Persistence of skin-resident memory T cells within an epidermal niche Ali Zaida,1, Laura K. Mackaya,1, Azad Rahimpoura, Asolina Brauna, Marc Veldhoenb, Francis R. Carbonea, Jonathan H. Mantonc, William R. Heatha,2, and Scott N. Muellera,2,3 aDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, VIC 3010, Australia; bLaboratory for Lymphocyte Signalling and Development, The Babraham Institute, Cambridge CB22 3AT, United Kingdom; and cDepartment of Electrical and Electronic Engineering, The University of Melbourne, Parkville, VIC 3010, Australia Edited by Rafi Ahmed, Emory University, Atlanta, GA, and approved February 26, 2014 (received for review December 2, 2013) Barrier tissues such as the skin contain various populations of indicating that this may be a common survival pathway for T cells immune cells that contribute to protection from infections. These residing in the epidermis. include recently identified tissue-resident memory T cells (TRM). In the skin, these memory CD8+ T cells reside in the epidermis after Results being recruited to this site by infection or inflammation. In this Migration of Epidermal TRM. To understand how TRM persist in the study, we demonstrate prolonged persistence of epidermal TRM skin and contribute to local protective immunity we sought to preferentially at the site of prior infection despite sustained mi- characterize the behavior of these memory T cells within their gration. Computational simulation of TRM migration within the natural environment. We used a well-established model of cu- skin over long periods revealed that the slow rate of random mi- taneous HSV infection (8) and MHC class I-restricted T-cell gration effectively constrains these memory cells within the region receptor (TCR) transgenic mice specific for an immunodo- + of skin in which they form. Notably, formation of TRM involved minant glycoprotein B epitope (gB498–505) (9). CD8 T cells from a concomitant local reduction in dendritic epidermal γδ T-cell num- these gBT-I mice expressing EGFP were transferred into re- bers in the epidermis, indicating that these populations persist in cipient C57BL/6 (B6) mice to track virus-specific T cells fol- mutual exclusion and may compete for local survival signals. Ac- lowing HSV infection using intravital two-photon microscopy of cordingly, we show that expression of the aryl hydrocarbon re- the skin. As we and others have observed previously (3, 10), γδ + ceptor, a transcription factor important for dendritic epidermal memory CD8 T cells were present in the epidermis and dis- T-cell maintenance in skin, also contributes to the persistence of skin played a unique dendritic morphology (Fig. 1A). These TRM TRM. Together, these data suggest that skin tissue-resident memory were highly dynamic, demonstrating active probing of their en- T cells persist within a tightly regulated epidermal T-cell niche. vironment with dendrites that rapidly extended and retracted (Movie S1). In many cases, multibranched dendrites formed, and intravital imaging HSV-1 infection | Langerhans cells | Brownian motion cells could be observed extending cellular projections in multiple directions simultaneously. he skin is a complex organ that acts as a primary barrier Closer examination of the location of these skin TRM revealed Tbetween the body and the environment. Multiple leukocyte a highly constricted localization to the basal epidermis (Fig. 1B), subsets reside within the main compartments of the skin, the immediately adjacent to and in contact with the basement dermis and the epidermis, as well as the hair follicles that are membrane that separates the dermis from the epidermis (Fig. contiguous with the epidermis. Populations of macrophages, 1C). Identification of cells in each compartment was achieved dendritic cells, mast cells, γδ T cells and αβ T cells are present in using second harmonic generation (SHG) fluorescence to dif- the dermis, and Langerhans cells (LCs) and dendritic epidermal ferentiate the collagen-rich dermis from the epidermis. This loca- γδ T cells (DETCs) lie in a strategic network in the epidermis tion defined the movement of the memory T cells, which extended IMMUNOLOGY + (1). We recently described a population of memory CD8 T cells dendritic projections laterally yet were not observed to probe that enter the epidermis and hair follicles during infection or inflammation and become long-lived populations of tissue-resi- Significance dent memory T cells (TRM)(2–5). These memory T cells are sequestered in this site and are distinct from circulating effector Tissue-resident memory T cells (TRM) form in the skin where memory (TEM) and central memory (TCM) populations (3, 6). they are retained and can protect against subsequent infection. + Memory CD8 T cells in the epidermis display a dendritic mor- Using a combination of intravital imaging and mathematical phology and move at a slower