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The Journal of Immunology, 2002, 168: 5359-5368.
Copyright © 2002 by The American Association of Immunologists

Characterization of Drug-Specific T Cells in Phenobarbital- Induced Eruption

Hideo Hashizume1, Masahiro Takigawa and Yoshiki Tokura

Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phenobarbital has a high potential to elicit adverse reactions including severe skin eruptions and systemic involvements among the worldwide-prescribed drugs. Although phenobarbital hypersensitivity is thought to be mediated by T cells specific to the drug, its precise mechanism remains not fully elucidated. To characterize T cells reactive with phenobarbital, we generated drug-specific T cell clones and lines from PBMCs of patients with phenobarbital hypersensitivity showing various degrees of cutaneous and extracutaneous involvements. Although the TCR V{beta} repertoire and phenotype in the T cell clones/T cell lines were heterogeneous among the patients, V{beta}13.1+ and V{beta}5.1+ clones or lines were raised from the individuals examined who possessed different HLA haplotypes. Histopathological examination suggested that V{beta}5.1+CD8+ T cells and V{beta}13.1+ T cells played a role in cutaneous and extracutaneous involvements, respectively. A V{beta}13.1+CD4+ clone was found to proliferate in response to the Ag with processing-impaired, fixed APCs. Most of the clones and lines belonged to the Th2 phenotype, producing IL-4 and IL-5 but not IFN-{gamma} upon phenobarbital stimulation. Clones/lines with Th1 or Th0 phenotypes also constituted minor populations. These observations clearly indicate the heterogeneity and a marked individual deviation of reactive T cell subsets among the patients in terms of CD4/8 phenotype, V{beta} repertoire, Ag recognition pattern, and cytokine production; and thus provide evidence whereby each pathogenic T cell subset contributes to special elements of clinical presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Anticonvulsants such as phenobarbital have a high risk in eliciting adverse hypersensitivity reactions characterized by not only cutaneous but also by extracutaneous involvements (1, 2). Toxic epidermal necrolysis (TEN)2 and Stevens-Johnson syndrome (SJS) are the most severe and acute life-threatening conditions with a high mortality rate. The patients with these conditions have severe malaise, high fever, and liver and renal dysfunction in addition to extensive epidermal bullae and epithelial detachment in cutaneous and mucous membranes (1, 3). The mild form of hypersensitivity reactions includes maculopapular eruption (MPE) and bullous eruption with minimum systemic organ involvements.

Multiple organ responses in phenobarbital hypersensitivity are suggested to be immunologically mediated, but the precise mechanism remains unelucidated. T cells from the patients sensitized with anticonvulsants are activated in vivo and in vitro (4, 5). By immunohistochemistry, both CD4+ and CD8+ T cells infiltrate in the lesional skin (2, 6, 7). These data indicate that pathogenic T cells reactive with phenobarbital may induce the various aspects of clinical manifestations. One of the important issues is the immunological characterization of drug-specific T cells in relation to the organ-specific involvements. Recent findings on the role of cytokines, chemokines, and their receptors in migration of lymphoid cell subsets with affinity to a particular organ suggest that a variety of clinical manifestations as reflected by multiple organ involvements are attributed to T cell subsets expressing different organ-specific homing molecules (8). Thus, it is possible that cutaneous lymphocyte Ag (CLA)+ and {alpha}E{beta}7+ T cells induce skin damage and mucous membrane injuries, respectively (9). Another concern is the mechanism in which the multiple subsets of pathogenic T cells with different immunological characteristics are generated in response to phenobarbital. The antigenic determinants generated by the drug and its metabolites seem to be taken up by APCs in at least two different ways and variously recognized by T cells. One way is processing of the drug-related Ag-protein complex by APC followed by the presentation of antigenic moieties in the context of MHC. The other is the presentation of drug without processing by APC as observed in sulfamethoxazole and lidocaine hypersensitivity (10). The generation of multiple T cell subsets may underlie these various recognition profiles.

In the present study, we compared immunological characteristics of T cells stimulated with phenobarbital for short-term and drug-specific T cell clones (TCC) in the hypersensitivity patients with different clinical involvements. Multiple subsets of drug-specific T cells with different surface Ag expression, TCR V{beta} usage, and cytokine profiles resided in skin and peripheral blood of the patients. These data strongly suggest that multiple organ involvements are mediated by functionally different T cell populations in phenobarbital hypersensitivity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and PBMC

One patient with TEN (patient A), three with SJS (B–D), five with MPE (E–I), and one with multiple bullous eruption (J) were enrolled in this study (Table IGo). They had a history of skin rash developed within 2 mo after the phenobarbital treatment was started and cleared by the cessation of the drug. Lymphocyte stimulation test (LST) with phenobarbital showed high (stimulation index (SI): >1.8) proliferating activity in 7 of 10 patients, confirming phenobarbital hypersensitivity. In other patients, the diagnosis was made on the basis of clinical features and courses. The patients with MPE and bullous eruption had minimal, if any, systemic involvements. Five normal individuals for controls were also investigated (mean age 33; male, 3; female, 2). All patients were informed about the purpose of this study and agreed to participate. PBMC were isolated from patients with a standard Ficoll-Hypaque method at least 2 wk after the drug allergy episodes. Patients with TEN and SJS were selected for generation of TCC. PBMC were taken several times from patient A during 2-year follow-up periods, and from patients B–D during 1-year follow-up periods.


