Expression of CD83 in tissue-resident regulatory T cells maintains local homeostasis and restricts effector cells in allergic asthma

Page 1/26 Expression of CD83 in tissue-resident regulatory T cells maintains local homeostasis and restricts effector cells in allergic asthma Anita Heiß Universitätsklinikum Erlangen Andreas Wild Universitätsklinikum Erlangen https://orcid.org/0009-0005-6008-2874 Alexander Steinkasserer Universitätsklinikum Erlangen Christine Kuhnt Universitätsklinikum Erlangen Christina Draßner Universitätsklinikum Erlangen Susanne Krammer Universitätsklinikum Erlangen https://orcid.org/0000-0002-1445-9555 Adriana Geiger Universitätsklinikum Erlangen Carol Geppert FAU-Uniklinikum Erlangen Stefan Schliep Universitätsklinikum Erlangen Article Keywords: Posted Date: January 12th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3787362/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License Page 2/26 Additional Declarations: There is NO Competing Interest. Page 3/26 Abstract Non-lymphoid tissue Tregs (NLT-Tregs) are critical for tissue homeostasis, inammation control, and induction of mucosal repair. Recent single-cell RNA sequencing data identied expression of CD83 as part of a NLT-Treg signature, however its biological signicance for this specialized Tregs was not yet fully understood. In our previous investigations, we found that conditional deletion of CD83 (CD83cKO) disrupts stability and differentiation of lymphoid Tregs and exacerbates autoimmune responses. The present study explores for the rst time the role of CD83 expression by lung-resident Tregs to understand its importance in barrier tissues. We report that CD83-decient lung Tregs are less differentiated but more activated, resulting in unrestrained T cell activation. Furthermore, using an allergic asthma model, CD83cKO mice showed an accelerated disease progression, with augmented eosinophilic inammation, driven by Th2-biased T cell responses. CD83cKO Tregs exhibited an enhanced responsiveness to IL-4, leading to insucient control of Th2-differentiation from naïve T cells. These ndings underscore the pivotal role of CD83 in the NLT-Treg-mediated modulation of inammation, especially in the context of Th2 responses. Overall, our results highlight CD83 as a key player in maintaining tissue homeostasis and modulating inammatory responses, suggesting potential therapeutic implications for inammatory disorders such as asthma. Introduction Regulatory T cells (Tregs) are vital components of our immune system, serving to suppress proinammatory immune responses, prevent autoimmune processes, and facilitate tissue homeostasis and regeneration following injury. Tregs can be further subdivided into two subsets of different ontogeny: thymic or natural Tregs (nTregs), which are generated during thymocyte differentiation, and inducible, peripheral Tregs (pTregs), which are generated in the periphery from CD4+ naïve T cells upon interaction with environmental antigens presented by mucosal tissue-resident dendritic cells in the presence of interleukin-2 (IL-2) and transforming growth factor (TGFß) [1]. By contrast, nTregs undergo several reprogramming steps during their development in the thymus before migrating into secondary lymphoid tissues (LTs), such as the spleen and the lymph nodes (LNs) as well as non-lymphoid tissues (NLTs), like visceral adipose tissue, skin, colon or the lungs [2]. NLT Tregs play a signicant role in maintaining tissue homeostasis, limiting inammation and allergic immune responses, thereby facilitating the induction of mucosal repair mechanisms. Notably, these NLT Tregs upregulate specic surface receptors, e.g., ST2 and KLRG1 and transcription factors such as GATA3 to fulll their roles within their specic environments. While KLRG1 plays an essential role in the late differentiation of Tregs and their ability to exert suppressive functions at sites of inammation [3, 4], ST2+ Tregs, following recruitment by the alarmin IL-33, initiate specic pathways to regulate inammation in different non-lymphoid tissues [2, 5– 8]. Furthermore, ST2+ Tregs are highly activated, strongly suppressive and show a Th2-biased phenotype by expressing GATA3 and producing Th2 cytokines [3, 7, 9]. Due to the specialized functions of different NLT-Tregs, which are imprinted by the respective tissue, identication of markers that uniformly distinguish these cells from their lymphoid counterparts is crucial for understanding their biology and Page 4/26 function. Recently, scRNA-Seq analyses of NLT-Tregs derived from skin and colon revealed an increased expression of the CD83 molecule when compared to Tregs from spleens and lymph nodes [10]. We have already demonstrated that a Treg-specic deletion of CD83 (CD83cKO) results in impaired stability and differentiation of Tregs, leading to exacerbated autoimmune responses and disturbed resolution of inammation [11]. Interestingly, these CD83cKO Tregs display reduced ST2 and KLRG1 expression already in lymphoid organs, and even more so in the lamina propria of the colon. The less differentiated state as well as impaired gut homing of CD83cKO Tregs into this specic NLT accounts for an exacerbated disease course in an adoptive transfer colitis model. Given the altered phenotype of CD83cKO cells with regard to NLT Treg markers, we hypothesize a crucial role of CD83 expression in Tregs involved in the homeostasis of tissues, especially of those in close contact with the environment, exerting barrier functions. To analyze this further, we investigated the phenotype of CD83cKO mice with special focus on the lungs. Our analysis revealed that lung-resident Tregs not only showed impaired late differentiation in CD83cKO mice, but were also highly activated and less able to prevent overshooting cytokine responses upon stimulation. In an inammatory lung specic model for asthmatic disease, we observed an increased Th2 inammation, excessive eosinophilia, and aggravated airway hyperresponsiveness (AHR). Mechanistically, CD83cKO Tregs displayed an increased response to IL-4, which directly led to an enhanced Th2 differentiation and proliferation. Collectively, we reveal that CD83 deletion in Tregs disrupts tissue homeostasis and leads to elevated pathogenic Th2 immune responses. Results Lung resident CD83cKO Tregs show a higher activation status and fail to suppress T cell activation. In our previous study, we observed a reduction of FoxP3 + Tregs in LTs as well as the lamina propria of CD83cKO mice [11]. Thus, we rst analyzed the frequency of splenic and lymph node derived LT-Tregs in comparison with Tregs isolated from the lungs. As expected, LT-Treg frequencies were signicantly reduced in CD83cKO, and a similar trend was observed in the lungs (Fig.1a). Regarding NLT-Treg markers, such as ST2 and KLRG1 in these organs, we observed a trend towards reduced ST2 surface level expressions in Tregs from the lungs, whereas the frequency of KLRG1+ CD83cKO Tregs was drastically decreased (Fig.1b, c). In contrast, Treg-specic receptors, such as ICOS and CD25 exhibited no changes upon CD83 deletion (Supplementary Fig.2a, b). However, GITR, a receptor associated with a higher activation status of T cells, was strongly upregulated in all tissues (Fig.1d). This is in line with our previous results, where CD83cKO Tregs exhibit an activated, proinammatory phenotype. Due to the signicant decrease in KLRG1+ Treg, with a concomitant elevated expression of GITR in CD83cKO mice, we examined the capacity of NLT-Tregs to suppress T cell activation and cytokine secretion. To this end, we stimulated cells from either the lungs or cervical lymph nodes (cLNs) with antiCD3/anti-CD28 and assessed the cytokine levels. Interestingly, we observed a generally elevated cytokine release of Th1, Th2, Th9, Th17 and Th22-specic cytokines in lung-derived lymphocytes from CD83cKO mice (Fig.2a). This was not the case for cLNs, except for increased secretion of IL-17A (Fig.2a, Supplementary Fig.3). Furthermore, the chemokine CCL20 was also signicantly increased upon T cell Page 5/26 stimulation of CD83cKO lung cells (Fig.2b). These results suggest that CD83 deletion in Tregs results in the failure to prevent activation of tissue-resident T cells, leading to escalating cytokine production and chemotactic signals. We reasoned that this phenotype might directly translate into a promotion of inammatory processes in the lung tissue. Thus, we examined the