cytometry were used. All TCR Vβ genes investigated (which included Vβ 2, Vβ 3, Vβ 5.1, Vβ 5.2/5.3, Vβ 5.3, Vβ 6.7, Vβ 8.1, Vβ 12, Vβ 13, and Vβ 17) were found to be expressed by the Vα 2.3+ CD4+ BAL T cells. In these cells, TCR Vβ usage did not differ significantly from the respective total BAL CD4+ T cell populations, showing no preferential Vβ use (data not shown). In another approach, Vβ- and Cβ-specific primers and the PCR technique were used to amplify cDNA synthesized from RNA of Vα 2.3+ CD4+ BAL T cells of patients. For Patients 1, 3, and 4, a polyclonal pattern was observed, with detectable signals from all 22 investigated Vβ chains (Table 4). In contrast, Patient 2 showed a more restricted Vβ usage, with strong signals for Vβ 7, Vβ 12, Vβ 13.1, and Vβ 18 in particular. In addition, weak signals were detected for Vβ 3, Vβ 5.2, Vβ 6, Vβ 10, Vβ 14, Vβ 17, and Vβ 19. When comparing the Vβ signals obtained for all four patients, an overall preference for Vβ 7 and Vβ 18 was noted (Table 4).
The TCR Jβ segments associated with Vβ 7, Vβ 12, Vβ 13.1, and Vβ 18, from Patient 2 were analyzed using a recently developed technique (22). With this method, a semiquantification of the Jβ gene segment usage in relation to the respective Vβ expressing cell population was obtained. A highly restricted Jβ segment usage was noted in all four samples (Fig. 1). Vβ 7 had an unusual preference for Jβ 2.1, while Vβ 12, Vβ 13.1, and Vβ 18 all preferentially used Jβ 2.3.
The previously described absolute positive correlation between lung restricted Vα 2.3+ CD4+ T cell expansions and expression of HLA-DR3(17), DQ2 in sarcoidosis patients with clinical disease, prompted us to suggest the presence of a specific sarcoidosis-associated antigen in the lungs of these (HLA-DR3+) sarcoidosis patients (19,20). In order to understand more about these lung accumulated Vα 2.3+ CD4+ T cells, presumably reacting to a specific antigen, we have analyzed the composition of the rest of the TCR molecules expressed by these cells.
Use of Jα segments by the α-chains of Vα 2.3+ CD4+ lung T cells was strongly biased, with a preference for certain Jα segments. Notably, in Patients 3 and 4 there was even a preference for the same Jα (Jα 1.8) gene segment. At the clonal level, identical VJ α-sequences were repeatedly found in each (i.e., four out of four) patient, most pronounced in Patient 2 who also showed evidence of restricted TCR Vβ and Jβ usage (see below). In several cases identical VJ α-nucleotide sequences were identified in different cDNA cloning experiments, rendering it unlikely that the results could be due to technical errors. Moreover, the same VJ α-nucleotide sequence (Jα 9.11), repeated twice in Patient 2, was also identified in Patient 1. These findings suggest that some degree of oligoclonal expansion of Vα 2.3+ CD4+ T cells has occurred in the lungs of this group of sarcoidosis patients.
Another highly relevant discovery was the identification of different nucleotide sequences that code for the same amino acid sequence, such as the Vα 2.3-Jα 1.8 sequences in Patient 4. Also, different amino acid sequences retaining very similar properties were found in several cases, both intra- and inter-individually. We consider these findings to provide strong evidence that the Vα 2.3+ CD4+ lung T cells, originally derived from different T cell clones, have been selected for their ability to interact with a specific antigen.
TCR Vβ usage by Vα 2.3+ CD4+ BAL T cells of five patients was also analyzed. In four of these patients, nonrestricted Vβ usage was noted, although Patient 5 was investigated only using a restricted panel of anti-TCR Vβ-specific Mab. Evidence for a polyclonal Vβ usage by the Vα 2.3+ CD4+ BAL T cells is also provided by our previous results, in which the panel of anti-TCR Vβ Mab showed more or less normal reactivities in all cases with Vα 2.3+ expansions (20). Similarly, two recently published reports showed a diverse TCR Vβ repertoire in lung accumulated T cells of sarcoidosis patients (17,30).
An exception was found in Patient 2, in whom a preference for Vβ 7, Vβ 12, Vβ 13.1, and Vβ 18 was noted. These β-chains are most likely derived from oligoclonal T cell populations, since the related Jβ usage showed a highly restricted pattern, with a strong bias for Jβ 2.1 in association with Vβ 7, and for Jβ 2.3 in association with Vβ 12, 13.1, and 18. Bellocq et al. also found Vβ 7+ lung T cells to be oligoclonal to a high degree (17). Interestingly, Patient 2 also had the most conspicuous TCR α-chain oligoclonality, with repeated nucleotide sequences of the Jα 9.11, Jα 15.7, and Jα 16.6 segments in particular. Thus, certain combinations of Vβ-Jβ segments, associated with certain Vα 2.3-Jα joinings, seemed to be the TCRs favourably used by the expanded CD4+ BAL T cells in Patient 2. Clinically, we did not find any difference between this patient and the others.
The duration of disease might influence the TCR usage in antigen-responding T cells. A very restricted TCR V gene usage in the initial immune response may exist, but, with time, more heterogenous T cell populations may become attracted to the inflammatory focus. In experimental autoimmune encephalomyelitis (EAE), cryptic myelin basic protein (MBP) determinants became immunogenic (i.e., determinant spreading) with time, allowing for a more heterogenous T cell response (31,32). In sarcoidosis, the onset of disease in the majority of patients is insidious, making it difficult to evaluate any possible influence of disease duration. The genetic background of the individuals may also influence TCR V gene usage in health (33,34), as well as in disease (21). In addition to HLA-DR3+, other HLA alleles, as well as other genes such as the Tap-genes (35), may be important here. Finally, although expanded lung T cells used a particular Vα gene and were strictly CD4+, oligoclonal to some degree, and seemingly restricted by HLA-haplotype, the possibility of a superantigen-driven T cell response in sarcoidosis cannot be completely excluded by the present results. It has recently been suggested that superantigens with a low affinity to its specific TCR V gene segment product would preferentially stimulate CD4+ cells, since these cells could, unlike CD8+ cells, strengthen the avidity to the MHC class II bearing superantigen presenting cell. Moreover, a bias in the usage of other TCR gene products, which would improve the binding to such antigen presenting cell, could also be expected (36).
In conclusion, we have examined the detailed composition of the TCR α- and β-chains of expanded Vα 2.3+ CD4+ lung T cells of HLA-DR3+ patients with pulmonary sarcoidosis. Evidence for a restricted α-chain usage was found, with convergence at the nucleotide level towards the same or similar amino acid sequences. On the other hand, the Vβ usage by the same cells was, with one exception, nonrestricted. These results indicate that oligoclonal T cells expand as a result of antigen recognition in the lungs of this group of sarcoidosis patients, with the TCR α-chain being the most important (T cell) component in such a specific interaction.
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We thank Dr. Anders Johannisson for FACS sortings and Dr. Vida Hodara for help with Vβ-Jβ analyses. This work was supported by the Swedish Heart-Lung Foundation, the Swedish Cancer Society, and the Karolinska Institute.
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Grunewald, J., Hultman, T., Bucht, A. et al. Restricted Usage of T Cell Receptor Vα/Jα Gene Segments with Different Nucleotide but Identical Amino Acid Sequences in HLA-DR3+ Sarcoidosis Patients. Mol Med 1, 287–296 (1995) doi:10.1007/BF03401553