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  		Table I. Patient profiles

 
Reagents, culture medium, and mAbs

Phenobarbital sodium (purity >98%) was purchased from WAKO (Tokyo, Japan). Culture medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS or 5% autologous serum, 25 mM HEPES, and 2 mM L-glutamine without any antibiotic to avoid nonspecific stimulation. Limulus amebocyte lysate assay (WAKO) revealed endotoxin concentrations <0.03 EU/ml in the reagents used for culture. mAbs used were unlabeled, or labeled with FITC, PE, or PerCP (Table IIGo).


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  		Table II. mAbs

 
Depletion of CD4 or CD8 subset

PBMC were suspended in 0.2 ml of PBS (pH 7.4) in a 15-ml conical tube and coincubated with human CD4 or CD8 DYNABEADS (M-450 CD4 or M-450 CD8; Dynal Biotech, Oslo, Norway) at the cell-to-bead ratio of 1:50 for 60 min on ice according to the manufacturer’s protocol. After incubation, PBS containing 1% FCS was added to a final volume of 8 ml, and the unbound cells were obtained by placing a tube in a magnetic particle concentrator (MPC-1; Dynal Biotech) for 2 min.

APC and fixation

EBV-transformed B cell lines were generated from freshly isolated patients’ PBMC by culturing with supernatants from EBV-producing cell line B95-8. Cyclosporin A (1 µg/ml; Sandoz Pharmaceutical, Basel, Switzerland) was added to prevent EBV-induced T cell growth. The EBV-infected B cells were used as APC in the following experiments. The cells were stored at -120°C until use. For fixation, cells in 0.5 ml medium without serum were mixed with 12.5 µl of 2% glutaraldehyde (final concentration 0.05%) for 30 s at room temperature (11). The reaction was stopped by adding 1 ml of 0.2 M L-glycine.

Generation of phenobarbital-specific TCC and T cell lines (TCL)

PBMC were stimulated with 50 and 100 µg/ml phenobarbital in culture medium at a cell density of 106 cells/well in a 24-well culture plate (Corning Glass, Corning, NY) for 7 days. The maximum concentration of phenobarbital used in this study was 100 µg/ml, because the inhibitory effect on the PHA-stimulated lymphocytic proliferation was occasionally observed above this concentration. These cells from bulk culture were seeded at 0.3 cells/well with 5 x 104 autologous mitomycin C-treated (50 µg/ml for 30 min) PBMC that were pulsed with 100 µg/ml of phenobarbital in a final volume of 100 µl in a 96-U bottom culture plate. Growing T cells were expanded with autologous or allogeneic mitomycin C-treated PBMC as feeder cells added at a 2 wk interval. Clonality was confirmed by immunofluorescence analysis with fluorescence-conjugated anti-CD3, -CD4, and -CD8 mAb and a battery of anti-TCR V{beta} mAbs. TCC and TCL were defined as monoclonal and oligoclonal T cell populations, respectively. Thus, cell lines raised were maintained in medium supplemented with 10–15 U/ml rIL-2 (Takeda Pharmaceutical, Tokyo, Japan).

In vitro stimulation with phenobarbital

PBMC at 2 x 105 cells/well were stimulated with phenobarbital at varying concentrations. Alternatively, TCC and TCL at 2 x 104 cells/well were cultured with mitomycin C-treated syngeneic APC at a ratio of 10:1 in the presence of phenobarbital. The culture was maintained at a final volume of 100 µl in a 96-well flat-bottom culture plate (Corning) at 37°C in 5% CO2 in air. For LST, cells were cultured for 48 h and [3H]TdR (0.6 µCi/well; Amersham, Arlington, IL) were added to the wells 12 h before harvest. The cells were collected on glass-fiber filters using a cell harvester (Cambridge Technology, Watertown, MA) and radioisotope uptake was measured in a liquid scintillation counter. SI was calculated as cpm with reagent/cpm without reagent. For examination of cell surface molecules including TCR V{beta}, culture periods varied depending on the experimental conditions. Cells were then subjected to flow cytometric and cell division analysis as below.

