The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woodfolk, J. A.
Right arrow Articles by Platts-Mills, T. A. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woodfolk, J. A.
Right arrow Articles by Platts-Mills, T. A. E.
The Journal of Immunology, 2001, 167: 5412-5419.
Copyright © 2001 by The American Association of Immunologists

Diversity of the Human Allergen-Specific T Cell Repertoire Associated with Distinct Skin Test Reactions: Delayed-Type Hypersensitivity-Associated Major Epitopes Induce Th1- and Th2-Dominated Responses1

Judith A. Woodfolk2 and Thomas A. E. Platts-Mills

Asthma and Allergic Diseases Center, Department of Internal Medicine, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Distinct immune responses in humans to the Trichophyton rubrum Ag, Tri r 2, are associated with different patterns of T cell epitope recognition based on in vitro proliferation to peptides derived from this 29-kDa protein. Specifically, the amino-terminal immunodominant epitope, peptide 5 (P5), stimulates strong T cell proliferative responses in subjects with delayed (DTH), but not immediate (IH) hypersensitivity skin tests. Evidence of a role for cytokines or changes in epitope recognition over time was examined in responses to Trichophyton using primary PBMC cultures established from seven IH and seven DTH subjects. Responses stimulated by Tri r 2 were dominated by the Th1 cytokine IFN-{gamma} (IFN-{gamma}:IL-5 >= 4:1) in five DTH subjects, even in the presence of Th2-dominated responses (IFN-{gamma}:IL-5 <= 3:1) to a subset of major epitopes. Paradoxically, P5 induced IL-5 and IL-10 production in DTH, but not IH subjects (p = 0.003 (IL-5), p = 0.024 (IL-10)), with no significant difference in IFN-{gamma} levels between the two groups. In cultures from IH responders, no IL-5 was measurable after stimulation with P6 and P7 (as well as P5); this region of the molecule was shown previously to stimulate markedly reduced T cell proliferation in these individuals. Repeat proliferation assays confirmed no change in the pattern of peptide recognition after >=20 mo in IH or DTH subjects. We conclude that T cell repertoires associated with distinct immune responses to Tri r 2 can be distinguished based on Th2 cytokine induction by DTH-associated major epitopes localizing to the amino-terminal region of the molecule.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Characterization of distinct CD4+ Th lymphocyte subsets by Mosmann et al. (1) in the 1980s opened the door to studies aimed at defining the role of T lymphocytes in the immune response. Ags derived from a variety of pathogens can elicit distinct immune responses, and studies in mice and humans have provided strong evidence for involvement of different CD4+ T lymphocytes in these responses (2, 3, 4, 5, 6). The dermatophyte fungus Trichophyton elicits distinct skin test reactions in different individuals. Immediate hypersensitivity (IH)3 responses are associated with chronic recurrent infections and the presence of serum IgG, and IgG4 Ab, in addition to IgE Ab, specific for Trichophyton Ags (7, 8, 9, 10). Furthermore, improvement of asthma symptoms in a subset of IH responders with late-onset asthma after treatment with the systemic antifungal fluconazole has provided convincing evidence for the role of Ags derived from Trichophyton in allergic disease (11). Delayed-type hypersensitivity (DTH) responses to Trichophyton are common among the general population (12). However, the association of DTH skin tests with acute infections that clear spontaneously has led to the view that these responses, which do not include IgE Ab, are protective (9, 13, 14, 15).

There is strong evidence for the role of Th2 responses to inhalant allergens in humans, as judged by the presence of serum IgE and IgG4 Ab and isolation of allergen-specific Th2 clones (16, 17, 18, 19). IH and DTH responses to Tri r 2 may reflect differential involvement of T lymphocyte subsets with a Th2 and Th1 phenotype, respectively. Indeed, we previously showed that short-term T cell lines established from IH and DTH responders had a Th2/Th0 and Th1 phenotype, respectively (20). However, studying cytokine production induced by whole Ag in T cell clones or lines does not provide insight into the diversity of T cell populations that constitute allergen-specific T cell repertoires. Cloning of Ags derived from Trichophyton rubrum has provided molecular tools for examining the T cell repertoire associated with distinct immune responses to Trichophyton (21). We recently reported major differences between IH and DTH responders in T cell repertoires specific for a 29-kDa allergen derived from T. rubrum, Tri r 2, which is a serine proteinase homologue (22). Those studies identified an amino-terminal immunodominant epitope (peptide 5 (P5)) specifically associated with DTH. The goal of our present study was to examine whether differences in T cell proliferative responses to Tri r 2 peptides in IH and DTH responders reflected differences in production of cytokines associated with distinct Th subsets or differences in stability of the T cell repertoire. Our results show that diminished T cell proliferation to the major epitope P5 in IH responders cannot be explained by decreased production of IFN-{gamma}, and that this epitope is associated with production of the Th2 cytokine IL-5 in DTH, but not IH responders.


