# Increased CD69 expression on activated eosinophils in eosinophilic chronic rhinosinusitis correlates with clinical findings

Yasutaka Yun, Akira Kanda, Yoshiki Kobayashi, Dan van Bui, Kensuke Suzuki, Shunsuke Sawada, Kazuyasu Baba, Masao Yagi, Mikiya Asako, Haruka Okazaki, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Yasutaka Yun, Akira Kanda, Yoshiki Kobayashi, Dan van Bui, Kensuke Suzuki, et al. Increased CD69 expression on activated eosinophils in eosinophilic chronic rhinosinusitis correlates with clinical findings. Allergology International, 2020, 69 (2), pp.232-238. 10.1016/j.alit.2019.11.002. hal-04358045

HAL Id: hal-04358045

https://hal.science/hal-04358045v1

Submitted on 21 Dec 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
1 **Title :**

2 Increased CD69 expression on activated eosinophils in eosinophilic chronic rhinosinusitis

3 correlates with clinical findings

4 **Author:**

5 Yasutaka Yun ¹,², Akira Kanda*§ ¹,³, Yoshiki Kobayashi ¹,³, Dan Van Bui ¹, Kensuke

6 Suzuki ¹, Shunsuke Sawada ¹, Kazuyasu Baba ¹,², Masao Yagi ¹, Mikiya Asako ¹,³, Haruka

7 Okazaki ⁴, Hiroki Ikeda ⁵, Shigeki Kawamura ⁶, Akihiko Nakamura ⁷, David Dombrowicz

8, Koichi Tomoda ¹, and Hiroshi Iwai ¹

9

10 **Affiliations**

11 1. Department of Otorhinolaryngology, Head & Neck Surgery, Kansai Medical University, Osaka, Japan

12 2. Department of Otorhinolaryngology, Takeda general hospital, Kyoto, Japan

13 3. Allergy Center, Kansai Medical University, Osaka, Japan

14 4. Okazaki E.N.T. clinic, Osaka, Japan

15 5. Ikeda E.N.T. clinic, Wakayama, Japan

16 6. Kawamura E.N.T. clinic, Osaka, Japan

17 7. Nakamura E.N.T. clinic, Osaka, Japan
19 8. University of Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, Lille,

20 France

21

22 <sup>§</sup> Authors share first authorship

23 <sup>*</sup> Corresponding author

24 Akira KANDA

25 Department of Otorhinolaryngology, Head and neck Surgery, Kansai Medical University.

26 2-5-1, Shinmachi, Hirakata City, Osaka, 573-1010, Japan. Phone: +81 72 804 0101. FAX:

27 072-804-2547. E-mail: akanda@hirakata.kmu.ca.jp

28

29 Author Contributions

30 Conceived and designed the experiments: YY, and AK. Performed the experiments: YY,

31 YK, HO, DV, MA, HI, SK, and AK. Analyzed the data: YY, HO, and AK. Wrote the
32 paper: YY, and AK. All authors read and approved the final manuscript.

33

34 Conflicts of Interest: The authors declare having no conflict of interest
**Abstract**

**Background:** Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis associated with asthma. CD69 is an important marker of activation for eosinophils. But, whether a correlation exist between the CD69 expression on eosinophils and clinical findings is unclear.

**Method:** We performed quantitative PCR and/or flow cytometry using tissue and purified eosinophils from the blood and nasal polyps of 12 patients with ECRS and from 8 patients without ECRS (controls). We assessed clinical findings including nasal polyp (NP) scores, sinus CT findings, and pulmonary function test results, and examined their possible association with the CD69 expression. We also performed CD69 cross-linking experiments in mouse eosinophils to investigate the functional role of CD69.

**Results:** Levels of cytokine mRNAs (IL-4, -5, -10, and -13) were significantly higher in purified NP eosinophils and tissues from patients with ECRS than the levels of those in controls. The expressions of major basic protein (MBP), eosinophilic cationic protein (ECP), eosinophilic-derived neurotoxin (EDN), eosinophil peroxidase (EPX) in cytotoxic granules, and CD69 mRNA were significantly higher in purified eosinophils from NPs than in those from blood. We also found a correlation between expression of CD69 and clinical findings. Moreover, we found EPX release from mouse eosinophils following CD69 cross-linking.

**Conclusions:** These data suggest that
53 increased CD69 expression by eosinophils is not only a biomarker for nasal obstruction

54 and pulmonary dysfunction, but also a potential therapeutic target for patients with ECRS

55 and asthma.

56

57 **Key Words**

58 Asthma, CD69, eosinophilic chronic rhinosinusitis (ECRS), eosinophil, nasal polyp

59

60 **Abbreviations:**

61 CRS, chronic rhinosinusitis; CRSwNP, chronic rhinosinusitis with nasal polyp; CRSsNP,

62 chronic rhinosinusitis without nasal polyp; ECRS, eosinophilic chronic rhinosinusitis;

63 NECRS; non-eosinophilic chronic rhinosinusitis; NP, nasal polyp; UT, uncinate process

64 tissue; PB, peripheral blood; JESREC, Japanese Epidemiological Survey of Refractory

65 Eosinophilic Chronic Rhinosinusitis; MBP, major basic protein; ECP, eosinophilic

66 cationic protein; EDN, eosinophilic-derived neurotoxin; EPX, eosinophil peroxidase; CD,

67 cluster of differentiation; NK, natural killer; MFI, mean fluorescence index
## 68 Introduction

69 Chronic rhinosinusitis (CRS) is a heterogeneous disease classified into two types based
70 on the presence (CRSwNP) or absence (CRSsNP) of nasal polyps (NP)¹. Approximately,
71 80% of patients with CRSwNP have infiltration of eosinophils into the NPs². Patients
72 who exhibit CRS with eosinophilia (or eosinophilic chronic rhinosinusitis, ECRS) are
diagnosed based on the Japanese Epidemiological Survey of Refractory Eosinophilic
73 Chronic Rhinosinusitis (JESREC) scoring system³. ECRS is characterized by ethmoid-
predominant sinusitis with eosinophilic infiltration. Kobayashi et al and Amelink et al
75 reported that 74% of patients with asthma have ECRS⁴, and that 53% of those with severe
76 asthma and 26.5% of those with mild-to-moderate asthma have NPs⁵. Consequently,
77 ECRS and asthma share macroscopic pathological and histological characteristics⁶, and
78 require comprehensive care according to the united airway concept⁷.