Flow cytometry (FCM) analysis

Cells were washed once with HBSS containing 1% BSA and 0.1% NaN3, and incubated with a panel of fluorescence-conjugated mAbs for 30 min at 4°C in the dark. After washing, the harvested cells were resuspended in HBSS and subjected to FCM. More than 5 x 104 cells per sample were analyzed on a FACScaliber flow cytometer (BD Biosciences, Heidelberg, Germany) by gating lymphocytes. For examining cells cultured for >14 days, propiodium iodide gating was set to exclude dead cells. Results were analyzed using a FlowJo software (ThreeStar, San Carlos, CA).

CFSE staining

CFSE (WAKO) was added to PBMC (107/ml) in ice-cold PBS at a final concentration of 5 µM, and the suspension was incubated at 37°C for 10 min as described previously (12). After washing in cold medium, the cells were cultured and processed for the cell-division analysis by FCM.

ELISPOT assay for cytokine production

Cells were adjusted at 1 x 104 cells/well in a 96-well filtration plate (Multiscreen; Millipore, Molsheim, France) precoated with purified anti-IL-4, -IL-5, -IL-10, or IFN-{gamma} mAb for 72 h at 37°C in 5% CO2 in air for 72 h. PBMC were incubated with phenobarbital at 25 and 50 µg/ml for 72 h. Alternatively, TCC and TCL were cocultured with mitomycin C-treated syngeneic APC at a ratio of 10:1 in the presence of phenobarbital at 100 µg/ml. After removing the cells, the plate was washed extensively with PBS, and then biotin-labeled anti-IL-4, -IL-5, -IL-10, or IFN-{gamma} mAb was added to each well at optimal concentrations. After incubation for 2 h at room temperature, the plates were washed, dried, and incubated for 2 h with the avidin-streptavidin complex at a concentration of 1:1000. A diaminobenzidine solution (Sigma Fast 3,3'-diaminobenzidine tablet sets; Sigma-Aldrich, St. Louis, MO) was used as a chromogen for visualization of the reaction. The black or dark brown spots that indicated footprints of the specific cytokine-producing cells were counted in a dissecting microscope.

Histology and immunohistochemistry of skin lesion

Punch-biopsy specimens (diameter, ~4-mm) from the acute skin lesions were fixed in 4% formalin and routinely stained with H&E for standard histology. Deparaffinized specimens were autoclaved in 10 mM citrate buffer (pH 6.0) for 10 min at 120°C to retrieve the antigenic epitopes and processed for CD4, CD8, and CD3 expression analysis by the avidin-biotin complex method. Alternatively, the specimens were snap-frozen in tissue-embedding medium and were processed for TCR V{beta}s analysis by the avidin-biotin complex method. A total of 4 µm of the skin specimens were cut and incubated with primary mAbs against TCR V{beta}s, and then reacted with appropriate peroxidase-conjugated secondary Abs after washing. For visualization, deoxyaminobentidine was used as a substrate. Nuclear staining was performed with hematoxylin. Substitution of the primary Ab with isotype-matched IgG and omission of the primary Ab served as controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation of a distinctive subset of patients’ lymphocytes in response to phenobarbital

As represented by patient A, phenobarbital elicited proliferation of PBMC in a concentration-dependent fashion up to 3.68 of SI at 100 µg/ml (Fig. 1GoA). The maximum proliferative response was also achieved at a concentration of either 50 or 100 µg/ml in other patients. Therefore, we used the drug at concentrations of 50 and 100 µg/ml in the following experiments. The proliferative response was completely inhibited by anti-HLA-DR mAb (G46-6) that binds the {alpha} subunit of MHC class II (13), but not by anti-HLA-A, -B, -C mAb, suggesting that the main response was restricted to MHC class II, and thus the majority of reactive cells were CD4+ T cells in patient A.



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  		FIGURE 1. Representative data of LST (A), cell division (B), and CD4, CD8, and CLA expression (C) in response to phenobarbital. A, PBMC freshly isolated from patient A were cultured for 48 h with varying concentrations of phenobarbital or with 100 µg/ml phenobarbital plus an anti-HLA-A, -B, -C mAb (anti-MHC class I) or anti-HLA DR mAb (anti-MHC class II). Vertical bars represent SD. *, p < 0.05, compared with control. **, p < 0.05, compared with 100 µg/ml phenobarbital. B, PBMC from patient A stimulated (open histogram) or nonstimulated (filled histogram) with phenobarbital (50 µg/ml) for 72 h were labeled CFSE and analyzed by FCM upon gating for CD3+ (top), CD4+ (middle), and CD8+ cells (bottom). C, PBMC from patients of A, B, E, I, and J were cultured with 50 µg/ml of phenobarbital for 7 (light gray bar), 14 (gray bar), or 28 days (dark gray bar) and analyzed for CD4, CD8, and CLA expression (oblique lines) by FCM. Open bars indicate the start of culture (day 0). The percentage of CD4+ or CD8+ cells and of cells double-positive for CLA and CD4 or CD8 (shaded area) in total cells is shown simultaneously at each bar.

