Materials and methods

Patients and nonallergic individuals

Ten nonatopic patients (median age, 31 y; nine females and one male) with ACD to Ni and 10 nonatopic, age-matched healthy volunteers (median age, 30 y; six females and four males) without history of cutaneous allergy to metals were included in the study. Both patients with ACD to metals and control subjects were tested with a standard patch test series, including NiSO₄ 5% in petrolatum (International Contact Dermatitis Research Group Series). Patch tests were applied under occlusion with Finn Chambers (Alpharma, Norgesplaster, Norway) on the upper back, and evaluated after 48 and 72 h. The reactivity was graded as follows: –, no reaction; +, redness, edema; ++, redness, edema, and papules; +++, redness, papules, and vesicles. To increase patch test sensitivity and definitively exclude contact sensitivity to the metal, healthy controls were also selected on the basis of a negative patch test to 5% NiSO₄ performed after 24 h pretreatment of the skin with 0.1% sodium lauryl sulfate (^{Seidenari et al. 1996}). Both patients and controls were not taking any medication for at least 15 d before blood donation. Peripheral blood samples were obtained after informed consent.

T cell purification and proliferation assays

T cell proliferation assays were performed in RPMI 1640 complemented with 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 0.05 mM 2-mercaptoethanol, 100 U penicillin per ml, 100 g streptomycin per ml (all from Life Technologies, Chagrin Falls, OH), and 5% pooled homologous plasma (complete medium). Peripheral blood mononuclear cells (PBMC) were separated by centrifugation over Ficoll-Hypaque (Lymphoprep, Nycomed-Pharma, Oslo, Norway) and left to adhere (6 10⁶cells per ml) in Petri dishes for 2 h at 37°C in complete medium. After extensive washing, adherent cells were incubated for 30 min in 0.2% ethylenediamine tetraacetic acid, removed from dishes by scraping, X-irradiated, and then used as antigen presenting cells (APC) at 10⁵ cells per well. The nonadherent fraction was depleted of CD19⁺, HLA-DR⁺, and CD4⁺ or CD8⁺ cells by incubation with immunomagnetic beads coated with specific monoclonal antibodies (MoAb) (Dynabeads M450, Dynal, Oslo, Norway). After two rounds of bead depletion, >98% pure CD8⁺ or >95% pure CD4⁺ T cells were obtained, as confirmed by flow cytometry analysis. In selected experiments, CD4⁺ T cells were further purified into CD45RA⁺ and CD45RO⁺ T cells by negative selection. To this end, purified CD4⁺ T cells were incubated with anti-CD45RO (UCHL-1) or anti-CD45RA (L48) MoAb (Becton Dickinson, San Jose, CA) for 1 h at 4°C, followed by anti-mouse IgG-conjugated immunomagnetic beads. Purified T cell populations were used as responder cells in proliferation assays performed in triplicate at 1.5 10⁵ cells per well in flat-bottom 96 well plates without or with 0.6–24 g NiSO₄ per ml (Sigma, St. Louis, MO). After 5 d, cocultures were pulsed with 5 Ci [³H]thymidine per ml (Amersham, Little Chalfont, U.K.) for about 16 h at 37°C, and then harvested onto fiber coated 96 well plates (Packard Instruments, Groningen, The Netherlands). Radioactivity was measured in a beta counter (Topcount, Packard Instruments). Results are given as the stimulation index, which represents the ratio of [³H]thymidine uptake in the presence and the absence of NiSO₄. MHC restriction of CD4⁺ and CD8⁺ T cell reactivity to NiSO₄ was evaluated by incubating APC with 10 g per ml of pooled anti-HLA-DR, -DP, and -DQ MoAb, anti-HLA class I MoAb, or mouse IgG (all from Becton Dickinson) for 30 min at 4°C and then for additional 30 min at 37°C prior to their use in proliferation assays.