79 In patients with ECRS with asthma, eosinophils play a critical role in the pathogenesis
80 of the diseases. Eosinophils must migrate from the bloodstream to the site of
81 inflammation to exert injury as effector cells by releasing cytokines, cytotoxic granules,
82 and chemokines⁸,⁹. During this process, eosinophils express activation markers such as
83 CD69, CD44, L-selectin, CD54, CD162, and CD11b⁹,¹⁰. CD69 is a type II transmembrane
84 protein, a member of the C-type lectin family, and a very early activation antigen (VEA)¹¹.
85
This molecule is expressed on infiltrated leukocytes such as T cells, B cells, NK cells, neutrophils, and eosinophils during chronic airway inflammation<sup>12,13,14</sup>. In terms of CD69 regulation in eosinophils, Walsh et al found that GM-CSF, IL-3, or IL-5 induce CD69 expression on peripheral blood eosinophils, but not in untreated ones<sup>15</sup>. Reports have shown that signaling through CD69 modulates the differentiation of T helper cells and induces platelet degranulation and aggregation<sup>11, 15</sup>. Although several CD69 ligands have been isolated, including galectin-1 and myosin light chain 9 and 12 (Myl9/12)<sup>11,16</sup>, the ligand on eosinophils has not been identified<sup>17</sup>. Whether CD69 is expressed on eosinophils in the NPs and in peripheral blood (PB) of patients with ECRS patients, and whether a correlation exist between CD69 expression on NP eosinophils and clinical findings of patients with ECRS (such as NP score, sinus CT findings, and pulmonary function) remains unclear.

For this report, we developed an approach for obtaining a high number of purified eosinophils from NPs, because conventional protocols lead to poor numbers. We also measured cytokine production, levels of cytotoxic granules, and CD69 expression on purified eosinophils, and determined correlations between their expression levels and clinical findings. Moreover, we investigated the functional role of CD69 signaling in mouse eosinophils.
104

105 **Methods**

106 *Patients*

107 We recruited patients from the Otorhinolaryngology and Head and Neck surgery of the

108 Takeda General Hospital and Kansai Medical University during a two-year period (2017-

109 2018). The diagnoses of patients with ECRS and non-ECRS (NECRS) patients were

110 based on those in the JESREC study. In all, 12 patients with ECRS and 8 controls

111 (NECRS) participated in the study. We obtained NPs and uncinate process tissues (UT)

112 during endoscopic sinus surgeries. Peripheral blood (PB) samples were collected before

113 surgical operations. A respiratory physicians diagnosed asthma based on the patient's

114 history of wheezing responsive to a bronchodilator. No systemic corticosteroids were

115 administered to the patients at least 4 weeks prior to their surgery. We used NP scores to

116 evaluate the severity of NPs in each patient¹⁸, and we defined sinus CT findings according

117 to the Lund-Mackay scale¹⁹. The examination of pulmonary function was performed

118 using SPIRO SIFT SP-770COPD (Fukuda Denshi, Tokyo, Japan) measuring the

119 following parameters: the ratio of peak expiratory flow (%PEF), ratio of predicted forced

120 expiratory volume in 1 s to forced vital capacity (FEV₁₀%), percentage of predicted mean

121 forced expiratory flow between 25 and 75% of forced vital capacity (%FEF₂₅-₇₅). **Table**
122 **1** and **Table S1** present patient characteristics.

123 The local ethics committees at the Takeda General Hospital (No 2016-002) and at the
124 Kansai Medical University (No.1313) approved this study. We obtained informed
125 consents from all participants.

126 *Mice*

127 We used the following mouse strains: IL-5 transgenic (Tg) mice (BALB/c background),
128 obtained from the co-author Dr. D. Dombrowicz (Institut Pasteur de Lille, Lille, France).
129 All mice were housed at 21°C-23°C with 40%-60% humidity in animal facilities with a
130 12-hour light/dark cycle and were provided food and water ad libitum. All animal
131 experiments were performed using protocols approved by the Kansai Medical University
132 Animal Ethics Committee (18-082).

133 *Human and mouse eosinophils preparation*

134 We separated human granulocytes from PB using the Ficoll-Paque technique (GE
135 healthcare, Uppsala, Sweden). After lysing red blood cells, eosinophils were isolated by
136 using a cell sorter, Aria III (BD science, New Jersey, U.S.A). Detailed protocols for the
137 human eosinophil purification and isolation from NP and mouse eosinophils purification
138 from splenocytes of IL-5 Tg mice are shown in the online supplemental material and in
139 the methods and supplementary Fig. S1.
140 Flow cytometric analysis

141 We performed flow cytometry experiments (Aria III and Cant II, BD Biosciences, New Jersey, U.S.A) using the FlowJo software (BD Biosciences, New Jersey, U.S.A).

143 We stained dissociated cells from human PB or NP with antibodies against CD3, CD14, CD16b, CD19, CD45, CD68, Siglec8, CD69, and/or control antibodies for 30 min at 4°C.

145 For CD69 mean fluorescence index (MFI) ratio on surface expression was calculated according as described 20.

147 The purified mouse eosinophils from mouse spleens were stained with Gr-1, CD45, SiglecF, CD69, and/or control antibodies for 30 min at 4°C. Eosinophils were identified by gated CD45⁺SiglecF⁺Gr-1int (Fig. S2). Table S2 lists detailed information regarding the antibodies for cell sorting.

151 Immunofluorescence staining and Quantitative PCR

152 We stained tissue sections with antibodies against siglec8, CD69 and control antibodies overnight at 4°C. Then, we incubated them with an appropriate secondary antibody for 1h at room temperature (as listed in the Table S2). Tissues were counterstained with Hoechst dye and mounted using Fluorescence Mounting Medium (Dako, CA, U.S.A). We captured images and analyzed them using a LSM 700 scope (Carl Zeiss, Jene, Germany).
We performed quantitative PCRs (qPCRs) following the extraction of mRNA from tissues and eosinophils. Relative gene expression was calculated using the comparative C~T~ method <sup>21</sup>. **Table S3** lists primer sequences. Detailed protocols for immunofluorescence staining and qPCR are shown in the online supplement.

**EPX releasing from mouse eosinophils by cross-linking of CD69**

We resuspended eosinophils (0.4M/200ul per well) purified from mouse spleens in PBS and incubated them with 1-100µg/ml Armenian Hamster anti-mouse CD69 mAb (Novus Biologicals, CO, U.S.A) or with 10µg/ml Armenian Hamster IgG (BioLegend, CA, U.S.A) as control antibody for 1 hour at 4°C. We then transferred the eosinophils into a 24-well plate coating with 20 µg/ml Goat anti-Armenian Hamster IgG (Jackson ImmunoResearch, PA, U.S.A) and incubated for 2 hours at 37°C. After centrifuge plate, we transferred the supernatants into a microtiter plate and analyzed them using an EPX detection kit as per the manufacturer's instructions (Cell Technology, CA, U.S.A).
Chemiluminescence was immediately measured with a luminometer, EnSpire (PerkinElmer, Mass, U.S.A).

