Immune Response of HLA-DQ Transgenic Mice to House Dust Mite Allergen p2: Identification of HLA-DQ Restricted Minimal Epitopes and Critical Residues

Clinical Immunology Vol. 97, No. 2, November, pp. 154 –161, 2000 doi:10.1006/clim.2000.4931, available online at http://www.idealibrary.com on

Immune Response of HLA-DQ Transgenic Mice to House Dust Mite Allergen p2: Identification of HLA-DQ Restricted Minimal Epitopes and Critical Residues Christopher J. Krco, Jerry Harders, Svetlana Chapoval, and Chella S. David 1 Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905

HLA-DQ8 (HLA-DQA1*0301; HLA-DQB1*0302) and HLA-DQ6 (HLA-DQA1*0103; HLA-DQB1*0602) genes were introduced into mouse class II (H-2A ␤o) knockout mice. Transgenic HLA-DQ8 and HLA-DQ6 mice were individually immunized and challenged using synthetic peptides representing HDM (Dermatophagoides pteronyssinus) allergen p2. HLA-DQ8 mice responded to p2 peptides 1–20, 41– 60, 51–70, 61– 80, 91–110, and 101–120. HLA-DQ6 mice responded to peptides 1–20, 11–30, 21– 40, 41– 60, and 51–70. Using single amino acid truncated 30-mer peptides, residues necessary for HLA-DQ8 recognition were identified spanning regions 3–12, 50 –70, and 91–120. A synthetic peptide comprising residues 3–12 was synthesized and a series of single alanine substitutions was introduced into the minimal peptide. Introduction of alanine residues at positions 3, 11, and 12 resulted in a significant loss of immune recognition. It was concluded that residues 4, 5, 7, 11, and 12 are critical for immune recognition by HLA-DQ8 mice. © 2000 Academic Press Key Words: house dust mite; HLA-DQ; transgenic; T lymphocyte; allergy; peptides; epitopes. INTRODUCTION

Hypersensitivity to various environmental stimuli is a worldwide problem affecting a large segment of the human population. House dust mites (HDM) are ubiquitous in the human environment (1). HDM are abundantly recovered from items in frequent human contact such as mattresses, upholstered furniture, carpets, stored clothing, and stuffed toys. Relative humidity has a significant impact upon the survival of HDM. Heat and low humidity is not conducive to HDM survival. Thus, in temperate climates, HDM populations increase in summer and decrease in winter. However, as a result of microenvironmental fluctuations, although HDM numbers may seasonally vary, the environmental levels of secreted HDM allergens remain 1 To whom correspondence should be addressed. Fax: 507-2660981. E-mail: [email protected].

1521-6616/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

relatively constant throughout the year. As a result, HDM atopic individuals suffer uninterrupted allergic HDM reactions. The health and economic consequences of HDM sensitivity are significant. It has been estimated that nearly 20% of the human population exhibits varying clinical symptoms of HDM sensitivity varying from dermatitis and rhinitis to extrinsic asthma (2, 3). Nearly 90% of patients with extrinsic asthma are also atopic for HDM (4). The incidence of asthma is rising partially as a consequence of humans living more sedentary, indoor lives (1). It has been approximately 30 years since it was established that Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) are major sources of perennial indoor aeroallergens (5). Since then, it has been established that there are nine major allergens in HDM preparations. The major two allergens, designated p1 and p2, have been cloned and sequenced (6 –9). Corresponding p1 and p2 allergens from Der p and Der f share ⬎80% sequence homology (8, 10, 11). Allergen p1 is 222 amino acids long and functions as a cysteine protease (2, 5). Allergen p2 has lysozymal activity and is 129 amino acids long (2, 5). Analysis of antigenic determinants expressed on the p2 allergen of Der p has revealed multiple IgG (12–14), IgE (15–18), and T cell (19, 20) determinants that collectively span the entire length of the molecule. Some determinants are more dominant than others (21) and IgE binding sites may be more restricted than IgG determinants (22). HLA-DR (23, 24), HLA-DQ (24 –26), and HLA-DP (27) associated responses, dependent upon CD4 ⫹ T cells (20, 21, 25), have been reported. IL-2, IL-4, IL-6, and ␥-interferon production can be measured following in vitro challenge using major HDM allergens (23, 28 –30). While HDM atopic as well as nonatopic individuals can elicit responses to HDM allergens, there is some evidence that the nature of the immune responses might be different (23, 25, 28). Clearly, allergen p2 of HDM is immunologically complex. Due in part to heterozygous, codominant HLA gene expression, it has been extremely difficult to as-

154

155

HLA-DQ RESTRICTED HOUSE DUST MITE ALLERGEN p2 EPITOPES

TABLE 1 Sequences of Synthetic Overlapping Peptides Representing a Major Der p Allergen (p2) Peptide 1–20 11–30 21–40 31–50 41–60 51–70 61–80 71–90 81–100 91–110 101–120 110–129