Frequency of Ni-specific T cells

Frequency of Ni-specific T cells was assessed by limiting dilution of purified CD4⁺ and CD8⁺ T cells (500–20,000 cells per well) in the presence of 6 g NiSO₄ per ml and 10⁴ irradiated autologous PBMC in U-bottomed 96 microplates in complete medium. For each dilution, 24 replicate cultures in the presence of NiSO₄ were compared with 12 replicate cultures performed in the absence of NiSO₄. [³H]Thymidine incorporation was measured after 5 d of culture. Ni-stimulated cultures in which the cpm exceeded the mean cpm plus 3 SD of the 12 corresponding antigen-free cultures were considered as positive. The distribution of Ni-reactive T cells was analyzed by plotting the fraction of the nonresponding cultures on the y axis and the cell input on the x axis. The frequency of Ni-specific T cells could be roughly calculated as the inverse of the number of T cells giving rise to 37% negative wells, as described by^{Lefkovitz & Waldman (1979)}.

T cell cloning, immunophenotype, cytokine production

CD4⁺ and CD8⁺ short-term T cell lines were cloned by limiting dilution in the presence of 2 10⁵ PBMC, 20 U rIL-2 per ml (kindly provided by Chiron Italia) and 1% phytohemagglutinin (Life Technologies) in U-bottomed 96 well microplates in complete medium containing 10% fetal bovine serum (Hyclone, Logan, UT). Clones were grown with rIL-2 (20 U per ml) and stimulated once a month with 1% phytohemagglutinin in the presence of feeder cells. Antigen specificity of T cell clones was assessed using autologous EBV-transformed B cell lines or PBMC as APC and 12 g NiSO₄ per ml. Immunophenoytpe of the clones was evaluated by double-color flow cytometry analysis using anti-CD4, anti-CD8, anti-TCR, and anti-TCR fluorescein isothyocyanate- or phycoerythrin-conjugated MoAb (Becton Dickinson). CLA expression was studied using the HECA-452 MoAb (kindly provided by Dr. Louis J. Picker, Laboratory of Experimental Pathology, Department of Pathology, University of Texas South-western Medical Center, Dallas, TX) or control rat IgM, followed by phycoerythrin-conjugated anti-rat IgM (Pharmingen, San Diego, CA). Cells were analyzed with a FACScan equipped with Cell Quest software (Becton Dickinson, Mountain View, CA). Supernatants from T cell clones (10⁶ cells per ml) stimulated in 24 wells with 10 ng phorbol 12-myristate 13-acetate (PMA) per ml and 1 g ionomycin per ml (Sigma), or with NiSO₄ (12 g per ml) plus 10⁶ autologous irradiated PBMC, were collected after 24–48 h culture, filtered, and stored at –80°C. IL-2, IL-4, IL-10, and IFN- content was measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), following the manufacturer's instructions.

Statistical analysis

Data were compared using the Mann–Whitney rank sum test (SigmaStat, Jandel, San Rafael, CA); p values 0.05 were considered significant.

Results

Patients included in our study had a positive history of eczematous dermatitis after contacting metals and a positive patch test to 5% NiSO₄ Table 1. Nonallergic individuals had no history of dermatitis to metals and a negative patch test to NiSO₄ even after pretreatment of the skin with 0.1% sodium lauryl sulfate, a procedure that increases the sensitivity of the patch test (^{Seidenari et al. 1996}). Neither the patients nor the healthy individuals had personal or family history of atopic diseases, and serum IgE were within normal limits. To assess the Ni-specific T cell response to NiSO₄, pure CD4⁺ T cell populations prepared from Ni-allergic and nonallergic subjects were compared in proliferation assays in the presence of autologous adherent cells and serial dilution of NiSO₄. As shown in Table 1 and Figure 1, both allergic and nonallergic displayed a strong CD4⁺ T cell proliferation to NiSO₄, without significant differences at each NiSO₄ concentration tested. NiSO₄ concentrations above 24 g per ml were toxic, and drastically reduced T cell proliferation. CD4⁺ T cell responses to Ni were markedly inhibited by blocking MHC class II receptors on APC with pooled anti-HLA-DR, -DP, and -DQ MoAb, but not with mouse IgG or anti-class I MoAb (not shown). Among nonallergic and allergic donors, both high and low responders were identified, without correlation either with the severity of the disease or with the patch test reactivity in the latter individual group Table 1. Peripheral blood samples were obtained either prior to or 2 wk after patch testing, but patch testing did not influence per se the magnitude of the CD4⁺ T cell responses, as no significant differences in the extent of the Ni-specific T cell proliferation were observed between assays performed before and after patch testing (not shown).