**Statistical analysis**

We presented data as means ± SEM and evaluated group differences for statistical significance using the Mann-Whitney U-test. We performed correlation analyses using
the Spearman rank correlation coefficient. We considered all p values <0.05 as statistically significant for all tests. All statistical analyses were performed using GraphPad Prism ver.8 (GraphPad Software, CA, U.S.A).

178

179 **Results**

180 *Expression of cytokine mRNAs in tissue from controls and patients with ECRS*

181 To investigate the cytokine profiles in the UTs and NPs of patients with ECRS, and in the
182 UTs of controls, we quantified cytokine mRNAs by qPCR. The expressions of IL-4, IL-
183 5, IL-10, and IL-13 mRNA in the UTs of patients with ECRS were significantly higher
184 than those in UTs from controls (Fig. 1). We observed similar results in terms of cytokine
185 expressions in the NPs of patients with ECRS and UTs of controls (Fig. 1). Although the
186 expressions of IFNγ mRNA among UT from patients with ECRS, NP from patients with
187 ECRS, and UT from controls were all similar, the expressions were 4.7- and 9.8-fold
188 higher in the UT and NP of patients with ECRS than in the UT of controls.

189 *Character of purified eosinophils from PB and NP*

190 To investigate differences in cytokine production between purified eosinophils from PB
191 and NP, we modified a conventional protocol for NP eosinophil purification. As shown in
192 **Fig. S3**, the number and percentage of CD45+ dissociated cells from NP using the tumor
193 dissociation kit was significantly higher than the number and percentage of the
194 conventional protocol: while the tumor dissociation kit yielded 1.93 ± 1.07×10⁷/g total
195 cells with 14% ± 9% CD45⁺ cells, we only obtained 0.76 ± 0.6 × 10⁷/g total cells and 7%
196 ± 4% CD45⁺ cells after the conventional protocol. Moreover, the % viability between the
197 two protocols was comparable: 76% ± 8% viability using the tumor dissociation kit and
198 75% ± 16% viability using the conventional protocol.

199 Using qPCR, we found significant differences in the levels of cytokines (IL-4, -5, -10,
200 and -13) and cytotoxic granules (MBP, ECP, EDN, and EPX) between purified
201 eosinophils from PB of controls and NP of patients with ECRS (Fig. 2A and 2B).
202 Interestingly, we found significant differences in levels of MBP, EDN, and EPX between
203 purified eosinophils from PB and NP of patients with ECRS (Fig. 2B); however, their
204 cytokine expressions were similar (Fig. 2A).

205 Expression of CD69 on infiltrating eosinophils

206 Next, we examined CD69 expression on eosinophils using qPCR and flow cytometry. As
207 shown in Fig.3A, infiltrating eosinophils isolated from the NP of patients with ECRS had
208 significantly higher expression of CD69 mRNA than the eosinophils from PB. According
209 to our flow cytometry analysis results, the MFI of CD69 was significantly increased on
210 eosinophils from NP compared with that in PB eosinophils from the same patients (Fig.
211 **3B**). We found similar results regarding CD69 expression on eosinophils using
212 immunofluorescence (Fig. 3C).

213 *Correlation between CD69 MFI on eosinophils and clinical findings*

214 We found that the CD69 MFI ratio on eosinophils was correlated with the number of
215 eosinophils infiltrating NPs (Fig. 4A). We also found similar results in terms of the NP
216 score (Fig. 4B) and sinus CT findings as measured by the Lund-Mackay scale (Fig. 4C).
217 Moreover, we found that the CD69 MFI ratio on eosinophils was negatively correlated
218 with pulmonary functions such as FEV~1.0~% and %FEF~25-75~ but not %PEF (Fig. 4D). By
219 contrast, we found no significant correlations between CD69 MFI ratios on T cells and
220 clinical findings (Fig.S4). In addition, we found the number of infiltrating eosinophils to
221 NPs and clinical findings correlated negatively with the %FEF~25-75~ (Fig.S5).

222 **Functional role of CD69 signaling in mouse eosinophils**

223 To investigate the functional role of CD69 signaling, we analyzed EPX release from
224 mouse eosinophils following cross-linking to CD69, since obtaining sufficient numbers
225 of human eosinophils during the protein assays is difficult. We found CD69 (6.1% ±
226 0.3%) on mouse eosinophils purified from splenocytes (Fig. 5A), but not on PB
227 eosinophils. Moreover, cross-linking of CD69 to mouse eosinophils from spleen induced
228 EPX release in a dose dependent manner (Fig. 5B).
229