velocity than T cells within the modeling of skin TRM that form after cutaneous herpes simplex dermis. These memory T cells are sequestered in the skin epithelial virus 1 infection, we reveal that these memory T cells persist at layer and do not recirculate to other tissues. In contrast, memory the site of infection for the life of a mouse owing to slow ran- + CD4 T cells are found within the dermis and at least a proportion dom migration. We also report that TRM compete with dendritic γδ of these cells are capable of recirculating around the body (3, 7). epidermal T cells in skin for local survival signals, suggesting In this study, we sought to further examine the mechanisms of that T cells compete for space within an epidermal niche. TRM persistence within the skin. We reveal that skin TRM persist Author contributions: F.R.C., W.R.H., and S.N.M. designed research; A.Z., L.K.M., A.R., A.B., at the site of their formation and despite displaying a sustained J.H.M., and S.N.M. performed research; M.V. contributed new reagents/analytic tools; mode of random migration, the slow rate of this movement lo- A.Z., L.K.M., J.H.M., and S.N.M. analyzed data; and S.N.M. wrote the paper. cally constrains these memory cells. Examination of other cells in The authors declare no conflict of interest. this environment showed that although epidermal TRM regularly This article is a PNAS Direct Submission. interact with LC, these interactions were not required for per- 1A.Z. and L.K.M. contributed equally to this work. sistence. In contrast, skin tissue-resident memory T cells replaced 2W.R.H. and S.N.M. contributed equally to this work. DETCs in the epidermis, seeming to compete for space within the 3To whom correspondence should be addressed. E-mail: [email protected]. epidermal niche. We found that expression of the aryl hydrocar- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bon receptor (AhR) is required for long-term persistence of TRM, 1073/pnas.1322292111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322292111 PNAS | April 8, 2014 | vol. 111 | no. 14 | 5307–5312 Downloaded by guest on September 24, 2021 Fig. 1. Migration of skin TRM at the site of prior infection. (A) gBT-I.GFP CD8+ T cells (green) imaged by two-photon microscopy in the skin 32 d after HSV infection. Examples of different cell morphol- ogies are magnified in i–iii. SHG (blue) delineates the collagen-rich dermis. Images correspond to Movie S1.(B)TRM are located within the basal epidermis + (Ep), adjacent to the SHG dermis (D). (C)TRM contact the basement membrane at the dermis–epidermal border. Tissue sections of skin containing gBT-I.DsRed + CD8 T cells were costained with anti-laminin-γ2 antibodies. (D) Mean velocity and (E) displacement of TRM migrating within the epidermis at the in- dicated times after HSV infection. ***P < 0.0001; ns, not significant. (F) Epidermal location defines the dendritic morphology of skin TRM.Shownisarepre- sentative example of an epidermal and a dermal gBT-I T-cell 10 d after HSV infection. Sphericity measure- ments of dermal and epidermal gBT-I T cells 10 d (acute) and 32 d (memory) after HSV infection are plotted. (G) Time-lapse imaging of gBT-I TRM migra- tion in the skin. (Left) The first frame of the movie; (Right) superimposed images taken at 3-min intervals over a 7.5-h period. Images correspond to Movie S4. (H) The average 2D surface area of individual TRM was calculated from 10 individual images per cell col- lected over a 60-min period. The total surface area covered per cell was calculated from superimposed images taken each minute for 1 h. The fold differ- ence between the average surface area at time 0 and 60 is shown. upward toward the apical epidermis and stratum corneum, nor in a matter of hours (Fig. 1G and Movie S4). We calculated the downward toward the dermis (Movie S2). This is in contrast to 2D surface area covered by TRM, given that they move within skin DETCs, whose dendrites probe the apical epidermis in the a roughly 2D environment. By examining cells over time, we steady state (11). TRM could also be observed within hair follicle estimated that the memory T cells scan an area approximately epithelial layers, as well as entering and exiting hair follicles via five times the size of an average cell per hour (in this experiment, the contiguous epidermis (Movie S3). 60 frames) (Fig. 1H). This suggests a substantial ability of TRM to To further examine the behavior of the skin TRM, we de- survey their immediate environment over time (10), particularly termined the migration velocity within the epidermis over time. given the restricted dimensions of this compartment and the T cells in the epidermis migrate considerably more slowly than T observation that skin TRM remain resident in the epidermis and cells within the dermis and have a dendritic as opposed to do not recirculate through other tissues (3).