in vivo relevance of our ndings in a lung specic inammation model of allergic asthma. CD83cKO mice develop exacerbated airway hyperresponsiveness (AHR) and enhanced eosinophilic inammation and elevated Th2 immunity. To induce asthmatic disease, mice were immunized twice with ovalbumin (OVA) complexed with alum and subsequently challenged by intranasal application of OVA alone, two weeks after the last immunization (Fig.3a). Mice that received an intranasal PBS-application served as control group. The lung function of mice was assessed using a non-invasive as well as an invasive method to determine airway hyperresponsiveness (AHR), to increasing doses of methacholine. OVA-treated CD83cKO mice exhibited signs of impaired lung function in both assessments (Fig.3c, Supplementary Fig.4a-b). We observed an increased enhanced pause (Penh) in OVA-treated CD83cKO mice during the 12.5mg/ml methacholine (Mch) challenge, indicating airway narrowing (Fig.3b). Furthermore, OVA-treated CD83cKO mice displayed a signicant impaired lung functionality, as evidenced by strongly increased resistance (Fig.3c) and elastance, as well as a signicant decrease in compliance of the lung (Supplementary Fig.4a-b). As these results suggested altered lung tissue functionality, we next assessed the lung tissues from asthma mice by histology. We selected small bronchioles characterized by a comparatively lesser thickness in both connective and smooth muscle tissues, as well as the extracellular matrix, in contrast to the more substantial dimensions observed in larger airways. An increased collagen deposition around the bronchioles in CD83cKO was observed, which was already evident in PBS-challenged mice and got even more prominent upon asthma induction (Fig.3d). Semiquantitative evaluation of these histological measurements revealed signicantly increased collagen thickness in the lung tissue of CD83cKO mice, indicating pathogenic morphological changes (Fig.3e). Supplementary data include complete histological images (see Supplementary Fig.5), conrming the absence of serositis or plaque formation in PBS mice and highlighting the OVA-dependent morphological changes not exclusively focally, but also in other regions of the lung. Additionally, we revealed enhanced thickness of the smooth muscle tissue surrounding the bronchioles in untreated CD83cKO mice, with a more pronounced effect in OVA-treated versus PBS-treated mice. Furthermore, OVA-treated CD83cKO mice exhibited elevated peribronchial and perivascular inltration of eosinophils and lymphocytes (Supplementary Fig.6). One hallmark of OVA-induced asthma is the inltration of inammatory eosinophils [12]. Therefore, we isolated single cells from the lungs as well as the bronchioalveolar lavage uids (BALFs) of asthmatic mice and assessed the cellular composition. Strikingly, we observed a massive inltration of eosinophils into the lungs of OVA-treated CD83cKO mice, which was even more pronounced in the BALFs, where eosinophils made up almost 80% of all inltrating immune cells (Fig.4a-c). Moreover, the frequency of the inammatory SiglecFhigh eosinophil subset was signicantly higher in lungs of CD83cKO mice. Importantly, this change in immune cell composition was not observed in respective cLNs, highlighting Page 6/26 the lung-specic effects of CD83-deletion (Supplementary Fig.8a-b). These results demonstrate that CD83-decient Tregs exacerbate the progression of asthma and enhance eosinophilic inammation. Exacerbated asthmatic disease in CD83cKO mice is driven by insucient control of Th2 immunity. In addition to eosinophils, we also examined the CD4+ T cell population in different tissues. Interestingly, CD83cKO mice did not exhibit any differences in CD4+ T cell frequencies when compared to the control (Ctrl) mice (Supplementary Fig.9a, Fig.5a). Furthermore, we assessed the expression of different surface receptors on both CD4+ and CD25+Foxp3+ Treg cells in the lung. Notably, CD83cKO mice displayed signicantly increased GITR expression levels even before OVA treatment, and this expression was further enhanced following treatment (Fig.5b-c, Supplementary Fig.9b-c). The elevated GITR expression has the potential to enhance Th2 cell activity in