Persistence of CD8+ skin-resident memory T cells in mice requires TCR signaling

Persistence of CD8+ skin-resident memory T cells in mice requires TCR signaling | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 January 2025 V1 Latest version Share on Persistence of CD8+ skin-resident memory T cells in mice requires TCR signaling Authors : Anders Funch 0000-0002-7576-3884 [email protected] , Julie Weber , Veronika Mraz , Martin Kongsbak-Wismann , Rebecca Kitt Davidson Lohmann , Mia Jee , Helen Vaher 0000-0002-8845-0326 , … Show All … , Kelvin Yeung 0000-0001-6083-3475 , Anne-Sofie Ø. Gadsbøll , Niels Ødum , Anders Woetmann , Jeanne Johansen , Carsten Geisler , and Charlotte Bonefeld 0000-0002-0523-6229 Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.173652866.63708399/v1 380 views 227 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Epidermal-resident memory CD8 + T (T RM ) cells play a significant role in fighting off pathogens. However, CD8 + T RM cells are also central in the pathogenesis of a variety of inflammatory skin diseases. It is unclear whether the generation and persistence of CD8 + T RM cells are dependent on the presence of cognate antigen and T cell receptor (TCR) signaling. Methods: We determined the generation and persistence of epidermal CD8 + T RM cells by flow cytometry and single-cell TCR sequencing in a well-characterized mouse model for allergic contact dermatitis. We examined the responses to four different contact allergens in combination with adoptive transfer and prime-pull experiments. We determined the presence of contact allergens in the skin by Western blot analysis. Results: We found that epidermal CD8 + T RM cells can develop in the absence of the cognate antigen and TCR signaling as determined by Nur77 induction, whereas persistence of epidermal CD8 + T RM cells requires presence of the cognate antigen and correlates with Nur77 expression. In the presence of contact allergen, a selective expansion of specific TCR clonotypes was seen. Conclusion: This study demonstrates that cognate antigen and TCR signaling are required for the persistence of allergen-specific CD8 + T RM cells in the skin. Persistence of CD8 + skin-resident memory T cells in mice requires TCR signaling Running title: Generation and persistence of epidermal CD8 + T RM cells Anders Boutrup Funch 1,2 * ¤ , Julie Friis Weber 1 , Veronika Mraz 1 , Martin Kongsbak-Wismann 1 , Rebecca Kitt Davidson Lohmann 1 , Mia Hamilton Jee 1 , Helen Vaher 1 , Kelvin Yeung 1 , Anne-Sofie Østergaard Gadsbøll 1 , Niels Ødum 1 , Anders Woetmann 1 , Jeanne Duus Johansen 2 , Carsten Geisler 1# and Charlotte Menné Bonefeld 1# * 1 LEO Foundation Skin Immunology Research Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. 2 National Allergy Research Centre, Department of Dermato-Allergology, Copenhagen University Hospital Herlev-Gentofte, Hellerup, Denmark. *Corresponding authors: Anders B. Funch or Charlotte M. Bonefeld, LEO Foundation Skin Immunology Research Center, Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Nørre Allé 14, DK-2200 Copenhagen, Denmark. E-mail [email protected] or [email protected] # Shared senior authors ¤ Present affiliation: Anders B. Funch, Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland. Acknowledgments Funding: This work was supported by the Carlsberg Foundation (grant CF23-0793), the LEO Foundation, the Independent Research Fund Denmark and the Danish Environmental Protection Agency as part of the Chemicals Act. Author contributions: Conceptualization of this study was done by ABF, CMB, CG, JDJ, NØ and AW. Methodology was done by ABF, CMB and CG. Investigation was done by ABF, JFW, MKW, VM, RKDL, KY, MHJ, ASØG and HV. Visualization was done by ABF, CG, JFW and MKW. Funding acquisition was done by ABF, CMB, CG and JDJ. Project administration was done by ABF and CMB. Supervision was done by CMB, CG and JDJ. Writing of original draft was done by ABF, CG and CMB. Writing review & editing was done by all authors. Conflict of interest: The authors declare no competing financial interests or other conflicts of interest. Data availability statement: The data that support the findings of this study are available on reasonable request from the corresponding author. Abstract Background: Epidermal-resident memory CD8 + T (T RM ) cells play a significant role in fighting off pathogens. However, CD8 + T RM cells are also central in the pathogenesis of a variety of inflammatory skin diseases. It is unclear whether the generation and persistence of CD8 + T RM cells are dependent on the presence of cognate antigen and T cell receptor (TCR) signaling. Methods: We determined the generation and persistence of epidermal CD8 + T RM cells by flow cytometry and single-cell TCR sequencing in a well-characterized mouse model for allergic contact dermatitis. We examined the responses to four different contact allergens in combination with adoptive transfer and prime-pull experiments. We determined the presence of contact allergens in the skin by Western blot analysis. Results: We found that epidermal CD8 + T RM cells can develop in the absence of the cognate antigen and TCR signaling as determined by Nur77 induction, whereas persistence of epidermal CD8 + T RM cells requires presence of the cognate antigen and correlates with Nur77 expression. In the presence of contact allergen, a selective expansion of specific TCR clonotypes was seen. Conclusion: This study demonstrates that cognate antigen and TCR signaling are required for the persistence of