Figure 1.

Ni-specific CD4⁺ T cells from allergic and nonallergic individuals proliferates in vitro to NiSO₄, whereas only allergic patients show Ni-specific CD8⁺ T cell responses. CD4⁺ T cells (A, from allergic patients;B, from nonallergic individuals) and CD8⁺ T cells (C, from allergic;D, from nonallergic) were purified from peripheral blood by negative selection with immunomagnetic beads, and used as responders (1.5 10⁵ cells per well) in proliferation assay together with autologous adherent cells (10⁵ cells per well). Results are shown as stimulation index, which represents the ratio between the mean [³H]thymidine incorporation of triplicate cultures tested in the presence and in the absence of NiSO₄. Differences in CD4⁺ T cell proliferation between allergic and nonallergic subjects were not significant (p > 0.05) at each NiSO₄ concentration.

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Table 1 - Characteristics of the subjects included in the study and results of purified CD4⁺ and CD8⁺ T cell proliferation to NiSO₄in vitro.

Full table

The CD45RO+ memory T cell subset mainly accounts for CD4+ T cell proliferation to NiSO4

To exclude the possibility that the CD4⁺ T cell responses observed in nonallergic subjects could have depended on in vitro T cell priming, CD4⁺ T cell proliferation to NiSO₄ was examined using purified CD45RO⁺ CD45RA^– (memory) and CD45RA⁺ CD45RO^– (naïve) subsets as responders. Separation of CD45RO⁺ and CD45RA⁺ cells was performed by negative selection using an indirect immunomagnetic technique with L48 and UCHL-1 MoAb, respectively, followed by anti-IgG-conjugated magnetic beads, and purification was assessed by two-color fluorescence-activated cell sorter analysis Figure 2a. Indeed, CD4⁺ CD45RO⁺ T cells from nonallergic individuals proliferated significantly to NiSO₄, demonstrating the presence of Ni-specific CD4⁺ memory T cells in nonallergic individuals Figure 2b. A similar pattern of CD4⁺ T cell responses were observed in allergic patients. CD4⁺ CD45RA⁺ T cells from both allergic and nonallergic individuals proliferated to NiSO₄ as well, although to a lesser extent. When CD4⁺CD45RO⁺ and CD4⁺CD45RA⁺ T cells from the same subjects where tested with the recall antigen, tetanus toxoid, a strong proliferative response of CD4⁺CD45RO⁺ T cells and a much lower, but still significant, proliferation of CD4⁺CD45RA⁺ T cells were observed (not shown). It has recently been demonstrated that in the absence of antigenic stimulation memory T cells may reacquire an unprimed (CD45RA⁺ CD45RO^–) phenotype, suggesting that the CD45RA/CD45RO switch is not unidirectional (^{Rothstein et al. 1991;Hamann et al. 1996}). Therefore, it is possible that the Ni-specific proliferation of CD4⁺ CD45RA⁺ T cells may depend on reversal of CD45 isoform switching of resting Ni-specific T cells rather than on in vitro T cell priming.

Figure 2.

Both allergic patients and nonallergic subjects harbor Ni-specific memory CD4⁺ CD45RO⁺ T lymphocytes. CD4⁺ T cells were purified by indirect immunomagnetic separation using UCHL-1 and L48 MoAb, and then used in proliferation assay. (A) Representative CD4⁺ T cell staining before (left panel) and after (middle and right panels) purification. Cells were labeled with fluorescein isothyocyanate-conjugated anti-CD45RA and phycoerythrin-conjugated anti-CD45RO. (B) Proliferative response of CD4⁺CD45RO⁺ and CD4⁺CD45RA⁺ T cell subsets from six nonallergic and six allergic individuals in the presence of 12 g NiSO₄ per ml using adherent cells as APC.