 
We confirmed the proliferation of CD4+ T cells in two different studies in patient A. First, CFSE, which covalently binds to cellular proteins and allows direct measurement of the cell division over several cell generations, was used to monitor the cell division induced specifically by Ag (12). Fig. 1GoB showed representative results obtained from patient A. After a 72-h culture, CD3+ T cells contained low but definitive percentages of dividing cells, as seen in the left peak, even in the absence of phenobarbital (Fig. 1GoB, top panel, filled histogram). However, when cultured with the drug, divided cells were increased in number (Fig. 1GoB, open histogram). The divided cells were restricted essentially to the CD4+ population (Fig. 1GoB, middle panel), whereas the CD8+ subset showed a minimal peak (Fig. 1GoB, bottom panel). Second, when PBMC deprived of CD8+ cells in patient A were cultured with phenobarbital for 14 days, >90% of the cells were CD25+. In contrast, only 17% of CD4-depleted, Ag-stimulated cells expressed CD25. Stimulation with phenobarbital induced the expressing CLA mainly on CD4+, but not on CD8+ cells in patient A (data not shown).

We monitored the phenotype of phenobarbital-reactive lymphocytes periodically in five patients (patients A, B, E, I, and J). In patients A, B, E, and I, CD4+ cells preferentially proliferated to expand such that the ratio of CD4 to CD8 was increased as the culture period was prolonged (Fig. 1GoC). In contrast, in patient J, CD8+ cells vigorously expanded. Preferential proliferating activity of the CD8+ subset and its MHC class I dependency in patient J were confirmed by CFSE analysis (data not shown). In patients A, E, I, and J, the percentage of CLA+ cells was significantly higher (6.2–27.4%) than phenobarbital-pulsed healthy controls or untreated patients’ PBMC (2.4 ± 1.4%) after a 7-day cultivation. The expression of CLA molecules was elevated in a culture time-dependent fashion in most cases.

The percentages of cells expressing activation Ag and early activation Ag were examined by enumerating CD25+ and CD69+ cells, respectively, in the drug-pulsed total lymphocytes. In patient A, the percentages of CD25+ cells were increased from 26% at day 7 to 50% at day 14 and 11.3% at day 28. In contrast, the percentages of CD69+ cells were decreased from 46% at day 7 to 11% at day 14. In patients B, C, and H, the similar expression pattern of early CD69+ and sequential CD25+ waves was found (data not shown), providing another supportive evidence for phenobarbital-induced activation of lymphocytes. These results demonstrated that a majority of phenobarbital-reactive T cells was CD4+ in patients with TEN, SJS, and MPE; and CD8+ in a patient with bullous eruption. In addition, the reactive T cells expressed CLA at high percentages.

Skewed usage of particular TCR V{beta}s in phenobarbital-specific T cells from patients with different MHC haplotypes.

We next investigated the TCR V{beta} usage by T cells reactive with phenobarbital in seven patients (Fig. 2Go). We tentatively defined the "skewed" usage of TCR V{beta} as the percentage of a particular V{beta} was above mean percentage +3 SD of the corresponding V{beta} in normal individuals. Although percentages of particular TCR V{beta}-bearing cells in freshly isolated patients’ PBMC were generally comparable to those in normal individuals, a slight increase in number was noted in V{beta}3+ and V{beta}9+ cells of patient A and V{beta}3+ cells of patients F and I. After a 7-day culture with phenobarbital, the TCR repertoire of reactive T cells tended to skew to several V{beta}s in all cases. Thus, proliferating T cells preferentially expressed V{beta}3 and V{beta}5.1 in five of seven patients (V{beta}3 in A, C, E, H, and J; and V{beta}5.1 in A, C, F, H, and J) (Fig. 2Go, bottom). Interestingly, we observed vigorous expansion of V{beta}5.1+ cells, especially in patients A and J, who suffered from blister formation. Such skewed usage in V{beta}s was not observed in patients’ PBMC after PHA stimulation or phenobarbital-stimulated normal PBMC.



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  		FIGURE 2. TCR V{beta} usage of phenobarbital-reactive T cells. Freshly isolated PBMC from normal individuals (n = 5), and patients A, C, E, F H, I, and J were cultured with 50 µg/ml of phenobarbital for 7 days and analyzed for TCR V{beta} usage by FCM. Each bar indicates the positive percentage of normal PBMC ({square}; error bar, SD), and freshly isolated () and cultured PBMC ({blacksquare}) from the patients. Number in parenthesis indicates percentage of the positive cells showing a prominent proportion. *, Skewed TCR V{beta} usage as the percentage above a mean value + 3 SD of the normal individuals. The frequency and percentage of the patients with the skewed usage in each TCR V{beta} are listed in the box. n.t., not tested.