Sequence D H C R F K L H V T S M

Q E H G E I E A K W E G

V I G K A E V C G N N D

D K S P V I D H Q V V D

V K E F Q K V Y Q P V G

K V P Q N A P M Y K V V

D L C L T S G K D I T L

C V I E K I I C I A V A

A P I A T D D P K P K C

sess the relative contributions of single HLA class II molecules to p2 reactivity. Using human HLA-DQ transgenic mice lacking endogenous mouse class II expression, we have described in vitro responses to HDM allergenic extracts. In these investigations we have identified sets of determinants recognized by HLA-DQ8 and HLA-DQ6 transgenic mice on the HDM p2 allergen. MATERIALS AND METHODS

Mice. The production of transgenic mice expressing HLA-DQ8 (HLA-DQA1*0301; HLA-DQB1*0302) and HLA-DQ6 (HLA-DQB1*0103; HLA-DQB1*0601) genes has been previously described (31). All mice were bred and maintained in accordance with guidelines established by an institutional animal care committee. Antigens. Sequences of synthetic overlapping peptides representing house dust mite D. pteronyssinus

N G H V A G P L Y K V A

H C R F K L N V T S M I

E H G E I E A K W E G A

I G K A E V C G N N D T

K S P V I D H Q V V D H

K E F Q K V Y Y P V G A

V P Q N A P M D K V V K

L C L T S G K I I T L I

V I E K I I C D A V A R

P I A T D D P K P K C D

G N V A G P L Y K V A

allergen p2 are summarized in Table 1. These and all other peptides used in these investigations were prepared at the Peptide Core Facility of the Mayo Clinic. Immunizations and in vitro cultures. Two hundred micrograms of peptide emulsified in complete Freund’s adjuvant was administered as subcutaneous injections into the tails and hind footpads of mice. Seven days postinjection the draining lymph node cells were challenged in vitro (32). The extent of T cell activation after 48 h of culture was determined by measuring the incorporation of [ 3H]thymidine. Results are expressed as ⌬ counts per minute (⌬ cpm) and are calculated as ⌬ cpm ⫽ (mean cpm of triplicate cultures containing antigen) ⫺ (mean cpm of triplicate cultures without antigen). Mean cpms of cultures containing medium varied between 1000 and 6000. Mean cpms of cultures containing Concanavalin A (positive control) were greater than 175,000.

FIG. 1. HLA-DQ8 and HLA-DQ6 transgenic mice respond to different sets of peptides representing allergen p2 of house dust mite. Mice were immunized and challenged in vitro with individual peptides. Draining lymph node cells were cocultured with medium alone, Concanavalin A, or immunizing peptide. Cell proliferation was assessed by determining the extent of [ 3H]thymidine incorporation. Data are expressed as ⌬ cpm and are calculated as ⌬ cpm ⫽ (mean cpm of triplicate cultures containing test antigen) ⫺ (mean cpm of triplicate cultures containing medium alone). Medium control cpms were less than 6000.

156

KRCO ET AL.

TABLE 2 Residues 3–12 of p2 Peptide 1–20 Are Critical for Immune Recognition in HLA-DQ8 Mice a Peptide

1

.

.

.

5

.

.

.

.

10

.

.

.

.

15

.

.

.

.

20

% Control

p2.1.1 p2.1.2 p2.1.3 p2.1.4 p2.1.5 p2.1.6 p2.1.7 p2.1.8 p2.1.9 p2.1.10 p.2.1 p2.1.11 p2.1.12 p2.1.13 p2.1.14 p2.1.15 p2.1.16 p2.1.17 p2.1.18 p2.1.19 p2.1.20

. . . . . . . . . . D D D D D D D D D D D

Q . . . . . . . . . Q Q Q Q Q Q Q Q Q Q Q

V V . . . . . . . . V V V V V V V V V V V

D D D . . . . . . . D D D D D D D D D D D

V V V V . . . . . . V V V V V V V V V V V

K K K K K . . . . . K K K K K K K K K K K

D D D D D D . . . . D D D D D D D D D D D

C C C C C C C . . . C C C C C C C C C C C

A A A A A A A A . . A A A A A A A A A A A

N N N N N N N N N . N N N N N N N N N N N

H H H H H H H H H H H H H H H H H H H H .

E E E E E E E E E E E E E E E E E E E . .

I I I I I I I I I I I I I I I I I I . . .

K K K K K K K K K K K K K K K K K . . . .

K K K K K K K K K K K K K K K K . . . . .

V V V V V V V V V V V V V V V . . . . . .

L L L L L L L L L L L L L L . . . . . . .

V V V V V V V V V V V V V . . . . . . . .

P P P P P P P P P P P P . . . . . . . . .

G G G G G G G G G G G . . . . . . . . . .

