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Long-Lived Th2 Clones Specific for Seasonal and Perennial Allergens Can Be Detected in Blood and Skin by Their TCR-Hypervariable Regions¹

Barbara Bohle^*, Herwig Schwihla, Huai-Zhong Hu^§, Roswitha Friedl-Hajek, Slawomir Sowka, Fátima Ferreira^||, Heimo Breiteneder, Carla A. F. M. Bruijnzeel-Koomen^¶, Roel A. de Weger^§, Geert C. Mudde^#, Christof Ebner^2,* and Frank C. Van Reijsen^¶

Divisions of ^* Immunopathology and Applied Experimental Pathology, Department of General and Experimental Pathology, University of Vienna, and Center of Applied Genetics, University of Agricultural Sciences, Vienna, Austria; Departments of ^§ Pathology and ^¶ Dermatology/Allergology, University Hospital Utrecht, Utrecht, The Netherlands; ^|| Institute of Genetics and General Biology, University of Salzburg, Salzburg, Austria; and ^# Novartis Research Institute, Vienna, Austria

	Abstract

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We investigated the longevity of allergen-specific Th cells derived from patients suffering from either allergic rhinitis or atopic dermatitis. T cell clones (TCC) specific for seasonal and perennial allergens were raised. To determine whether these TCC were long-lived in vivo, PBMC and allergen-specific polyclonal T cell lines, collected and established inside a period of up to 4 years, were screened for the TCC of interest. For this purpose, a T cell tracing protocol was established in which oligonucleotides specific for the TCR ß-chain hypervariable junctional region were used as tools to identify each particular TCC. Seven pollen-specific TCC and two house dust mite-specific TCC, with a Th2-like cytokine production pattern in vitro, were demonstrated to be long-lived memory T cells in vivo. Specificity of the tracing protocol was ascertained by TCR sequence analysis. We conclude that allergen-specific TCC can persist for years, evidence for which can be monitored in blood, but also in the target organ of the allergic disorder. The data indicate that in vitro-characterized, allergen-specific, long-lived TCC may well reflect a repertoire of T lymphocytes of pathogenetic importance in vivo.

	Introduction

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In industrialized countries, about 20% of the population suffer from atopic allergy (1). Atopic individuals share elevated levels of serum IgE specific for environmental Ags. Inhaled proteins, e.g., from tree or grass pollen or house dust mite (Dermatophagoides pteronyssimus, Dpt),³ are a major cause of type I allergy. The aberrant, atopic response to these allergens may lead to clinical manifestations such as allergic rhinitis, allergic asthma, or atopic dermatitis (AD).

Production of IgE is promoted by IL-4 and inhibited by IFN- (2). Hence, the isolation of blood-derived, Dpt- and pollen-specific Th2-like clones, secreting high amounts of IL-4 but little or no IFN-, implies an important role for Th2 cells in the induction and maintenance of elevated serum IgE levels in atopic subjects (3, 4, 5). Moreover, the isolation of allergen-specific Th2 clones from skin of AD patients indicates that these clones can be physically involved in the development of allergic inflammatory reactions (6, 7, 8, 9).

T cells recognize antigenic peptides through their TCR (10). In 95 to 97% of circulating T cells, the TCR is a heterodimer comprising an - and a ß-chain (11). Both peptides are encoded by a variable (V), a joining (J), and a constant (C) gene segment, whereas only the ß-chain has a diversity (D) gene segment located between V and J (12). Additional diversity in both TCR chains is created by loss or template-independent addition of random nucleotides at the junctions of the V(D)J segments (13). The structure of the TCR is similar to that of Ig, containing hypervariable loops that correspond to the complementarity-determining regions (CDR) (14). In contrast to the CDR1 and CDR2 loops, which are encoded by germline V segment sequences, the CDR3 loops are encoded by the V(D)J junctional regions. The diversity of the TCR is focused in the V(D)J regions, which therefore play an important role in Ag recognition. Recently, crystallographic analysis of the complex between a human TCR, a viral peptide, and the MHC binding site confirmed the hypothesis that the CDR3 loops of both V and Vß contact the peptide (15). However, in previous studies we failed to find structural constraints on CDR3 regions of different allergen-specific TCC based on sequence analysis (16, 17), e.g., when comparing V(D)J junctional regions of different TCC specific for one T cell epitope, a striking heterogeneity in length and amino acid composition was found (17).