asthma, contributing to airway inammation, as has previously been reported [13, 14]. To further pursue the notion of enhanced Th2 responses, we examined effector T cells and observed a signicant increase in GATA3+Foxp3− T cells in OVA-treated CD83cKO mice (Fig.5d). When we analysed the expression of additional Th2 markers on effector T cells versus Tregs, we found signicantly increased frequencies of ST2+/FoxP3+ Tregs as well as ST2+/FoxP3− effector cells. Remarkably, lung Tregs from CD83cKO mice exhibited a substantial reduction in Foxp3 expression even without challenge. (Fig.5e-f). Since ST2 serves as the receptor for IL33, an alarmin released during tissue damage that contributes to tissue homeostasis and the local expansion of NLT-Tregs at the site of inammation, we assessed circulatory IL33 levels in the serum of asthmatic mice. Our results show a strong tendency towards higher IL33 levels in OVA-treated CD83cKO mice (Supplementary Fig.10). These data suggest that CD83cKO mice have a higher state of inammation and are unable to regulate the activity of Th2-specic effector cells in allergic asthma. This notion was further corroborated by cytokine secretion of lung derived immune cells upon ex vivo restimulation with OVA. While cells from PBS-treated mice did not react to OVA restimulation, we observed a strikingly increased release of the Th2-related cytokines IL-5 and IL-13 in the cultures of CD83cKO cells from OVA-challenged mice (Fig.5g). Additionally, there was an increased production of chemokines, such as CCL17, CCL22, and CXCL5, upon restimulation with OVA. Collectively, we reasoned that the dysfunctional state of CD83-decient Tregs, which is characterized by reduced terminal differentiation and higher expression of GITR, renders these cells unable to conne Th2responses. Consequently, CD83cKO mice develop an exacerbated disease course which is driven by massive eosinophilia. CD83-decient Tregs fail to conne Th2 differentiation and proliferation. To test whether the inability to suppress Th2 responses was specic to the used in vivo model or a Treg-intrinsic defect, we performed Th skewing experiments under Th2 conditions, specically examining the proliferation and differentiation of CD4+ T cells. Therefore, total CD4+ T cells were isolated from CD83cKO or control mice, labeled with CellTraceTMViolet and cultivated in the presence of OVA/LPS-pulsed dendritic cells (DCs). As we aimed to dissect the effect of Treg-specic deletion of CD83 on Th2 polarization, we set up our co-cultures using the following conditions: (i) anti-CD3 antibodies were added to initiate a clonal T cell proliferation, (ii) Page 7/26 anti-IFN-γ antibodies added to mitigate Th1 differentiation, and (iii) “full Th2-differentiation” was achieved by the addition of IL-4. In these co-culture experiments, we observed that Treg frequencies remained unaffected (Fig.6b), although there was a trend towards higher frequencies in cultures with CD83cKO T cells. Notably, while we detected no differences in the expression of Th2 master transcription factor GATA3, when T cells were only activated with anti-CD3, mitigating IFN-γ signaling and thus, Th1 differentiation, already resulted in a signicantly higher proportion of GATA3+ T cells (Fig.6c). This effect was drastically enhanced in the presence of IL-4, where we observed an increase of GATA3+ T cells, along with a higher replication index in CD83cKO mice, suggesting a more vigorous induction of Th2 differentiation (Fig.6c-d). When we examined activation markers expressed by CD4+ T cells, we found that GITR was signicantly increased already under control conditions (anti-CD3 only). This increase became increasingly more pronounced upon addition of anti-IFN-γ and IL-4, paralleling the pattern of GATA3-expression (Fig.6e). We detected similar behavior for CD25, which was also strikingly increased in CD83cKO cultures under “full” Th2 conditions. In line with the profound Th2 differentiation of CD83cKOderived T cells, IL-13 cytokine levels were signicantly elevated in the supernatants of these cultures (Fig.6g). Taken together, these data suggest that CD4+ T cells from CD83cKO mice are more prone to Th2 differentiation, and this potential is fully unlocked upon stimulation with IL-4. Sorted CD83cKO Tregs display a higher IL-4 responsiveness with a preserved GITR phenotype. From the above described data, we concluded that IL-4 might have an important function in uncovering the effect of CD83-deletion on Treg biology. IL-4 is known to have context-dependent effects on Tregs: i.e. while IL-4 stabilizes nTregs in vitro [15], it can inhibit the differentiation of iTregs from naive CD4+ T cells in allergic airway disease [16].Additionally, it can promote the conversion of Tregs into exFoxp3-Th2 effector cells [17]. Since IL-4 signaling is predominantly mediated via IL-4Rα, we rst assessed the expression of the respective Il4ra transcript in sorted Tregs. Thereby, we detected signicantly upregulated Il4ra transcripts in CD83cKO Tregs compared to wildtype Tregs (Fig.7a). To examine the effect of IL-4 stimulation in CD83cKO in more detail, we stimulated sorted Tregs with IL-4 (Fig.7). As expected from literature reports [15], Foxp3 expression was stabilized and increased upon IL-4 stimulation, but showed no signicant differences when comparing CD83cKO cells to controls cells (Fig.7b). When we analyzed surface receptors on Tregs, the results mirrored those obtained in the coculture experiments, with an increase in CD25 and GITR expression in CD83cKO Tregs following IL-4 stimulation, indicating enhanced activation (Fig.7c, d). In addition, we examined additional activation markers, including ICOS and CD69 (Fig.7e, however they were not inuenced by the Treg-specic CD83 deletion. To ensure that the observed changes are indeed depending on IL-4 stimulation, we included experiments with IL-4-blocking antibodies before addition of IL-4 which abrogates the observed effects (Supplementary Fig.12). Taken together, CD83 deletion results in an elevated IL-4-responsiveness leading to a highly activated phenotype. Altogether, our data disclosed a signicant role of CD83 in NLT-Tregs and especially for the suppression of Th2-responses. CD83 deletion in Tregs impairs their regulatory activity towards tissue-resident T cells in the lungs, leading to an increased proinammatory activated phenotype in the lung and impairing tissue homeostasis. Furthermore, CD83-decient Tregs are more responsive to IL-4 stimulation, probably Page 8/26 subverting their capacity to suppress Th2 immune responses. This phenotype directly translates into development of aggravated disease symptoms upon induction of Th2-dependent asthma symptoms and provides valuable insights into the key modulatory role of CD83 for Treg biology and function. Discussion Most NLT-Tregs, particularly those on barrier sites like the skin, the colon, or the lungs undergo priming in the LTs before migrating to the periphery, displaying specic transcriptomic adaptations. These Tregs undergo nal expressional changes, such as including the upregulation KLRG1, ST2, and GATA3 in the NLTs, developing into effector Tregs that surveil the tissue to suppress pathogenic responses at sites of inammation or injury [2, 10]. Interestingly, recent single-cell sequencing analyses revealed that CD83 is upregulated in these barrier tissue resident Tregs [10]. Our previous ndings have already demonstrated that CD83 deletion in Tregs confers an enhanced proinammatory phenotype and leads to impaired late differentiation with compromised resolution of inammatory events. More importantly, splenic CD83cKO Tregs already displayed a reduction of ST2 and KLRG1 expression, which implied an even more pronounced impact of CD83-deletion on tissue Tregs. Within the present study, we provide evidence that lung-resident T cells from CD83cKO mice respond to stimulation aCD3/aCD28 with a drastic general increase of cytokine secretion. Since this effect was not observed in cLNs from the same animals, we conclude that it is indeed NLT-specic and propose a crucial role of CD83 in regulating Treg function in barrier tissues. Notably, we observed the most prominent dysregulation of cytokines related to Th17responses. Additionally, lung lymphocytes of CD83cKO animals secreted increased amounts of CCL20, a chemokine that is important for the recruitment of DCs and Th17 T cells. IL-17 is essential for lung