 
The TCR V{beta} usage of phenobarbital-reactive T cells was further analyzed at the clonal level in four patients with TEN (A) and SJS (B–D) (Table IIIGo). Twenty-nine of established 68 TCCs/TCLs from the patients (A, 14 TCCs; B, 5 TCCs; C, 2 TCCs; D, 5 TCCs and 3 TCLs) were confirmed to be phenobarbital-specific by showing proliferation activity (SI, >1.5) following stimulation with phenobarbital. No clone was established without phenobarbital stimulation.


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  		Table III. Profiles of phenobarbital-specific TCC

 
Whereas the usage of TCR V{beta} by these clones was heterogeneous among the patients, we again found the preferential usage of particular V{beta}, despite different MHC haplotypes among the patients. As underlined in the third column of Table IIIGo, V{beta}13.1 was used by CD4+ TCCs in patients A–C, and by a CD8+ TCC in patient D. V{beta}5.1 was used by CD8+ TCCs in patients A and B, by a CD4+ TCC in patient C, and a TCL in patient D. Less preferential usage was noted in V{beta}17 and V{beta}21.3 by CD4+ TCCs of patients A and B. These data suggested that at least two types of T cell recognition of antigenic determinants were critical in clinical manifestations in phenobarbital hypersensitivity. First, there seems to be promiscuous Ag recognition by CD4+ and CD8+ phenobarbital-reactive T cells since the antigenic epitopes are formed between the drug and the different classes and haplotypes of MHC (14). The second type of recognition may be specific for each individual characterized by the interaction between particular V{beta}s and unique MHC haplotypes. Therefore, cutaneous and extracutaneous manifestations in phenobarbital hypersensitivity can be explained in part by T cells with a variety of V{beta}s with the different recognition patterns.

Phenotype and TCR V{beta} analysis of lymphocytes infiltrating lesional skin

Histologic examination of the acute skin lesions from seven patients (A–D and H–J) revealed the common features of lichenoid tissue reactions showing dermal perivascular lymphocytic infiltration and basal cell damage (6). The degrees of lymphocytic infiltration and epidermal damages seemed to be closely associated with clinical features, moderate lymphocytic infiltration, and massive epidermal necrosis in TEN (patient A) and bullous eruption (J); marked epidermal lymphocytic infiltration in SJS (B–D); and mild epidermal and moderate dermal lymphocytic infiltration in MPE (H and I).

In patients with TEN (A), SJS (D), MPE (H), and multiple bullous eruption (J), we investigated which T cell subset infiltrated in the lesional skin by immunohistochemical staining (Table IVGo). In all patients, although CD8+ cells outnumbered CD4+ cells (<20%) in the epidermis, more CD8+ cells were associated with more severe epidermal damage. In contrast, numbers of CD4+ and CD8+ cells were comparable around dermal vessels. We performed further analyses for the TCR repertoire of infiltrating lymphocytes in patients H and J (Table IVGo). In patients H and J, >=10% of infiltrating cells were V{beta}5.1+ and less numbers of cells expressed V{beta}3 in the epidermis (Fig. 3Go, A–C). In the dermis, infiltrating cells in the vicinity of the vessels of the deeper dermis were positive for various V{beta}s in patient H. The TCR V{beta} usage of dermal infiltrating cells were more limited in patient J. Again, V{beta}5.1+ and V{beta}3+ cells were the main populations in the dermal infiltrate. These findings were in accordance with the observation that PBMC from these patients preferentially expressed V{beta}5.1 and V{beta}3 upon phenobarbital stimulation. Accumulation of V{beta}5.1+ cells in the skin lesions seemed to be a specific event because T cells resided in normal skin do not preferentially use this V{beta} (15). Collectively, although heterogeneous T cell subsets infiltrated skin lesion, CD8+ and V{beta}5.1+ cells might locate in and close to the epidermis in phenobarbital-induced eruption.


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  		Table IV. Immunophenotype and TCR V{beta} usage of infiltrating cells in skin lesions

 


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  		FIGURE 3. Immunohistochemical analysis for phenotype and TCR V{beta} usage of infiltrating cells in the skin lesions from patients H (A) and J (B and C). Arrows show positive staining. Note V{beta}5.1+ cells were located in epidermis and upper reticular dermis in both cases. A, V{beta}5.1 expression from patient H with MPE; B, V{beta}3+; C, V{beta}5.1+ from patient J with multiple blister formation (indicated as Blister).

