Krótkoterminowe efekty działania immunoterapii jadem osy na ekspresję receptora dla interleukiny 7 (IL-7) na komórkach T CD4+ krwi obwodowej

Introduction

Venom immunotherapy (VIT) remains the most effective tool in causative treatment of systemic allergic reactions to hymenoptera venom [1]. In general, the effects of allergen-specific immune therapy (SIT) are based on the induction of tolerance to specific allergens in sensitized patients [2]. There is a large body of evidence indicating that tolerance to allergens achieved in the course of SIT can be strongly related with modulation of activities of regulatory T cells [3]. In fact, an increased expansion of T cells with immunosuppressive potential was observed in the course of VIT [4]. Many of the long-term immune effects of SIT have traditionally been linked to modulation of CD4+ T cells, mainly those with a regulatory phenotype characterized by the high expression of CD25 and/or FoxP3 [3]. Recently, VIT has been shown to upregulate proliferation of regulatory T cells (Treg) delineated by the CD4+CD127(IL-7R)-CD25(IL-2R)+ phenotype and the expression of Foxp3 [4]. However, much less is known about complexity of mechanisms associated with allergen-specific immunotherapy and that justifies importance of continuous studies [5]. IL-7/IL-7R(CD127) axis is critical for all T cells’ homeostasis [6], however, more recent data additionally underline a significant influence of IL-7 on allergen-induced memory CD4+ T cells [7]. Moreover, the chronic activation status of immune cells in the course of viral infection or asthma was associated with a significant decrease in IL-7R expression on T cells, the process being efficiently restored by glucocorticoid treatment [8]. However, despite numerous studies on allergy immunotherapy, there are no data on the effects of this kind of treatment on IL-7R levels in CD4+ T cells in wasp venom allergic patients.

Aim

Here, we intended to analyze whether ultra-rush wasp venom specific immunotherapy might influence IL-7R expression on CD4+ T cells in allergic subjects.

Material and methods

Patients

This pilot study was conducted on ten wasp venom-sensitive patients subjected to ultra-rush immune therapy using Venomenhal Wespe (HAL Allergy). EDTA-anticoagulated peripheral blood was collected from patients at three time points: up to 1 h prior to the first injection, after 24 h, and at day 120.

Flow cytometry

Immunostaining with fluorochrome-labeled monoclonal antibodies was performed on 150 µl of blood samples, using 5 µl of each selected antibody: anti-CD4 FITC and anti-CD127 (IL-7R) PE (BD Bioscience). 25-minute incubation was followed by erythrocyte lysis with the use of FACS Lysing Solution (BD Bioscience), and subsequent double washing of cells in phosphate-buffered saline (PBS, Biomed Lublin). Flow cytometric data acquisition was conducted on FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analyses were performed with the use of FlowJo software (Three Star Inc.) XX was used to perform analysis of the collected flow cytometric data. Proper gating of CD4+CD127+ cells was based on unstained and fluorescence minus one (FMO) controls (Figure 1).

Statistical analysis

Wilcoxon matched pairs test (due to non-normal distribution) was used for statistical analysis of the data. Obtained data were presented as frequency and mean fluorescence intensity (MFI) of CD127 marker within CD4+ T cells. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA).

Results

In general, we did not reveal any significant changes in IL-7R frequencies within CD4+ T cells in the course of first 3 months of wasp-venom immunotherapy (Figures 2 A, C; Table 1). However, with regard to the MFI of IL-7R, we found an insignificant decrease in the expression of IL-7R on CD4+ T cells at day 1 (MFI = 72.20) as compared to day 0 (MFI = 96.40) (p = 0.0547). In addition, at day 120 we observed a return of IL-7R MFI to baseline levels (MFI = 106.5) (Figures 2 B, C; Table 1).

Discussion

Here, we intended to investigate effects of wasp venom ultra-rush desensitization therapy on the expression of IL-7R (CD127) in CD4+ T cells. First, we did not find any significant changes in frequency of CD4+CD127+ T cells at any of the time points during allergen-specific immune therapy. Interestingly however, non-significant decline in mean fluorescence intensity of CD127 expression on CD4+ cells was observed. Noteworthy, similar results were observed in asthmatic patients, where shortly (6- and 24-hours) after intrabronchial allergen provocation with HDM (house dust mite), a significant decrease in IL-7R was observed in CD4+ T-cells [8]. Noteworthy, allergen challenge in patients allergic to dust mite is also associated with a decline in frequency of CD4+CD25+CD127- regulatory T cells [9]. Considering a well-known essential role of IL-7R in the T cell survival and homeostasis [10–12], complementary studies will be necessary to investigate changes of T cells function in response to desensitization therapies. It was shown previously that in certain conditions IL-7R levels positively correlate with the frequency of regulatory T cells [13]. Additionally, a low expression of IL-7R is one of the surface markers that enable to distinguish T cells with a regulatory phenotype [14, 15]. These results seem to be in concert with previous studies indicating the effect of venom immunotherapy (VIT) on allergen-specific regulatory T cells generation, and subsequent restoration of normal tolerance [16]. Further research might reveal whether IL-7R expression is followed by regulatory T cells distribution and how these factors influence the outcome of wasp venom ultra-rush desensitization therapy. Another crucial argument justifying the need for more comprehensive studies on IL-7R is the fact some changes at later stages of the immunotherapy [17], therefore prolonged monitoring will be required.

Conclusions

Our findings suggest that wasp venom SIT at early stages of therapy does not seem to affect frequencies of CD4+CD127+ T cells. However, late effects of VIT on T cell phenotype will have to be analyzed. Therefore, further studies are warranted to establish the long-term effects of hymenoptera VIT on IL-7/IL-7R axis and its role in the induction of allergen tolerance.

Conflict of interest

The authors declare no conflict of interest.

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