Previously, the existence of long-lived Dpt-specific T cells was demonstrated by analysis of blood-derived TCC from one patient suffering from perennial rhinitis (18). Here, we investigated the longevity of TCC specific for seasonal and perennial allergens, in blood and skin, respectively. For these purposes, we developed a T cell tracing system in which the hypervariable region of the TCR ß-chain was used as a fingerprint. Essentially, by means of an oligonucleotide complementary to the VDJ region, each TCC was traced in one or more autologous, polyclonal T cell populations. Using this system, TCC were detected up to 4 yr after isolation. The data demonstrate that long-lived TCC, specific for different environmental allergens, can be detected in both the blood and target organ of allergic individuals.

	Materials and Methods

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Patients

Six patients were included in this study. Peripheral blood was collected from five patients (three were birch pollen allergic and two were grass pollen allergic). Type I allergy was documented by typical case histories, positive skin prick test, and positive RAST (Radio-Allergo-Sorbent Test). One adult subject with AD diagnosed in accordance with the criteria of Hanifin and Rajka (19) was included in this study.

Allergens

Dpt was a kind gift from Haarlems Allergenen Laboratorium, Haarlem, The Netherlands. Recombinant birch pollen (Betula verrucosa) major allergen 1 (rBet v 1) and recombinant timothy grass pollen (Phleum pratense) major allergen 1 (rPhl p 1) were obtained from Biomay, Linz, Austria (20, 21).

Allergen-specific T cell lines (TCL) and PBMC

PBMC were isolated by Ficoll-Hypaque densitiy gradient centrifugation. Pollen-specific short-term TCL were established from PBMC as previously described (22). Isolation of Dpt-specific TCL from skin was described earlier (6, 8). Skin punch biopsies (3 mm) were obtained from clinically noninvolved skin, atopy patch test (APT) reaction sites, and lesional skin of one AD patient after injection with 1% lidocain as a local anesthetic. APT was performed as described elsewhere (23, 24). Briefly, clinically noninvolved skin from the back was stripped 10 times with adhesive tape, after which Dpt (80 µl, 10,000 allergen units/ml) was applied using Leucotest patches (Beiersdorf AG, Hamburg, Germany). APT was read after 12, 24, 36, and 48 h and was considered positive when erythema and papules were present.

Allergen-specific TCC

In total, 10 allergen-specific TCC were included in this study. Isolation, phenotype, and cytokine production of each TCC have been reported previously (22, 25, 26), and characteristics of the TCC are summarized in Table I.

Total RNA of TCC, TCL, and PBMC was isolated by the single-step guanidinium thiocyanate/phenol chloroform extraction method (27). Total RNA (0.5–5 µg) was used in first-strand cDNA synthesis, using an oligo d(T)₁₆ (Perkin-Elmer, Norwalk, CT) or a common TCR-Cß primer (3'- CACCCAAAAGGCCACACTGGTGT-5') and murine leukemia virus reverse transcriptase (Perkin-Elmer).

TCR ß-chain sequencing

First, the Vß gene usage of allergen-specific TCC was determined with 26 subfamily-specific primers and the same Cß primer used in cDNA synthesis, according to the method described by Hu et al. (28). Sequences of the Vß subfamily-specific primer were described by Hu et al. for Vß 1–20 (28) and Genevée et al. for Vß 21–24 (29). PCR reaction products of the pollen-specific TCC were sequenced as previously described (17). In the case of Dpt-specific TCC, sequencing of the PCR products was performed by Dye Deoxy Terminator cycle sequencing (Applied Biosystems Inc., Foster City, CA) with the Cß primer, followed by sequence analysis with a Biosystems 373A automated DNA sequencer (Applied Biosystems). All obtained sequences were then compared either with gene databank entries (GenBank, Los Almos, NM; EMBL, Heidelberg, Germany) or with available published TCR V and J segments (28, 29, 30, 31, 32, 33, 34, 35, 36). The CDR3 region was taken as the number of amino acids between the last germline-encoded residue of the V segment and the conserved phenylalanine residue of the J segment.