allergen-specific CD8 + T RM cells in the skin. Key words Tissue-resident memory T cells, epidermis, skin, T cell receptor, contact allergen, allergic contact dermatitis, Nur77, Ki67 Introduction Naïve CD8 + T cells are activated and start to proliferate in the secondary lymphoid organs after recognition of cognate antigen presented by major histocompatibility complex-class I (MHC I) molecules by their T cell receptor (TCR) [1]. Following clonal expansion, the resulting CD8 + effector T (T EFF ) cells enter the circulation and subsequently infiltrate inflamed tissues [2]. Here, some of the T EFF cells have the potential to differentiate into CD8 + tissue-resident memory T (T RM ) cells [3,4]. In the skin, CD8 + T RM cells are mainly located to the epidermis, where they serve as first line protection against pathogens [3-8]. However, CD8 + T RM cells are also involved in the pathogenesis of a variety of inflammatory skin diseases, where they target self-proteins or non-harmful substances such as contact allergens [9-11]. Accordingly, pathogenic T RM cells have been identified in psoriasis [12,13], alopecia areata [14], vitiligo [15], atopic dermatitis [16] and allergic contact dermatitis (ACD) [10,17-22]. Thus, a deeper understanding of the mechanisms behind the generation and persistence of T RM cells could open new avenues for the development of treatments for inflammatory skin diseases and vaccines against infectious diseases. Infiltration of circulating T EFF cells into the skin is guided by the local inflammatory response independently of the presence of the cognate antigen [7,20,23-27]. However, whether the generation and persistence of CD8 + T RM cells in the skin is antigen-dependent is still debated. Some studies found that inflammation was sufficient for persistence of CD8 + T RM cells in the skin [23,24,28], whereas others found that presence of cognate antigen leading to TCR signaling was required for efficient generation and persistence of skin-resident CD8 + T RM cells [25-27]. The objective of the present study was to investigate whether cognate antigen and TCR signaling are required for the generation and persistence of epidermal-resident CD8 + T RM cells. Nr4a1 (Nur77) is an immediate early gene up-regulated by TCR stimulation in thymocytes and T cells [29]. Nur77 expression in T cells is dependent on the presence of the cognate antigen and MHC but is not affected by IL-2 or inflammatory stimuli in vivo [30,31]. Accordingly, Nur77 expression is used as a specific marker for TCR signaling [25,31-39]. By determining Nur77 expression, we examined whether TCR signaling is required for the generation and persistence of epidermal CD8 + T RM cells using a well-characterized mouse model for ACD. This model closely mirrors ACD in humans, where CD8 + T RM cells play a central role in the inflammatory response seen after re-exposure of the skin to the contact allergen [18,20,22,40]. We found that epidermal CD8 + T RM cells can develop in the absence of the cognate antigen and Nur77 induction, whereas persistence of epidermal CD8 + T RM cells requires presence of the cognate antigen and correlates with expression of Nur77. Thus, this study supports that TCR signaling and presence of cognate antigen in the skin are required for the persistence of allergen-specific epidermal CD8 + T RM cells. Mice Female C57Bl/6J mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and housed in a specific pathogen-free environment at the animal facility at the Department of Experimental Medicine, University of Copenhagen in accordance with the national animal protection guidelines (license number 2022-15- 0201-01340). Allergic contact dermatitis model Mice were sensitized on the dorsum of the ears with the given contact allergens. Depending on the experiment, mice were euthanized and analyzed 5, 30, 90, 180 or 365 days after sensitization as described in the Supplemental Materials. Flow cytometry and Western blotting Flow cytometry and Western blotting were performed as described in the Supplemental Materials. Single-cell TCR sequencing Single-cell TCR sequencing was performed on epidermal CD8 + T cells isolated from ears of mice sacrificed 30 or 180 days after sensitization to 1-fluoro-2,4-dinitrobenzene (DNFB) as detailed in the Supplemental Materials. Adoptive transfer experiment Adoptive transfer experiments were performed as previously described [20] and as detailed in the Supplemental Materials. Statistical analysis Statistical comparisons were performed using GraphPad Prism version 10.2.2. Gaussian distributions were tested using D’Agostino and Pearson- or Shapiro-Wilk normality test for smaller data sets. Statistical significance was tested using Students unpaired t-tests (for 2 comparisons) or One-way ANOVA (for >2 comparisons). For conditions without Gaussian distribution a Mann-Whitney U (for 2 comparisons) or a Kruskal-Wallis test (for >2 comparisons) was performed. Post-hoc adjustment for multiple comparisons were done using Bonferroni’s multiple comparisons test. Significance levels are illustrated in all figures as ns (not significant) = p > 0.05; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. Results CD8 + T RM cells persist in the epidermis for at least one year after sensitization to DNFB To investigate whether presence of cognate antigen in the skin correlates with expression of Nur77 by the epidermal CD8 + T RM cells, we took advantage of the knowledge that the experimental contact allergen DNFB remains in the epidermis for at least one month after exposure of the skin to DNFB [19,41]. We exposed mice to DNFB diluted in olive oil:acetone (OOA) or to OOA alone as control on both ears for three consecutive days (Fig. 1A). Single cell suspensions were prepared from the epidermis of the ears and analyzed by flow cytometry 30 days after sensitization. We found a large population of CD8 + TCRβ + T cells of which the vast majority was CD69 + CD103 + T RM cells in mice exposed to DNFB (Fig. 1B and C, upper row and Fig. S1). In contrast, control mice had very few CD8 + TCRβ + T cells in the epidermis (Fig. 1C, upper row). A distinct subpopulation of the CD8 + T RM cells that expressed Nur77 was found in mice exposed to DNFB (Fig. 1D and E, upper row). Likewise, a distinct subpopulation of the CD8 + T RM cells that expressed the proliferation marker Ki67 [42,43] was found in mice exposed to DNFB (Fig. 1D-F, upper row). We next extended the period between the exposure to DNFB and the analysis of the epidermal cells to 180 and 365 days, respectively. We observed a decrease in the total numbers of CD8 + T RM cells from day 30 to day 180. However, a stable number of CD8 + T RM cells was found in the epidermis 180 and 365 days after exposure to DNFB (Fig. 1B and C, middle and lower row). A significant and stable number of the CD8 + T RM cells expressed Nur77 both 30, 180 and 365 days after exposure to DNFB, whereas the number of Ki67 + CD8 + T RM cells decreased with time (Fig. 1D-F, middle and lower row). Exposure to DNFB induces long-term persistence of DNP adducts in the epidermis If Nur77 expression by the CD8 + T RM cells was a consequence of TCR signaling elicited by the cognate antigen, the above observations indicated that DNFB should be present in the epidermis for an extended period. After exposure of the skin to DNFB, DNFB almost exclusively reacts with primary amines on lysine residues leading to the formation of 2,4-dinitrophenol (DNP) lysine protein complexes [44], here referred to as DNP adducts. To determine the longevity of DNP adducts in the skin, we treated mice with OOA or DNFB on the ears for three consecutive days (Fig. 2A). Five and 180 days after the treatment, epidermal ear sheets were isolated, homogenized in lysis buffer and subjected to Western blotting, targeting DNP adducts with specific anti-DNP antibodies. As expected, a strong DNP signal appeared on day five in the lysates from DNFB-treated mice, whereas no signal was detected in the lysates from OOA-treated mice (Fig. 2B). Interestingly, we could clearly detect DNP adducts in the epidermis 180 days after DNFB treatment although at a reduced level compared to five days after DNFB treatment (Fig. 2C). This indicated that some DNP adducts were highly stable and existed in the epidermis for at least 180 days after exposure of the skin to DNFB. Selective expansion of specific TCR clonotypes with time If the stability of the various DNP adducts differed and persistence of the CD8 + T RM cells depended on TCR signaling mediated by specific DNP adducts, it should be expected that selective expansion of specific clonotypes took place in the skin with time after DNFB exposure. To evaluate this, we treated mice with DNFB on the ears for three consecutive days and prepared single cell suspensions from the epidermis of the ears 30 and 180 days after the treatment (Fig. 3A). We isolated the CD8 + T cells by FACS and performed single-cell TCR sequencing and repertoire analysis [45,46]. We identified from 235 to 669 unique clonotypes from each of the mice with minimal clonotype overlap between the individual mice (Fig. 3B). The relative abundance of cells in the clones demonstrated that bigger clones dominated the TCR repertoire at day 180 compared to day 30 (Fig. 3C). Likewise, the frequency distribution of the clonotypes clearly demonstrated that larger clones dominated the repertoire at day 180 compared to day 30. Thus, on day 30, the 118 largest clones amounted to 50% of the total number of CD8 + T cells on average (Fig. 3D), whereas on day 180, only the 48 largest clones were required to reach 50% of the total number of CD8 + T cells (Fig. 3E). Finally, as a measure for the distribution of number of cells per clonotype, we calculated the Gini coefficient. The Gini coefficient yields a value between 0 and 1, where 0 represents a perfectly even distribution of cells per clone, and a higher value represents a less even distribution [45,46]. As shown in Fig. 3F, a substantial and statistically significant shift toward unevenness at day 180 compared to day 30 was observed. Collectively, these data demonstrated that a selective expansion of specific TCR clonotypes took place with time. Nur77 expression correlates with CD8 + T RM cell persistence Next, we wanted to determine whether a correlation between Nur77 expression and the generation of a stable pool of CD8 + T RM cells was a general trait for contact allergens. To investigate this, we exposed mice to three chemically different and clinically relevant contact allergens, namely cinnamaldehyde (cinnamal), para-phenylenediamine (PPD) and methylisothiazolinone (MI) with OOA as control (Fig. 4A). To facilitate the generation of CD8 + T RM cells, we depleted the CD4 + T cells during sensitization as previously described [47]. Mice were euthanized either 30 or 180 days after exposure to the contact allergens and single cell suspensions from the epidermal ear sheets were prepared and analyzed by flow cytometry. Exposure of the skin to each of the three contact allergens induced a significant population of