91 63 26 3 1 2 1 1 1 1 100 103 98 108 105 112 119 127 93 4 3

HLA-DQ8 mice were immunized with 100 ␮g of peptide p2.1 and the T cell response measured at 48 h (⌬ cpm 40,790) was set as 100% control value. Draining lymph node cells were challenged with individual truncated peptides for 48 h and the ⌬ cpms determined were expressed as a percentage of control responses relative to p2.1 peptide response. a

RESULTS

HLA-DQ8 and HLA-DQ6 mice recognize different sets of determinants on allergen p2 of Der p. Results of immunizing and challenging HLA-DQ8 and HLADQ6 mice with individual synthetic peptides representing the p2 molecule are summarized in Fig. 1. HLA-DQ8 mice responded to peptides 1–20 (⌬ cpm 36,389), 41– 60 (⌬ cpm 46,871), 51–70 (⌬ cpm 49,958), 61– 80 (⌬ cpm 24,381), 91–110 (⌬ cpm 47,711), and 101–120 (⌬ cpm 54,256). HLA-DQ8 mice were unresponsive to all other p2 peptides. HLA-DQ6 mice responded to in vitro challenge using peptides 1–20 (⌬ cpm 27,182), 11–30 (⌬ cpm 73,053), 21– 40 (⌬ cpm 54,151), 41– 60 (⌬ cpm 50,531), and 51–70 (⌬ cpm 23,315). HLA-DQ6 mice were hyporesponsive to the remaining p2 peptides. From these results it is possible to localize HLA-DQ8 restricted determinants to residues 1–20, 40 –70, and 90 –120. In contrast, HLA-DQ6 restricted determinants reside within residues 11–30 and 40 – 60. Residues 3–12 of p2 peptide 1–20 are critical for immune recognition in HLA-DQ8 mice. In order to identify residues within peptide 1–20 necessary for T cell recognition in HLA-DQ8 mice, a series of truncated peptides (Table 2) was synthesized. HLA-DQ8 mice were immunized with full-length p2 peptides (designated p2.1). Draining lymph node cells were challenged using individual truncated peptides. HLA-DQ8

mice responded to challenge using full-length p2.1 peptide (⌬ cpm 90,160). Removal of one residue from the NH 2-terminus (p2.1.1) did not alter (91% of control, full-length peptide) the response by HLA-DQ8 T cells. However, removal of successive NH 2-terminus residues (peptides 2.1.2 and p2.1.3) progressively diminished (63 and 26% of control responses) the measured in vitro responses. HLA-DQ8 T cells were unresponsive (⬍5% of control response) to in vitro challenges using any of the remaining NH 2-terminus series of truncated peptides (peptides 2.1.4 through 2.1.10). In contrast, eight COOH-terminus residues (peptides 2.1.11 through 2.1.18) could be removed without diminishing the HLA-DQ8 restricted responses (93–127% of control peptide response). However, removal of residues 11 or 12 of peptide p2.1 abrogated T cell recognition (3– 4% of control peptide response). From these results it can be concluded that residues 3–12 of allergen p2 molecule are important for T cell recognition in HLA-DQ8 T cells. Residues 50 –70 of p2 peptide 41.70 are critical for immune recognition by HLA-DQ8 mice. Results of testing synthetic overlapping p2 peptides indicated that sequential 41– 60 and 51–70 peptides elicited comparable T cell responses in HLA-DQ8 mice (Fig. 1). In order to identify likely immunodominant residues, a series of truncated 30-mer peptides (Table 3) was utilized. HLA-DQ8 mice responded vigorously to in vitro challenge using intact peptide p2.6A (⌬ cpm 90,160).

157

HLA-DQ RESTRICTED HOUSE DUST MITE ALLERGEN p2 EPITOPES

TABLE 3 Residues 50 –70 of p2 Peptide 41–70 Are Critical for Immune Recognition in HLA-DQ8 Mice a Peptide

41

.

.

.

45

.

.

.

.

50

.

.

.

.

55

.

.

.

.

60

.

.

.

.

65

.

.

.

.

70

% Control

p2.6A.1 p2.6A.2 p2.6A.3 p2.6A.4 p2.6A.5 p2.6A.6 p2.6A.7 p2.6A.8 p2.6A.9 p2.6A.10 p2.6A.11 p2.6A.12 p2.6A.13 p2.6A.14 p2.6A.15 p2.6A.16 p2.6A.17 p2.6A.18 p2.6A.19 p2.6A.20 p2.6A.21 p2.6A.22 p2.6A p2.6A.23 p2.6A.24 p2.6A.25 p2.6A.26 p2.6A.27 p2.6A.28 p2.6A.29 p2.6A.30 p2.6A.31 p2.6A.32 p2.6A.33 p2.6A.34 p2.6A.35 p2.6A.36 p2.6A.37 p2.6A.38 p2.6A.39 p2.6A.40 p2.6A.41 p2.6A.42 p2.6A.43 p2.6A.44

. . . . . . . . . . . . . . . . . . . . . . F F F F F F F F F F F F F F F F F F F F F F F

E . . . . . . . . . . . . . . . . . . . . . E E E E E E E E E E E E E E E E E E E E E E E

A A . . . . . . . . . . . . . . . . . . . . A A A A A A A A A A A A A A A A A A A A A A A

V V V . . . . . . . . . . . . . . . . . . . V V V V V V V V V V V V V V V V V V V V V V V

Q Q Q Q . . . . . . . . . . . . . . . . . . Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q

N N N N N . . . . . . . . . . . . . . . . . N N N N N N N N N N N N N N N N N N N N N N N

T T T T T T . . . . . . . . . . . . . . . . T T T T T T T T T T T T T T T T T T T T T T T

K K K K K K K . . . . . . . . . . . . . . . K K K K K K K K K K K K K K K K K K K K K K K

T T T T T T T T . . . . . . . . . . . . . . T T T T T T T T T T T T T T T T T T T T T T .