Tracing of T cell clones in polyclonal T cell lines and PBMC

TCC were traced in TCL or PBMC using a three-step analysis, based on identification of their TCR ß-chain hypervariable region. First, cDNA from TCL or PBMC was subjected to PCR with the 5'Vß family primer, corresponding to the TCC to be traced, and the common 3'Cß primer. Reaction conditions were as follows: one cycle of 94°C for 3 min, 93°C for 45 s, 55°C for 50 s, and 72°C for 50 s, followed by 34 cycles of 93°C for 45 s, 55°C for 50 s, and 72°C for 50 s, with a final extension period of 7 min. Second, the purified PCR products were subjected to a seminested re-PCR with the Vß primer and the respective Jß primer. This seminested re-PCR was again run for 35 cycles. Third, tracing of pollen-specific TCC in PBMC and short-term blood-derived TCL was performed by seminested re-PCR using the Vß family primer and a primer corresponding to the VDJ region (Table II) of each particular TCC. To ensure specificity, the annealing temperature for each VDJ primer was optimized and the following negative controls were performed. First, we performed PCR with cDNA obtained from TCC sharing the TCR Vß family with the TCC of interest but having different VDJ regions. Second, every single PCR step was controlled in parallel by samples to which no cDNA was added. Third, we evaluated whether the TCC under investigation were detectable in cDNA from PBMC and TCL of different patients. Finally, we tested whether PCR products were obtained when a relevant Vß primer (i.e., Vß2 for TCC WD22 and WD25) was combined with VDJ primers specific for other TCC. Tracing of Dpt-specific TCC in skin-derived TCL was performed by probe hybridization with [-³²P]ATP-labeled VDJ region-specific oligonucleotides (Table II) overnight at 55°C.

To prove that the PCR products were identical to the TCR ß-chain VDJ region of the allergen-specific TCC under investigation, the experimental procedure shown in Figure 1 was used. The Bet v 1-specific TCC, WD25, was chosen because of its long hypervariable sequence (Table II). Vß-Jß PCR products were purified and cloned into the TA cloning vector pCRII (Invitrogen Corp., San Diego, CA). Plasmid DNA of isolated Escherichia coli clones was screened for WD25 by PCR using the 5'Vß primer and the VDJ-specific primer under optimized conditions: one cycle of 94°C for 3 min, 62°C for 50 s, and 72°C for 50 s, followed by 34 cycles of 94°C for 45 s, 62°C for 50 s, and 72°C for 50 s, with a final extension time of 7 min. Inserts amplified with these primers were sequenced.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Experimental procedure for sequencing the TCC-derived PCR product. First-strand cDNA synthesis was performed using a common TCR-Cß primer followed by amplification with the respective Vß primer. Using seminested re-PCR, a mixture of TCR ß-chain-derived products sharing the same Vß and Jß families was generated. These Vß-Jß products were separated by ligation and transformation into E. coli. Inserts that were amplified by screening the colonies with PCR using the TCC-specific Vß-VDJ primer combination were subsequently sequenced.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Two slightly different approaches were followed for the tracing of TCC in PBMC and blood-derived short-term TCL vs skin-derived TCL. In both methods, we used oligonucleotides specific for the hypervariable VDJ region of each TCC as a fingerprint (Table II). For detection of allergen-specific TCC in PBMC, the best results were obtained when the TCC-specific oligonucleotide was used as a primer in a second seminested re-PCR of Vß-Jß re-PCR products. Five Bet v 1-specific TCC and 2 Phl p 1-specific TCC, isolated from peripheral blood between 1991 and 1994, were demonstrated to be still present in freshly isolated PBMC as well as in allergen-stimulated TCL that had been collected and established in 1995 or 1996 (Fig. 2). No relevant PCR products could be amplified when TCC-specific primer sets were used in the PBMC and TCL of nonmatching patients. In addition, in individual WD, combining the Vß2 primer with VDJ-specific primers for TCC other than WD22 and WD25 did not result in detectable PCR products (second PCR step, Fig. 1).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Pollen-specific TCC can be found in the peripheral blood of allergic patients several years after their first isolation. Each TCC was traced by a three-step analysis. The TCC-specific PCR products amplified in the second seminested re-PCR using primers specific for the TCR Vß familiy and the VDJ region of each clone are shown. Seven TCC isolated between 1991 and 1994 were detected in PBMC (P) and in allergen-stimulated TCL (T) collected from the patients in 1995 and 1996. +, Indicates positive control, Vß-VDJ-specific message amplified from the TCC from which the VDJ primer sequence originated; -, negative control, no amplification in a TCC sharing the TCR Vß family but differing in the hypervariable region.