230

**Discussion**

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

In this study, we found significantly higher mRNA expressions of the Treg-associated cytokine (IL-10) and Th2 cytokines (IL-4, IL-5, and IL-13) in both UT and NP tissues from patients with ECRS compared with the same expressions in controls. In addition, expression of the Th1 cytokine (IFNγ) was increased in patients tissues compared to that in control tissues. As demonstrated by our results, Tomassen et al used a cluster analysis of biomarkers to show that CRS is a heterogeneous inflammatory disease. Therefore, patients tend to have endotypes corresponding to diverse inflammatory profiles containing Th1 and Th2 cytokines²². To better characterize eosinophils into the inflamed sites, we also developed a purification protocol using a cell sorter following a density gradient technique. Our protocol resulted in a 4-fold increase in the number of CD45⁺ cells compared to the number obtained after the conventional protocol, with comparable cell viabilities. We found only one other study demonstrating eosinophil purification from NPs in patients with ECRS: A report by Miyake et al, in which authors directly purified eosinophils from dissociated cells from NPs using a cell sorter without density gradient technique. However, they did not mention the total corrected cell numbers or the cell viability²³.
Next, we also found high mRNA expression of cytokines in the purified eosinophils from NPs in ECRS patients, supporting the existence of a difference in cytokine expression in the tissues compared with the expressions in controls. These data suggest that eosinophils are the source of cytokines in the patients with ECRS. Moreover, we found a significant difference in mRNA levels of the eosinophil-derived cytotoxic granules (MBP, EDN, and EPX) between eosinophils in the PB and NP from patients with ECRS. Although the cytokine levels between these groups were similar, higher levels of cytotoxic granules indicate eosinophil activation, since eosinophil-specific granules are associated with airway remodeling during the allergic disorders <sup>24</sup>. However, Miyata reported similar granule proteins (EDN, ECP, and EXP) contained in eosinophils from both NPs and PB <sup>23</sup>. This discrepancy between mRNA and protein in eosinophils may result from type of eosinophil in each experiment. Eosinophils come in two different types (hypodense and normodense eosinophils), the former and later reflect released and contained rich granules, respectively <sup>25</sup>. Since the ratio of hypodense eosinophils (reflecting activated eosinophils) exists dominantly during allergic inflammations <sup>26 27 28</sup>, it is possible that the protein of granules between these eosinophils be similar. By contrast, our results, showing high mRNA expressions of these cytotoxic granules in the tissue eosinophils, suggests hypodense but not normodense eosinophils, reflecting activation.
Interestingly, we also found over 30-fold increase in CD69 expression on eosinophils between NPs and PB from ECRS. Similarly, we found upregulated CD69 protein expression. Thus, the degree of CD69 expression on eosinophils may be used as a biomarker for eosinophilic inflammation. Then, we also investigated the association between the CD69 MFI ratio on eosinophils and the severity of symptoms among patients with ECRS having asthma, and we found that expression of CD69 (as an activation marker) in eosinophils from NPs was positively correlated with the NP score and sinus CT findings, and negatively correlated with pulmonary function (as measured by FEV₁₀% and %FEF₂₅-₇₅). By contrast, we could not find any correlation between the CD69 MFI ratio on T cells and these clinical findings. In ECRS, FEV₁₀% is a noninvasive marker of airway remodeling during asthma²⁹, and %FEF₂₅-₇₅ is associated with bronchial hyperresponsiveness in asthma³⁰, ³¹. Studies reported that the FEV₁₀% and %FEF₂₅-₇₅, but not the %PEF, were lower in patients with ECRS than in controls³²,³³. Therefore, CD69 expression on the eosinophils (but not in T cells isolated from the NP of patients with ECRS) is a potential biomarker for the severity of patients with ECRS.

Moreover, we found EPX release after cross-linking of CD69 onto mouse eosinophils, suggesting that the signaling contributes to development of remodeling during the eosinophilic airway inflammation. By contrast, Walsh et al reported that CD69 signaling
283 induced apoptosis, but not EPX release <sup>15</sup>. The discrepancies between their and our results

284 could be due to differing incubation times; they analyzed results after 48 hours and we

285 after 2 hours. Hosokawa et al showed that airway inflammation and airway

286 hyperresponsiveness (AHR) were diminished in CD69 deficient mice, and systemic

287 treatment with anti-CD69 antibody in an asthma mouse model improved symptoms

288 compared with treatment in naïve animals and treatment with isotype control antibodies<sup>34</sup>.

289 These findings suggest that CD69 plays a role as a marker of activation, and that it

290 regulates airway inflammation<sup>35</sup>.

291 In conclusion, we found that activated eosinophils from the NPs of patients with ECRS

292 expressed CD69, but not eosinophils from the PB of the same patients. In addition, CD69

293 expression on eosinophils was significantly correlated with the number of eosinophils

294 infiltrating NPs, the NP score, sinus CT findings, and measurements of pulmonary

295 function such as FEV<sub>1.0</sub>% and %FEF<sub>25-75</sub>. Moreover, we found that CD69-expressing

296 eosinophils released EPX following activation (cross-linking to CD69). Thus, our results

297 suggest that CD69 is a biomarker for the severity of nasal obstructions and for the degree

298 of pulmonary dysfunction as well as a novel therapeutic target for patients with ECRS

299 and asthma.

300 Acknowledgements
301 This research was supported by grants from the Japanese MEXT (Ministry of Education,

302 Culture, Sports, Science and Technology: KAKENHI, no.15K10793, no.16K08951, and

303 no.19K18749); the Fund of Academic Society for Research in Otolaryngology, Kansai

304 Medical University; a research grant D2 from Kansai Medical University; The branding

305 program as a world-leading research university on intractable immune and allergic

306 diseases. The authors would like to thank Enago (www.enago.jp) for the English language

307 review.
308 References

309 1. Fokkens WJ, Lund VJ, Mullol J, Bachert C, Alobid I, Baroody F, et al. European Position Paper on Rhinosinusitis and Nasal Polyps 2012. Rhinol Suppl 2012; 23:3 p preceding table of contents, 1-298.

312 2. Wang ET, Zheng Y, Liu PF, Guo LJ. Eosinophilic chronic rhinosinusitis in East Asians. World J Clin Cases 2014; 2:873-82.

314 3. Tokunaga T, Sakashita M, Haruna T, Asaka D, Takeno S, Ikeda H, et al. Novel scoring system and algorithm for classifying chronic rhinosinusitis: the JESREC Study. Allergy 2015; 70:995-1003.

317 4. Kobayashi Y, Asako M, Ooka H, Kanda A, Tomoda K, Yasuba H. Residual exhaled nitric oxide elevation in asthmatics is associated with eosinophilic chronic rhinosinusitis Journal of Asthma 2015; 52:1060-4.

320 5. Amelink M, de Groot JC, de Nijs SB, Lutter R, Zwinderman AH, Sterk PJ, et al. Severe adult-onset asthma: A distinct phenotype. Journal of Allergy and Clinical Immunology 2013; 132:336-41.

323 6. Kanda A, Kobayashi Y, Asako M, Tomoda K, Kawauchi H, Iwai H. Regulation of Interaction between the Upper and Lower Airways in United Airway Disease. Med Sci (Basel) 2019; 7.

326 7. Kobayashi Y, Yasuba H, Asako M, Yamamoto T, Takano H, Tomoda K, et al. HFA-BDP Metered-Dose Inhaler Exhaled Through the Nose Improves Eosinophilic Chronic Rhinosinusitis With Bronchial Asthma: A Blinded, Placebo-Controlled Study. Front Immunol 2018; 9:2192.

330 8. Hogan SP, Rosenberg HF, Moqbel R, Phipps S, Foster PS, Lacy P, et al. Eosinophils: biological properties and role in health and disease. Clin Exp Allergy 2008; 38:709-50.

332 9. Shah SA, Ishinaga H, Takeuchi K. Pathogenesis of eosinophilic chronic rhinosinusitis. J Inflamm (Lond) 2016; 13:11.

334 10. Johansson MW. Activation states of blood eosinophils in asthma. Clin Exp Allergy 2014; 44:482-98.

336 11. Cibrian D, Sanchez-Madrid F. CD69: from activation marker to metabolic gatekeeper. Eur J Immunol 2017; 47:946-53.