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

T cell responses in peptide-stimulated PBMC cultures were analyzed in seven individuals with IH and seven with DTH skin tests to recombinant Tri r 2 (r-Tri r 23). Clinical and serologic characteristics of these skin test groups are described in detail elsewhere (22). Briefly, patients were classified as IH responders based on a positive immediate skin test to Trichophyton extract and to purified r-Tri r 2, and the presence of serum anti-Tri r 2 IgE Ab. DTH responders had a DTH skin test response to Trichophyton extract and r-Tri r 2, and no measurable anti-Tri r 2 IgE Ab. Skin testing of human subjects and drawing of blood samples for T cell studies were approved by the University of Virginia human investigation committee.

Preparation of Ags and peptides

r-Tri r 2 (29 kDa) was produced in Escherichia coli strain BL21 as a GST fusion protein using the pGEX-4T-3 expression vector (21). rGST produced by the same method was used as a negative control in skin test and in vitro lymphocyte cultures. r-Tri r 2 and rGST were absorbed with polymyxin B agarose (Sigma, St. Louis, MO) before use in cell cultures. A set of synthetic peptides, including twenty-seven 20 mers (designated P1 through P27) and one 19 mer (P28), which were designed for mapping T cell antigenic determinants of Tri r 2, was used to study cytokine responses and T cell proliferative responses in primary PBMC cultures. Peptides were designed with a 10-aa overlap spanning the full-length primary amino acid sequence of Tri r 2 (22).

PBMC cultures

PBMC were isolated from 100 ml blood by density gradient centrifugation over Ficoll-Paque, according to methods previously described (22). Conditions for induction of cytokines in peptide-stimulated PBMC cultures were as follows: cells were cultured in 24-well plates at 4 x 106 cells/well in the presence of Tri r 2 peptides (7.5 µM). Complete medium contained RPMI 1640 (Life Technologies, Gaithersburg, MD) with L-glutamine, 10% heat-inactivated autologous human serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Culture supernatants were harvested on day 5 and stored at -70°C before assay. Positive control wells included r-Tri r 2 and PHA, while unstimulated, GST-stimulated, and medium-only wells served as negative controls. Repeat proliferation assays were conducted on one IH and one DTH responder using methods described previously (22). Briefly, PBMC (3 x 105/well in 96-well plates) were stimulated with 7.5 µM peptide for 5 days in complete medium containing 10% autologous serum (non-heat-inactivated). Each assay incorporated 12 replicate cultures for each of 28 peptides and 60 unstimulated wells as negative controls. Each of four 96-well plates consisted of 84 wells containing 7 peptides and 12 unstimulated wells. Plate 5 contained an additional 12 control wells and quadruplicate cultures of GST, r-Tri r 2, tetanus toxoid, and PHA. There was no significant difference in the interval between T cell proliferation studies conducted previously and cytokine studies for DTH (mean, 17 mo (range 9–24 mo)) and IH (mean, 18 mo (range 13–23 mo)) responders.

Chemiluminescent ELISA for IFN-{gamma}, IL-4, IL-5, and IL-10

Cytokines were measured using matched mAb pairs (Pierce-Endogen, Woburn, MA). The assay was conducted at room temperature. Briefly, primary Ab (1 µg/well in 100 µl) diluted in PBS, pH 7.4, was added to 96-well opaque plates (ISC BioExpress, Kaysville, UT) and incubated overnight at room temperature. Plates were washed five times with PBS containing 0.1% Tween 20 between each subsequent step. After washing, plates were blocked for 1 h with PBS, pH 7.4, containing 1% BSA. Culture supernatants were added to the plate in duplicate (100 µl/well) and incubated for 2 h. Plates were then incubated for 1 h with secondary Ab at 10 ng/ml (100 µl/well). After a further 30-min incubation with HRP-streptavidin conjugate (1/320,000 dilution, 100 µl/well), the assay was developed by adding 100 µl/well ELISA Super Signal Pico (Pierce-Endogen) and counted in a Topcount NXT Microplate Luminescent Counter (Packard, Meriden, CT). Values were extrapolated from standard curves established in complete medium on the same assay plate. Sensitivity was 2 pg/ml for IL-4, IL-5, and IFN-{gamma}, and 4 pg/ml for IL-10.

Statistical analysis

Linear regression analysis was used to determine relationships between cytokine levels, serum Ab titers, and T cell proliferation (stimulation index (r-Tri r 2) or standardized index (peptides)). The standardized index was derived as described previously (22). Briefly, data for 28 peptides (12 replicate wells each) were log transformed. The standardized index value was calculated by subtracting the mean proliferation value for 12 unstimulated wells from the mean proliferation value for each peptide contained on the same assay plate (22). Cytokine levels in peptide-stimulated cultures were compared between the two skin test groups using two-sample Student’s t test for IFN-{gamma} (assuming normal distribution of values) and the nonparametric Mann-Whitney U test for independent variables for IL-5 and IL-10. Statistical tests were conducted using the SPSS (Chicago, IL) 10.1 software package.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of cytokines by r-Tri r 2 and antigenic peptides in primary PBMC cultures