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Dpt-specific TCC 4:3.1 is present in different skin-derived TCL of the AD patient. Although all TCL express the Vß6-Jß2.4 combination as visualized in an agarose gel (a), only TCL 4:2*90, 4:3*90, 4:5*90, 4:22*93 and 4:01*93 contain TCC 4:3.1 as determined by probe hybridization with the TCC-specific VDJ probe (b).

To control the specificity of our T cell tracing protocol, we followed the experimental procedure shown in Figure 1. Using seminested re-PCR, a mixture of peripheral TCR Vß-Jß products was obtained and transformed into E. coli to isolate monoclonal products after ligation. Plasmid DNA was prepared of E. coli clones and screened for TCC WD25 (Table I) using a primer corresponding to the VDJ region (Table II). The inserts of positive E. coli clones were sequenced and found to display identity with the original sequence.

In addition, TCC NO 29 II was isolated again by limiting dilution of a blood sample collected 1 yr after the first isolation (TCC NO 5 III). Again, the clonal relation of these TCC was proven by identity of their VDJ region nucleotide sequences (Table II).

	Discussion

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In vitro characterization of allergens concerning allergenicity, antigenicity, epitope composition, etc. requires the use of allergen-specific TCC, which can be isolated from peripheral blood or from tissues that are infiltrated by lymphocytes during an inflammatory process (22, 25, 37, 38, 39, 40). The properties of TCC concerning membrane marker expression, cytokine production, and TCR gene usage can be characterized by routine analysis. In vitro propagation of TCC is based on the selection of single cells and their expansion to suitable cell cultures by the use of mitogenic stimuli and growth factors (recombinant cytokines). It has always been an issue of debate whether these culture conditions allow the isolation of TCC that are representative for the T cells involved in the pathogenesis of the studied disorder. To evaluate the in vivo relevance of allergen-specific TCC that we isolated and characterized in earlier studies (22, 25, 26), we investigated whether these TCC were long-lived in vivo. Evidence for the existence of long-lived, allergen-specific TCC has been provided by Wedderburn et al., who detected Dpt-specific TCC in peripheral blood of one patient suffering from perennial rhinitis, 6 yr after the original isolation of the TCC (18). In the current study, we expanded this observation: we used a TCC tracing protocol based on the nucleotide sequence of the TCR ß-chain hypervariable region, and allergen-specific TCC were identified in blood and skin. These TCC displayed cytokine production patterns typical for allergen-specific T cell populations in allergic individuals; i.e., the majority belonged to the Th2-like subset (Table I) (3, 4, 5, 22, 25, 37, 38, 39, 40).

We demonstrate that allergen-specific TCC can be long-lived in vivo, since they were detected in either blood or skin up to 4 yr after their original isolation. Oligonucleotides specific for the hypervariable region of the TCR ß-chain were used to identify particular TCC (16, 17). We show that this experimental approach may be used efficiently to search for distinct T lymphocytes either in peripheral blood or other tissues (skin). The tracing protocols for skin- and blood-derived TCC were different with regard to the final step. It was possible to trace skin-derived, Dpt-specific TCC by oligonucleotide probing of re-PCR products. Similar results were obtained when the TCC-specific oligonucleotide was used as a primer in a second re-PCR (data not shown). For tracing blood-derived, pollen-specific TCC, however, oligonucleotide probing was not successful. Here, a second re-PCR with the VDJ-specific primer was required. The need for different approaches may result from a higher frequency of allergen-specific T cells in the target organ than in the periphery. In addition, perennial allergens may induce a higher precursor frequency of allergen-specific T lymphocytes than seasonal allergens.