338 12. Martin P, Gomez M, Lamana A, Matesanz Marin A, Cortes JR, Ramirez-Huesca M, et al. The leukocyte activation antigen CD69 limits allergic asthma and skin contact hypersensitivity. J Allergy Clin Immunol 2010; 126:355-65, 65.e1-3.

341 13. Julius P, Luttmann W, Knoechel B, Kroegel C, Matthys H, Virchow JC, Jr. CD69 surface expression on human lung eosinophils after segmental allergen provocation. Eur Respir J 1999; 13:1253-9.
344 14. Hartnell A, Robinson DS, Kay AB, Wardlaw AJ. CD69 is expressed by human eosinophils activated in vivo in asthma and in vitro by cytokines. Immunology 1993; 80:281-6.

346 15. Walsh GM WM, Symon FA, Willars GB, Wardlaw AJ. Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor. blood 1996; 87:2815-21.

349 16. Kimura MY, Hayashizaki K, Tokoyoda K, Takamura S, Motohashi S, Nakayama T. Crucial role for CD69 in allergic inflammatory responses: CD69-Myl9 system in the pathogenesis of airway inflammation. Immunol Rev 2017; 278:87-100.

352 17. Hayashizaki K, Kimura MY, Tokoyoda K, Hosokawa H, Shinoda K, Hirahara K, et al. Myosin light chains 9 and 12 are functional ligands for CD69 that regulate airway inflammation. Sci Immunol 2016; 1:eaaf9154.

355 18. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA, et al. Rhinosinusitis: developing guidance for clinical trials. J Allergy Clin Immunol 2006; 118:S17-61.

358 19. Lund VJ, Mackay IS. Staging in rhinosinusitus. Rhinology 1993; 31:183-4.

359 20. Marquez ME, Deglesne PA, Suarez G, Romano E. MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal. Int J Lab Hematol 2011; 33:194-200.

362 21. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nature Protocols 2008; 3:1101.

364 22. Tomassen P, Vandeplas G, Van Zele T, Cardell LO, Arebro J, Olze H, et al. Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers. J Allergy Clin Immunol 2016; 137:1449-56 e4.

367 23. Miyata J, Fukunaga K, Kawashima Y, Watanabe T, Saitoh A, Hirosaki T, et al. Dysregulated fatty acid metabolism in nasal polyp-derived eosinophils from patients with chronic rhinosinusitis. Allergy 2019.

370 24. Rosenberg HF, Dyer KD, Foster PS. Eosinophils: changing perspectives in health and disease. Nat Rev Immunol 2013; 13:9-22.

372 25. Legrand F, Woerly G, Driss V, Capron M. Innate immune function of eosinophils: from antiparasite to antitumor cells. Methods Mol Biol 2008; 415:215-40.

374 26. Conesa A, Tassinari P, Rivera H, De Sanctis JB, Bianco N, Aldrey O. Hypodense eosinophils: characterization of surface molecule expression. Allergy Asthma Proc 2002; 23:117-24.

377 27. Frick WE, Sedgwick JB, Busse WW. Hypodense eosinophils in allergic rhinitis. J Allergy Clin Immunol 1988; 82:119-25.

379 28. Kuo HP, Yu TR, Yu CT. Hypodense eosinophil number relates to clinical severity, airway hyperresponsiveness and response to inhaled corticosteroids in asthmatic subjects. Eur
381 Respir J 1994; 7:1452-9.

382 29. Barisione G, Crimi E, Bartolini S, Saporiti R, Copello F, Pellegrino R, et al. How to interpret reduced forced expiratory volume in 1 s (FEV1)/vital capacity ratio with normal FEV1. Eur Respir J 2009; 33:1396-402.

385 30. Alberts WM, Ferris MC, Brooks SM, Goldman AL. The FEF25-75% and the clinical diagnosis of asthma. Ann Allergy 1994; 73:221-5.

387 31. Contoli M, Bousquet J, Fabbri LM, Magnussen H, Rabe KF, Siafakas NM, et al. The small airways and distal lung compartment in asthma and COPD: a time for reappraisal. 2010; 65:141-51.

390 32. Tanaka S, Hirota T, Kamijo A, Ishii H, Hatsushika K, Fujieda S, et al. Lung functions of Japanese patients with chronic rhinosinusitis who underwent endoscopic sinus surgery. Allergol Int 2014; 63:27-35.

393 33. Uraguchi K, Kariya S, Makihara S, Okano M, Haruna T, Oka A, et al. Pulmonary function in patients with eosinophilic chronic rhinosinusitis. Auris Nasus Larynx 2018; 45:476-81.

395 34. Miki-Hosokawa T, Hasegawa A, Iwamura C, Shinoda K, Tofukuji S, Watanabe Y, et al. CD69 controls the pathogenesis of allergic airway inflammation. J Immunol 2009; 183:8203-15.

398 35. Weller PF, Spencer LA. Functions of tissue-resident eosinophils. Nat Rev Immunol 2017; 17:746-60.
401 **Figure Legends**

402 **Fig 1. mRNA expression of cytokines in tissues of patients with ECRS and controls**

403 Quantitative PCR analysis of IL-4, IL-5, IL-10, IL13, and IFN-γ mRNA expression levels
404 in tissues of patients with ECRS and in controls. NECRS: non-eosinophilic chronic
405 rhinosinusitis, controls. ECRS: eosinophilic chronic rhinosinusitis. UT: uncinate process
406 tissue. NP: nasal polyp. Relative gene expression levels were calculated by the
407 comparative C~T~ method with UT of controls as the reference. Data are expressed as means
408 ± SEM (ECRS and controls are n = 12, 8). P values determined by the Mann-Whitney U-
409 test (*P* < 0.05).

410 **Fig 2. mRNA expression of cytokines and cytotoxic granules on purified eosinophils**
411 **from peripheral blood (PB) and nasal polyps (NP)**

412 (A) Quantitative PCR analysis of IL-4, IL-5, IL-10, IL13, and IFN-γ mRNA expression
413 levels on purified eosinophils from patients with ECRS and controls (B) Quantitative
414 PCR analysis of MBP, EDN, ECP, and EPX mRNA expression levels on purified
415 eosinophils from patients with ECRS and controls. PB Eos: Eosinophils from peripheral
416 blood. NP Eos: eosinophils from nasal polyp. Relative gene expression levels were
417 calculated by the comparative C~T~ method with PB of NECRS as the reference. We
418 expressed data as means ± SEM (ECRS and controls are n = 8, 5). P values determined
419 by the Mann-Whitney U-test (*P* < 0.05).