Cytokines induced by r-Tri r 2. Measuring T cell proliferative responses to peptides derived from Tri r 2, we previously demonstrated differences in the recognition of T cell epitopes between subjects with IH and DTH skin tests (22). Seven IH (mean age, 59 years) and seven DTH (mean age, 38 years) subjects were available for further study (Table IGo). Initial studies examined kinetics of IL-5 and IFN-{gamma} production in response to r-Tri r 2 and a subset of Tri r 2 peptides (P5, P9, and P25). Stimulation with whole Ag or peptides induced dose-dependent increases in IL-5 and IFN-{gamma}; maximal levels were achieved for r-Tri r 2 and peptides after 4 and 6 days, respectively. Previous studies showed that optimal patterns of T cell proliferation using peptides were achieved after 5 days of culture, and this time point was selected for cytokine studies. Tri r 2 stimulated production of IFN-{gamma}, IL-5, and IL-10 in both IH and DTH responders, with no significant difference in mean cytokine levels or in the ratio of Th1 (IFN-{gamma}) and Th2 (IL-5) cytokines between the two skin test groups (Table IGo). IL-4 was not measurable in any subjects. Cytokine levels were not significantly correlated with stimulation index in r-Tri r 2-stimulated cultures. Production of IFN-{gamma} was positively correlated with serum anti-Tri r 2 IgG Ab levels in the DTH group (r = 0.877, p = 0.01), but was inversely related in the IH group (r = -0.401, p = 0.372) (Table IGo).


View this table:
[in this window]
[in a new window]
  		Table I. Cytokine production in PBMC cultures stimulated with r-Tri r 21

 
Cytokines induced by Tri r 2 peptides. Production of IFN-{gamma}, IL-5, and IL-10 was measured in response to 28 peptides derived from Tri r 2. A high cell density (4 x 106/well) was used to allow for the low expected frequency of Ag-specific T cells in peripheral blood and to optimize conditions for detection of cytokines in early (i.e., day 5) cultures. Each subject showed a unique pattern of cytokine production (Fig. 1Go). Cytokine levels were generally lower in peptide- compared with r-Tri r 2-stimulated cultures; however, some peptides induced IFN-{gamma} at levels comparable with whole Ag (DTH responders, P4, P5, and P9; IH responders, P4 and P9) (Fig. 1Go). Although r-Tri r 2 stimulated Th1-dominated responses (IFN-{gamma}:IL-5 >= 4:1) in the majority of DTH (n = 5) and IH (n = 7) subjects, Th2-dominated responses (IFN-{gamma}:IL-5 <= 3:1) were induced by a subset of peptides in a number of these individuals (three DTH and four IH subjects) (Fig. 1Go). Two DTH responders gave Th2-dominated responses in cultures stimulated with whole Ag and peptides. Surprisingly, the DTH-associated major epitopes induced Th2-dominated peptide responses (denoted by arrows in Fig. 1Go). This response localized to the amino-terminal region of Tri r 2 in DTH, but not IH responders. Furthermore, Th2-dominated responses to peptides were observed in all three DTH subjects with a history of allergic disease (subjects 1, 2, and 7) (Table IGo, Fig. 1Go). Production of IL-10 was generally low in peptide-stimulated cultures, with the exception of two IH responders who gave IL-10-dominated responses (>20 pg/ml) to a subset of peptides (subjects 12 and 14) (Fig. 1Go).



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 1. Profiles of cytokine production in PBMC cultures stimulated with Tri r 2 peptides. Production of IFN-{gamma}, IL-5, and IL-10 was measured in seven DTH (A) and seven IH (B) responders. The final two columns in each plot represent cultures stimulated with rGST (G) and r-Tri r 2 (T). Arrows denote peptides that stimulated strong T cell proliferative responses in previous studies (standardized index >2). P5 (•->) and peptides stimulating Th2-dominated responses (**) are indicated. Cytokines were not measurable in unstimulated wells (no peptide or Ag), with the exception of two individuals in which low levels of IFN-{gamma} were detected. IL-10 was not measured in cultures derived from two subjects (8 and 10) owing to insufficient sample.

 
Previous studies showed that the amino-terminal peptide, P5, induced significantly higher T cell proliferation in DTH responders compared with IH subjects (22). To determine whether diminished T cell proliferation in IH subjects was related to elaboration of cytokines, levels of IFN-{gamma}, IL-5, and IL-10 were compared between the two skin test groups. P5 induced both IL-5 and IL-10 production in cultures established from DTH, but not IH responders (p = 0.003 (IL-5) and p = 0.024 (IL-10)) (Fig. 2Go). Surprisingly, levels of IFN-{gamma} were not significantly different between skin test groups (p = 0.322). The highest levels of IFN-{gamma} (445 pg/ml) induced by P5 corresponded to the strongest proliferative response previously observed to this peptide (standardized index = 4.34, subject 6) (Fig. 2Go). Mean levels of IFN-{gamma} for cultures stimulated with peptides ranged from 3 pg/ml (P26) to 68 pg/ml (P4) for IH responders, and from 4 pg/ml (P26) to 78 pg/ml (P5) for DTH responders (Fig. 3GoA). Mean levels of IL-5 ranged from 2 pg/ml (19 peptides) to 35 pg/ml (P16) for IH subjects, and from 2 pg/ml (P28) to 44 pg/ml (P5) for DTH subjects. The most striking difference between the two groups was the absence of measurable IL-5 in cultures established from IH responders that were stimulated with P5, P6, and P7 (Fig. 3GoB).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 2. Comparison of cytokines induced in P5-stimulated cultures derived from IH and DTH responders. Numbers indicate standardized indexes for T cell proliferation. IL-10 was not measured in two IH responders owing to insufficient sample.