 
Processing was required to induce proliferation of CD4+V{beta}2+ TCC, but not CD4+V{beta}13.1+ TCC

It is suggested that "processing" or "covalent binding" of drug to MHC on APC is not necessary for T cell activation in sulfonamide and lidocaine hypersensitivities (10, 16). Therefore, we examined requirement of processing by APC in phenobarbital hypersensitivity in clones A0 and A5 from patient A. Both clones expressed CD3, CD4, HLA-DR, and CLA to a various degree (Fig. 4GoA) 72 h after drug stimulation. V{beta}2+ A0 and V{beta}13.1+ A5 clones proliferated vigorously in response to phenobarbital presented by unfixed APC (Fig. 4GoB). When stimulated with the drug in the presence of glutaraldehyde-fixed EBV-transformed autologeous B cells, only A5 proliferated as vigorously as these stimulated with unfixed APC (Fig. 4GoB).



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  		FIGURE 4. Phenotypes (A) and proliferation in response to unfixed and fixed APC plus phenobarbital (B) of A0 and A5 clones. A, The expression of TCC surface molecules at the start of culture was analyzed by FCM. Numbers indicate the percentage of positive cells in the total. B, Clones A5 and A0 were stimulated for 48 h with APC fixed ({blacksquare}) or nonfixed ({square}) with glutaraldehyde in the presence (100 µg/ml) or absence of phenobarbital. Each bar indicates [3H]TdR incorporation (cpm), and vertical bars represent SD. *, p < 0.0012; **, p < 0.04; ***, p < 0.005.

 
Because fixed EBV-transformed autologeous B cells were capable of presenting superantigen toxic shock syndrome toxin-1, but not conventional Ag-purified protein derivative, to T cells in PBMC from healthy individuals to proliferate (data not shown), it was unlikely that the fixation was imperfect for inhibition of the processing or altered the expression/synthesis of surface protein including MHC.

Th2 cytokine production by phenobarbital-stimulated PBMC and phenobarbital-specific TCC

To clarify the involution of Th1/2 paradigm in phenobarbital hypersensitivity, the cytokine production of fresh PBMC from two patients and of phenobarbital-specific TCCs was examined by the ELISPOT assay. PBMC from patients A and G produced IL-4 and IL-5, but no IFN-{gamma}, upon stimulation with phenobarbital compared with individuals without phenobarbital hypersensitivity (Fig. 5GoA). Data of the ELISPOT assay in TCCs were shown in Table IIIGo and in detail in Fig. 5GoB. Twelve of 16 clones (11 clones from patient A and 5 clones from patient B) preferentially produced IL-4 and/or IL-5, representing Th2 phenotype (Table IIIGo). In patient A, most of the clones produced IL-4/IL-5 upon stimulation, reflecting the cytokine production of fresh PBMC. For example, clones A5 that were negative for CD69 produced significant numbers of IL-4 and/or IL-5 spots only following phenobarbital stimulation (Fig. 5GoB). A0, A10, and A11 that continuously expressed CD69 produced both IL-4 and IFN-{gamma} even without stimulation, indicative of Th0 phenotype, but shifted to the Th2 cytokine production upon stimulation. In contrast, in A9 and A14, the production of IL-4/IL-5 was unchanged or down-regulated, whereas that of IFN-{gamma} was up-regulated by phenobarbital stimulation, showing a shift from Th2 to Th0 cytokine profile. Alternatively, only one clone, B3, seemed to belong to the Th1 subset because the production of IFN-{gamma}, but not IL-4 or IL-5, was observed. These data again showed heterogeneity of the cytokine profile in phenobarbital-specific T cell clones despite the fact that fresh blood cells showed Th2 cytokine production.



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  		FIGURE 5. Cytokine profiles of freshly isolated PBMC and TCC assessed by ELISPOT assay. A, A total of 1 x 104 cells/well of freshly isolated PBMC from patients A and G were cultured with phenobarbital for 72 h. Each bar indicates the number of cytokine spots/1 x 104 cells. Error bar, SD. P, patient and C, healthy individuals. *, p < 0.01, compared with controls. B, TCC were cultured with mitomycin C-treated syngeneic APC at the ratio of 10:1 for 72 h in the presence of 50 µg/ml phenobarbital (+) or its absence (-). Clone names are shown in the left column. Each bar indicates the number of cytokine spots/1 x 104 cells. {blacksquare}, IFN-{gamma}; , IL-4; and {square}, IL-5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we tried to elucidate the relationship between clinical manifestations and participation of T cells with the particular immune phenotype. An important issue to be addressed was how pathogenic T cells mediated phenobarbital hypersensitivity in the context of TCR V{beta} usage, Ag recognition, and cytokine production.