Using our T cell tracing protocol, seven pollen allergen-specific TCC were identified in blood of five patients suffering from seasonal pollen-induced rhinitis (Fig. 2). Along with the systematic search for TCC, we found additional evidence for long-lived allergen-specific TCC: grass pollen-specific TCC NO 29 II, obtained by limiting dilution of peripheral blood in 1993, was found to be identical with TCC NO 5 III isolated and expanded from a blood sample collected 1 yr later (Table I). Together, these findings suggest that in vitro-propagated TCC are representative of detectable allergen-specific clonotypes in peripheral blood.

A role for aeroallergens as triggering factors for AD is indicated by the fact that they can elicit eczematous skin reactions in 50% of allergen-sensitized AD patients when applied epicutaneously in an atopy patch test (APT) (41, 42). We hypothesize that the APT reaction is initiated by the focusing of aeroallergens on IgE-bearing epidermal dendritic cells (43), which bind IgE monomerically with high affinity receptors for IgE (44, 45) and are highly efficient allergen-presenting cells (46). Stimulation of allergen-specific Th2 cells in the dermis, and subsequent release of Th2 cell-derived cytokines, will lead to an inflammatory reaction in which skin-infiltrating eosinophils are involved (24). Detection of the APT-derived, Dpt-specific TCC 4:3.1 and 4:7.4 in skin of the same patient several years later (Table II, Fig. 3) demonstrates that Th2 cell clonotypes that infiltrate the skin can be long-lived and implies that these TCC are prominent allergen-specific T cells in the skin of this patient. Moreover, the finding that both TCC were traced in lesional skin and even in clinically noninvolved skin could mean that these long-lived, allergen-specific T lymphocytes carry out an immune surveillance of the skin. It remains to be resolved, however, whether the T cells are retained in the skin or constantly extravasate from the circulatory system. In both cases, allergen-specific T lymphocytes would be able to interact immediately with resident APC, thus eliciting the allergic skin reaction when contact with Ag occurs (47).

It has been reported that memory T cells require repeated stimulation to avoid cell death (48). In the case of house dust mite-induced atopic disease, Ag contact is obviously associated with perennial inhalation or skin contact with Dpt itself. In the case of pollinosis, however, the seasonal exposure and the occurrence of cross-reactive plant-derived Ags in food could be responsible for sustained survival of T lymphocytes (49, 50). Another factor contributing to the observed longevity of allergen-specific T lymphocytes could be a reduced susceptibility to Fas/Fas ligand-mediated apoptosis in Th2 cells (51, 52).

In conclusion, we demonstrate the existence of allergen-specific, long-lived Th2-like cells in vivo in subjects suffering from allergic disease. These long-lived T lymphocytes may be specific for perennial as well as for seasonal allergens and can be detected in peripheral blood, but also in situ by using special molecular biologic methods. Together, the data indicate that in vitro-characterized, long-lived, allergen-specific TCC are good representatives of allergen-reactive Th2 lymphocytes in vivo.

	Acknowledgments

 
We are grateful to U. Siemann and A. G. van Ieperen-van Dijk for their excellent technical assistance. We thank the Red Cross Blood Bank, Utrecht, The Netherlands, for providing human blood group AB⁺ serum; and Novartis Research Institute, Vienna, Austria, for providing human rIL-2 and rIL-4.

	Footnotes

 
¹ This study was supported by Grants S06704-MED and S06707-MED from the Fonds zur Förderung der Wissenschaftlichen Forschung, Vienna, Austria. The study was facilitated in part by financial support from Novartis Research Institute, Vienna, Austria.

² Address correspondence and reprint requests to Dr. Christof Ebner, Department of General and Experimental Pathology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna. E-mail adress:

³ Abbreviations used in this paper: Dpt, Dermatophagoides pteronyssimus; Der p 1, allergen of Dpt; Bet v 1, birch pollen (Betula verrucosa) allergen; Phl p 1, major grass pollen (Phleum pratense) allergen; TCC, T cell clone; TCL, T cell line; AD, atopic dermatitis; CDR, complementarity determining region; APT, atopy patch test.

Received for publication July 9, 1997. Accepted for publication November 3, 1997.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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