Genetic restriction of antigen-presentation dictates allergic sensitization and disease in humanized mice

Abstract

Background: Immunoglobulin(Ig)E-associated allergies result from misguided immune responses against innocuous antigens. CD4⁺ T lymphocytes are critical for initiating and perpetuating that process, yet the crucial factors determining whether an individual becomes sensitized towards a given allergen remain largely unknown.

Objective: To determine the key factors for sensitization and allergy towards a given allergen.

Methods: We here created a novel human T cell receptor(TCR) and human leucocyte antigen (HLA)-DR1 (TCR-DR1) transgenic mouse model of asthma, based on the human-relevant major mugwort (Artemisia vulgaris) pollen allergen Art v 1 to examine the critical factors for sensitization and allergy upon natural allergen exposure via the airways in the absence of systemic priming and adjuvants.

Results: Acute allergen exposure led to IgE-independent airway hyperreactivity (AHR) and T helper(Th)2-prone lung inflammation in TCR-DR1, but not DR1, TCR or wildtype (WT) control mice, that was alleviated by prophylactic interleukin(IL)-2-αIL-2 mAb complex-induced expansion of Tregs. Chronic allergen exposure sensitized one third of single DR1 transgenic mice, however, without impacting on lung function. Similar treatment led to AHR and Th2-driven lung pathology in >90% of TCR-DR1 mice. Prophylactic and therapeutic expansion of Tregs with IL-2-αIL-2 mAb complexes blocked the generation and boosting of allergen-specific IgE associated with chronic allergen exposure.

Conclusions: We identify genetic restriction of allergen presentation as primary factor dictating allergic sensitization and disease against the major pollen allergen from the weed mugwort, which frequently causes sensitization and disease in humans. Furthermore, we demonstrate the importance of the balance between allergen-specific T effector and Treg cells for modulating allergic immune responses.

Keywords: Aeroallergen; Airway hyperreactivity; Allergen-specific TCR; IL-2-αIL-2 complexes; Mugwort allergy; Sensitization; T regulatory cells; Tolerance.

Fig. 1

Expression and function of chimeric Art v 1-specific TCR and HLA-DR1. A, Flow cytometry analysis of TCR Vβ18 and HLA-DR1 expression on CD4⁺ and CD45R/B220⁺ PB lymphocytes isolated from WT, DR1, TCR and TCR-DR1 mice. Markers indicate negative control mAbs, numbers show percentage of cells. n = 4 per group of four analyses. B, Percentages of CD3⁺CD4⁺Foxp3⁺ cells in lymph nodes of WT, DR1, TCR and TCR-DR1 mice. Symbols represent individual mice. C, Dose dependent proliferation of TCR-DR1 splenocytes upon incubation with rArt v 1-protein, or Bet v 1_142–153-peptide (negative control). kcpm, kilo counts per minute of incorporated [³H]-thymidine. Shown are representative SEMs of triplicate cultures of one out of four independent experiments. ***p < .001, Kruskal-Wallis test and Mann-Whitney-U test followed by post hoc Bonferroni correction.

Fig. 2

Rapid induction in TCR-DR1 mice of airway hyperreactivity and inflammation in the absence of allergen-specific antibody production. A, Experimental design for exposure of mice (WT, DR1, TCR, or TCR-DR1) to 1% nebulized mugwort-pollen extract (3 mg extract/mouse) in a whole body plethysmograph (Buxco) for 16 min on three consecutive days (green arrow heads). Allergen-specific airway hyperreactivity was determined at the end of the 3rd exposure on day 3 (WBP, whole body plethysmography), lung resistance (Rl) on day 4. B, Shown are mean enhanced pause (Penh) values for each mouse (individual symbols). C, Lung resistance (RI) was determined on day 4 (Buxco, Finepoint Software). D, and E, Shown are cytospin preparations (modified Wright-Giemsa stain, size bars 50 μm) and mean numbers of BALF cells. F, Shown are H&E stainings of lung tissues (insets peripheral lung tissues) analyzed by light microscopy, size bars 100 μm. G, and H, Shown are the percentages of CD3⁺CD4⁺IL13⁺ and CD3⁺CD4⁺IFN-γ⁺ T cells in lung suspensions of PBS or mugwort extract challenged TCR-DR1 mice. Each symbol represents an individual mouse. I, Cytokine ratios of PHA-restimulated lung cells comparing fold-induction of IL-4, IL-5 and IL-13 with IFN-γ. J, Allergen-specific serum Ig levels (day 4). Symbols represent individual mice. Data show the summary (B, E) or are representative (C, D, F, G) of 12 (except 22 for TCR-DR1) mice per group of four independent experiments (B, D, E, F, H, I, J), or three mice per group of three independent experiments (C), or four mice per group (except three for WT) of two independent experiments (J), or 12 (except 4 for PBS) mice per group of two independent experiments (G, H, I). *p < .05, **p < .01, ***p < .001; ns, not significant; ANOVA and Tukey's multiple comparison test or Student's t-test (H, I), respectively.