A A A A A A A A A . . . . . . . . . . . . . A A A A A A A A A A A A A A A A A A A A A . .

K K K K K K K K K K . . . . . . . . . . . . K K K K K K K K K K K K K K K K K K K K . . .

I I I I I I I I I I I . . . . . . . . . . . I I I I I I I I I I I I I I I I I I I . . . .

E E E E E E E E E E E E . . . . . . . . . . E E E E E E E E E E E E E E E E E E . . . . .

I I I I I I I I I I I I I . . . . . . . . . I I I I I I I I I I I I I I I I I . . . . . .

K K K K K K K K K K K K K K . . . . . . . . K K K K K K K K K K K K K K K K . . . . . . .

A A A A A A A A A A A A A A A . . . . . . . A A A A A A A A A A A A A A A . . . . . . . .

S S S S S S S S S S S S S S S S . . . . . . S S S S S S S S S S S S S S . . . . . . . . .

I I I I I I I I I I I I I I I I I . . . . . I I I I I I I I I I I I I . . . . . . . . . .

D D D D D D D D D D D D D D D D D D . . . . D D D D D D D D D D D D . . . . . . . . . . .

G G G G G G G G G G G G G G G G G G G . . . G G G G G G G G G G G . . . . . . . . . . . .

L L L L L L L L L L L L L L L L L L L L . . L L L L L L L L L L . . . . . . . . . . . . .

E E E E E E E E E E E E E E E E E E E E E . E E E E E E E E E . . . . . . . . . . . . . .

V V V V V V V V V V V V V V V V V V V V V V V V V V V V V V . . . . . . . . . . . . . . .

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D . . . . . . . . . . . . . . . .

V V V V V V V V V V V V V V V V V V V V V V V V V V V V . . . . . . . . . . . . . . . . .

P P P P P P P P P P P P P P P P P P P P P P P P P P P . . . . . . . . . . . . . . . . . .

G G G G G G G G G G G G G G G G G G G G G G G G G G . . . . . . . . . . . . . . . . . . .

I I I I I I I I I I I I I I I I I I I I I I I I I . . . . . . . . . . . . . . . . . . . .

D D D D D D D D D D D D D D D D D D D D D D D D . . . . . . . . . . . . . . . . . . . . .

P P P P P P P P P P P P P P P P P P P P P P P . . . . . . . . . . . . . . . . . . . . . .

92 96 94 101 92 77 84 84 83 73 64 64 56 93 57 52 60 53 58 55 48 40 100 39 39 47 49 52 44 48 48 49 38 22 17 18 14 15 12 23 14 32 6 6 6

HLA-DQ8 mice were immunized with 100 ␮g of peptide p2.6A and the T cell response measured at 48 h (⌬ cpm 70,473) was set as 100% control value. Draining lymph node cells were challenged with individual truncated peptides for 48 h and the ⌬ cpms determined were expressed as a percentage of control responses relative to p2.6A peptide response. a

Truncating the first 10 NH 2-terminal residues (peptides p2.6A.1 through p2.6A.10) did not abrogate T cell recognition (⬎72% of control peptide response). However, removal of any of the first 20 COOH-terminal residues resulted in drastic reductions of T cell activation (12–52% of control peptide response). From the pattern of T cell responses measured it was concluded that residues 50 –70 contain residues necessary for immune recognition by HLA-DQ8 mice.

Residues 99 –120 of p2 peptide 91–120 are critical for immune recognition by HLA-DQ8 mice. Synthetic p2 peptides 91–110 and 101–120 elicited nearly identical responses in HLA-DQ8 mice (Fig. 1). In an attempt to identify immunostimulatory residues, mice were immunized using a 30-mer peptide (p2.11A) representing residues 91–120 (Table 4). This peptide is immunogenic in HLA-DQ8 mice (⌬ cpm 64,407). Since removal of all but six of the COOH-terminal residues (peptides

158

KRCO ET AL.

TABLE 4 Residues 99 –120 of p2 Peptide 91–120 Are Critical for Immune Recognition in HLA-DQ8 Mice a Peptide

91

.

.

.

95

.

.

.

.

100

.

.

.

.

105

.

.

.

.

110

.

.

.

.

115

.

.

.

.