<caption>420 **Fig 3. Eosinophils infiltrating the nasal polyps of patients with ECRS were activated**</caption>

421 (A) Quantitative PCR analysis of CD69 mRNA expression levels from peripheral blood
422 (PB) eosinophils and nasal polyp (NP) eosinophils from patients with ECRS. Relative
423 gene expression levels were calculated by the comparative C~T~ method with PB of controls
424 as the reference. P values determined by the Mann-Whitney U-test (*P* < 0.05, NECRS
425 and ECRS are n = 5, 8). (B) Histograms of CD69 expression on eosinophils in NP (red)
426 and PB (blue) from patients with ECRS. Representative data from patient #18. The gray
427 bar is isotype control. MFI of CD69 comparing eosinophils in PB to those in NPs. Result
428 shown as MFI subtracting corresponding to the isotype control. P values determined by
429 the Mann-Whitney U-test (*P* < 0.05, n = 12). (D) Immunofluorescence images of fixed
430 NP sections stained with antibodies against CD69 and Siglec8, and corresponding isotype
431 controls. Representative data from patient #18. 400X, Scale bar 10µm.

<caption>432 **Fig 4. Eosinophil activation correlated with clinical findings and pulmonary function results**</caption>

433 (A) Positive correlation between CD69 MFI ratio on eosinophils with numbers of
434 infiltrating eosinophils to NPs of patients with ECRS (P < 0.05, r = 0.7273, n = 12). (B)
435 Positive correlation between CD69 MFI ratio on eosinophils with nasal polyp scores (P
436
437 < 0.05, r = 0.624, n = 12). (C) Positive correlation between CD69 MFI ratio on eosinophils with Lund-Mackay CT scale (P < 0.05, r = 0.6103, n = 12). (D) Negative correlation between CD69 MFI ratio on eosinophils with FEV<sub>1.0</sub>% and %FEF<sub>25-75</sub> (reflecting asthma severity), but not with %PEF (FEV<sub>1.0</sub>%: P < 0.05, r = -0.6783, n = 12, %FEF<sub>25-75</sub>: P < 0.05, r = -0.5874, n = 12). We expressed correlations as Spearman's rank correlation coefficients. PB Eos: Eosinophils from peripheral blood. NP Eos: eosinophils from nasal polyp.

444 **Figure 5. CD69 expression and EPX release by cross-linking of CD69**

445 (A) Expression of CD69 on eosinophils purified from mouse spleen IL-5 Tg mouse and peripheral blood eosinophils. (B) indicates EPX release after cross-linking of CD69 on mouse eosinophils purified from mouse spleen IL-5 Tg (n = 4). We expressed data as means ± SEM. P values determined by the Mann-Whitney U-test (*P* < 0.05).

449

450 **Table 1.**

<table><thead><tr><th></th><th>non-ECRS</th><th>ECRS</th><th>P value</th></tr></thead><tbody><tr><td>Patients,n</td><td>8</td><td>12</td><td></td></tr><tr><td>Age (y), median</td><td>45.5±19.7</td><td>58.7±10.2</td><td></td></tr><tr><td>Male / female, n</td><td>3 / 5</td><td>9 / 3</td><td></td></tr><tr><td>Blood eosinophils counts (/µl)</td><td>245.5±151.9</td><td>374.8±192.6</td><td></td></tr><tr><td>Total IgE</td><td>358.5±461.7</td><td>761.2±1332.8</td><td></td></tr><tr><td>Asthma, n</td><td>1</td><td>10</td><td>P&lt;0.05</td></tr><tr><td>NP, n</td><td>0</td><td>12</td><td></td></tr><tr><td>Tissue eosinophils counts (/HPF)</td><td></td><td>227.1±146.6</td><td></td></tr></tbody></table>

451

452
1 **Supplementary Data**

2 **Title :**

3 Increased CD69 expression on activated eosinophils in eosinophilic chronic rhinosinusitis

4 correlates with clinical findings

5 **Author:**

6 Yasutaka Yun, Akira Kanda, Yoshiki Kobayashi, Dan Van Bui, Kensuke Suzuki,

7 Shunsuke Sawada, Kazuyasu Baba, Masao Yagi, Mikiya Asako, Haruka Okazaki, Hiroki

8 Ikeda, Shigeki Kawamura, Akihiko Nakamura, David Dombrowicz, Koichi Tomoda, and

9 Hiroshi Iwai

10

11 **Material and methods**

12 *Human and mouse eosinophil purification*

13 For eosinophil purifications from nasal polyps (NP), as shown in the **Fig.S1A**, we used a

14 dissociator from a tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany)

15 or a conventional protocol using collagenases, hyaluronidases, and DNases. These

16 dissociated cells were separated by granulocytes including eosinophils and other cells

17 using density gradient centrifugation with Ficoll-Paque (1.077 mg/dl). We removed dead

18 cells from separated granulocytes in the bottom layer using the Dead cell removable kit
(Miltenyi Biotec, Bergisch Gladbach, Germany). Then, we isolated eosinophils using a cell sorter. Eosinophils from NP and PB were identified by gated CD45⁺Siglec8⁺CD68⁻ CD16b⁻CD3⁻CD19⁻ and CD45⁺Siglec8⁺CD14⁻CD16b⁻CD3⁻ CD19⁻, respectively (Fig. S1B). Isolated eosinophils from NP and PB did not contain mast cells or basophils, respectively (Fig. S1C and D). To assess viability, or survival rate, we stained cells with 7-AAD and Annexin V.

To purify eosinophils from splenocytes of IL-5 Tg mice (hyper eosinophilic mice) we followed a published protocol¹. Briefly, we used negative selection with biotin-conjugated anti-CD19, anti-CD90.2, and anti-CD8a antibodies (BD PharMingen, CA, U.S.A) and anti-biotin-conjugated magnetic beads. Purity was greater than 95%.

### Immunofluorescence staining

NPs and UTs were fixed in 10% formaldehyde and embedded in paraffin. Sections were sliced at 4-µm thicknesses. For antigen retrieval, sections were activated in Histo VT One solution (Nacalai Tesque, Kyoto, Japan) for 15 min at 121°C. Sections were then incubated with a blocking solution of phosphate-buffered saline containing 10% Goat serum (Sigma-Aldrich, Miss, U.S.A) and 0.5% tween-20 (Sigma-Aldrich, Miss, U.S.A) for 1 hour at room temperature.