 


View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 3. Mean levels of IFN-{gamma} and IL-5 in peptide-stimulated cultures. Vertical bars represent SEM. Boxes denote six peptides (P6, P7, P8, P24, P25, P26) spanning two distinct regions of the molecule used previously to predict patient classification (22 ). The amino-terminal region of the molecule associated with differences in IL-5 production is denoted ({leftrightarrow}) ; *, p = 0.003 (P5) and p = 0.025 (P7).

 
Relationship between T cell proliferation and IFN-{gamma}, IL-5, and IL-10 production

The region of Tri r 2 spanned by P11 through P15 was associated with decreased production of IFN-{gamma} and IL-5 in both skin test groups (Fig. 3Go, A and B); these peptides failed to induce T cell proliferation in cultures established from IH subjects in previous studies (22). Production of IFN-{gamma}, IL-5, and IL-10 was positively correlated with T cell proliferation (r = 0.384, r = 0.444, r = 0.327 (p < 0.0005), respectively) (Fig. 4Go). However, several peptides that stimulated strong T cell proliferative responses (standardized index >2) did not induce IFN-{gamma} or IL-5 production (Figs. 1Go, 4GoA, and 4B); conversely, several peptides that induced >20 pg/ml IFN-{gamma} did not stimulate T cell proliferation (standardized index <0.5) (Fig. 4GoA). Thus, T cell proliferation and production of Th1 and Th2 cytokines were not consistently associated.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 4. Correlation between T cell proliferation and cytokine production in peptide-stimulated PBMC cultures. Points in A and B represent data for each of the 28 peptides in cultures established from seven IH ({circ}) and seven DTH (•) responders. Data in C are for seven DTH and five IH responders. Dashed lines (A and B) are arbitrary reference lines corresponding to IFN-{gamma} and IL-5 levels of 20 pg/ml.

 
Stability of the Tri r 2-specific T cell repertoire

To determine whether the disparity between T cell proliferative responses and cytokine production could be explained by loss of recognition of antigenic determinants over time, T cell epitopes were mapped by standard proliferation assay using methods previously described. Experiments were conducted in one IH (age 76) and one DTH (age 30) responder, and results were compared with those obtained 20 and 22 mo earlier, respectively. The results showed that the pattern of T cell epitope recognition was consistent for each subject, with no evidence of selective loss or increased recognition of peptides over time (Fig. 5Go).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 5. Long-term stability of T cell epitope recognition in IH and DTH subjects. T cell proliferative responses were measured in one DTH (A) and one IH (B) responder at 22- and 20-mo intervals, respectively. Standardized indexes for different time points are compared for each Tri r 2 peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary goal of these experiments was to examine cytokine production associated with allergen-specific T cell repertoires in humans with distinct skin test reactions to Trichophyton. Differences in production of Th1 and Th2 cytokines associated with IH and DTH were complex. First, culture of PBMC in the presence of the whole protein stimulated both IFN-{gamma} and IL-5 production in DTH and IH responders; however, the majority of these responses were dominated by IFN-{gamma} irrespective of skin test response. Second, r-Tri r 2 stimulated Th2-type responses in two subjects with DTH, but not in any of the IH responders. Finally, production of IL-5 was a major feature of T cell responses to DTH-associated immunodominant epitopes (including P5) in the DTH, but not in the IH skin test group. The unique ability for Tri r 2, unlike other allergens, to elicit distinct skin test reactions in different individuals provides a model for studying the relevance of the Th1/Th2 paradigm to the human system. In keeping with this, we initially hypothesized that differences in T cell proliferation to Tri r 2 peptides between IH and DTH responders would be explained by association of Th1 and Th2 cytokines with DTH and IH responses, respectively. Surprisingly, our findings are contradictory to this theory and suggest that dichotomous immune responses in the human do not adhere to classical Th1 or Th2 profiles. It is our opinion that the complexity of the observed cytokine responses, in large part, reflects the genetic heterogeneity of humans compared with other animal systems that have been used to establish the Th1/Th2 paradigm. Indeed, in murine models of contact sensitivity, regulation of IL-5 production by MHC has been demonstrated (23, 24). Interestingly, in those studies, delayed skin reactions were observed in congenic mouse strains producing high or low levels of IL-5. Similarly, in our studies, levels of IL-5 production were highly variable in subjects with delayed skin tests. Nevertheless, despite the complexity of T cell responses in humans, distinctions between IH and DTH responses, as judged by both T cell proliferation and cytokine production, can be clearly defined.

