MHC class II restriction for T cell proliferative response to mite antigen

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1 MHC class II restriction for T cell proliferative response to mite antigen Hitoshi Matsuoka, MD; Hisamitsu Uno, MD; Kiyohide Kawano, MD; Kazunori Tsuda, MD; and Hirohito Tsubouchi, MD Background: We investigated the immunoreguratory role of the major histocompatibility complex in peripheral blood lymphocytes proliferative response to mite antigen. Method: Peripheral blood lymphocytes of Japanese asthmatic patients were incubated with antigen obtained from Dermatophagoides pteronyssinus with a molecular weight of about 15,000, with and without 0.05 g/ml of monoclonal antibody against HLA class I or class II for seven days at 37 C humidified in 5% CO 2 and 95% air. Results: High and low responders to the mite body antigen were found among the patients while there were no high responders among the healthy individuals tested. In the mite-sensitive asthmatic patients, CD4 T cells were the population that responded to the antigen. Depletion of CD8 T cells from the peripheral blood lymphocytes of mite-insensitive individuals caused high responsiveness to the antigen, indicative that the mite-specific CD4 T cells controlled the high responsiveness and the antigen-specific CD8 T cells, the low responsiveness. Anti- HLA-DR monoclonal antibody inhibited responsiveness. In contrast, anti-hla-dq monoclonal antibody produced high responsiveness in low responders to mite antigen. Conclusion: High T cell proliferative responsiveness to mite antigen was restricted by HLA-DR antigen through CD4 T cells in high responders, whereas HLA-DQ antigen is a restriction antigen of low responsiveness through CD8 T cells in low responders and non-atopic individuals. Ann Allergy Asthma Immunol 1996;77: From the Second Department of Internal Medicine, Miyazaki Medical School, Miyazaki, Japan. Received for publication December 30, Accepted for publication in revised form March 13, INTRODUCTION Dermatophagoides pteronyssinus (Dp) is one of the most common species of mite in house dust. 1 3 Mite-specific IgE production may be critical for the onset of allergic diseases, especially bronchial asthma. Intensive serologic studies have characterized two dominant allergenic fractions from Dp; Der I with a molecular weight (MW) of about 24,000 and Der II with one of about 15, T cell dependency of IgE antibody production in response to aero-allergens such as Dermatophagoides has been demonstrated 7 9 ; however, T cell immunity to mite allergens has been little studied. For T cell activation, co-recognition of the antigen in association with the membrane proteins encoded by the major histocompatibility complex (MHC) is essential. The HLA-D region loci (DR, DQ, and DP) are reported to function as restriction elements in the presentation of extrinsic antigen. The evidence for this is serologic inhibition in antigen-dependent activation of cloned T cells. 10,11 Increased responsiveness to Cryptomeria japonica pollen antigen, is reported to be associated primarily with HLA-linked genes. 12 We have reported elsewhere that the Dp bodyderived allergen (Der p II) induces peripheral blood lymphocyte proliferation only in mite-sensitive asthmatic patients. 13 We extended that study by investigating T cell immunity in terms of the T cell proliferative response to Der p II in atopic and nonatopic Japanese individuals, and have clarified the role of HLA antigens in the immune response to antigen. MATERIALS AND METHODS Patients and Healthy Subjects Asthmatic patients with high anti-dp IgE antibody titer in their sera were studied as mite-sensitive asthmatic patients. Nonasthmatic healthy persons were the controls. Preparation of Antigen We have reported elsewhere the preparation of antigen derived from mite bodies. 