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CD8+ T cells from Ni-allergic patients, but not from healthy individuals, proliferate in vitro in response to NiSO4

CD8⁺ T cells are thought to be critical in the development of murine CH (^{Gocinski & Tigelaar 1990;Bour et al. 1995;Xu et al. 1996}). Hapten-specific CD8⁺ T cell clones have been isolated from patients with ACD to urushiol (^{Kalish & Johnson 1990}), and, more recently, from patients with ACD to Ni (^{Vollmer et al. 1997;Werfel et al. 1997}), suggesting their contribution to the expression of the human disease as well. Thus, purified CD8⁺ T cells obtained from both allergic and nonallergic individuals were compared in terms of proliferation to NiSO₄. Results showed that CD8⁺ T cells from all allergic individuals proliferated to Ni (stimulation index > 3), whereas nonallergic subjects failed to respond to the hapten Figure 1, Table 1, suggesting that the presence of Ni-specific CD8⁺ T cells is required for the expression of the disease.

The frequency of Ni-specific CD4+ T cells is comparable in allergic and nonallergic subjects, whereas the frequency of CD8+ T cells is higher in patients

The frequency of peripheral blood derived Ni-specific T cells was determined by limiting dilution analysis. In agreement with the proliferation data, the estimated frequency of Ni-specific CD4⁺ T cells from allergic and nonallergic individuals did not show significant differences, ranging in both subject groups from 1:1700 to 1:6200. In contrast, the frequency of Ni-specific CD8⁺ T cells ranged from 1:2600 to 1:31,000 in allergic patients, and was extremely low or undetectable in nonallergic subjects Figure 3, Table 2.

Figure 3.

Frequency of Ni-specific CD4⁺ and CD8⁺ T cells. Results of the limiting dilution assays performed with purified CD4⁺ and CD8⁺ T cells from six allergic (A, CD4⁺;C, CD8⁺) and six nonallergic individuals (B, CD4⁺;D, CD8⁺). T cells (500–20,000 cells per well) were plated with 10⁴ irradiated adherent PBMC in U-bottomed microplates in the presence of 6 g NiSO₄ per ml, and evaluated for [³H]thymidine uptake as described in Materials and Methods. For each Ni concentration, 24 replicates in the presence of NiSO₄ and 12 without NiSO₄ were plated. Wells showing [³H]thymidine incorporation exceeding the mean 3 SD of the controls were considered positive. The estimated frequencies roughly correspond to the condition showing 37% of negative replicates, and are shown in Table 2.

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Table 2 - Allergic patients and nonallergic individuals show similar frequency of Ni-specific CD4⁺ T lymphocytes, but differ in the frequency of Ni-responsive CD8⁺ T lymphocytes.

Full table

Ni-specific CD4+ T cell clones derived from peripheral blood of allergic and nonallergic individuals are both CLA+ but display a different pattern of IL-10 and IFN- release