120

% Control

p2.11A.1 p2.11A.2 p2.11A.3 p2.11A.4 p2.11A.5 p2.11A.6 p2.11A.7 p2.11A.8 p2.11A.9 p2.11A.10 p2.11A.11 p2.11A.12 p2.11A.13 p2.11A.14 p2.11A.15 p2.11A.16 p2.11A.17 p2.11A.18 p2.11A.19 p2.11A.20 p2.11A.21 p2.11A.22 p2.11A p2.11A.23 p2.11A.24 p2.11A.25 p2.11A.26 p2.11A.27 p2.11A.28 p2.11A.29 p2.11A.30 p2.11A.31 p2.11A.32 p2.11A.33 p2.11A.34 p2.11A.35 p2.11A.36 p2.11A.37 p2.11A.38 p2.11A.39 p2.11A.40 p2.11A.41 p2.11A.42 p2.11A.43 p2.11A.44

. . . . . . . . . . . . . . . . . . . . . . T T T T T T T T T T T T T T T T T T T T T T T

W . . . . . . . . . . . . . . . . . . . . . W W W W W W W W W W W W W W W W W W W W W W W

N N . . . . . . . . . . . . . . . . . . . . N N N N N N N N N N N N N N N N N N N N N N N

V V V . . . . . . . . . . . . . . . . . . . V V V V V V V V V V V V V V V V V V V V V V V

P P P P . . . . . . . . . . . . . . . . . . P P P P P P P P P P P P P P P P P P P P P P P

K K K K K . . . . . . . . . . . . . . . . . K K K K K K K K K K K K K K K K K K K K K K K

I I I I I I . . . . . . . . . . . . . . . . I I I I I I I I I I I I I I I I I I I I I I I

A A A A A A A . . . . . . . . . . . . . . . A A A A A A A A A A A A A A A A A A A A A A A

P P P P P P P P . . . . . . . . . . . . . . P P P P P P P P P P P P P P P P P P P P P P .

K K K K K K K K K . . . . . . . . . . . . . K K K K K K K K K K K K K K K K K K K K K . .

S S S S S S S S S S . . . . . . . . . . . . S S S S S S S S S S S S S S S S S S S S . . .

E E E E E E E E E E E . . . . . . . . . . . E E E E E E E E E E E E E E E E E E E . . . .

N N N N N N N N N N N N . . . . . . . . . . N N N N N N N N N N N N N N N N N N . . . . .

V V V V V V V V V V V V V . . . . . . . . . V V V V V V V V V V V V V V V V V . . . . . .

V V V V V V V V V V V V V V . . . . . . . . V V V V V V V V V V V V V V V V . . . . . . .

V V V V V V V V V V V V V V V . . . . . . . V V V V V V V V V V V V V V V . . . . . . . .

T T T T T T T T T T T T T T T T . . . . . . T T T T T T T T T T T T T T . . . . . . . . .

V V V V V V V V V V V V V V V V V . . . . . V V V V V V V V V V V V V . . . . . . . . . .

K K K K K K K K K K K K K K K K K K . . . . K K K K K K K K K K K K . . . . . . . . . . .

V V V V V V V V V V V V V V V V V V V . . . V V V V V V V V V V V . . . . . . . . . . . .

M M M M M M M M M M M M M M M M M M M M . . M M M M M M M M M M . . . . . . . . . . . . .

G G G G G G G G G G G G G G G G G G G G G . G G G G G G G G G . . . . . . . . . . . . . .

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D . . . . . . . . . . . . . . .

D D D D D D D D D D D D D D D D D D D D D D D D D D D D D . . . . . . . . . . . . . . . .

G G G G G G G G G G G G G G G G G G G G G G G G G G G G . . . . . . . . . . . . . . . . .

V V V V V V V V V V V V V V V V V V V V V V V V V V V . . . . . . . . . . . . . . . . . .

L L L L L L L L L L L L L L L L L L L L L L L L L L . . . . . . . . . . . . . . . . . . .

A A A A A A A A A A A A A A A A A A A A A A A A A . . . . . . . . . . . . . . . . . . . .

C C C C C C C C C C C C C C C C C C C C C C C C . . . . . . . . . . . . . . . . . . . . .

A A A A A A A A A A A A A A A A A A A A A A A . . . . . . . . . . . . . . . . . . . . . .

64 59 65 64 60 65 70 60 50 70 71 167 44 57 38 35 30 8 22 12 14 15 100 67 56 64 58 65 67 70 39 33 47 32 15 29 26 18 24 10 28 5 7 10 9

a HLA-DQ8 mice were immunized with 100 ␮g of peptide p2.11A and the T cell response measured at 48 h (⌬ cpm 64,407) was set as 100% control value. Draining lymph node cells were challenged with individual truncated peptides for 48 h and the ⌬ cpms determined were expressed as a percentage of control responses relative to p2.11A peptide response.

p2.11A.29 through p2.11A.44) greatly reduced T cell responses (⬍50% of control peptide response) while removal of NH 2-terminal residues had less dramatic effects, it was inferred that residues 99 –120 contained important residues necessary for HLA-DQ8 restricted responses. Identification of residues within peptide 3–12 is critical for immune recognition. Previously summarized results were consistent with the notion that residues 3–12 were a minimal length determinant sufficient for immune recognition by HLA-DQ8 T cells. As a prelude to developing candidate antagonists, agonists, and al-

tered peptide ligands for in vivo desensitization, a synthetic peptide (p2.1MN) comprising residues 3–12 was synthesized (Table 5). A series of single alanine substitutions was introduced into the p2.1MN peptide. HLADQ8 mice were immunized using the minimal-length 3–12 peptide and challenged in vitro using alanine substituted peptides. Under conditions in which HLADQ8 mice responded to peptide p2.1MN (⌬ cpm 41,662), the introduction of alanine residues at positions 3, 11, and 12 resulted in a significant loss of immune recognition (⬍7% of control peptide response). Substitution of alanine residues at positions 4, 5, and 7

159

HLA-DQ RESTRICTED HOUSE DUST MITE ALLERGEN p2 EPITOPES

TABLE 5 Residues 4, 5, 7, 11, and 12 of p2 Peptide 3–12 Are Critical for Immune Recognition by HLA-DQ8 Mice a Peptide

3

.