13 Briefly, mites were cultured and isolated using to the method of Miyamoto et al. 14 The mites collected were homogenized in PBS (0.01 M, ph 7.2) and applied to a Sephadex G-75 column ( cm). The elution buffer was 0.01 M phosphate buffered saline, and molecular weight fractions were obtained. IgE binding activity was measured for each fraction using pooled sera from mite-sensitive asthmatic patients and the enzyme linked immunosorbent assay (ELISA). The fraction with high IgE binding activity, prepared for Dp body-derived antigen, was designated Dp2. The molecular weight of Dp2 was about 15,000, compatible with that of Der p II. Preparation of Peripheral Blood Lymphocyte Peripheral blood lymphocytes (PBLs) were separated from heparinized peripheral blood by Ficoll-Conray density gradient centrifugation (specific gravity 1.077). After being washed, the cells were resuspended in RPMI1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf se- VOLUME 77, SEPTEMBER,

2 rum, penicillin 100 u/ml, streptomycin 100 g/ml, L-glutamine 20 mm, and anti-pplo 100 U/mL (RPMI-10% fetal calf serum). Monoclonal Antibody Murine monoclonal antibody against human MHC class I (anti-hla-abc) was purchased from the Australian Monoclonal Development Pty., Astarmon, Australia. Anti-human MHC class II (anti-hla-dr and anti-hla- DQ), Leu2b (anti CD8), and Leu3a (anti-cd4) antibodies were purchased from Becton-Dickinson, Mountain View, CA. Ten microliters of monoclonal antibody was added to each well containing with Dp2 (final concentration 0.05 g/ml). Cell Fractionation Peripheral blood lymphocytes suspended in 10 ml of 5% fetal calf serum/phosphate buffered saline were placed in a plastic dish (90 20 mm, Fischer, Canada) and incubated for 60 minutes at 37 C in humidified 5% CO 2 and 95% air. After extensive washing, the dish-adhering cells were removed by scraping with a rubber policeman, and used as the macrophage fraction. The nonadhering cells were used as macrophage-depleted fraction. T and B cells were separated from the nonadhering cells by the nylon wool column method. The nylon wool nonadhering cells were collected as T cells which were divided into CD4 T and CD8 T cell fractions by the panning method of Wysocki and Sato 15 using anti Leu2a or anti-leu3a antibody. Antigen-Specific Peripheral Blood Lymphocyte Proliferative Response In a flat-bottomed, 96-well microtiter plate (Nunc, Roskilde Denmark), 200 L of a PBL suspension containing cells was suspended in each well, to which 20 L of antigen-containing or antigen-free RPMI-10% fetal calf serum was added. Optimal antigen concentration and incubation time were determined in our previous study 13 : peripheral lymphocyte proliferation to Dp2 showed maximum response on day 8 at the antigen concentration of 10 g/ml. After seven days of incubation at 37 C in humidified 5% CO 2 and 95% air, 1 Ci of 3 H- thymidine was added to each well for the final 12 hours. The incorporation of 3 H-thymidine in triplicate cultures was counted by liquid scintillation spectrometry. Data analysis was based on counts per minute (cpm) or a stimulation index (SI). The stimulation index was calculated as (cpm in the presence of the antigen)/(cpm in the control culture). Statistical Analysis Student s t test was used for the statistical analysis. Table 1. T Cell Population Responsed to Dp2 Cells Patient Dp2 Whole CD8( ) CD4( ) (SI) * (SI) * (SI) ,807 (27.32) 1,058 30,159 (28.56) 986 1,015 (1.03) ,800 (3.44) 1,201 5,968 (4.97) 1,102 2,060 (1.78) ,488 (4.27) 483 2,580 (5.34) 785 1,397 (1.78) Three mite-sensitive asthmatic patients were tested. Peripheral blood lymphocytes were cultured with/without Dp2 (10 g/ml) as described in Materials and Methods. The results were expressed as mean cpm and stimulation index. * P.01, P values are for the T cell proliferation with Dp2 antigen of the CD8( ) cells compared with that of the CD4( ) cells. RESULTS T Cell Population Responding to the Mite Antigen, Dp2 To determine the subset of the responding T cell population, PBLs depleted of CD4 T cells or CD8 T cells were incubated with Dp2 (Table 1). Three mite-allergic asthmatic patients whose PBLs showed high proliferative responses to Dp2 were tested. An enhanced proliferative response to Dp2 was observed in CD8 PBLs. The SI of the CD8 PBLs is 1.5 to 2.0 times that of the entire PBLs. In contrast, CD4 PBLs did not respond to Dp2, indicating that the CD4 T cells were responsible for the proliferative response. To examine the effects of CD8 T cells on the proliferative response to Dp2, we depleted the CD8 T cells from the PBL of asthmatic patients who were low responders to Dp2 (Table 2). The whole PBLs from seven asthmatic patients had low proliferative responses to Dp2. The depletion of CD8 T cells from the PBLs induced a high response to Dp2. When CD8 T cells were autologously reconstituted to CD8 PBLs, high responsiveness to Dp2 was inhibited (Fig 1). CD8 T cells from low responders to Dp2 did not inhibit the high responsiveness to candida antigen in asthmatic patients who showed high proliferative responses to candida but not to mite (data not shown). Macrophage Dependence in Dp2- Induced Lymphocyte Proliferative Response In order to define whether T cells or macrophages contribute as restriction elements in the proliferative response to Dp2, macrophages and T cells were separated from either high responder of low responder to the antigen, and reconstituted to test the response. Neither T cells nor macrophages from five high responders showed proliferative response to the antigen; however, reconstitution of T cells and macrophages restored high responsiveness to Dp2 (Fig 2). HLA Class II Antigen Restriction in Dp2-Induced Lymphocyte Proliferative Response To evaluate the role of HLA antigens in mite antigen-induced T cell proliferative response, we incubated PBLs from mite-sensitive asthmatic patients whose PBLs showed high proliferative responses to Dp2 with the antigen 192 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY

3 Table 2. Effect of the CD8 T Cell in the Proliferative Response to Dp2 Cells Patient Dp2 Whole CD8( ) * (SI) * (SI) ,017 (1.10) 905 1,948 (2.05) 2 1,031 1,412 (1.37) 980 3,419 (3.48) ,202 (1.87) 853 2,815 (3.30) (0.75) 907 3,428 (3.78) (1.67) 753 2,470 (3.28) (1.02) 761 4,451 (5.85) ,343 (1.69) 654 1,590 (2.43) Seven asthmatic patients who were low responders to Dp2 were tested. Peripheral blood lymphocytes (PBLs) and CD8 PBLs were cultured with/without Dp2 as described in Materials and Methods. The results were expressed as mean cpm and stimulation index (SI). * P.01, P values are for T cell proliferation with the Dp2 antigen of whole cells compared with that of CD8 cells. Figure 1. Suppression by CD8 positive T cells in proliferative response to Dp2. Peripheral blood lymphocytes were separated from four nonatopic individuals. By panning method using mouse monoclonal anti-human CD8 antibody, nonadherent cells were used as CD8 T cells depleted PBLs and adherent cells were used as CD8 T cells. Peripheral blood lymphocytes (1 105/well) were cultured with Dp2 (10 g/ml) as described in Materials and Methods. The results were expressed as stimulation index. The number in each column and the bar on the top of column represent the mean cpm with the standard deviation, respectively *P.01. Figure 2. Macrophage dependence in T cell proliferative response to Dp2. Five mite-sensitive asthmatic patients were tested. Macrophages were isolated from PBLs by plastic dish adherence. T cells were collected by nylon wool column method. The results were expressed as stimulation index. The number in each column and the bar on the top of column represent the mean cpm with standard deviation, respectively *P.01. with/without anti-hla class I or anti- HLA class II MoAb. As shown in Figure 3, anti-hla-dr MoAb blocked the proliferative response to the antigen whereas MoAb to HLA class I did not. The role of HLA class II subregion antigens in the interaction between macrophages and T