Obviously, it could be argued that delayed skin responses induced by Trichophyton do not reflect classical DTH, but an alternative response. Delayed reactions to Trichophyton are typically maximal at 24–48 h after injection of Ag into the skin and, like classical DTH, are characterized by erythema and induration. Variations in the manifestations of DTH responses in patients with concurrent dermatophytosis have been reported, including contact hypersensitivity to Trichophyton extract applied to the skin surface and eczematous responses to Ag injected intradermally (12, 25). Furthermore, other types of delayed cell-mediated responses associated with diverse Ags have been described in guinea pigs or humans, including cutaneous basophil hypersensitivity and the Jones-Mote reaction (26, 27, 28). Despite characterization of Langerhans cells and T cells at the site of dermatophyte infection, immunohistologic studies of DTH skin tests to Trichophyton extract have not been conducted (29). Thus, an important next step would be to determine the characteristics of T cells and other cell types in these delayed reactions. Preliminary studies by our group showed that eosinophils were a major component of the cellular infiltrate in delayed skin tests to purified Trichophyton Ags (unpublished observations). These findings raise the intriguing possibility of a role for IL-5 in DTH to Trichophyton. Indeed, in murine models, there is evidence of a role for both eosinophils and IL-5 in different forms of delayed responses (23, 24, 30). Furthermore, there is mounting evidence to support a role for both type 1 and type 2 cytokines in delayed skin reactions associated with Th1-mediated (nickel sensitivity) and Th2-mediated (atopic dermatitis) allergic conditions (31, 32, 33, 34, 35). Our findings are consistent with these observations and underscore the complexity of cellular mechanisms that govern allergic and nonallergic skin test responses. Identification of cell surface markers and intracellular cytokines in Trichophyton-induced delayed skin tests could provide insight into the relevance of T cell subsets and eosinophils at reaction sites; however, such studies may be complicated by expression of Th1 markers (e.g., CXCR3) on both eosinophils and T lymphocytes (36, 37).

There is strong evidence that fungal Ags contribute to development of late-onset asthma in a subset of patients with IH to Trichophyton (11). Production of IL-5 to major epitopes in DTH responders suggests that the lack of association between DTH and asthma cannot be attributed to a protective classical Th1 response to Trichophyton. It seems likely that differences in Ag-specific T cell populations associated with DTH and IH are defined, not solely at the cytokine level, but at the level of TCR specificity (as previously shown), and by other T cell properties. There is evidence to suggest that expression of the skin-specific homing Ag, cutaneous lymphocyte-associated Ag, is down-regulated in T cells specific for Tri r 2 from patients with chronic dermatophytosis (38, 39). Thus, differences in homing of Ag-specific T cells to anatomical sites could explain the different features of skin infection and manifestations of allergic disease associated with distinct immune responses to Trichophyton.

In our system, a subset of peptides induced responses dominated by IL-5 even in the presence of IFN-{gamma}-dominated responses to the whole Ag, suggesting that the frequency of responding cells can modulate cytokine production. It can be assumed that in cultures stimulated with a single peptide, fewer T cells are activated, resulting in lower concentration of cytokines; these conditions may allow cells with a Th2 phenotype to dominate the response. It has previously been reported that the Th1/Th2 balance in Ag-specific systems is dependent on the total number of cells activated (40). Thus, it is reasonable to suggest that the cytokine milieu induced after stimulation with whole Ag reflects complex cellular interactions induced by activation of T cells with multiple specificities and, possibly, different helper phenotypes.

Previous studies by our group showed that T cell lines specific for Trichophyton Ags that were established from IH responders had a Th2/Th0 phenotype (20). In that study, a subset of the cell lines derived from IH subjects produced higher amounts of IFN-{gamma} compared with IL-4 or IL-5. The presence of IFN-{gamma} in peptide-stimulated cultures established from IH responders is consistent with previous studies that report induction of IFN-{gamma} by a variety of allergens or peptides (17, 18, 19, 41, 42, 43). However, the most striking finding associated with IH was a lack of measurable IL-5 in cultures stimulated with peptides spanning the amino-terminal region of Tri r 2, including P5, P6, and P7 (Figs. 2Go and 3Go). This region was previously shown to stimulate markedly reduced T cell proliferation in IH compared with DTH responders (22). Our present results show that decreased T cell proliferation to P5 cannot be explained by diminished IFN-{gamma} production.

We previously proposed that T cell proliferative responses to P5 might promote the development of DTH, thereby preventing progression to chronic dermatophytosis (22). Our cytokine findings suggest that, if this is the case, induction of a Th0 response to P5, as opposed to a Th1 response, may be required to confer protection. More importantly, our results raise the question of how allergen-specific vaccines should be designed for treatment of allergic disease in general. Although IH and DTH responses to Tri r 2 are associated with distinct T cell reactivity to the amino-terminal region of the molecule, it remains to be determined whether administration of a subunit vaccine or a combination of relevant peptides corresponding to this portion would have a protective effect in allergic subjects. IL-10 is a potent antiinflammatory cytokine that inhibits T cell proliferative and cytokine responses, and there is mounting evidence for its role in T cell anergy associated with allergen-specific immunotherapy (44, 45, 46, 47). Furthermore, IL-10 has been shown to inhibit DTH responses induced by Th1 clones in mice (48). We hypothesized that production of IL-10 would be increased in cultures established from IH responders who exhibited decreased T cell proliferation to P5. Two IH responders (subjects 12 and 14) showed production of high levels of IL-10 in peptide-stimulated cultures; however, no IL-10 was measurable in P5-stimulated cultures derived from any of the IH responders. Overall, IL-10 production was positively correlated with proliferation. These findings do not provide strong evidence of a role for IL-10 in IH responses to Tri r 2.