5

.

.

.

.

10

.

.

% Control

2.1MN 2.1MN.A3 2.1MN.A4 2.1MN.A5 2.1MN.A6 2.1MN.A7 2.1MN.A9 2.1MN.A10 2.1MN.A11 2.1MN.A12

V A . . . . . . . .

D . A . . . . . . .

V . . A . . . . . .

D . . . A . . . . .

C . . . . A . . . .

A . . . . . . . . .

N . . . . . A . . .

N . . . . . . A . .

H . . . . . . . A .

E . . . . . . . . A

100 102 3 40 87 55 85 63 6 1

HLA-DQ8 mice were immunized with 100 ␮g of peptide 2.1MN and the T cell response measured at 48 h (⌬ cpm 41,662) was set as 100% control value. Draining lymph node cells were challenged with individual truncated peptides for 48 h and the ⌬ cpms determined were expressed as a percentage of control responses relative to 2.1MN peptide response. a

resulted in a 50% reduction in T cell responses. From these results it was inferred that residues 4, 5, 7, 11, and 12 are critical for immune recognition by HLADQ8 mice. DISCUSSION

House dust mite allergen p2 is a nonglycosylated, 129-amino-acid-long molecule having lysozymal activity (2, 5). Approximately 90% of sera from house dust mite sensitive patients contain antibodies (IgE, IgG) reactive with allergen p2 (4). Although several IgE binding sites have been identified on p2 (4, 13, 17, 22), residues 65–78 contain an immunodominant IgE determinant (22). IgG antibody binding sites have been detected throughout the length of the p2 molecule (12–14). T cells from atopic donors respond to in vitro challenge using HDM p2 allergen (19, 20, 24, 25). Through the use of overlapping synthetic peptides it has been estimated that there is significant patient variability in the number and location of T cell determinants (23, 25, 28). Immunodominant T cell regions have been reported to be clustered around residues 11–50, 61– 86, 78 –104, and 111–129 (19). HLA association studies have determined that both HLA-DR and HLA-DQ restricted T cells can be isolated from atopic donors (20, 24 –26). HLA-DR1 restricted responses to residues 29 – 42 and 11–129 have been reported (20). HLA-DRB1*1101 recognition of residues 22– 40 and 82–100 has also been demonstrated. HLA-DQB1*0301 confers reactivity to residues 16 –31 and 111–129 while HLA-DQB1*0602 responds to residues 20 –33. HLA-DQ8 transgenic mice responded to p2 peptides 1–20, 41– 60, 51–50, 61– 80, 91–110, and 101–120. HLA-DQ6 mice responded to in vitro challenge using peptides 1–20, 11–30, 21– 40, 41– 60, and 51–70. Thus,

while recognition of some peptides is common to both HLA-DQ8 and HLA-DQ6 molecules (peptides 1–20, 41– 60, and 51–70), other peptides are uniquely recognized by either HLA-DQ8 (peptides 91–110 and 101– 120) or HLA-DQ6 (11–30 and 21– 40). HLA-DQ7 and HLA-DQ8 molecules have identical ␣-chains (HLADQA1*0301) but express different ␤-chains (HLADQB1*0301 versus HLA-DQB1*0302). The HLA-DQ7 and HLA-DQ8 ␤-chains differ at four residues within the second exon (33) and this allelism exerts a major effect upon the HDM p2 peptides recognized. Although HLA-DQB1*0301 recognition of peptides 16 –31 and 111–129 has been reported, the HLA-DQ8 transgenic mice did not respond to peptides encompassing these regions. However, HLA-DQB1*0302 gene does confer responsiveness to peptides 11–30 and 21– 40, which also elicit responses by HLA-DQB1*0301 and HLADQB1*0602 (24, 25). Through the application of truncated peptides, we have identified minimal epitopes for HLA-DQ8 restricted T cells. Within residues 3–12 we have identified critical T cell recognition epitopes. The alanine substituted peptides may have antagonist and agonist properties applicable to in vivo desensitization protocols aimed at redirecting immune responses from allergic Th2 reactions toward Th1 pathways. Our data support the concept that HLA-DQ allelism has a significant impact upon the nature of p2 determinants recognized. We have recently published data demonstrating that HLA-DQ8 mice are more susceptible than HLA-DQ6 mice to in vivo lung inflammation and airway hypersensitivity following challenge using allergens (34). In view of the different sets of immunogenic peptides recognized by the two HLA-DQ transgenic lines of mice, the use of human class II transgenic mice may prove to be a valuable research tool in characterizing the pathogenesis of allergy and asthma as

160

KRCO ET AL.

well as determining the efficacy of various modes of immunotherapy. ACKNOWLEDGMENTS We thank Julie Hanson and her staff for outstanding mouse husbandry. We thank Dr. Dan McCormick and his staff (Jane Liebenow and Denise Walker) for superb technical support in synthesizing and purifying the peptides used in these investigations. We thank Mary Brandt for her secretarial assistance. This work was supported by NIH Grants AI 14764 and CA24473.