cells was investigated in the presence of Dp2. Peripheral blood lymphocytes from eight mite-sensitive asthmatic patients were incubated with Dp2 and anti HLA-DR or -DQ MoAb. In the PBLs of the high responders, MoAb to the HLA-DR framework inhibited the T cell proliferative response whereas MoAb to HLA-DQ did not. These results indicate that DR-bearing macrophages were responsible for the Dp2-induced T cell proliferative response in mitesensitive asthmatic patients whose PBLs showed high proliferative responses to Dp2. When PBLs of the low responders to Dp2 were incubated with MoAb to HLA class II, anti-hla-dq antibody induced a high proliferative response to the antigen but MoAb to HLA-DR did not (Table 3). MHC Restriction Between T Cells and APCs in the Proliferative Response to Dp2 Major histocompatibility complex restriction between T cells and macrophages was investigated in one mitesensitive individual (P1) and in four non-atopic individuals (P2, P3, P4, and P5). Peripheral blood lymphocytes from P1 showed a high response to Dp2, whereas the PBLs from the four non-atopic individuals showed low responses to the antigen. The macrophages derived from P2 and P3, whose HLA-DR antigens were identical to those of P1, induced a high proliferative response in cooperation with the T cells of P1; those of P4 and P5, which had haploidentical HLA-DR antigens with P1, failed to induce a high response to P1. Macrophages derived from P1 did not induce high responses to the T cells of all the low responders irrespective of the HLA-DR antigens (Table 4). VOLUME 77, SEPTEMBER,

4 DISCUSSION House dust mite is the most prevalent and important source of inhaled allergens for inducing allergic disease. 2 Dermatophagoides is one of the most common species of house dust mite. 1,3 Many efforts have been made to define the major immunodominant determinants to the mite allergen in order to gain an understanding of the immunopathogenesis of mite allergy. 4,16,17 Several important allergens, the main antigens of anti-mite IgE antibodies in allergic individuals, have been found. Included in the components that strongly bind IgE are those in fractions ranging from 13,000 to 95,000 MW. Studies using monoclonal antibodies have characterized two major allergens derived from the mite of the genus Dermatophagoides. Der I with a MW of about 25,000 4 and Der II with one of about 15, Previously, we fractionated a mite body-derived allergen designated Dp2, using the binding activity of the anti-mite IgE antibody. The molecular weight of Dp2 is about 15,000 and is compatible with that of Der II. 13 In the study reported here, the T cell proliferative response to Dp2 and the roles of MHC antigens in the response were investigated. The T cell response to Dp crude mite antigen, 10 P1 11 and antigens derived from Dermatophagoides farinae (Df), 18 have been studied recently. Results indicate that these antigens can induce T cell immunity. Kimura et al 19 studied the T cell proliferative response and IL-2 production in the fraction derived from Dp. They reported that the molecular weight of about 95,000 had a sharp high peak response and differed from the weights of the IgE-binding molecules. In our study, we investigated T cell immunity to the fraction responding to IgE with Dp2. O Hehir et al 10 showed that the immune response of human T cell clones to Df antigen was regulated by HLA antigens in patients with perennial rhinitis. The T cell proliferative response to the mite antigen Dp2 in mite-sensitive asthmatic patients is evidence that the Figure 3. Effect of anti MHC antibody in T cell proliferative response to Dp2. Seven mite-sensitive asthmatic patients who were high responders to Dp2 were investigated. Peripheral blood lymphocytes ( /ml) were cultured with Dp2 (10 g/ml) with monoclonal anti-human MHC antibody, anti-hla class I (0.05 g/ml) and HLA-DR (0.05 g/ml), for seven days. The results were expressed as stimulation index. The number in each column and the bar on the top of column represent the mean cpm with standard deviation, respectively *P.01. Table 3. Effect of Anti-MHC Antibody in the Proliferative Response of T Cell to Dp2 Antibody Patient Dp2 None Anti-DR (SI) Anti-DQ (SI) (0.70) 610 (1.20) 1,500 (2.87) ,195 (4.80) 255 (0.41) 1,723 (3.60) ,760 (2.12) 695 (1.54) 1,980 (2.62) ,280 (1.32) 780 (0.90) 2,113 (2.14) ,940 (2.15) 562 (0.60) 1,950 (2.01) (1.13) 761 (1.01) 2,390 (3.05) ,120 (1.13) 680 (0.72) 2,455 (3.22) 8 1, ,735 (9.62) 5,575 (5.67) 12,940 (13.62) Eight mite-sensitive asthmatic patients were tested. T cells collected by the nylon wool column method were with Dp2 with/without anti-hla-dr (0.05 g/ml) or anti HLA-DQ antibody (0.05 g/ml) as described in Materials and Methods. The results were expressed as mean cpm and stimulation index (SI). A SI over 2 was regarded as being positive for the T cell response. Dp2-induced T cell response was a macrophage-dependent response and that the T cells that proliferated in the presence of the antigen were the CD4 T cell subset. The depletion of CD8 T cells induced high responsiveness in low responders, and reconstitution of CD8 T cell subset inhibited high responsiveness to Dp2. The CD4 T cells of the low responders therefore may have the capacity for high responsiveness to the antigen, and CD8 T cell may suppress the T cell response to the antigen in low responders in an antigen-specific manner, which was demonstrated by the fact that CD8 cells from Dp2 low responders did not inhibit the responsiveness to candida antigen in the same individuals. We could not show the mechanism of CD8 T cell-related CD4 T cell suppression in this study in the context of HLA Class I and HLA-DQ antigen. Sasazuki et al, however, have reported there are high and low responders to 194 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY

5 Table 4. Cooperation Between T Cells and Allogenic Macrophage Sharing HLA-DR Antigen Dp2 T cell Macrophage (SI) P1(DR4,DR8) P ,039 (7.68) P1 P2(DR4,DR8) 983 5,121 (5.21) P1 P3(DR4,DR8) 1,095 3,624 (3.31) P1 P4(DR1,DR4) (0.89) P1 P5(DR4, ) (0.93) P2 P ,015 (1.32) P3 P1 1,168 1,097 (0.94) P4 P1 1,054 1,306 (1.24) P5 P ,136 (1.18) One mite-sensitive individual (P1) and four nonatopic individuals (P2, SI, P4, and P5) were investigated. P1 was a high responder and the other four individuals were low responders to Dp2. T cells were collected by the nylon wool column method and macrophages were isolated from PBLs by plastic adherence. HLA-DR haplotype; P1(DR4,DR8) P2(DR4,DR8) P3(DR4, DR8) P4(DR1, DR4) P5(DR4, ). The results were expressed as mean cpm and stimulation indexsi). A SI over 2 was regarded as being positive for T cell response. the streptococcal cell wall antigen (SCW) in a human population. 20 They established an SCW-specific CD4 T cell lines 21 and used them to analyze the mechanism of low responsiveness to SCW at the cellular level. They showed that DQ molecules as well as DR molecules were used as restriction molecules in low responders and CD4 cell lines were a mixture of T cells restricted by DR or DQ molecules. In low responders, a small portion of CD8 T cells was also found. Experiments using these cell lines suggested that CD4 suppressor-inducer T cells, which activate CD8 suppressor T cells to suppress the proliferative response of CD4 T cells, recognized SCW antigen in the context of DQ molecule. HLA-DR upregulates the immune response and HLA-DQ down regulates it through induction of CD8 T cells by DQ restricted CD4 suppressor-inducer T cells. We therefore assume that CD4 suppressor-inducer T cells which activate DP2-specific CD8 suppressor T cells to suppress the proliferative response of antigen-specific CD4 T cells recognized Dp2 antigen in the context of HLA-DQ molecule. They reported the existence of autologous cytotoxic CD8 T cells in the proliferative response to the SCW 22 and showed that the CD8 T cell line does not lyse autologous CD4 T cell lines. They therefore suggested that the suppressive effect of CD8 T cell on the proliferation of SCW specific CD4 T cells may be mediated by cytotoxicity against monocytes. We have shown that the mite-induced CD4 T cell response is dependent on macrophages as the antigen presenting cells. The same mechanism may function in CD8 -related CD4 suppression in the mite-induced T cell response. Although almost all humans in their daily environment are exposed to mites, only a few develop asthma. A lack or decrease of antigen-specific CD8 T cells may be involved in the onset of atopic asthma. Macrophages and T cells were separated from high and from low responders, then reconstituted in order to study the response to mite antigen. The reconstitution test showed that macrophages from low responders induced a high response to the T cells from high responder when they had identical HLA-DR haplotypes. Concerning human CD4 T cells, the HLA-D region loci is reported to be able to conduct all the functions of restriction elements on the presentation of extrinsic antigen. In Cryptomeria japonica pollen (CP) allergy, Matsushita et al 12 reported that HLA-DR is most likely to be a product of the human immuneresponse genes to CP-Ag and the HLA-DQ a product of immune-suppress genes. In mite allergy, O Heir et al 10 and Yessel et al 11 studied the MHC class II restriction in human T cell clones induced by mite antigen and reported that recognition of the antigen might be restricted by elements associated with DRw52 and DRw53 supertypic specificity. Their study, however, dealt only with MHC restriction in high responsiveness and used T cell clones derived from high responders. We studied the T cell response to Dp antigen using fresh PBLs from both high and low responders. In our study, anti HLA class I MoAb failed to inhibit high responsiveness. It is intriguing that anti-hla-dq antibody induced high responses in low responders. That observation suggests that antigen recognition is regulated by immune-response genes of the HLA-DR products through CD4 T cells in high responders to Dp2. In contrast, the HLA-DQ products in low responders to Dp2, may act as immune-suppressor genes in antigen-dependent proliferation through CD8 T cells. Major histocompatibility complex class II in mice act as restriction elements in the activation of helper or suppressor T cells when extrinsic antigens are challenged In these systems, antigens are presented to helper T cells in association with the I-A product on antigen presenting cells, whereas suppressor T cell are stimulated by antigens in the context of the I-E on antigen presenting cells. I-A is comparable to HLA-DQ and I-E to HLA-DR. Although the human HLA-D region is much more complicated than the murine H-2 I region, it appears that in both mice and humans, one of the two molecules encoded by the duplicated class II loci controls the immune response and the other controls immune suppression. Last, our present findings should facilitate characterization of T cell immunity to the mite. Further analysis of the T cell response involved in cytokine production and the organ-specific affinity of T cell immunity should VOLUME 77, SEPTEMBER,

6 show the mechanism for the onset of atopic disease to mites and lead to the establishment of new therapies. REFERENCES 1. Voorhorst R, Spieksma FThM, Varekamp H, et al. The house-dust mite (Dermatophagoides pteronyssinus) and allergens it produces. Identity with the house-dust allergen. J Allergy 1967;39: Voorhorst R, Spieksma BMI, Spieksma FThM. Is a mite (Dermatophagoides sp.) the producer of the house-dust allergen? Allergy Asthma 1964;10: Ishii A, Shimomura H, Hashiguchi J, Kabayama Y. Biochemical and allergenic properties of the house dust mite extract Dermatophagoides pteronyssinus. Allergy 1982;37: Chapman MD, Heymann PW, Wilkins SR, et al. Monoclonal immunoassay for major dust mite (Dermatophagoides) allergens, Der PI and Der fi, and quantitative analysis of the allergen content of mite and house dust extracts. J Allergy Clin Immunol 1987; 80: Anderson A. Isolation and characterization of an allergen-rich fraction derived from cultures of Dermatophagoides pteronyssinus. Int Arch Allergy Appl Immunol 1989;89: Yasueda H, Mita H, Yui T, Shida T. Isolation and characterization of two allergens from Dermatophagoides farinae. Int Arch Allergy Appl Immunol 1986;81: Gurbindo C, Tricas L, Lauzurica P, et al. T cell dependence of human IgE response: quantitative and functional studies. Int Arch Allergy Appl Immunol 1985;77: Kanowith-Klein S, Saxon A. Regulation of ongoing IgE synthesis by human T cell supernatant derived from atopic and nonatopic donors. Int Arch Allergy Appl Immunol 1986;80: Halvorsen R, Bosnes V, Thorsby E. T cell response to a Dermatophagoides farinae allergen preparation in allergic and healthy controls. Int Arch Allergy Appl Immunol 1986;80: O Heir RE, Eckels DD, Frew AJ, et al. MHC class II restriction specificity of cloned human T lymphocytes reactive with Dermatophagoides farinae (house dust mite). Immunology 1988; 64: Yssel H, Johnson KE, Schneider PV, et al. T cell activation-inducing epitopes of the house dust mite allergen Der pi, proliferation and lymphokine production patterns by Der pispecific CD4 T cell clones. J Immunol 1992;148: Matsushita S, Muto M, Suemura M, et al. HLA-linked nonresponsiveness to Cryptomeria japonica pollen antigen I. Nonresponsiveness is mediated by antigen-specific suppressor T cell. J Immunol 1987;138: Matsuoka H, Uno H, Kawano K, et al. The immune response specific to HLA-linked nonresponsiveness Dermatophagoides pteronyssinus body antigen in asthmatics. Jpn J Allergol 1988;37: Miyamoto J, Ishii A, Sasa M. A successful method for mass culture of the house dust mite Dermatophagoides pteronyssinus (Trouess-art, 1947). Jpn J Exp Med 1978;45: Wysocki LJ, Sato VL. Panning for lymphocytes: a method for cell selection. Proc Natl Acad Sci USA 1978; 75: Lind D. Purification and partial characterization of two major allergens from the house dust mite Dermatophagoides pteronyssinus. J Allergy Clin Immunol 1985;76: Tovey ER, Baldo BA. Comparison by electroblotting of IgE-binding components in extracts of house dust mite bodies and spent mite culture. J Allergy Clin Immunol 1987;79: Gavaillon JM, Fitting C, Guinnepain MT, et al. Lymphocyte proliferative response to the purified Dermatophagoides farinae major allergen in untreated and hyposensitized atopic patients. Allergy 1988;43: Kimura JY, Ohta N, Ishii A, et al. Functional characterization of lymphocyte response to fractionated house dust mite antigens (Dermatophagoides pteronyssinus) in atopic and nonatopic individuals. Immunol 1990;70: Sasazuki T, Kaneoka H, Nishimura Y, et al. An HLA-linked immune suppression gene in man. J Exp Med 1980; 152:297s 313s. 21. Hirayama K, Matsushita S, Kikuchi I, et al. HLA-DQ is epistatic to HLA-DR in controlling the immune response to schistosomal antigen in humans. Nature 1987;6121: Yoshizumi H, Kamikawaji N, Nishimura Y, Sasazuki T. Generation of a novel CD8 cytotoxic T lymphocyte that requires soluble factor to lyse autologous antigen-presenting cells. Eur J Immunol 1993;23: Bazevanis CN, Ishii N, Nagy ZA, Klein J. H-2-controlled suppression of T cell response to lactate dehydrogenase B. J Exp Med 1982;156: Nagy ZA, Baxevanis CN, Klein J. Cross-reactivity of suppressor T cells specific for lactate dehydrogenase B and IgG2a myeloma protein. J Immunol 1983;130: Oliveira DBG, Bloackwell N, Virohis AE, Axelrod RA. T helper and suppressor cells are restricted by the A and E molecules, respectively, in the antigen system. Immunogenetics 1985;22: Request for reprints should be addressed to: Hitoshi Matsuoka Second Department of Internal Medicine Miyazaki Medical School 5200 Kihara Kiyotake-Cho Miyazakigun Miyazaki Japan 196 ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY