3448  |    LETTERS DOI: 10.1111/all.15424 SEB- induced IL- 13 production in CLA+ memory T cells defines Th2 high and Th2 low responders in atopic dermatitis To the Editor, Staphylococcus aureus, memory skin- homing cutaneous lymphocyte- associated antigen (CLA)+ T cells and IL- 13 constitute relevant play- ers in atopic dermatitis (AD) pathogenesis.1 Since circulating CLA+ T cells reflect cutaneous abnormalities present in human inflammatory skin diseases,2 an ex vivo coculture model made of purified circu- lating CLA+/− effector and central memory T cells and autologous lesional epidermal cells was established. We show a CLA- dependent production of IL- 13 upon activation with staphylococcal enterotoxin B (SEB) that allows the differentiation of the Th2 high and Th2 low groups, with distinct clinical correlations between both groups, within a clinically homogeneous population of adult non- treated moderate- to- severe AD patients. Our results showed that IL- 13, together with IL- 4, IL- 17A, IL- 22, CCL17, and CCL22, was preferentially produced by circulating memory CLA+ T cells upon activation with SEB in the presence of autologous lesional epidermal cells (Figure 1A). Interestingly, SEB ac- tivation of the CLA+/Epi cocultures resulted in a predominant IL- 13 production among the Th2 cytokines (IL- 13, IL- 4, lL- 5) (Figure 1B). The amount of IL- 5 and IFN- γ produced by SEB- activated CLA− T cells was higher or similar than that by CLA+ T cells, respectively, suggesting their relationship to extracutaneous sites. This model is stimulus- specific since polyclonal activators such as PMA/Ionomycin and CD3/CD28 are not CLA- specific (Figure S1A), and epidermal cells contributed to the T- cell activation (Figure S1B). Patients were stratified based on the median of the IL- 13 re- sponse in the SEB- induced CLA+ T- cell AD cocultures (Figure 1C), and we found differentiated T- cell responses to SEB between the Th2 high and the Th2 low groups (Figure 1D). Although both groups were clinically homogeneous (Figure S1C), this stratification suggested differential immunological mechanisms between both groups, since they not only differed in terms of in vitro stimulation, but also in terms of severity, plasma markers, IgE levels against S. aureus and mRNA expression from cutaneous lesions. In the Th2 high group, in contrast to the Th2 low, the IL- 13 re- sponse by SEB CLA+ T cells directly correlated with EASI score and plasma levels of CCL17 and sIL- 2R (Figure 2A- C). This group also showed a direct correlation between anti- S. aureus IgE lev- els and SEB- induced CLA+ T- cell- mediated IL- 13 response in vitro (Figure 2D). The mRNA expression from lesional skin biopsies was similar between both groups (Figure S2A), but the IL- 13 produced by SEB- stimulated CLA+/Epi cocultures directly correlated with CCL26 (Figure 2E) and inversely correlated with LCN2 mRNA expression in the Th2 high group (Figure 2F). Additionally, the IL- 13/IL- 17A and IL- 13/IFN- γ ratios in the SEB- stimulated CLA+ T- cell cocultures were higher in the Th2 high than the Th2 low group (Figure S2B), support- ing the type 2 signature and the lowered type 17 and type 1 immu- nity in the Th2 high group, which may facilitate S. aureus infection.3 The study has a few limitations. We did not study the presence of S. aureus in the skin of the AD patients and the number of patients was not very high but we found consistent significant results on the relationship between the SEB- induced CLA+ T- cell IL- 13 response and clinical features of the patients. The novelty of our results relies on the separation of the Th2 high and low populations, corresponding with disease activity, based on the CLA+ T- cell IL- 13 response to SEB, which are key mediators in AD pathogenesis.4 Interestingly, the existence of Th2 high and low groups in non- treated moderate- to- severe AD patients has been shown by serum proteomic profiling.5 In conclusion, we consider that this new and translational approach allows obtaining readouts on cytokine production that complement current studies based on transcriptomics and flow cytometry and may help to explore the complex heterogeneity of AD pathophysiology from a more func- tionally point of view. This is an open access article under the terms of the Creative Commons Attribution- NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.     | 3449LETTERS F I G U R E 1  Production of AD- associated mediators by SEB- activated cocultures of CLA+ T cells and lesional epidermal cells and stratification into the Th2 high and Th2 low groups. (A) Quantification (pg/ml) of IL- 13, IL- 4, IL- 5, IL- 17A, IL- 22, IFN- γ, CCL17, and CCL22 in 24- hour cocultures in basal conditions or stimulated with SEB (n = 35 for IL- 13/4/17A and IFN- γ, n = 30 for IL- 5, n = 29 for IL- 22, and n = 20 for CCL17/22). (B) Th2 cytokines produced by SEB- induced CLA+ T- cell cocultures presented by column bars and the median ± 95% CI. (C) IL- 13 levels in AD (n = 35) and control subjects (n = 8). Dotted line indicates the median of SEB- induced CLA+ T- cell IL- 13 response in AD. (D) Cytokine response (pg/ml) by SEB- activated CLA+ T- cell cocultures was compared between the Th2 high and the Th2 low groups. Abbreviations: AD, atopic dermatitis; CLA, cutaneous lymphocyte- associated antigen T cells; Epi, epidermal cells suspension; HC, healthy controls; M, untreated; SEB, staphylococcal enterotoxin B. **: p < .01; ***: p < .001; ****: p < .0001 13989995, 2022, 11, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/all.15424 by Readcube (Labtiva Inc.), W iley Online Library on [11/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3450  |    LETTERS FUNDING INFORMATION The study was funded by FIS/ISCIII (Ministerio de Economía y Competitividad e Instituto de Salud Carlos III) 2021 (PI21/01179 and PI21/00335) and Fondo Europeo del Desarrollo Regional (FEDER). Additionally, Sans- De San Nicolàs L was granted by a PhD fellowship from the Agency for Management of University and Research Grants of the Generalitat de Catalunya (FI- SDUR 2020); De Jesús- Gil C was granted by a PhD fellowship from the Agency for Management of F I G U R E 2  In the Th2 high group, SEB- triggered CLA+/Epi IL- 13 response directly correlates with EASI, CCL17, sIL- 2R, and anti- S. aureus IgE plasma levels and CCL26 mRNA expression in cutaneous lesions and inversely correlates with LCN2 mRNA expression in cutaneous lesions. IL- 13 (pg/mL) from 24- hour cocultures was correlated with (A) EASI (n = 17 for Th2 high and n = 15 for Th2 low), (B) plasma CCL17 (n = 18 for Th2 high and n = 17 for Th2 low), (C) plasma sIL- 2R (n = 18 for Th2 high and n = 17 for Th2 low), (D) anti- S. aureus IgE plasma levels (n = 17 for Th2 high and n = 17 for Th2 low), (E) CCL26 (n = 11 for Th2 high and n = 10 for Th2 low), and (F) LCN2 mRNA expression in lesional skin biopsies (n = 11 for Th2 high and n = 10 for Th2 low). Abbreviations: CLA, cutaneous lymphocyte- associated antigen T cells; Epi, epidermal cells suspension; M, untreated; SEB, staphylococcal enterotoxin B. ns: p > .05; *: p < .05; ***: p < .001 Th 2 hi gh (A) (B) (C) Th 2 lo w Th 2 hi gh (D) (E) (F) Th 2 lo w 13989995, 2022, 11, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/all.15424 by Readcube (Labtiva Inc.), W iley Online Library on [11/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License     | 3451LETTERS University and Research Grants of the Generalitat de Catalunya (FI- 2018), co- financed with FEDER; García- Jiménez I was granted by a PhD fellowship from the Universitat de Barcelona (PREDOCS- UB 2020). ACKNOWLEDG EMENTS We are grateful to all individuals who participated in our study. CONFLIC T OF INTERE S T Antonio Guilabert is a consultant for Sanofi, Almirall, and AbbVie. Laia Curto- Barredo is a consultant for Sanofi, AbbVie, Leo Pharma, and Lilly. Esther Serra- Baldrich is a consultant for Sanofi, Almirall, Leo Pharma, Pfizer, Galderma, and Lilly. Michael D. Howell is an em- ployee and shareholder of DermTech. The rest of authors declare no conflict of interests. Lídia Sans- De San Nicolàs1 Ignasi Figueras- Nart2 Montserrat Bonfill- Ortí2 Carmen De Jesús- Gil1 Irene García- Jiménez1 Antonio Guilabert3 Laia Curto- Barredo4 Marta Bertolín- Colilla4 Marta Ferran4 Esther Serra- Baldrich5 Anna Zalewska- Janowska6 Yui- Hsi Wang7,8 Michael D. Howell9 Ramon M. Pujol4 Luis F. Santamaria- Babí1 1Grup d’Immunologia Translacional, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona (UB), Parc Científic de Barcelona (PCB), Barcelona, Spain 2Departament de Dermatologia, Hospital de Bellvitge, Universitat de Barcelona (UB), L’Hospitalet de Llobregat, Spain 3Departament de Dermatologia, Hospital General de Granollers, Granollers, Spain 4Departament de Dermatologia, Hospital del Mar, Institut Hospital del Mar d’Investigacions Mèditques (IMIM), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain 5Departament de Dermatologia, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain 6Psychodermatology Department, Rheumatology and Clinical Immunology, Medical University of Lodz, Lodz, Poland 7Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA 8Type 2 Inflammation and Fibrosis Cluster, Immunology and Inflammation Research, Sanofi, Cambridge, Massachusetts, USA 9DermTech, Inc, La Jolla, California, USA Correspondence Luis F. Santamaria- Babí, Translational Immunology, Parc Científic de Barcelona, Baldiri i Reixac, 10, 08028 Barcelona, Spain. Email: luis.santamaria@ub.edu ORCID Lídia Sans- De San Nicolàs https://orcid. org/0000-0002-2828-2842 Luis F. Santamaria- Babí https://orcid.org/0000-0002-1674-6654 R E FE R E N C E S 1. Langan SM, Irvine AD, Weidinger S. Atopic dermatitis. Lancet. 2020;396(10247):345- 360. 2. DeJesús- Gil C, Sans- de San Nicolàs L, García- Jiménez I, et al. The translational relevance of human circulating memory cutaneous lymphocyte- associated antigen positive T cells in inflammatory skin disorders. Front Immunol. 2021;12:1- 7. 3. Simpson EL, Villarreal M, Jepson B, et al. Patients with atopic der- matitis colonized with staphylococcus aureus have a distinct phe- notype and endotype. J Invest Dermatol. 2018;138(10):2224- 2233. 4. Santamaria- Babi LF. Atopic dermatitis pathogenesis: lessons from immunology. Dermatol Pract Concept. 2022;12(1):1- 6. 5. Thijs JL, Strickland I, Bruijnzeel- Koomen CAFM, et al. Moving toward endotypes in atopic dermatitis: identification of patient clusters based on serum biomarker analysis. J Allergy Clin Immunol. 2017;140(3):730- 737. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. 13989995, 2022, 11, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/all.15424 by Readcube (Labtiva Inc.), W iley Online Library on [11/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License