When stimulated in vitro with phenobarbital, PBMC in four patients with TEN, SJS, and MPE responded to show preferential proliferation of CD4+ cells, whereas CD8+ cells tended to die out under prolonged cultivation. In contrast, in a patient (J) with bullous eruption, in which massive epidermal necrosis was found histologically, the growth potential of CD8+ cells seemed to be superior to CD4+ cells in long-term culture. By clonal level analysis, most of TCCs/TCLs established from patients with TEN and SJS (A–C) belonged to CD4+ subsets, and CD8+ TCCs/TCLs constituted small populations. In contrast, CD8+ TCCs/TCLs were also raised preferentially from a patient with SJS (D) in addition to small numbers of CD4+ TCC/TCL. Although we could not generate TCC/TCL from a patient (J) with severe epidermal damage, CD8+ cells expanded in vitro vigorously, following phenobarbital-stimulation. In drug hypersensitivity in Ag-specific TCC/TCL, CD4+ TCCs are preferentially generated (17), indicating the major role of this cell type in the pathogenesis. However, it is not excluded that the culture conditions in our studies favored CD4+ cell growth and that other cells participated in the reactions as well. In this context, the emergence of CD8+ clones in patients with the severe forms might strengthen their functional significance. Skin pathology common to our patients was epidermal injuries with basal cell damage and dermal perivascular lymphocytic infiltration, so-called lichenoid tissue reactions (18, 19, 20). In confirmation of the previous study in anticonvulsant hypersensitivity (6, 7), CD4+ and CD8+ T cell subsets seemed to elicit different pathological changes, because CD8+ cells located at and close to the epidermis and both CD4+ and CD8+ cells at the perivascular area of the dermis. Establishment of drug-specific CD8+ TCCs from patients sensitized with penicillin emphasizes an important role of cells with the cytotoxic phenotype in eliciting bullous eruptions (21). Furthermore, CD8+ cell infiltration was still predominant even at a resolving phase of the eruptions in patient A (data not shown). These results suggested that both CD4+ and CD8+ cells contributed to the pathogenesis, although the size of each fraction among the phenobarbital-reactive cells varied depending on individual patients and that the preferential location of CD8+ subsets was characteristic in phenobarbital-induced skin manifestations.

The present study showed that multiple T cell subsets with shared V{beta} were generated from individuals of different haplotypes who showed essentially the same pathological findings upon phenobarbital stimulation. These included V{beta}3+ and V{beta}5.1+ T cells in short-term culture and V{beta}5.1+, V{beta}13.1+, V{beta}2+, V{beta}17+, and V{beta}21.3+ cells in TCCs/TCLs. We could raise V{beta}13.1+ clones but no V{beta}3+ clones in TCC analysis; whereas V{beta}3+ cells expanded prominently, but V{beta}13.1+ cells did not outgrow in short-term culture. The change in growth potential during culture periods has been frequently observed (22) and might be due in part to the in vitro microenvironmental conditions in TCC generation. These data indicated that a variety of clinical manifestations in skin and other organs was mediated by multiple T cell subsets that respond to different antigenic determinants generated through catalysis of drug by hepatic and extrahepatic enzymes or via bindings of drug to the particular regions in cryptic self peptides of MHC class II molecules as observed in nickel (23) and penicillin (24). Of particular importance is the relationship between V{beta}5.1+ and V{beta}3+ T cells of CD4 and CD8 phenotypes and skin pathology. First, large numbers of V{beta}5.1+ and V{beta}3+ cells infiltrated in and close to the epidermis and at the dermal perivascular areas in MPE (patient H) and multiple bullous eruption (patient J). Second, short-term drug stimulation resulted in the appearance of these two subsets at high frequencies in five of seven patients (A, C, F, H, and J). Finally, phenobarbital-specific V{beta}5.1+ TCCs/TCLs were generated from all the patients with TEN and SJS. Therefore, infiltration of the V{beta}5.1+ and V{beta}3+ subsets might be relevant to the various types of cutaneous manifestations, and V{beta}5.1+CD8+ cells, in particular, to epidermal damage. The use of certain TCR V{beta} by phenobarbital-specific T cells was not very convincing as the oligoclonality of the drug-specific clones seemed to be less stringent in our data. The clonal-level analysis suggested that V{beta}13.1+ cells, although not found in the lesional infiltrate, contributed to the extracutaneous manifestations.