In the following experiments, a large number of Ni-specific T cell clones were prepared from peripheral blood of allergic patients and nonallergic donors and compared for the pattern of cytokine release and CLA expression. These clones were all CD4⁺, CD8^–, TCR⁺, and TCR^– (not shown). Ni-specific CD4⁺ T cell clones (n = 50) from nonallergic individuals displayed lower IFN- (mean 3.56 vs 4.72 ng per ml, p = 0.03), and increased IL-10 release (1.04 vs 0.4 ng per ml, p = 0.006) when compared with T cell clones (n = 50) prepared from allergic patients, whereas IL-4 production did not differ significantly (0.4 vs 0.2 ng per ml, nonallergic and allergic, respectively) Figure 4. Comparing the resulting pattern of cytokine release, CD4⁺ clones from nonallergic individuals showed a lower percentage of Th1 clones (58% vs 76%) and a similar number of Th0 clones (26% vs 24%), whereas only 4% of T cell clones from nonallergic subjects, and none from allergic subjects, showed a clear cut Th2 pattern. Interestingly enough, IL-10 was the predominant cytokine produced by 15 of 50 (30%) and by three of 50 (6%) of the Ni-specific CD4⁺ T cell clones isolated from nonallergic and allergic individuals, respectively. These T cell clones exhibited high IL-10 production and very low or undetectable IFN- or IL-4 release, and could not fulfil the criteria for the Th1 or Th2 pattern. Representative IL-10^high producing T cell clones are shown in Table 2III. In the remaining T cell clones from both individual groups, IL-10 release did not segregate with IFN- or IL-4 production, and was detected in either Th2, Th0, as well as Th1 subsets, as previously reported (^{Del Prete et al. 1993}). The same pattern of cytokine release was observed when T cell clones were stimulated with PMA and ionomycin or autologous APC and NiSO₄, although lower amounts of cytokines were released with the latter stimulation (Table 2III).

Figure 4.

Ni-specific CD4⁺ T cell clones from allergic patients and nonallergic individuals display a distinct pattern of cytokine release. Culture supernatants were collected 48 h after stimulation with 10 ng PMA per ml and 1 g ionomycin per ml, and content of IFN- (A), IL-4 (B), and IL-10 (C) was measured by enzyme-linked immunosorbent assay. Ni-specific T cell clones from nonallergic donors (n = 50) released a lower amount of IFN- (p = 0.03) and higher IL-10 (p = 0.006), compared with T cell clones from allergic patients (n = 50). No significant differences were observed for IL-4 release.

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CLA is thought to be the major homing receptor for T cell targeting to the skin, being selectively expressed on the majority of skin-infiltrating T cells during ACD (^{Picker et al. 1991;Bos et al. 1993;Santamaria Babi et al. 1995}). Therefore, differences in the CLA expression on Ni-specific T cells could be crucial in determining the clinical outcome of the immune response to the hapten. Figure 5 shows that the large majority of Ni-specific CD4⁺ T cell clones prepared from peripheral blood of both allergic and nonallergic donors were CLA⁺, thus possessing the potential for recirculating through the skin.

Figure 5.

Ni-specific CD4⁺ T cell clones from both allergic and nonallergic individuals express the CLA skin homing receptor. T cell clones were stained with rat IgM (white bars) or HECA-452 MoAb (black bars) followed by phycoerythrin-conjugated anti-rat IgM, and examined by flow cytometry. Results are shown as mean fluorescence intensity.

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Ni-specific CD8+ T cell clones from allergic individuals express a Tc1 phenotype and are CLA+

To better characterize the Ni-specific CD8⁺ T cell responses, 20 Ni-specific CD8⁺ T cell clones (CD8⁺, CD4^–, TCR⁺, and TCR^–) isolated from five allergic patients were examined for cytokine release and nine of them for CLA expression. Results indicated that the great majority of hapten-specific CD8⁺ T cell clones were CLA⁺ and produced IFN- but not IL-4, thus belonging to the Tc1 subset Figure 6. Several attempts to isolate CD8⁺ T cell clones from nonallergic subjects failed, in agreement with their very low frequency in these individuals.

Figure 6.

Ni-specific CD8⁺ T cell clones from Ni-allergic patients are CLA⁺ and belong to the Tc1 subset. (A) CLA expression was evaluated as described in Figure 5. (B) Cytokine release was measured by enzyme-linked immunosorbent assay in culture supernatants 48 h after stimulation with PMA and ionomycin.

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Discussion

In murine models, it has been demonstrated that the expression of CH to haptens is primarily regulated through the activities of distinct T cell subsets. Using CD4⁺- or CD8⁺-Ab-depleted mice (^{Gocinski & Tigelaar 1990}) and MHC class II or class I knockout mice (^{Bour et al. 1995}), it has been shown that hapten-specific IFN--producing CD8⁺ T cells are crucial for the development of CH and exert a major effector function. In contrast, CD4⁺ T cells appear to perform a predominant regulatory activity by releasing type 2 cytokines and IL-10 (^{Xu et al. 1996}). So far no studies have compared the reciprocal role of CD4⁺ and CD8⁺ T cells in human disease, but indirect evidence suggests an important role for CD8⁺ T cells in the cutaneous reactions to haptens. In particular, ACD to Ni has been documented in patients with CD4⁺ idiopathic lymphopenia (^{Goodrich et al. 1993}) and in HIV⁺ patients with very low CD4⁺ T cell counts (^{Viraben et al. 1994}). Moreover, hapten-specific CD8⁺ T cells have been isolated from peripheral blood of patients with ACD to urushiol, a strong sensitizer contained in poison ivy (^{Kalish & Johnson 1990}), and, more recently, from the peripheral blood and the skin of patients with ACD to Ni (^{Vollmer et al. 1997;Werfel et al. 1997}).

Ni is a widely diffused metal and it is the most common cause of ACD (^{Peltonen 1979}); however, in spite of the large diffusion of the hapten and the extremely high possibility of contacting Ni-releasing materials in professional settings and in everyday life, most people are apparently protected from the development of ACD to Ni. To assess whether the expression of the disease could be related to the expansion of distinct T cell subsets and/or a different pattern of cytokine release, we compared the characteristics of Ni-specific T cell responses in Ni-allergic patients and in nonallergic, healthy individuals. Our results showed that Ni-specific CD8⁺ T cell responses could be detected only in allergic patients. Ni-specific CD8⁺ T cell clones prepared from these patients displayed a Tc1 pattern of cytokine release and were CLA⁺, suggesting their active intervention in the expression of the dermatitis. The lack of functional CD8⁺ T cell responses in nonallergic individuals could have different explanations. First, it might be the consequence of a reduced capacity to generate MHC class I-restricted hapten-epitopes. Because haptens recognition by CD8⁺ T cells depends on the interaction of the chemical with MHC class I-loaded peptides (^{Martin et al. 1992}), a different repertoire of bound peptides could greatly influence the generation of appropriate numbers of antigenic stimuli. Ni-allergy, however, has not been definitely associated with particular HLA class II or class I alleles (^{Emtestam et al. 1993}). Alternatively, the expansion of effector CD8⁺ T cells might be prevented by regulatory mechanisms, including hapten-specific suppressor T cells. In contrast to CD8⁺ T cell responses, both allergic and nonallergic individuals were shown to harbor in the peripheral blood Ni-specific CD4⁺ T cells belonging to the CD45RO⁺ memory subset. Both the frequency of Ni-specific CD4⁺ T cells and the magnitude of the Ni-specific CD4⁺ T cell responses to different NiSO₄ concentrations were similar in allergic and nonallergic subjects. These findings suggest that CD4⁺ T cell priming to Ni occurs also in nonallergic subjects, and can be related to the continuous exposure to this ubiquitous metal. In keeping with our results, it has been shown that T cell responses to Dermatophagoides farinae and Parietaria judaica can be detected in both allergic and nonallergic subjects (^{Cavaillon et al. 1988;Sallusto et al. 1993}). In such a case, the expression of the allergic disease has been related to the cytokine pattern of allergen-specific CD4⁺ T cells that were shown to belong to the Th2 subset in atopic individuals and to the Th1 subset in healthy subjects (^{Wierenga et al. 1991}).

Our results showed that the great majority of Ni-specific CD4⁺ T cells form both allergic and nonallergic subjects were CLA⁺ and thus they do not differ in their capacity to target to the skin. In contrast, a distinct pattern of cytokine release was observed. Ni-specific CD4⁺ T cell clones from nonallergic subjects exhibited lower IFN- production and augmented IL-10 release compared with T cell clones from allergic patients. In contrast to our data, no differences in IFN- production were observed by^{Kapsenberg et al. (1992)} when Ni-specific T cell clones obtained from two allergic patients and from a single nonallergic donor were compared. In 15 of 50 (30%) Ni-specific CD4⁺ T cell clones obtained from nonallergic individuals, and in three of 50 (6%) clones from patients with ACD to Ni, a unique pattern of cytokine release was detected, consisting of high IL-10 and low or undetectable IFN-, IL-4, and IL-2 release. These IL-10^high Ni-reactive CD4⁺ T cell clones closely resembled a newly identified CD4⁺ T cell subset, named T regulatory cells 1 (Tr1), generated in vitro in the presence of IL-10 from both human peripheral blood and murine spleen cells. Tr1 cells produce a high amount of IL-10 and low quantities of IL-2, IL-4, and IFN-, and display an immunosuppressive function both in vitro and in vivo (^{Groux et al. 1997}). Work is currently in progress in our laboratory to better define the functional role of these Ni-specific IL-10^high T cell clones. In humans, IL-10 has been identified as a potent anti-inflammatory cytokine produced by both Th1 and Th2 CD4⁺ T cell clones (^{Del Prete et al. 1993}), which inhibits T cell proliferation of both subsets, and regulates the development of Th1 cells by blocking IL-12 driven Th1 responses (^{D'Andrea et al. 1993}). The immunosuppressive effects of IL-10 mostly depend on the inhibitory activity on the APC functions of dendritic cells and macrophages (^{Fiorentino et al. 1991;Enk et al. 1993;Hsieh et al. 1993}). Strong evidence exists demonstrating that IL-10 provides fundamental regulatory mechanisms for both the induction and the effector phases of murine CH. IL-10 mRNA is enhanced in the skin of allergic patients challenged with NiSO₄ (^{Szepietowski et al. 1997}) as well as in the skin of sensitized mice challenged with the relevant hapten (^{Ferguson et al. 1994}). Neutralization of IL-10 with local injections of specific MoAb prolongs murine CH beyond its natural course (^{Ferguson et al. 1994}); IL-10 injection during the sensitization process or before challenge with the hapten markedly decreases CH responses, and induces hapten-specific unresponsiveness (^{Enk et al. 1994}). Finally, CH to haptens is enhanced in mice with targeted disruption of the IL-10 gene (^{Berg et al. 1995}). Whether Ni-specific IL-10 producing CD4⁺ T cells could have a direct role in preventing clinical expression of ACD, e.g., by downregulating Ni-specific CD8⁺ responses, needs to be further investigated. In keeping with this hypothesis, it has been shown that IL-10 inhibits murine as well human CD8⁺ T cell proliferation and IFN- production (^{Bejarano et al. 1992;Macatonia et al. 1993}). Also IL-4 has been described as an important regulatory cytokine in murine CH (^{Gautam et al. 1992;Asada et al. 1997}), but other experiments using IL-4 knockout mice have produced contrasting data, with CH reactions either depressed (^{Weigmann et al. 1997}) or similar (^{Berg et al. 1995}) when compared with CH reactions in littermate controls. Finally, it has been shown that skin infiltrating Ni-specific CD4⁺ T cells produced higher amount of IL-4 than Ni-specific T cell clones derived from peripheral blood (^{Werfel et al. 1997}). Although we could not detect significant differences in IL-4 production between Ni-specific CD4⁺ T cell clones from allergic and nonallergic individuals, we cannot exclude a contribution of IL-4 in the modulation of ACD.

In conclusion, our results indicate that the expansion of Ni-specific CD8⁺ T cells is directly correlated to the expression of ACD to Ni, whereas Ni-specific CD4⁺ T cells may have a predominant regulatory function, possibly via release of IL-10. Strategies to selectively target these distinct T cell populations should prove to be helpful in immunotherapeutic approaches to ACD.

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Acknowledgments

We would like to thank Dr. L. Picker for the donation of the HECA-452 MoAb and F. Nasorri for the excellent technical help. This work was supported by the European Community (Biomed 2 program, grant BMH4-CT98-3713), the Ministero della Sanità, the Ministero dell'Università e della Ricerca Scientifica e Tecnologica, and by the Istituto Superiore di Sanità (AIDS project).