15.

16.

17. REFERENCES 1. Hart, B. J., The biology of allergenic domestic mites. Clin. Rev. Allergy Immunol. 13, 115–133, 1995. 2. O’Hehir, R. E., Hoyne, G. F., Thomas, W. R., and Lamb, J. R., House dust mite allergy: From T-cell epitopes to immunotherapy. Eur. J. Clin. Invest. 23, 763–772, 1993. 3. O’Brien, R. M., Thomas, W. R., and Wooten, A. M., T cell responses to the purified major allergens from the house dust mite Dermatophagoides pteronyssinus. J. Allergy Clin. Immunol. 89, 1021–1031, 1992. 4. Kobayashi, I., Sakiyama, Y., Tame, A., Kobayashi, K., and Matsumoto, S., IgE and IgG 4 antibodies from patients with mite allergy recognize different epitopes of Dermatophagoides peronyssinus group II antigen (Der p2). J. Allergy Clin. Immunol. 97, 638 – 645, 1996. 5. Stewart, G. A., Dust mite allergens. Clin. Rev. Allergy Immunol. 13, 135–150, 1995. 6. Thomas, W. R., Stewart, G. A., Simpson, R. J., Chua, K. C. K., Plozza, T. M., Dilworth, R. J., Nisbet, A., and Turner, K. J., Cloning and expression of DNA coding for the major house dust mite allergen Der p1 in Escherichia coli. Int. Arch. Allergy Appl. Immunol. 85, 127–129, 1988. 7. Chua, K. Y., Doyel, C. R., Simpson, R. J., Turner, K. J., Stewart, G. A., and Thomas, W. R., Isolation and cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay. Int. Arch. Allergy Appl. Immunol. 91, 118 –123, 1990. 8. Dilworth, R. J., Chua, K. Y., and Thomas, W. R., Sequence analysis of cDNA coding for a major house dust mite allergen, Der f I. Clin. Exp. Allergy 21, 25–32, 1991. 9. Trudinger, M., Chua, K. Y., and Thomas, W. R., cDNA encoding the major mite allergen Der f II. Clin. Exp. Allergy 21, 33– 40, 1991. 10. Chua, K. Y., Huang, C. H., Shen, H. D., and Thomas, W. R., Analysis of sequence polymorphism of a major mite allergen, Der p2. Clin. Exp. Allergy 26, 829 – 837, 1996. 11. Chua, K. Y., Stewart, G. A., Thomas, W. R., Simpson, R., Dilworth, R. J., Plozza, T., and Turner, K. J., Sequence analysis of cDNA coding for a major house dust mite allergen Der p I. Homology with cysteine proteases. J. Exp. Med. 167, 175–182, 1988. 12. Tame, A., Sakiyama, Y., Kobayashi, I., Terai, I., and Kobayashi, K., Differences in titers of IgE, IgG 4 and other IgG subclass anti-Der p2 antibodies in allergic and non-allergic patients. Clin. Exp. Allergy 26, 43– 49, 1996. 13. Oshika, E., Kuroki, Y., Sakiyama, Y., and Matsumoto, S., Measurement of IgG subclass antibodies to the group II antigen of Dermatophagoides pteronyssinus (Der p II) in sera from children with bronchial asthma. Ann. Allergy 69, 427– 432, 1992. 14. Oshika, E., Kuroki, Y., Sakiyama, Y., Matsumoto, S., and Akino, T., A study of the binding of immunoglobulin G and immuno-