Fig. 3

Severe lung pathology in TCR-DR1 mice upon intranasal exposure to aqueous mugwort pollen extract. A, Experimental design: Intranasal exposure of mice (WT, DR1, TCR, or TCR-DR1) to four doses of aqueous mugwort pollen extract (450 μg in 20 μl) at bi-weekly intervals. B, Allergen-specific airway hyperreactivity was measured 24 h after the last exposure by inhalation of 1% nebulized aqueous mugwort pollen extract aerosol and the mean enhanced pause (Penh) was determined by unrestricted whole body plethysmography (Buxco). Symbols represent individual mice. C, Shown are flow cytometric analyses of BALF cells gated on the indicated cell populations. D, H&E and E, immunohistochemical CD3 stainings of lung tissues analyzed by light microscopy, size bars 100 μm. F, Allergen-specific serum Ig levels (day 44). G, Percentages of CD3⁺CD4⁺Foxp3⁺ positive T cells after exposure. Data show the summary (B, F, G) or are representative (C, D, E) of 12 (for wild-type (WT), 11 for DR1, 13 for TCR and 22 for TCR-DR1) mice per group of three independent experiments (B–E) or 15 (for WT, 17 for DR1, 13 for TCR and 28 for TCR-DR1) mice per group of five independent experiments (F), or 12 for TCR-DR1 (except 8 for WT and 7 each for DR1 and TCR, respectively) mice of two independent experiments (G). *p < .05, **p < .01, ***, p < .001. ANOVA and Tukey's multiple comparison test.

Fig. 4

IL-2-αIL-2 mAb-dependent in vivo expansion of Treg cells mitigates subsequent sensitization to mugwort extract. A, Scheme for induction and phenotypic analysis of Treg cells induced by IL-2-αIL-2 (1 μg mIL-2 complexed to 5 μg JES6-1 mAb). B, Summary of CD3⁺CD4⁺CD25⁺ PB T cells co-expressing Foxp3 of sham (PBS) or IL-2-αIL-2 treated TCR-DR1 mice obtained on day 6. Symbols represent individual mice. C, Scheme to test the influence of IL-2-αIL-2 complexes on AHR in the presence or absence of αIL-10 or control mAb TCR-DR1 mice. D, IL-2-αIL-2 complex treatment leads to reduced AHR upon aerosol sensitization and challenge with 1% nebulized mugwort pollen extract (3 mg/mouse) of TCR-DR1 mice, which is only modestly reversed by αIL-10 mAb in vivo. Data show the summary (B, D) of 33 IL-2-αIL-2 and 30 PBS treated mice per group (B) and 18 (for IL-2-αIL-2 complex and isotype control mAb, 17 for isotype control mAb, and 9 for IL-2-αIL-2 complex and αIL-10 mAb) treated mice (D) that were analyzed in five (D) and four (except one for αIL-10 mAb treated mice) (D) independent experiments. E, Scheme for the prophylactic treatment of mice with IL-2-αIL-2 complexes and F, corresponding allergen-specific serum Ig levels. G, Scheme for the therapeutic treatment of mice with IL-2-αIL-2 complexes and H, corresponding allergen-specific serum Ig levels. Serum IgE levels of mice treated according to (E) or (F) with IL-2-αIL-2 complexes or PBS used as control substance. Art v 1-specific IgE levels determined by ELISA and expressed in arbitrary units (OD 405 nm) are shown. Symbols represent individual mice. Data show the summary of 10 (G) or 20 (H) (except 18 for PBS treated) mice per group analyzed in two (G) or three (H) independent experiments. *p < .05, **p < .01, ***p < .001, ns, not significant. Student's t-test and ANOVA, followed by Tukey's multiple comparison test.