tissue homeostasis and bacterial clearance but excessive release of IL-17 can contribute to brosis and pathogenic airway remodeling in the lung including collagen depositions [18]. Intriguingly, we observed morphological changes in the lungs of naïve CD83cKO mice, with epithelial hyperplasticity, hypertrophy of the smooth muscle tissue, and increased collagen deposition surrounding the bronchi even unchallenged mice. These data suggest that deletion of CD83 in Tregs disturbs their capability to maintain tissue integrity even in the absence of inammation. We also revealed that CD83-decient Tregs, while exhibiting a phenotype of less terminally differentiated cells, express higher levels of the costimulatory molecule GITR. Interestingly, we observed signicantly elevated levels of GITR on CD83decient Tregs, both in the steady state and in asthma, highlighting the central role of CD83 in Treg biology. GITR is constitutively expressed on nTregs and upregulated on activated CD4+ T cells [19]. GITRmediated signaling is involved in plasticity of Tregs towards a Th9 and Th2 phenotype [13, 20, 21], alters their stability [22], and limits Treg suppressive capacity, leading to the exacerbation of autoimmune diseases and increased resistance to tumors [21, 23]. A recent publication also proved the importance of GITR signaling for the establishment of Th2-driven asthmatic disease [13]. This perfectly aligns with our in vivo data in the OVA-induced asthma model, with CD83cKO animals developing more severe disease symptoms, which rely on excessive Th2 responses and concomitant eosinophilia. Moreover, T cells in the lungs of asthmatic CD83cKO mice expressed elevated levels of ST2 and we observed a clear trend towards elevated levels of its ligand IL-33 in the sera of those mice. Stimulation with IL-33 led to Page 9/26 suppressive function of lung-derived Tregs and aggravation of allergen-driven inammation, which was associated with pathological Th2-like Tregs, secreting IL-5 and IL-13, thereby enhancing airway inammation [24]. In line with this, we observed that lung lymphocytes from CD83cKO also secreted drastically increased amounts of IL-5 and IL-13 upon restimulation with OVA. While IL-13 is known to induce tissue remodeling, airway hyperresponsiveness and mucus secretion in the lung [25, 26], IL-5 is a key stimulus for recruitment and survival of eosinophils [27]. Concomitantly, we observed increased collagen deposition around the bronchi as well as massive eosinophilia in the lung tissue and the BALF. In asthma, Th2-type cells are recruited to the site of inammation in the lung via chemokines such as CCL17 and CCL22 [28, 29], which are both upregulated in asthmatic CD83cKO mice. Lung lymphocytes from asthmatic mice exhibited an increased production of CXCL5 upon restimulation with OVA, which is associated with neutrophil recruitment, but also triggers the specic granule content release of eosinophils [30, 31]. Collectively, these data suggest that CD83-deletion renders Tregs less effective to suppress Th2 responses, leading to an exacerbated asthmatic disease. When we further analyzed whether CD83-decient Tregs are able to control Th2 differentiation, we observed that the more the commitment to Th1 differentiation is inhibited, the stronger is the inability of CD83-cKO Tregs to prevent Th2 responses. Experiments using isolated Tregs, disclosed that CD83cKO cells exhibit an elevated expression of IL-4Rα, leading to an increased IL-4 responsiveness and increased induction of CD25 and GITR. This is in line with IL-4 hyperresponsiveness leading to STAT6-dependent inhibition of Foxp3 expression, and subsequently, the development of pathogenic Th2 responses [8, 32]. Interestingly, a soluble form of CD83 (sCD83) with well-described immunomodulatory functions [33], has been shown to trigger apoptosis in Th2 cells in a model for allergic rhinitis [34]. Since Tregs up-regulate and stably express CD83 after stimulation [35], and sCD83 can be generated from the membrane-bound form, this adds to the complexity of Treg-mediated control of Th2 responses. In summary, our ndings underscore the crucial role of CD83 regarding the suppressive function of NLTTregs and maintenance of tissue homeostasis. Deletion of CD83 in Tregs results in a highly activated and pro-inammatory phenotype, characterized by elevated expression levels of GITR. In the lungs of CD83cKO mice, we observe impaired late differentiation and a pronounced failure in suppressive function of NLT-Tregs, consequently promoting pathogenic Th2 immunity in an IL-4 dependent manner. Collectively, we provided new evidence that CD83 is a crucial component of Treg-mediated suppression of inammation, especially of Th2 responses (Fig.8). Material and methods Animals. All of the mice were maintained on the C57BL/6 strain background. Floxed CD83 animals were generated in our laboratory as described previously [36]. Foxp3-Cre animals were provided by A. Rudensky (University of Washington, Seattle, Washington, USA). For Treg-specic depletion of the CD83 gene, oxed Page 16/26 23. Amoozgar Z et al (2021) Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas. Nat Commun 12(1):2582 24. Chen C-C et al (2017) IL-33 dysregulates regulatory T cells and impairs established immunologic tolerance in the lungs. J Allergy Clin Immunol 140(5):1351–1363e7 25. Karo-Atar D et al (2016) A protective role for IL-13 receptor α 1 in bleomycin-induced pulmonary injury and repair. Mucosal Immunol 9(1):240–253 2. Kuperman DA et al (2002) Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 8(8):885–889 27. Pelaia C et al (2019) Interleukin-5 in the pathophysiology of severe asthma. Front Physiol 10:1514 2. Lukacs NW (2001) Role of chemokines in the pathogenesis of asthma. Nat Rev Immunol 1(2):108– 116 29. Ackland J et al (2021) Interrupting the conversation: implications for crosstalk between viral and bacterial infections in the asthmatic airway . Front Allergy, : p. 72 30. Garcia JCH (2020) Eosinophils as a Biomarker in Asthma and COPD , in Update in Respiratory Diseases . IntechOpen 31. Liu C et al (2018) Role of epithelial chemokines in the pathogenesis of airway inammation in asthma. Mol Med Rep 17(5):6935–6941 32. Cheru N, Haer DA, Sumida TS (2023) Regulatory T cells in peripheral tissue tolerance and diseases. Front Immunol 14:1154575 33. Grosche L et al (2020) The CD83 molecule–an important immune checkpoint. Front Immunol 11:721 34. Wu Y-J et al (2020) Soluble CD83 alleviates experimental allergic rhinitis through modulating antigen-specic Th2 cell property. Int J Biol Sci 16(2):216 35. Kreiser S et al (2015) Murine CD83-positive T cells mediate suppressor functions in vitro and in vivo. Immunobiology 220(2):270–279 3. Krzyzak L et al (2016) CD83 modulates B cell activation and germinal center responses. J Immunol 196(9):3581–3594 37. Grund JC et al (2023) Vitamin D3 resolved human and experimental asthma via B lymphocyte– induced maturation protein 1 in T cells and innate lymphoid cells. J Allergy Clin Immunology: Global 2(3):100099 3. Kölle J et al (2022) Targeted deletion of Interleukin-3 results in asthma exacerbations . Iscience, 25(6) 39. Wild AB et al (2019) CD83 orchestrates immunity toward self and non-self in dendritic cells . JCI insight, 4(20) 40. Bustin SA et al (2009) The MIQE Guidelines: M inimum I nformation for Publication of Q uantitative Real-Time PCR E xperiments. Oxford University Press Figures Page 17/26 Figure 1 CD83cKO mice show a higher activation status and an impaired non-lymphoid-tissue (NLT) function. FACS analysis of Tregs from spleen (SPL) mesenteric lymph node- (mLN), and lungs of 8–12-week-old mice. a Frequencies of FoxP3+ Tregs among all living CD45+ in the respective organs b-d Surface receptor expression of ST2, KLRG1, and GITR on Tregs. (Ctrl n=4-10, CD83cKO n=4-10, pool of 3 independent experiments). In all graphs, data are represented as mean ± SEM and two-tailed Mann–Whitney U-test was used to analyze the data. Page 18/26 Figure 2 Lung resident lymphocytes of CD83cKO mice show escalating cytokine responses. Lung and cervical lymph node (cLNs) derived lymphocytes were stimulated for 48 h with anti-CD3/anti-CD28. a Supernatant cytokine and b chemokine analysis of stimulated lung and cLN lymphocytes from 8–12-week-old mice. (Ctrl n=20-21, CD83cKO n= 20-21, pool of 6 independent experiments). In all graphs, data are represented as mean ± SEM and two-tailed