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

globulin E from children with bronchial asthma to peptides derived from group II antigen of Dermatophagoides pteronyssinus. Pediatr. Res. 33, 209 –213, 1993. Schuurman, J., Lousens, T. E., Perdok, G. J., Parren, P. W. H. I., and Aalberse, R. C., Mouse/human chimeric IgE antibodies directed to the house dust mite allergen Der p2. Int. Arch. Allergy Immunol. 107, 465– 466, 1995. Smith, A. M., and Chapman, M. D., Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: Contribution of disulfide bonds to the antigenic structure of the major house dust allergen Der p2. Mol. Immunol. 33, 399 – 405, 1996. Van’t Hof, W., van den Berg, M., Driedijk, P. C., and Aalberse, R. C., Heterogeneity in the IgE binding to a peptide derived from the house dust mite allergen Der p II indicates that the IgE response is highly polyclonal. Int. Arch. Allergy Immunol. 101, 437– 441, 1993. Chua, K. Y., Greene, W. K., Kehal, P., and Thomas, W. R., IgE studies with large peptides expressed from Der p II constructs. Clin. Exp. Allergy 21, 161–166, 1991. O’Hehir, R. E., Verhoef, A., Panagiotopoulou, E., Keswani, S., Hayball, J. D., Thomas, W. R., and Lamb, J. R., Analysis of human T cell responses to the group II allergen of Dermatophagoides species: Localization of major antigenic sites. J. Allergy Clin. Immunol. 92, 105–113, 1993. Van Neerven, R. J. J., van t’Hof, W., Ringrose, J. H., Jansen, H. M., Aalberse, R. C., Wierenga, E. A., and Kapsenberg, M. L., T cell epitopes of house dust mite major allergen Der p II. J. Immunol. 151, 2326 –2335, 1993. Hoyne, G., Bournes, T., Kristensen, N., Hetzel, N., and Lamb, J., From epitopes to peptides to immunotherapy. Clin. Immunol. Immunopathol. 80, S23–S30, 1996. Van’t Hot, W., Drieduk, P. C., van den Berg, M., Beck-Sickinger, A. G., Jung, G., and Aalberse, R. C., Epitope mapping of the dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides. Mol. Immunol. 28, 1225–1232, 1991. Okano, M., Nagano, T., Nakada, M., Masuda, M., Kino, Y., Yaseuda, H., Nose, Y., Nishimura, Y., and Ohta, N., Epitope analysis of HLA-DR-restricted helper T-cell responses to Der p II, a major allergen molecule of Dermatophagoides pteronyssinus. Allergy 51, 29 –35, 1996. Verhoef, A., Higgins, J. A., Thorpe, C. J., Marsh, S. G. E., Hayball, J., Lamb, J. R., and O’Heir, R. E., Clonal analysis of the atopic immune response to the group 2 allergen of Dermatophagoides spp.: Identification of HLA-DR and -DQ restricted T cell epitopes. Int. Immunol. 12, 1589 –1597, 1993. Van Neerven, R. J. J., van de Pol, M. M., Wierenga, E. A., Aalberse, R. C., Jansen, H. M., and Kapsenberg, M. L., Peptide specificity and HLA structure do not dictate lymphokine production by allergen-specific T lymphocyte clones. Immunology 82, 351–356, 1994. O’Brien, R. M., Thomas, W. R., Nicholson, I., Lamb, J. R., and Tait, B. D., An immunogenetic analysis of the T-cell recognition of the major house dust mite allergen Der p2.: Identification of the T-cell recognition of the major house dust mite allergen Der p2: Identification of high- and low-responder HLA-DQ alleles and localization of T-cell epitopes. Immunology 86, 176 –182, 1995. Higgins, J. A., Lamb, J. R., Marsh, S. G. E., Tonks, S., Hayball, J. D., Rosen-Bronson, S., Bodmer, J. D., and O’Hehir, R. E., Peptide-induced nonresponsiveness of HLA-DP restricted hman T cells reactive with Dermatophagoides ssp. (house dust mite). J. Allergy Clin. Immunol. 90, 749 –756, 1992.

HLA-DQ RESTRICTED HOUSE DUST MITE ALLERGEN p2 EPITOPES 28. McHugh, S. M., Wilson, A. B., Deighton, J., Lachmann, P. J., and Ewan, P. W., The profiles of interleukin (IL)-2, IL-6, and interferon-gamma production by peripheral blood mononuclear cells from house-dust-mite-allergic patients: A role for IL-6 in allergic disease. Allergy 49, 751–759, 1994. 29. Byron, K. A., O’Brien, R. M., Varigos, G. A., and Wooton, A. M., Dermatophagoides pteronyssinus II-induced interleukin-4 and interferon-␥ expression by freshly isolated lymphocytes of atopic individuals. Clin. Exp. Allergy 24, 878 – 883, 1994. 30. Werfel, T. A., Morita, M., Grewe, H., Renz, U., Wahn, J., Kruutman, and Kapp, A., Allergen specificity of skin-infiltrating T cells is not restricted to a type-2 cytokine pattern in chronic skin lesions of atopic dermatitis. J. Invest. Derm. 107, 871– 876, 1996. 31. Zhou, P., Smart, M. K., Cheng, S., Savarirayan, S., Inoko, H., and David, C. S., HLA-DQ ␤ chain can present mouse endogenous Received July 26, 2000; accepted August 9, 2000

161

provirus MTV-9 product and clonally delete TCR V ␤5 ⫹ and V ␤11 ⫹ T cells in transgenic mice. Immunogenetics 35, 219 –223, 1992. 32. Krco, C. J., Pawelski, J., Harders, J., McCormick, D., Griffiths, M., Luthra, H. S., and David, C. S., Characterization of the antigenic structure of human type II collagen. J. Immunol. 156, 2761–2768, 1996. 33. Sanjeevi, C. B., Lybrand, T. P., DeWeese, C., Landin-Olsson, M., Kockum, I., Dahlquist, G., Stenger, D., and Lernmark, A., Polymorphic amino acid variations in HLA-DQ are associated with systematic physical protein changes and occurrence of IDDM. Diabetes 44, 125–131, 1995. 34. Chapoval, S. P., Nabozny, G. H., Marietta, E. V., Raymond, E. L., Krco, C. J., Andrews, A. G., and David, C. S., Short ragweed allergen imduces eosinophilic lung diseasde in HLA-DQ transgenic mice. J. Clin. Invest. 103, 1707–1717, 1999.