In several of the individuals studied in this work, T cell proliferation and cytokine production can be dissociated for a subset of peptides (Figs. 1Go and 4Go). A similar phenomenon has been described previously using clonal Th cell populations stimulated with altered peptide ligands (49). It is generally accepted that two signals are required for optimal T cell activation (provided by TCR engagement and ligation of a distinct coreceptor). Suboptimal signaling to T cells could occur as a result of low affinity/avidity interactions of specific Tri r 2 peptide-MHC complexes with the TCR, with subsequent uncoupling of distinct signaling pathways required for cell division and cytokine production. Proliferation assays were repeated to investigate whether the disparity between cytokine production and T cell proliferation resulted from loss of epitope recognition over time. The results showed a remarkably consistent pattern over prolonged periods, and also confirmed the reproducibility of the system used. We previously speculated that decreased proliferation to P5 could result from narrowing of the T cell repertoire during aging given the significantly older age of IH responders (22). Our present findings suggest that the allergen-specific T cell repertoire is extremely stable, even in advanced age, and that changes in the repertoire would require many years to develop.

In summary, we have identified distinct features of allergen-specific T cell repertoires associated with immediate and delayed skin reactions based on cytokine production. The amino-terminal major epitope associated with delayed skin tests induces IL-5 production only in T cells from DTH responders. The results demonstrate marked diversity of the allergen-specific T cell repertoire between different subjects. Furthermore, they suggest that, within the same individual, the allergen-specific T cell repertoire comprises T cells with multiple specificities (perhaps with varied affinity or avidity for different epitopes) and with Th1 and/or Th2 characteristics, not closely related to the nature of the skin test response. Stability of the T cell repertoire over time indicates that memory T cells associated with distinct immune response are long-lived, and that decreased T cell proliferative responses to DTH-associated major epitopes in subjects with immediate hypersensitivity are unlikely to be due to selective loss of Ag-specific T cells. Our findings demonstrate the value of detailed studies on T cell responses to allergens, and have important implications for the design of Ag-specific therapies in humans.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI-20565 and AI-34607. Back

2 Address correspondence and reprint requests to Dr. Judith A. Woodfolk, Asthma and Allergic Diseases Center, University of Virginia Health System, PO Box 801355, Charlottesville, VA 22908. E-mail address: jaw4m{at}virginia.edu Back

3 Abbreviations used in this paper: IH, immediate hypersensitivity; DTH, delayed-type hypersensitivity; P, peptide; r-Tri r 2, recombinant Tri r 2. Back