Trafficking of pathogenic T cells by means of some essential molecules such as CLA (2), cytokine receptors, and chemokine receptors is crucial for elicitation of cutaneous inflammation (8, 25). In four of five patients, CLA+ T cells expanded during in vitro short-term culture in the presence of phenobarbital and about one-third of phenobarbital-specific TCCs/TCLs generated from four patients expressed CLA at >10%. Because CLA, a counter receptor for E-selectin, is expressed on cells with a potential to migrate to the skin, the interaction of these two adhesion molecules may participate in the skin changes of phenobarbital hypersensitivity (26). CLA-negative populations might migrate into skin by expressing other ligands for cytokines and chemokines such as monokine induced by IFN-{gamma}, IP-10, IL-8, cutaneous T cell-attracting chemokine, and thymus and activation-regulated chemokine (8), although the present study did not address in detail the expression of cytokine or chemokine receptors in phenobarbital-reactive cells. Alternatively, it was possible that some of these populations attacked organs other than skin by expressing different organ-specific adhesion molecules and chemokine receptors on their cell surface, resulting in liver dysfunction and blood abnormalities. For example, pathogenetic roles of V{beta}13.1+ clones were strongly suggested by their establishment at high frequencies from different patients. However, virtual absence of T cells with this phenotype among the infiltrate in skin lesions indicated that V{beta}13.1+ cells might have had little potential of skin-homing.

T cell recognition of antigenic moieties on APC is not so stringent as has been suggested, and promiscuous under certain settings. Allele-unrestricted presentation of haptenic small compounds by MHC class II molecules is recently described in the interactions between T cells specific for lidocaine, sulfamethoxazole, and amoxicillin and APC (16, 27). Direct binding of nickel to particular amino acids of cryptic MHC peptides results in conformational change of the MHC-peptide-metal complex so as to be recognized by nickel-specific T cells irrespective of APC alleles (28). Furthermore, a monomorphic surface molecule, CD39, can function as additional recognition structures on haptenated target cells for HLA-A1-restricted hapten-specific CD8+ T cells (29). Therefore, the establishment of T cell clones with shared V{beta}s at high frequencies from different patients might reflect promiscuous recognition of phenobarbital presented by APC in various ways.

As we addressed herein, phenobarbital-related Ag recognition without processing by APC took place in special T cell subsets responsible for the hypersensitivity. For example, CD4+V{beta}13.1+ TCC from patient A proliferated with processing-impaired APC in the presence of phenobarbital. Proliferation of this clone was inhibited by addition of anti-DR mAb, suggesting that the Ags would directly bind to the {alpha}-chain of HLA-DR molecule (13). This type of Ag recognition is observed in sulfamethoxazole and lidocaine hypersensitivity in which the drugs become immunogenic by simply interacting with the MHC-peptide complex (30). Another concern is cross-reactivity of drug-reactive T cells to multiple neoantigenic determinants. For example, T cells respond polyclonally to several antibiotics including penicillin G in patients sensitized with {beta}-lactams (31). The CDR3 region of TCR in {beta}-lactam-reactive TCC established from these patients has no common sequence that can recognize the penicilloyl group, indicating the generation of cross-reactive V{beta}s upon drug stimulation. This possibility is also likely in phenobarbital hypersensitivity.

Fresh PBMC from two patients and the majority of established clones from three individuals produced IL-4/5 and IL-10, but not IFN-{gamma} in response to phenobarbital. This may explain blood eosinophilia, a hallmark of Th2 cell activation, observed in several patients. Furthermore, the cytokine profiles of other drug-specific TCCs upon stimulation were various, that is, enhanced production of both Th1 and Th2 cytokines, and shifts from Th0 to Th2 and from Th2 to Th0 cytokine production. A previous report has suggested that in vivo polarized expression of cytokines are dependent on the chronology of the clinical reaction (32). Although we could not confirm this finding, the pattern of cytokine profiles in TCCs/TCLs may figure the variation of clinical manifestations in phenobarbital hypersensitivity.

The present data suggests that a spectrum of the clinical presentations ranging from severe forms such as TEN and SJS to mild ones with minimum systemic involvements in phenobarbital hypersensitivity can be explained by pathogenic T cells with different immune characteristics. The drug-reactive T cells may arise through presentation of a variety of antigenic determinants derived from phenobarbital and its metabolites by APC with and without Ag processing. These T cell subsets bearing different TCR V{beta}s are likely to express homing receptors or chemokine receptors for either skin or other organs and produce Th1 and/or Th2 cytokines at the migrated organs. Future challenges will include identification of antigenic determinants specific for each clinical manifestation and elucidation of the molecular mechanism in the interaction between these determinants and pathogenic T cells.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Hideo Hashizume, Department of Dermatology, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu, 431-3192, Japan. E-mail address: hihashiz{at}hama-med.ac.jp Back

2 Abbreviations used in this paper: TEN, toxic epidermal necrolysis; CLA, cutaneous lymphocyte-associated Ag; FCM, flow cytometry; LST, lymphocyte stimulation test; MPE, maculo-papular eruption; SI, stimulation index; SJS, Stevens-Johnson syndrome; TCC, T cell clone; TCL, T cell line. Back

Received for publication January 14, 2002. Accepted for publication March 14, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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