Allergy Asthma Immunol Res.  2018 Nov;10(6):662-674. 10.4168/aair.2018.10.6.662.

Decreased CRTH2 Expression and Response to Allergen Re-stimulation on Innate Lymphoid Cells in Patients With Allergen-Specific Immunotherapy

• Mitthamsiri W
• Pradubpongsa P
• Sangasapaviliya A
• Boonpiyathad T

• KMID: 2441815
• DOI: http://doi.org/10.4168/aair.2018.10.6.662

Abstract

PURPOSE
Group 2 innate lymphoid cells (ILC2s) have been implicated in the pathogenesis of allergic disease. However, the effect of allergen-specific immunotherapy (AIT) on ILCs remains to be clarified. The aim of this study was to evaluate the levels of ILC subsets in allergic rhinitis (AR) patients in response to house dust mite (HDM)-specific immunotherapy.
METHODS
We enrolled 37 AR patients undergoing AIT (16 responders and 11 non-responders) for 2 years, 35 HDM AR patients and 28 healthy subjects. Peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry to identify ILC subsets. Stimulation of ILC2s with recombinant allergen-specific protein was used to determine ILC2's activation (CD69 expression).
RESULTS
Responder AIT patients and healthy subjects had a decreased frequency of circulating ILC2s compared to non-responder AIT and AR patients. Conversely, ILC1s from responder AIT patients and healthy subjects showed increased frequency compared to non-responder AIT and AR patients. The frequency of ILC3s natural cytotoxicity receptor (NCR)+ and NCR− in responder AIT patients was significantly lower compared to AR patients and healthy subjects. The ILC1: ILC2 proportion in responder AIT patients was similar to that of healthy subjects. PBMCs from patients who were responders to AIT had a significantly lower expression of the activation marker CD69 on ILC2s in response to allergen re-stimulation compared to AR patients, but no difference compared to non-responder AIT patients and healthy subjects.
CONCLUSIONS
We propose that AIT might affect ILC responses. The activation of ILC2s was reduced in AR patients treated with AIT. Our results indicate that a relative ILC1/ILC2 skewed response is a possible key to successful AIT.

Keyword

House dust mites; Der p 1; allergen immunotherapy; innate immunity; CRTH2; allergic rhinitis

MeSH Terms

Desensitization, Immunologic
Flow Cytometry
Healthy Volunteers
Humans
Immunity, Innate
Immunotherapy*
Lymphocytes*
Pyroglyphidae
Rhinitis, Allergic

Figure

• 

  Fig. 1 ILCs gating strategy use to describe ILC1s, ILC2s and ILC3s from peripheral blood. Representative flow cytometry dot plot demonstrating ILC gating strategy, ILC were defined as CD45+ Viable Lin−CD127+CD161+. ILCs were characterized into 3 groups based on CRTH2 and c-Kit, ILC1s (CRTH2−c-Kit−), ILC2s (CRTH2+), and ILC3s (CRTH2−c-Kit+). NKp44 in humans is used to gate ILC3s NCR+ (NKp44+) and ILC3s NCR− (NKp44−). ILC, innate lymphoid cell; NCR, natural cytotoxicity receptor.

• 

  Fig. 2 ILC1s and ILC2s from human peripheral blood. (A) Representative dot plots of ILCs population, and mean of ILC1s and ILC2s. Frequency of ILC1s (B) and ILC2s (C) were expressed as percentages of the ILCs (mean ± SD) in healthy (n = 28), AR (n = 35), non-responder AIT (n = 11), and responder AIT (n = 26). The data were analyzed by analysis of variance and Tukey's multiple comparison test. ILC, innate lymphoid cell; SD, standard deviation; AR, allergic rhinitis; AIT, allergen-specific immunotherapy. *P < 0.05, †P < 0.001, ‡P < 0.0001.

• 

  Fig. 3 ILC3s NCR+ and NCR− from the human peripheral blood. (A) Representative dot plots and mean of ILC3s NCR+ and NCR−. Frequency of ILC3s NCR+ (B) and NCR− (C) were expressed as percentages of the ILCs (mean ± SD) in healthy (n = 28), AR (AR, n = 35), non-responder AIT (n = 11) and responder AIT (n = 26). (D) Proportions of ILC subsets in each group. The data were analyzed by analysis of variance and Tukey's multiple comparison test. ILC, innate lymphoid cell; NCR, natural cytotoxicity receptor; SD, standard deviation; AR, allergic rhinitis; AIT, allergen-specific immunotherapy. *P < 0.01, †P < 0.001.

• 

  Fig. 4 Activated ILC2s decreased in responder AIT patients. (A) Representative overlay histogram graph of CD69 expression on ILC2s between baseline and 3 days after stimulation with the recombinant antigen-specific protein. (B) Fold change in the mean fluorescence intensity of CD69 expression on ILC2s from healthy (n = 20), AR (AR, n = 20), non-responder AIT (n = 9), and responder AIT (n = 16) in response to allergen-specific stimulation. The data were analyzed by analysis of variance and Tukey's multiple comparison test. ILC, innate lymphoid cell; AIT, allergen-specific immunotherapy; AR, allergic rhinitis. *P < 0.01, †P < 0.001.

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