### qPCR
37 mRNA was extracted using RNeasy Micro Kit (Qiagen, Hilden, Germany) or PureLink™

38 RNA Micro Kit (Invitrogen, CA, U.S.A). We performed reverse transcription to the first-

39 strand cDNA using PrimeScript RT Master Mix (Takara, Shiga, Japan), and performed

40 quantitative PCRs (qPCRs) using Rotor-Gene SYBR Green PCR Kit (Qiagen, Hilden,

41 Germany) on a Rotor-Gene Q HRM (Qiagen, Hilden, Germany).
42 Supplementary figure legends

43 Supplementary Figure 1. Modified protocol to purify eosinophils from nasal polyps
44 (NPs).

45 (A)We dissociated NPs using a human tumor dissociation kit. After dissociating the NPs,
46 the buffer diluted cell samples (the cell suspensions) were separated using the Ficoll-
47 Paque technique by centrifuge at 300 g for 30 min. The 2nd layer contained the NP
48 mononuclear cells, and the lowest layer contained granulocytes and eosinophils. To
49 eliminate dead cells, we resuspended the lower layer in a suspension of the Dead Cell
50 Removal kit. We applied the cell suspensions onto magnetic columns, and collected the
51 effluent containing the live cells, following cell sort. (B) We then used a flow cytometer
52 to identify eosinophils using a gating strategy. We showed representative data from one
53 of the patients with ECRS. Eosinophils from NPs were gated as cell populations with
54 CD45+siglec8+CD68-CD16b-CD3-CD19-. Eosinophils from PB were gated as cell
55 populations with CD45+siglec8+CD14-CD16b-CD3-CD19-. Magnification, 400x. (C)(D)
56 Isolated eosinophils from NP and PB were stained using CD117, CD123 and
57 corresponding isotypes. NP Eos: eosinophils from nasal polyp. PB Eos: eosinophils from
58 peripheral blood.

59 Supplementary Figure 2. Gating strategy for mouse eosinophils
60 (A)(B) Gating strategy for the identification of mouse eosinophils from spleen and PB
61 using flow cytometry. Mouse eosinophils were gated as cell populations with
62 CD45⁺siglecF⁺Gr-1int.

63 **Supplementary Figure 3. Comparison between modified and conventional protocols**
64 We compared both protocols in terms of dissociation cell numbers, CD45 positive cells,
65 and cell survival rates. Data are expressed as means ±SEM (n = 12). P-values were
66 determined by the Mann–Whitney U-test.

67 **Supplementary Figure 4. Comparison of T cell activation and clinical findings**
68 (A-D) We found no correlations among CD69 MFI ratios on T cells, infiltrating number
69 of eosinophils, and clinical findings. We assessed correlations using the Spearman`s rank
70 correlation coefficient.

71 **Supplementary Figure 5. Correlation between number of infiltrating eosinophils and clinical findings**
72
73 (A)(B) We found no correlation between the number of NP infiltrating eosinophils and
74 the NP scores or sinus CT findings (as defined by Lund-Mackay CT scale). (C) The
75 number of NP infiltrating eosinophils correlated with the %FEF25-75. While other
76 pulmonary functions lacked correlations. We applied Spearman`s rank correlation
77 coefficients to assess correlations.
78

79 Supplementary tables

80 Supplementary Table 1. Detail character information of NECRS and ECRS

<table><thead><tr><td>No.</td><td>Age</td><td>sex</td><td>Phenotype</td><td>Tisseu Eos (HPF)</td><td>Eos (%)</td><td>Blood Eos (μg)</td><td>OCS</td><td>INCS</td></tr></thead><tbody><tr><td>1</td><td>21</td><td>M</td><td>NECRS</td><td>NA</td><td>6.0</td><td>468</td><td>-</td><td>+</td></tr><tr><td>2</td><td>49</td><td>M</td><td>NECRS</td><td>NA</td><td>2.9</td><td>240.7</td><td>-</td><td>+</td></tr><tr><td>3</td><td>67</td><td>F</td><td>NECRS</td><td>NA</td><td>5.4</td><td>297</td><td>-</td><td>+</td></tr><tr><td>4</td><td>49</td><td>F</td><td>NECRS</td><td>NA</td><td>4.7</td><td>408.9</td><td>-</td><td>+</td></tr><tr><td>5</td><td>62</td><td>M</td><td>NECRS</td><td>NA</td><td>3.1</td><td>161.2</td><td>-</td><td>+</td></tr><tr><td>6</td><td>70</td><td>F</td><td>NECRS</td><td>NA</td><td>1.3</td><td>41.6</td><td>-</td><td>+</td></tr><tr><td>7</td><td>31</td><td>F</td><td>NECRS</td><td>NA</td><td>7.4</td><td>325.6</td><td>-</td><td>+</td></tr><tr><td>8</td><td>15</td><td>F</td><td>NECRS</td><td>NA</td><td>0.5</td><td>21</td><td>-</td><td>+</td></tr><tr><td>9</td><td>73</td><td>M</td><td>ECRS</td><td>158</td><td>7.9</td><td>402.9</td><td>-</td><td>+</td></tr><tr><td>10</td><td>68</td><td>M</td><td>ECRS</td><td>120</td><td>3.0</td><td>123</td><td>-</td><td>+</td></tr><tr><td>11</td><td>51</td><td>F</td><td>ECRS</td><td>163</td><td>4.8</td><td>211.2</td><td>-</td><td>+</td></tr><tr><td>12</td><td>62</td><td>M</td><td>ECRS</td><td>320</td><td>4.6</td><td>211.6</td><td>-</td><td>+</td></tr><tr><td>13</td><td>40</td><td>F</td><td>ECRS</td><td>292</td><td>4.8</td><td>321.6</td><td>-</td><td>+</td></tr><tr><td>14</td><td>46</td><td>M</td><td>ECRS</td><td>123</td><td>10.3</td><td>700.4</td><td>-</td><td>+</td></tr><tr><td>15</td><td>70</td><td>M</td><td>ECRS</td><td>629</td><td>11.3</td><td>542.4</td><td>-</td><td>+</td></tr><tr><td>16</td><td>66</td><td>M</td><td>ECRS</td><td>328</td><td>9.6</td><td>662.4</td><td>-</td><td>+</td></tr><tr><td>17</td><td>68</td><td>M</td><td>ECRS</td><td>230</td><td>5.5</td><td>258.5</td><td>-</td><td>+</td></tr><tr><td>18</td><td>51</td><td>F</td><td>ECRS</td><td>193</td><td>8.3</td><td>581</td><td>-</td><td>+</td></tr><tr><td>19</td><td>56</td><td>M</td><td>ECRS</td><td>95</td><td>5.8</td><td>330.6</td><td>-</td><td>-</td></tr><tr><td>20</td><td>53</td><td>M</td><td>ECRS</td><td>74</td><td>3.1</td><td>151.9</td><td>-</td><td>+</td></tr></tbody></table>

81

82 NECRS: non-eosinophilic chronic rhinosinusitis, ECRS: eosinophilic chronic

83 rhinosinusitis, HPF: high power field, OCS: oral corticosteroids, INCS: intranasal

84 corticosteroids, NP: nasal polyp, Eos: eosinophils, NA: not applicable.

85
<table><caption>86 Supplementary Table 2. List of antibodies</caption><thead><tr><th>Flow cytometric analysis</th><th>Clone</th><th>Supplier</th><th>Dilution</th><th>Isotype</th></tr></thead><tbody><tr><td>Antibody</td><td></td><td></td><td></td><td></td></tr><tr><td>mouse anti-human CD45 FITC</td><td>HI30</td><td>Invitrogen</td><td>1:200</td><td>mouse IgG1</td></tr><tr><td>mouse anti-human CD125 PE</td><td>A14</td><td>BD Biosciences</td><td>1:200</td><td>mouse IgG1</td></tr><tr><td>mouse anti-human CD68 PerCP</td><td>Y1/82A</td><td>BioLegend</td><td>1:200</td><td>mouse IgG2b</td></tr><tr><td>mouse anti-human CD14 PerCP</td><td>HCD14</td><td>BioLegend</td><td>1:200</td><td>mouse IgG1</td></tr><tr><td>mouse anti-human CD69 PE/Cy7</td><td>FN50</td><td>BioLegend</td><td>1:200</td><td>mouse IgG1</td></tr><tr><td>mouse anti-human Siglec8 APC</td><td>7C9</td><td>BioLegend</td><td>1:200</td><td>mouse IgG1</td></tr><tr><td>mouse anti-human CD16b APC-Vio770</td><td>REA589</td><td>Miltenyi Biotec</td><td>1:200</td><td>human IgG1</td></tr><tr><td>mouse anti-human CD3 BV421</td><td>UCHT1</td><td>BD Biosciences</td><td>1:200</td><td>mouse IgG1</td></tr><tr><td>mouse anti-human CD19 BV510</td><td>5E8</td><td>BD Biosciences</td><td>1:200</td><td>mouse IgG2b</td></tr><tr><td>Hamster anti-mouse CD69 PE</td><td>H1.2F3</td><td>BD Parmingen</td><td>1:100</td><td>Ar Hamster IgG1</td></tr><tr><td>Rat anti-mouse Gr-1 PerCP</td><td>RB6-8C5</td><td>BD Parmingen</td><td>1:200</td><td>Rat IgG2a</td></tr><tr><td>Rat anti-mouse SiglecF Alexa Fluor 647</td><td>E50-2440</td><td>BD Parmingen</td><td>1:200</td><td>Rat IgG2a</td></tr><tr><td>Rat anti-mouse CD45 V500</td><td>30-F11</td><td>BD Horizon</td><td>1:200</td><td>Rat IgG2b</td></tr></tbody></table>

87

<table><thead><tr><th>Immunofluorescence staining</th><th>Clone</th><th>Supplier</th><th>Dilution</th><th>Isotype</th></tr></thead><tbody><tr><td>1<sup>st</sup> antibody</td><td></td><td></td><td></td><td></td></tr><tr><td>Rabbit anti-human Siglec8</td><td>polyclonal</td><td>GeneTex</td><td>1:400</td><td>Rabbit IgG</td></tr><tr><td>mouse anti-human CD69</td><td>FN50</td><td>BioLegend</td><td>1:400</td><td>mouse IgG</td></tr><tr><td>2<sup>nd</sup> antibody</td><td></td><td></td><td></td><td></td></tr><tr><td>conjugated with CF<sup>TM</sup> 488A Goat Anti-rabbit IgG</td><td></td><td>Biotium</td><td>1:200</td><td></td></tr><tr><td>conjugated with CF<sup>TM</sup> 647A Goat Anti-mouse IgG</td><td></td><td>Biotium</td><td>1:200</td><td></td></tr></tbody></table>

88
<table><caption>89 Supplementary Table 3. Primer List</caption><thead><tr><th></th><th>Forward</th><th>Reverse</th></tr></thead><tbody><tr><td>IL-4</td><td>5'-GCAGCTGATCCGATTCCTGA-3'</td><td>5'-CTCTGGTTGGCTTCCTTCAC-3'</td></tr><tr><td>IL-5</td><td>5'-AACTGTGCAAGGGGGTACTG-3'</td><td>5'-TCTCCGTCTTTCTTCTCCACA-3'</td></tr><tr><td>IL-10</td><td>5'-GAGATGCCTTCAGCAGAGTGAA-3'</td><td>5'-ATCTCAGACAAGGCTTGGCAAC-3'</td></tr><tr><td>IL-13</td><td>5'-CTGACAGCTGGCATGTACTGTG-3'</td><td>5'-TGTCTCGGACATGCAAGCTG-3'</td></tr><tr><td>IFNγ</td><td>5'-TTCAGCTCTGCATCGTTTTG-3'</td><td>5'-TCTTTTGGATGCTCTGGTCA-3'</td></tr><tr><td>MBP</td><td>5'-GAAAGATGGGGCTGTTGAGT-3'</td><td>5'-CTTCTCACCAGGAGGTAGCG-3'</td></tr><tr><td>EDN</td><td>5'-TCTCACAGGAGCTACAGCGCG-3'</td><td>5'-AACATGTTTGCTGGTGTCTGC-3'</td></tr><tr><td>ECP</td><td>5'-GCAGACAGACCAGGAAGGAG-3'</td><td>5'-GCAGACAGACCAGGAAGGAG-3'</td></tr><tr><td>EPX</td><td>5'-CCCGGTGGAATGGAGACAAA-3'</td><td>5'-TCGATGCCCCCTTCATACAC-3'</td></tr></tbody></table>
<table><tr><td>CD69</td><td>5'-CTGGACTTCAGCCCAAAATGC-3'</td><td>5'-TCAGTCCAACCCAGTGTTCC-3'</td></tr><tr><td>GAPDH</td><td>5'-TTCACCACCATGGAGAAGGC-3'</td><td>5'-AGGAGGCATTGCTGATGATCT-3'</td></tr></table>
<figure><img src="image_3.png" /></figure>
A.

<figure><img src="image_4.png" /></figure>

B.

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
A.

<figure><img src="image_16.png" /></figure>

B.

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>
<figure><img src="image_21.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_22.png" /></figure>
<figure><img src="image_23.png" /></figure>
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>
<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>