Received for publication July 2, 2001. Accepted for publication August 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mosmann, T. R., H. Cerwinski, M. W. Bond, M. A. Giedlin, R. L. Coffman. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 156:1695.[Abstract]
  2. Abbas, A. K., K. M. Murphy, A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[Medline]
  3. Salgame, P., M. Yamamura, B. R. Bloom, R. L. Modlin. 1992. Evidence for functional subsets of CD4+ and CD8+ T cells in human disease: lymphokine patterns in leprosy. R. L. Coffman, ed. Regulation and Functional Significance of T-Cell Subsets, Chemical Immunology Series 44. S. Karger, Basel.
  4. Munk, M. E., M. Emoto. 1995. Functions of T-cell subsets and cytokines in mycobacterial infections. Eur. Respir. J. 20:668S.
  5. Kemp, M., T. G. Theander, A. Kharazmi. 1996. The contrasting roles of CD4+ T cells in intracellular infections in humans: leishmaniasis as an example. Immunol. Today 17:13.[Medline]
  6. Romani, L., P. Puccetti, F. Bistoni. 1996. Biological role of Th cell subsets in candidiasis. Chem. Immunol. 63:115.[Medline]
  7. Jones, H. E., J. H. Reinhardt, M. G. Rinaldi. 1974. Immunologic susceptibility to chronic dermatophytosis. Arch. Dermatol. 110:213.[Medline]
  8. Hay, R. J., J. Brostoff. 1977. Immune responses in patients with chronic Trichophyton rubrum infections. Clin. Exp. Dermatol. 2:373.[Medline]
  9. Woodfolk, J. A., J. B. Slunt, B. Deuell, M. L. Hayden, T. A. E. Platts-Mills. 1996. Definition of a Trichophyton protein associated with delayed hypersensitivity in humans: evidence for immediate (IgE and IgG4) and delayed type hypersensitivity to a single protein. J. Immunol. 156:1695.
  10. Deuell, B., L. K. Arruda, M. L. Hayden, M. D. Chapman, T. A. E. Platts-Mills. 1991. Trichophyton tonsurans allergen I: characterization of a protein that causes immediate but not delayed hypersensitivity. J. Immunol. 147:96.[Abstract]
  11. Jr Ward, G. W., J. A. Woodfolk, M. L. Hayden, S. Jackson, T. A. E. Platts-Mills. 1999. Treatment of adult onset asthma with fluconazole. J. Allergy Clin. Immunol. 104:541.[Medline]
  12. Wood, S. R., C. N. D. Cruickshank. 1962. The relation between trichophytin sensitivity and fungal infection. Br. J. Dermatol. 74:329.[Medline]
  13. Jones, H. E., J. H. Reinhardt, M. G. Rinaldi. 1974. Model dermatophytosis in naturally infected subjects. Arch. Dermatol. 110:369.[Medline]
  14. Kerbs, S., J. Greenberg, K. Jesrani. 1977. Temporal correlation of lymphocyte blastogenesis, skin test responses, and erythema during dermatophyte infections. Clin. Exp. Immunol. 27:526.[Medline]
  15. Sorensen, G. W., H. E. Jones. 1976. Immediate and delayed hypersensitivity in chronic dermatophytosis. Arch. Dermatol. 112:40.[Abstract]
  16. Parronchi, P., D. Macchia, M. Piccinni, M. Biswas, C. Simonelli, E. Maggi, M. Ricci, A. A. Ansari, S. Romagnani. 1991. Allergen- and bacterial antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production. Proc. Natl. Acad. Sci. USA 88:4538.[Abstract/Free Full Text]
  17. Wierenga, E. A., M. Snoek, H. M. Jansen, J. D. Bos, R. A. van Lier, M. L. Kapsenberg. 1991. Human atopen-specific types 1 and 2 T helper cell clones. J. Immunol. 147:2942.[Abstract]
  18. Van Neerven, R. J., M. M. van de Pol, F. J. van Milligen, H. M. Jansen, R. C. Aalberse, M. L. Kapsenberg. 1994. Characterization of cat dander-specific T lymphocytes from atopic patients. J. Immunol. 152:4203.[Abstract]
  19. Spiegelberg, H. L., L. Beck, D. D. Stevenson, G. Y. Ishioka. 1994. Recognition of T cell epitopes and lymphokine secretion by rye grass allergen Lolium perenne I-specific human T cell clones. J. Immunol. 152:4706.[Abstract]
  20. Slunt, J. B., E. A. Taketomi, J. A. Woodfolk, M. L. Hayden, T. A. E. Platts-Mills. 1996. The immune response to Trichophyton tonsurans: distinct T cell cytokine profiles to a single protein among subjects with immediate and delayed hypersensitivity. J. Immunol. 157:5192.[Abstract]
  21. Woodfolk, J. A., L. M. Wheatley, R. V. Piyasena, D. C. Benjamin, T. A. E. Platts-Mills. 1998. Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity: sequence homology to two families of serine proteinases. J. Biol. Chem. 273:29489.[Abstract/Free Full Text]
  22. Woodfolk, J. A., S. J. Sung, D. C. Benjamin, J. K. Lee, T. A. E. Platts-Mills. 2000. Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2. J. Immunol. 165:4379.[Abstract/Free Full Text]
  23. Dieli, F., G. Sireci, D. Lio, C. T. Bonanno, A. Salerno. 1993. Major histocompatibility complex regulation of interleukin-5 production in the mouse. Eur. J. Immunol. 23:2897.[Medline]
  24. Dieli, F., G. L. Asherson, C. T. Bonanno, G. Sireci, A. Salerno. 1995. Major histocompatibility complex control of the class of the immune response to the hapten trinitrophenyl. Immunology 84:355.[Medline]
  25. Tagami, H., S. Watanabe, S. Ofuji, K. Minami. 1977. Trichophytin contact sensitivity in patients with dermatophytosis. Arch. Dermatol. 113:1409.[Abstract]
  26. Richerson, H. B., H. F. Dvorak, S. Leskowitz. 1970. Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote reaction, general characteristics. J. Exp. Med. 108:823.
  27. Mitchell, E. B., P. W. Askenase. 1983. Basophilia in human disease. Clin. Rev. Allergy 1:427.[Medline]
  28. Graziano, F. M., L. Gunderson, L. Larson, P. W. Askenase. 1983. IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs. J. Immunol. 131:2675.[Abstract]