CD8 + T RM cells on day 30 (Fig. 4B and C). Importantly, only mice exposed to MI had a significant number of Nur77 + CD8 + T RM cells on day 30 (Fig. 4D and E). Mice exposed to cinnamal and MI had a significant number of Ki67 + CD8 + T RM cells on day 30 (Fig. 4F). On day 180, only mice exposed to MI continued to have a significant population of CD8 + T RM cells, whereas the CD8 + T RM cells were not maintained in mice exposed to cinnamal or PPD (Fig. 4G and H). Mice exposed to MI maintained a significant number of Nur77 + CD8 + and Ki67 + CD8 + T RM cells at day 180 (Fig. 4I-K). Taken together, these data demonstrated that Nur77 expression correlated with persistence of CD8 + T RM cells. In contrast to Nur77, expression of Ki67 was seen in both short-lived and long-lived CD8 + T RM cells . Persistence of CD8 + T RM cells in the epidermis requires presence of the cognate antigen The presence of Nur77 + and Ki67 + CD8 + T RM cells in the epidermis even one year after exposure of the skin to DNFB as depicted in Figure 1, suggested that the CD8 + T RM cells constitutively received signals through their TCR and proliferated. In theory, this could be due to the continuous presence of the cognate antigen in the epidermis or due to cross-reactive antigens or to homeostatic mechanisms independent of the exposure to any antigen [48,49]. Although our data indicated that DNP adducts were present in the epidermis for more than 180 days after exposure of the skin to DNFB, we had not formally proven that Nur77 expression and persistence of epidermal CD8 + T RM cells were dependent on the presence of the cognate antigen. To directly investigate this, we next performed adoptive transfer experiments. We exposed donor mice to either DNFB or OOA for three consecutive days. The donor mice were euthanized on day five and cells from the superficial cervical lymph nodes draining the ears were isolated. Five x 10 7 donor lymph node cells were transferred i.v. to untreated recipient mice. An additional group of mice that was not subjected to cell transfer, and thus referred to as “non-transfer” mice, was included as control. To pull the transferred T EFF cells from the circulation to the skin, recipient mice were exposed to sodium lauryl sulphate (SLS) on both ears on day five immediately after the donor cell injection and once a day for the two following days (Fig. 5A). The non-transfer group of mice were similarly treated with SLS for three days. The mice were euthanized on day 30 or 90, and epidermal single cell suspensions were prepared from the ears and analyzed by flow cytometry. In accordance with previous studies [20], recipient mice that received lymph node cells from DNFB-treated donors showed a significant formation of epidermal CD8 + T RM cells on day 30 compared to the recipient group that received cells from donors treated with OOA and to the non-transfer group (Fig. 5B and C, upper row). The absence of Nur77 expression in all groups demonstrated that TCR signaling did not take place in accordance with the absence of the cognate antigen (DNFB) in the recipient and non-transfer mice (Fig 5D, upper row). Staining for Ki67 demonstrated that even in the absence of antigen, a significant number of T RM cells proliferated on day 30 in the recipient mice that received lymph node cells from DNFB-treated donors (Fig. 5E, upper row). On day 90, the number and frequency of CD8 + T RM cells in the recipient mice that received lymph node cells from DNFB-treated donors had declined and did not differ in frequency and numbers from CD8 + T RM cells found in the control groups (Fig. 5B and C, lower row). Likewise, the expression of Nur77 and Ki67 was equal in the three groups on day 90 (Fig. 5D and E, lower row). These data indicated that CD8 + T RM cells did not persist in the absence of cognate antigen. Given that the persistence of CD8 + T RM cells in the epidermis was dependent on the presence of the cognate antigen, addition of DNFB to the ears should rescue the persistence of the CD8 + T RM cells in recipient mice that had received lymph node cells from DNFB-treated donors. In preliminary experiments, we found that a single treatment with a low dose (0.01%) of DNFB did not in itself induce skin inflammation (Fig. S2). To determine if presence of the cognate antigen could rescue the persistence of epidermal CD8 + T RM cells, we treated non-transfer control mice and recipient mice that received donor cells from DNFB- or OOA-treated mice with a single low dose of 0.01% DNFB at day 7 in addition to the standard SLS treatment given day 5-7 (Fig. 5A). Epidermal single cell suspensions were prepared from the ears on day 90 and analyzed by flow cytometry. The low dose of DNFB did not induce CD8 + T RM cells in the non-transfer group or in the recipient group that received cells from OOA-treated donors. However, a significant number of CD8 + T RM cells were found in the recipient group that received cells from DNFB-treated donors (Fig. 5F and G). A significant number of these CD8 + T RM cells were Nur77 + supporting that the cognate antigen was present and that signaling through the TCR took place (Fig. 5H). Likewise, a significant number of the CD8 + T RM cells expressed Ki67 (Fig. 5I). Taken together, these experiments demonstrated that the persistence of epidermal CD8 + T RM cells required the presence of the cognate antigen and signaling through the TCR. Discussion In this study, we show that CD8 + T RM cells can be generated in the epidermis in the absence of cognate antigen but that the persistence of CD8 + T RM cells requires the presence of the cognate antigen. We further demonstrate that some contact allergens persist in the epidermis for extended periods. We found that a fraction of the CD8 + T RM cells expressed Nur77 for more than 180 days after exposure of the skin to DNFB. This indicates that constitutive TCR signaling took place in the CD8 + T RM cells. In agreement, we could detect TCR ligands in the form of DNP adducts for at least 180 days after exposure of the skin to DNFB. At first sight, it might seem surprising that cognate antigen/DNP adducts linger in the epidermis for such an extended period [18]. However, this is in accordance with previous studies that found DNP adducts for at least 30 days after exposure to DNFB [19,41]. When evaluating the contact allergens cinnamal and PPD, we found that CD8 + T RM cells were formed and could be detected 30 days after exposure to the contact allergens. However, the CD8 + T RM cells did not express Nur77 on day 30, and on day 90, they had disappeared. In contrast, exposure of the skin to MI induced CD8 + T RM cells that expressed Nur77 on day 30. The MI-induced CD8 + T RM cells persisted on day 90, where a fraction of them still expressed Nur77. This indicates that MI, like DNFB but in contrast to cinnamal and PPD, persists for an extended period in the skin. In the adoptive transfer experiments, CD8 + T RM cells were found for at least 30 days after transfer of primed T cells to naïve mice that had not experienced antigen. We found no induction of Nur77 in the absence of cognate antigen in the adoptive transfer experiments in accordance with Nur77 being a specific marker for TCR signaling [25,30-39]; however, a fraction of the CD8 + T RM cells expressed the proliferation marker Ki67 [42,43]. This is in accordance with previous studies, which found Ki67 expression in skin-resident CD8 + T RM cells independently of antigen presentation [25,27], and it suggests that cytokines can support CD8 + T RM cell proliferation for at least one month after an inflammatory incident. In contrast to the generation of CD8 + T RM cells, persistence of CD8 + T RM cells for more than one month required the presence of the cognate antigen. Thus, whereas the CD8 + T RM cells had vanished after 90 days in recipient mice treated only with SLS, addition of a low dose of DNFB rescued persistence of the CD8 + T RM cells in the adoptive transfer experiments. Importantly, presence of the cognate antigen in the adoptive transfer experiments correlated with induction of Nur77. It is puzzling why some studies find that persistence of CD8 + T RM cells is dependent on the presence of cognate antigen, whereas other studies find that persistence of CD8 + T RM cells is independent on the presence of cognate antigen. In the present study, we show that the generation of CD8 + T RM cells can take place in inflamed skin in complete absence of the cognate antigen but that persistence of CD8 + T RM cells is dependent on the cognate antigen. It is known that IL-15 plays a central role in memory CD8 + T cells homeostasis [50-53] and that an increased production of IL-15 is seen following skin injury and inflammation [52,54,55]. This might explain the antigen-independent but inflammation-dependent survival of the CD8 + T RM cells in the initial period after recruitment of the cells to the inflamed skin. However, as inflammation fades, the production of IL-15 probably drops below the limit required for CD8 + T RM cell persistence. Interestingly, it has been shown that survival of CD8 + T RM cells becomes less dependent of IL-15 after repeated encounter with the cognate antigen [56], which could explain the persistence of CD8 + T RM cells in the absence of obvious inflammation but in the presence of cognate antigen. TGFβ also plays a significant role in generation and persistence of CD8 + T RM cells in the skin [53,57]. Interestingly, a recent study proposed that TCR signaling renders CD8 + T RM cell more fit and better able to compete for TGFβ and thereby survive for a long time in the skin [57]. In conclusion, this study demonstrates that CD8 + T RM cells can develop in the skin in the absence of cognate antigen, but that TCR signaling is required for the persistence of allergen-specific CD8 + T RM cells in the skin. As inflammation might sustain CD8 + T RM cell survival, future studies should elucidate whether chronic skin inflammation allows for the persistence of CD8 + T RM cells in the absence of cognate antigen. REFERENCES 1. N. Zhang, M. J. Bevan ”CD8(+) T cells: foot soldiers of the immune system,” Immunity 35, no. 2 (2011):161-168.2. L. Kok, D. Masopust, T. N. Schumacher ”The precursors of CD8(+) tissue resident memory T cells: from lymphoid organs to infected tissues,” Nat Rev Immunol 22, no. 5 (2022):283-293.3. A. W. Ho, T. S. Kupper ”T cells and the skin: from protective immunity to inflammatory skin disorders,” Nat Rev Immunol 19, no. 8 (2019):490-502.4. S. N. Christo, S. L. Park, S. N. Mueller, et al., ”The Multifaceted Role of Tissue-Resident Memory T Cells,” Annu Rev Immunol 42, no. 1 (2024):317-345.5. T. Gebhardt, L. M. Wakim, L. Eidsmo, et al., ”Memory T cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus,” Nat Immunol 10, no. 5 (2009):524-530.6. X. Jiang, R. A. Clark, L. Liu, et al., ”Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity,” Nature 483, no. 7388 (2012):227-231.7. H. Shin, A. Iwasaki ”A vaccine strategy that protects against genital herpes by establishing local memory T cells,” Nature 491, no. 7424 (2012):463-467.8. Y. Pan, T. Tian, C. O. Park, et al., ”Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism,” Nature 543, no. 7644 (2017):252-256.9. G. E. Ryan, J. E. Harris, J. M. Richmond ”Resident Memory T Cells in Autoimmune Skin Diseases,” Front Immunol 12, no. (2021):652191.10. M. A. Lefevre, M. Vocanson, A. Nosbaum ”Role of tissue-resident memory T cells in the pathophysiology of allergic contact dermatitis,” Curr Opin Allergy Clin Immunol 21, no. 4 (2021):355-360.11. L. Migayron, R. Merhi, J. Seneschal, et al., ”Resident memory T cells in nonlesional skin and healed lesions of patients with chronic inflammatory diseases: Appearances can be deceptive,” J Allergy Clin Immunol 153, no. 3 (2024):606-614.12. S. Cheuk, M. Wikén, L. Blomqvist, et al., ”Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis,” J Immunol 192, no. 7 (2014):3111-3120.13. T. R. Matos, J. T. O’Malley, E. L. Lowry, et al., ”Clinically resolved psoriatic lesions contain psoriasis-specific IL-17-producing αβ T cell clones,” J Clin Invest 127, no. 11 (2017):4031-4041.14. L. Xing, Z. Dai, A. Jabbari, et al., ”Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition,” Nat Med 20, no. 9 (2014):1043-1049.15. K. Boniface, C. Jacquemin, A. S. Darrigade, et al., ”Vitiligo Skin Is Imprinted with Resident Memory CD8 T Cells Expressing CXCR3,” J Invest Dermatol 138, no. 2 (2018):355-364.16. P. M. Brunner, R. O. Emerson, C. Tipton, et al., ”Nonlesional atopic dermatitis skin shares similar T-cell clones with lesional tissues,” Allergy 72, no. 12 (2017):2017-2025.17. O. Gaide, R. O. Emerson, X. Jiang, et al., ”Common clonal origin of central and resident memory T cells following skin immunization,” Nat Med 21, no. 6 (2015):647-653.18. J. D. Schmidt, M. G. Ahlstrom, J. D. Johansen, et al., ”Rapid allergen-induced interleukin-17 and interferon-gamma secretion by skin-resident memory CD8(+) T cells,” Contact Dermatitis 76, no. 4 (2017):218-227.19. P. Gamradt, L. Laoubi, A. Nosbaum, et al., ”Inhibitory checkpoint receptors control CD8+ resident memory T cells to prevent skin allergy,” J Allergy Clin Immunol no. (2019).20. A. B. Funch, V. Mraz, A. Gadsbøll, et al., ”CD8(+) tissue-resident memory T cells recruit neutrophils that are essential for flare-ups in contact dermatitis,” Allergy 77, no. 2 (2022):513-524.21. A. Gadsbøll, M. H. Jee, A. B. Funch, et al., ”Pathogenic CD8(+) Epidermis-Resident Memory T Cells Displace Dendritic Epidermal T Cells in Allergic Dermatitis,” J Invest Dermatol 140, no. 4 (2020):806-815.e805.22. A. B. Funch, M. G. Ahlström, J. D. Johansen, et al., ”Neutrophil infiltration in allergic contact dermatitis to nickel,” Br J Dermatol 190, no. 4 (2024):569-570.23. L. K. Mackay, A. T. Stock, J. Z. Ma, et al., ”Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation,” Proc Natl Acad Sci U S A 109, no. 18 (2012):7037-7042.24. S. L. Park, A. Zaid, J. L. Hor, et al., ”Local proliferation maintains a stable pool of tissue-resident memory T cells after antiviral recall responses,” Nat Immunol 19, no. 2 (2018):183-191.25. T. N. Khan, J. L. Mooster, A. M. Kilgore, et al., ”Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection,” J Exp Med 213, no. 6 (2016):951-966.26. A. Muschaweckh, V. R. Buchholz, A. Fellenzer, et al., ”Antigen-dependent competition shapes the local repertoire of tissue-resident memory CD8+ T cells,” J Exp Med 213, no. 13 (2016):3075-3086.27. M. Abdelbary, S. J. Hobbs, J. S. Gibbs, et al., ”T cell receptor signaling strength establishes the chemotactic properties of effector CD8(+) T cells that control tissue-residency,” Nat Commun 14, no. 1 (2023):3928.28. S. Wijeyesinghe, L. K. Beura, M. J. Pierson, et al., ”Expansible residence decentralizes immune homeostasis,” Nature 592, no. 7854 (2021):457-462.29. B. A. Osborne, S. W. Smith, Z. G. Liu, et al., ”Identification of genes induced during apoptosis in T lymphocytes,” Immunol Rev 142, no. (1994):301-320.30. A. E. Moran, K. L. Holzapfel, Y. Xing, et al., ”T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse,” J Exp Med 208, no. 6 (2011):1279-1289.31. N. Kamenjarin, K. Hodapp, F. Melchior, et al., ”Cross-presenting Langerhans cells are required for the early reactivation of resident CD8(+) memory T cells in the epidermis,” Proc Natl Acad Sci U S A 120, no. 34 (2023):e2219932120.32. B. B. Au-Yeung, J. Zikherman, J. L. Mueller, et al., ”A sharp T-cell antigen receptor signaling threshold for T-cell proliferation,” Proc Natl Acad Sci U S A 111, no. 35 (2014):E3679-3688.33. J. F. Ashouri, A. Weiss ”Endogenous Nur77 Is a Specific Indicator of Antigen Receptor Signaling in Human T and B Cells,” J Immunol 198, no. 2 (2017):657-668.34. K. P. Li, B. H. Ladle, S. Kurtulus, et al., ”T-cell receptor signal strength and epigenetic control of Bim predict memory CD8(+) T-cell fate,” Cell Death Differ 27, no. 4 (2020):1214-1224.35. J. F. Ashouri, L. Y. Hsu, S. Yu, et al., ”Reporters of TCR signaling identify arthritogenic T cells in murine and human autoimmune arthritis,” Proc Natl Acad Sci U S A 116, no. 37 (2019):18517-18527.36. M. K. Chanda, C. E. Shudde, T. L. Piper, et al., ”Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry,” J Immunol Methods 506, no. (2022):113290.37. J. M. Bartleson, A. A. Viehmann Milam, D. L. Donermeyer, et al., ”Strength of tonic T cell receptor signaling instructs T follicular helper cell-fate decisions,” Nat Immunol 21, no. 11 (2020):1384-1396.38. J. S. Low, Y. Farsakoglu, M. C. Amezcua Vesely, et al., ”Tissue-resident memory T cell reactivation by diverse antigen-presenting cells imparts distinct functional responses,” J Exp Med 217, no. 8 (2020).39. T. A. E. Elliot, E. K. Jennings, D. A. J. Lecky, et al., ”Nur77-Tempo mice reveal T cell steady state antigen recognition,” Discov Immunol 1, no. 1 (2022):kyac009.40. P. L. Scheinman, M. Vocanson, J. P. Thyssen, et al., ”Contact dermatitis,” Nat Rev Dis Primers 7, no. 1 (2021):38.41. E. Ono, V. Lenief, M. A. Lefevre, et al., ”Topical corticosteroids inhibit allergic skin inflammation but are ineffective in impeding the formation and expansion of resident memory T cells,” Allergy 79, no. 1 (2024):52-64.42. T. Scholzen, J. Gerdes ”The Ki-67 protein: from the known and the unknown,” J Cell Physiol 182, no. 3 (2000):311-322.43. S. Bruno, Z. Darzynkiewicz ”Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells,” Cell Prolif 25, no. 1 (1992):31-40.44. J.-P. Lepoittevin, A. Goossens. Molecular Basis for the Recognition of Haptens by T Lymphocytes. In: Lepoittevin J-P, Basketter DA, Goossens A, Karlberg A-T, eds. Allergic Contact Dermatitis: The Molecular Basis. Berlin, Heidelberg: Springer Berlin Heidelberg; 1998:112-128.45. J. Chiffelle, R. Genolet, M. A. Perez, et al., ”T-cell repertoire analysis and metrics of diversity and clonality,” Curr Opin Biotechnol 65, no. (2020):284-295.46. M. Arunkumar, C. E. Zielinski ”T-Cell Receptor Repertoire Analysis with Computational Tools-An Immunologist’s Perspective,” Cells 10, no. 12 (2021).47. A. B. Funch, J. F. Weber, R. K. D. Lohmann, et al., ”CD4(+) T cells inhibit the generation of CD8(+) epidermal-resident memory T cells directed against clinically relevant contact allergens,” Contact Dermatitis 88, no. 6 (2023):425-437.48. L. F. Su, B. A. Kidd, A. Han, et al., ”Virus-specific CD4(+) memory-phenotype T cells are abundant in unexposed adults,” Immunity 38, no. 2 (2013):373-383.49. C. Haluszczak, A. D. Akue, S. E. Hamilton, et al., ”The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion,” J Exp Med 206, no. 2 (2009):435-448.50. M. K. Kennedy, M. Glaccum, S. N. Brown, et al., ”Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice,” J Exp Med 191, no. 5 (2000):771-780.51. J. P. Lodolce, D. L. Boone, S. Chai, et al., ”IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation,” Immunity 9, no. 5 (1998):669-676.52. T. Adachi, T. Kobayashi, E. Sugihara, et al., ”Hair follicle-derived IL-7 and IL-15 mediate skin-resident memory T cell homeostasis and lymphoma,” Nat Med 21, no. 11 (2015):1272-1279.53. L. K. Mackay, A. Rahimpour, J. Z. Ma, et al., ”The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin,” Nat Immunol 14, no. 12 (2013):1294-1301.54. L. K. Mackay, E. Wynne-Jones, D. Freestone, et al., ”T-box Transcription Factors Combine with the Cytokines TGF-β and IL-15 to Control Tissue-Resident Memory T Cell Fate,” Immunity 43, no. 6 (2015):1101-1111.55. J. Suwanpradid, M. J. Lee, P. Hoang, et al., ”IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses,” Front Immunol 12, no. (2021):713304.56. J. M. Schenkel, K. A. Fraser, K. A. Casey, et al., ”IL-15-Independent Maintenance of Tissue-Resident and Boosted Effector Memory CD8 T Cells,” J Immunol 196, no. 9 (2016):3920-3926.57. T. Hirai, Y. Yang, Y. Zenke, et al., ”Competition for Active TGFβ Cytokine Allows for Selective Retention of Antigen-Specific Tissue- Resident Memory T Cells in the Epidermal Niche,” Immunity 54, no. 1 (2021):84-98.e85. Figure legends Figure 1. CD8 + T RM cells persist in the epidermis for at least one year after sensitization to DNFB (A) Experimental setup: Mice were exposed to OOA or 0.15% DNFB in OOA on day 0-2 on both ears and epidermal cells were isolated for flow cytometric analysis day 30, 180 and 365. (B) Representative plots of TCR β and CD8 α expression of live cells (left column) and CD103 and CD69 expression of CD8α + TCRβ + cells (right column). (C) Absolute numbers of CD8α + TCRβ + cells in the given subsets per two ears in mice exposed to OOA (white circles/bars) and DNFB (light blue circles/bars). (D) Representative plots of Nur77 (yellow) and Ki67 (dark blue) expression by CD69 + CD103 + CD8α + TCRβ + cells. (E-F) Absolute numbers of CD69 + CD103 + CD8α + TCRβ + cells expressing (E) Nur77 and (F) Ki67 per two ears. Bars; mean number of cells. Dots; number of cells from each mouse. Error bars; standard deviation (SD). Statistical comparisons; Students unpaired t-tests or one-way ANOVA. Figure 2. Exposure to DNFB induces long-term persistence of DNP adducts in the epidermis (A ) Experimental setup: Mice were exposed to OOA or 0.15% DNFB on day 0-2 and the epidermal ear sheets were analyzed for the presence of DNP adducts on day 5 or 180 by Western blotting. (B) Western blot showing DNP adducts from epidermal homogenates isolated on day five from 4 OOA-treated (lane 1-4) and 4 DNFB-treated (lane 5-8) mice. Membrane exposure time 1 minute. (C) Western blot showing DNP adducts from epidermal homogenates isolated on day 180 from 4 OOA-treated (lane 1-4) and 4 DNFB-treated (lane 5-8) mice. Membrane exposure time is 40 minutes. Vinculin was used as a loading control. Figure 3. Selective expansion of specific TCR clonotypes with time (A) Experimental setup: Mice were exposed to 0.15% DNFB on day 0-2 and CD8 + T cells were isolated from the epidermal ear sheets and analyzed by scTCR-seq at day 30 and 180. (B) TCR repertoire richness and overlap. (C) Abundance of cells in each of the indicated clonal sizes relative to the total number of cells in all clones identified in each mouse. (B-C) M1-4 and M5-9 indicate 4 and 5 individual mice analyzed at day 30 and day 180 after DNFB exposure, respectively. (D-E) Frequency distribution of clonotypes for each mouse at (D) day 30 and at (E) day 180. The clonotype index is ordered by decreasing clonotype size. The average number of clones required to reach 50% of the total number of cells in all clones identified in each mouse is given in red and indicated with the stippled lines. (F) The Gini coefficient of the TCR clonotypes at day 30 and day 180. Violin plots; distribution and mean number of cells. Dots; number of cells from each mouse. Statistical comparisons; Students unpaired t-tests. Figure 4. Nur77 expression correlates with CD8 + T RM cell persistence (A) Experimental setup: Mice were treated with anti-CD4 mAb injections i.v. on day -1, 0 and 5 and exposed to the indicated contact allergens or OOA as control on day 0-2. Epidermal cells were isolated for flow cytometric analysis day 30 and 180. (B) Representative plots of TCR β and CD8 α expression of live cells (left column) and CD103 and CD69 expression of CD8α + TCRβ + cells (right column) at day 30. (C) Absolute numbers of CD8α + TCRβ + cells in the given subsets per two ears in mice exposed to OOA (white circles/bars) and the given contact allergen (colored circles/bars). (D) Representative plots of Nur77 (yellow) and Ki67 (dark blue) expression by CD69 + CD103 + CD8α + TCRβ + cells. (E-F) Absolute numbers of CD69 + CD103 + CD8α + TCRβ + cells expressing (E) Nur77 and (F) Ki67 per two ears. (G) Representative plots of TCR β and CD8 α expression of live cells (left column) and CD103 and CD69 expression of CD8α + TCRβ + cells (right column) at day 180. (H) Absolute numbers of CD8α + TCRβ + cells in the given subsets per two ears in mice exposed to OOA (white circles/bars) and the given contact allergen (colored circles/bars). (I) Representative plots of Nur77 (yellow) and Ki67 (dark blue) expression by CD69 + CD103 + CD8α + TCRβ + cells. (J-K) Absolute numbers of CD69 + CD103 + CD8α + TCRβ + cells expressing (J) Nur77 and (K) Ki67 per two ears. Bars; mean number of cells. Dots; number of cells from each mouse. Error bars; standard deviation (SD). Statistical comparisons; Students unpaired t-tests or one-way ANOVA. Figure 5. Persistence of CD8 + T RM cells in the epidermis requires presence of the cognate antigen (A) Experimental setup: Mice were exposed to OOA or DNFB on day 0-2 on the ears. Lymphocytes were isolated from the draining lymph nodes on day five from DNFB- and OOA-treated donor mice, and 5 x 10 7 lymphocytes were transferred i.v. to untreated recipient mice. Recipient mice and a non-transfer control group of mice were treated on the ears with 2.0% SLS diluted in OOA day 5-7. Epidermal cells were isolated for flow cytometric analysis day 30 and 90. (B) Representative plots of TCR β and CD8 α expression of live cells at day 30 (upper row) and day 90 (lower row) in the given groups of mice treated with SLS. (C) Absolute numbers of CD8 + T RM cells in the given group of mice per two ears at day 30 (upper row) and day 90 (lower row). (D-E) Absolute numbers of CD8 + T RM cells expressing (D) Nur77 and (E) Ki67 per two ears at day 30 (upper row) and day 90 (lower row). (F) Representative plots of TCR β and CD8 α expression of live cells at day 90 in the given groups of mice treated with SLS + 0.01% DNFB. (G) Absolute numbers of CD8 + T RM cells in the given group of mice per two ears at day 90. (H-I) Absolute numbers of CD8 + T RM cells expressing (H) Nur77 and (I) Ki67 per two ears at day 90. Non-transfer (NT). Bars; mean number of cells. Dots; number of cells from one mouse Error bars; standard deviation (SD). Statistical comparisons; Students unpaired t-tests or one-way ANOVA. Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Information & Authors Information Version history V1 Version 1 10 January 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords allergens and epitopes allergic contact dermatitis animal models barrier dermatology t cells Authors Affiliations Anders Funch 0000-0002-7576-3884 [email protected] Kobenhavns Universitet View all articles by this author Julie Weber Kobenhavns Universitet View all articles by this author Veronika Mraz Kobenhavns Universitet View all articles by this author Martin Kongsbak-Wismann Kobenhavns Universitet View all articles by this author Rebecca Kitt Davidson Lohmann Kobenhavns Universitet View all articles by this author Mia Jee Kobenhavns Universitet View all articles by this author Helen Vaher 0000-0002-8845-0326 Kobenhavns Universitet View all articles by this author Kelvin Yeung 0000-0001-6083-3475 Kobenhavns Universitet View all articles by this author Anne-Sofie Ø. Gadsbøll Kobenhavns Universitet View all articles by this author Niels Ødum Kobenhavns Universitet View all articles by this author Anders Woetmann Kobenhavns Universitet View all articles by this author Jeanne Johansen Gentofte Hospital Hud og allergiafdeling View all articles by this author Carsten Geisler Kobenhavns Universitet View all articles by this author Charlotte Bonefeld 0000-0002-0523-6229 Kobenhavns Universitet View all articles by this author Metrics & Citations Metrics Article Usage 380 views 227 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Anders Funch, Julie Weber, Veronika Mraz, et al. Persistence of CD8+ skin-resident memory T cells in mice requires TCR signaling. Authorea . 10 January 2025. DOI: https://doi.org/10.22541/au.173652866.63708399/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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