Mann–Whitney U-test was used to analyze the data. Page 19/26 Figure 3 CD83cKO mice showed increased AHR and pathological changes in lung morphology. a Graphical overview experimental procedure for the in vivo allergic asthma mouse model. b Non-invasive AHR measurement by whole-body plethysmography. Enhanced pause (Penh) has been calculated at baseline and after challenge with metacholine (Mch): 0 mg/ml: p=0.4926; 25 mg/ml: p=0.1067; 50 mg/ml: p=0.0826; cInvasive AHR analysis was performed and the resistance (Rrs) of the lung was calculated Page 20/26 upon methacholine challenge: 0 mg/ml: p=0.938; 12.5 mg/ml: p=0.4068. d Representative Sirius Red stainings of lung tissue from PBS or OVA-treated Ctrl and CD83cKO mice. Green arrows demonstrate enlarged bronchial epithelium and blue arrows indicate collagen deposition. e Thickness of the peribronchial collagen layer was quantied by dividing the collagen thickness beneath the bronchial epithelium by the bronchial diameter. Statistical analysis of AHR data was performed using a two-way ANOVA test with Tukey´s correction test. Relative collagen thickness is represented as mean ± SEM and two-tailed Mann–Whitney U-test was used to analyze the data. (Ctrl n=4-5, CD83cKO n= 4-5, one representative experiment out of two). Figure 4 CD83cKO mice developed an exacerbated eosinophilia in allergic asthma. FACS analysis of immune cells in the bronchoalveolar lavage-uid (BALF) and lung tissue from asthmatic and control mice. a Page 21/26 Frequencies of CD45+ immune cells, b total CD11b+ cells as well as c eosinophils in the BALFs and lungs. d Frequencies of SiglecFhigh-inammatory eosinophils in lung tissue. (Ctrl n=4-5, CD83cKO n= 4-5, one representative experiment out of two). In all graphs, data are represented as mean ± SEM and two-tailed Mann–Whitney U-test was used to analyze the data. Figure 5 Page 22/26 Asthmatic lungs derived from CD83cKO mice resulted in enhanced Th2 effector T cell responses and a highly activated phenotype. a-f FACS analysis of lung-resident CD4+ T cells and Tregs from asthmatic and control mice: a CD4+ T cell frequencies. Percentage of GITR+ cells among b CD4+ T cells and c on Tregs. d Th2 effector cells (Foxp3-GATA3+) frequencies in the CD4+ T cell population. e Representative FACS plots of ST2 and FoxP3 populations among CD4+ cells. f Analysis of ST2+ Foxp3effector cells, ST2+Foxp3+ Tregs and St2-Foxp3+ Tregs among lung-resident T cells, and median expression of FoxP3 on tissue-resident Tregs. g Cytokine and chemokine analysis of lung lymphocytes re-stimulated with OVA for 24 h. In all graphs, data are represented as mean ± SEM and two-tailed Mann–Whitney U-test was used to analyze the data. (Ctrl n=4-5, CD83cKO n= 4-5, one representative experiment out of two) Page 23/26 Figure 6 T cells from CD83cKO mice revealed a higher proliferation rate and a shewing towards Th2 differentiation. a Schematic overview of DC/T cell coculture: OVA/LPS-stimulated DCs are co-cultured with CellTraceTMViolet (CTV)-labeled CD4+ T cells from wild-type or CD83cKO mice in the presence of stimuli (anti-CD3/anti-IFNγ/IL-4) for 4 days b Frequencies of Tregs (CD25+CD4+Foxp3+) among T cells, c GATA3+ T cells. d Replication index of T cells from co-cultures. e Median uorescence of GITR and f CD25 on CD4+ T cells. g Assessment of IL-13 production in supernatants of co-cultures (Ctrl n=9, Page 24/26 CD83cKO n= 9, pool of 3 independent experiments). In all graphs, data are represented as mean ± SEM and two-tailed Mann–Whitney U-test was used to analyze the data. Figure 7 Sorted splenic Tregs derived from CD83cKO mice show an enhanced IL-4 responsiveness and an increased GITR expression. a Gene expression analysis of IL-4Rα of sorted Tregs (CD3+CD4+YFP+) from Page 25/26 spleens of Ctrl and CD83cKO mice. b-f FACS analysis of sorted Tregs without any treatment (non-treated controls, NTC) or after stimulation with IL-4 for 24 h. b Median uorescence of Foxp3, c CD25, d GITR, e ICOS and f CD69 were determined on Foxp3+ Tregs in relation to NTCs. (Ctrl n=6, CD83cKO n= 6-7, pool of 3 independent experiments). In all graphs, data are represented as mean ± SEM and two-tailed Mann– Whitney U-test was used to analyze the data. Figure 8