  29. Szepes, E., M. Magyarlaki, Z. Battyani, I. Schneider. 1993. Immunohistological characterization of the cellular infiltrate in dermatophytosis. Mycoses 36:203.[Medline]
  30. Teixeira, M. M., A. Talvani, W. L. Tafuri, N. W. Lukacs, P. G. Hellewell. 2001. Eosinophil recruitment into sites of delayed-type hypersensitivity reactions in mice. J. Leukocyte Biol. 69:353.[Abstract/Free Full Text]
  31. Eerfel, T., M. Hentschel, A. Knapp, H. Renz. 1997. Dichotomy of blood- and skin-derived IL-4 producing allergen-specific T cells and restricted V{beta} repertoire in nickel-mediated contact dermatitis. J. Immunol. 158:2500.[Abstract]
  32. Kapsenberg, M. L., E. A. Wierenga, F. E. M. Stiekema, A. B. C. Tiggelman, J. D. Bos. 1992. Th1 lymphokine production profiles of nickel-specific CD4+ T lymphocyte clones from nickel contact allergic and nonallergic individuals. J. Invest. Dermatol. 98:59.[Medline]
  33. Spergel, J. M., E. Mizoguchi, H. Oettgen, A. K. Bhan, R. S. Geha. 1999. Roles of Th1 and Th2 cytokines in a murine model of allergic dermatitis. J. Clin. Invest. 103:1103.[Medline]
  34. Grewe, M., K. Gyufku, K. Schopf, J. Krutman. 1994. Lesional expression of interferon-{gamma} in atopic eczema. Lancet 343:25.[Medline]
  35. Tsicopoulos, A., Q. Hamid, A. Haczku, M. R. Jacobson, S. R. Durham, J. North, C. J. Corrigan, Q. Meng, R. Moqbel. 1994. Kinetics of cell infiltration and cytokine messenger RNA expression after intradermal challenge with allergen and tuberculin in the same atopic individuals. J. Allergy Clin. Immunol. 94:764.[Medline]
  36. Jinquan, T., C. Jing, H. H. Jacobi, C. M. Reimert, A. Millner, S. Quan, J. B. Hansen, S. Dissing, H.-J. Malling, P. S. Skov, L. K. Poulsen. 2000. CXCR3 expression and activation of eosinophils: role of IFN-{gamma}-inducible protein-10 and monokine induced by IFN-{gamma}. J. Immunol. 165:1548.[Abstract/Free Full Text]
  37. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A. E. Koch, B. Moser, C. R. Mackay. 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101:746.[Medline]
  38. Ludwig, R. J., J. A. Woodfolk, M. Grundmann-Kollmann, R. Enzensberger, M. Podda, U. Runne, T. A. E. Platts-Mills, R. Kaufmann, and T. M. Zollner. 2001. Chronic dermatophytosis in lamellar ichthyosis: relevance of a Th2 immune response to Trichophyton. Br. J. Dermatol. In press.
  39. Zollner, T. M., M. Podda, R. Kaufmann, T. A. Platts-Mills, and J. A. Woodfolk. 2001. Increased incidence of skin infections in atopy: evidence for an antigen-specific homing defect? Clin. Exp. Allergy. In press.
  40. Grakoui, A., D. L. Donermeyer, O. Kanagawa., K. M. Murphy, P. M. Allen. 1999. TCR-independent pathways mediate the effects of antigen dose and altered peptide ligands on Th cell polarization. J. Immunol. 162:1923.[Abstract/Free Full Text]
  41. Hales, B. J., H. Shen, W. R. Thomas. 2000. Cytokine responses to Der p 1 and Der p 7: house dust mite allergens with different IgE-binding activities. Clin. Exp. Allergy 30:934.[Medline]
  42. Looney, R. J., D. Pudiak, S. I. Rosenfeld. 1994. Cytokine production by mite-specific T cells from donors with mild atopic disease. J. Allergy Clin. Immunol. 93:476.[Medline]
  43. Byron, K. A., R. M. O’Brien, G. A. Varigos, A. M. Wootton. 1994. Dermatophagoides pteronyssinus II-induced interleukin-4 and interferon-{gamma} expression by freshly isolated lymphocytes of atopic individuals. Clin. Exp. Allergy 24:878.[Medline]
  44. Del Prete, G., M. DeCarli, F. Almerigogna, M. G. Giudizi, R. Biagiatti, S. Romagnani. 1993. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J. Immunol. 150:353.[Abstract]
  45. Akdis, C. A., T. Blesken, M. Akdis, B. Wuthrich, K. Blaser. 1998. Role of interleukin-10 in specific immunotherapy. J. Clin. Invest. 102:98.[Medline]
  46. Schandene, L., C. Alonso-Vega, F. Willems, C. Gerard, A. Delvaux, T. Velu, R. Devos, M. de Boer, M. Goldman. 1994. B7/CD28-dependent IL-5 production by human resting T cells is inhibited by IL-10. J. Immunol. 152:4368.[Abstract]
  47. Joss, A., M. Akdis, A. Faith, K. Blaser, C. A. Akdis. 2000. IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway. Eur. J. Immunol. 30:1683.[Medline]
  48. Li, L., J. F. Elliott, T. R. Mosmann. 1994. IL-10 inhibits cytokine production, vascular leakage, and swelling during T helper 1 cell-induced delayed-type hypersensitivity. J. Immunol. 153:3967.[Abstract]
  49. Evavold, B. D., P. M. Allen. 1991. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science 252:1308.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 QJMHome page
S.L. Seneviratne, A.P. Black, L. Jones, A.S. Bailey, and G.S. Ogg
The role of skin-homing T cells in extrinsic atopic dermatitis
QJM, January 1, 2007; 100(1): 19 - 27.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
J. A. Woodfolk
Allergy and Dermatophytes
Clin. Microbiol. Rev., January 1, 2005; 18(1): 30 - 43.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. J. Reefer, R. M. Carneiro, N. J. Custis, T. A. E. Platts-Mills, S.-S. J. Sung, J. Hammer, and J. A. Woodfolk
A Role for IL-10-Mediated HLA-DR7-Restricted T Cell-Dependent Events in Development of the Modified Th2 Response to Cat Allergen
J. Immunol., March 1, 2004; 172(5): 2763 - 2772.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woodfolk, J. A.
Right arrow Articles by Platts-Mills, T. A. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woodfolk, J. A.
Right arrow Articles by Platts-Mills, T. A. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS