Chemokine Receptor−Dependent Control of Skin Tissue−Resident Memory T Cell Formation

This information is current as Ali Zaid, Jyh Liang Hor, Susan N. Christo, Joanna R. of September 25, 2021. Groom, William R. Heath, Laura K. Mackay and Scott N. Mueller J Immunol published online 30 August 2017 http://www.jimmunol.org/content/early/2017/08/30/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 30, 2017, doi:10.4049/jimmunol.1700571 The Journal of Immunology

Chemokine Receptor–Dependent Control of Skin Tissue–Resident Memory T Cell Formation

Ali Zaid,*,1,2 Jyh Liang Hor,*,†,2 Susan N. Christo,*,2 Joanna R. Groom,‡,x,{ William R. Heath,*,† Laura K. Mackay,*,† and Scott N. Mueller*,†

Infection or inﬂammation of the skin recruits effector CD8+ T cells that enter the epidermis and form populations of long-lived

tissue-resident memory T (TRM) cells. These skin TRM cells migrate within the constrained epidermal environment by extending multiple dynamic dendritic projections and squeezing between keratinocytes to survey the tissue for pathogens. In this study, we examined the signals required for this distinctive mode of T cell migration by inhibiting key cytoskeletal components and

performing intravital two-photon microscopy to visualize TRM cell behavior. We found that TRM cell motility and dendrite formation required an intact actomyosin cytoskeleton and the Rho-associated coiled-coil containing kinases. We also identiﬁed

an essential role for microtubules for maintaining skin TRM cell shape and cellular integrity. We reveal a role for pertussis toxin– Downloaded from sensitive signaling for TRM cell dendritic morphology and migration that is independent of CXCR3 or CXCR6, or the skin- selective chemokine receptors CCR10 and CCR8. However, we found that CXCR6 and CCR10 expression by CD8+ T cells was

required for the optimal formation of memory T cell populations, in particular TRM cell populations in the skin. The Journal of Immunology, 2017, 199: 000–000.

cells are important for protection from pathogens and in tissues without exiting into the bloodstream. TRM cells have http://www.jimmunol.org/ tumors. Following the clearance of a pathogen, populations been observed in most tissues and organs in mice, and in many T of memory T cells develop with different functional, tissues in humans. phenotypic, and migratory properties (1). Subsets of memory T cells, CD8 and CD4 memory T cell populations are found in tissues, referred to as central and effector memory T cells, recirculate via often with clear differences in the localization of these two subsets the blood through secondary lymphoid organs, returning to the within the microarchitecture of tissues such as the skin, intestines,

circulation via the lymphatics. Effector memory T cells can also lungs, and reproductive tract (1, 3). TRM cells are deﬁned on the migrate through nonlymphoid tissues before returning to lymph basis of expression of the markers CD69 and the integrin aE chain, nodes (LN) via lymphatics. Notably, nonlymphoid tissues also CD103. Additionally, TRM cells have been shown to express a by guest on September 25, 2021 contain a substantial proportion of nonrecirculating, tissue-resident transcriptional proﬁle distinct from that of recirculating memory memory T (TRM) cells (2). These cells can persist for long periods T cells and related to that of populations of tissue-resident innate

lymphoid cells (4, 5). Notably, TRM cells in different nonlymphoid *Department of Microbiology and Immunology, The University of Melbourne, at the tissues possess unique gene expression signatures, indicating that Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000 Aus- local signals shape the development of memory T cells resident in tralia; †Australian Research Council Centre of Excellence in Advanced Molecular Imag- ing, The University of Melbourne, Melbourne, Victoria 3000, Australia; ‡Division of different tissues. Immunology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria Upon re-exposure to a pathogen, T cells can contribute to x RM 3052, Australia; Division of Molecular Immunology, Walter and Eliza Hall Institute early protection by releasing cytokines that stimulate local innate of Medical Research, Parkville, Victoria 3052, Australia; and {Department of Med- ical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia immunity and help to recruit T cells from the blood (6, 7). Integral 1Current address: Institute for Glycomics, Grifﬁth University, Gold Coast Campus, to this ability to respond rapidly upon reinfection, TRM cells Queensland, Australia. within tissues perform key local immunosurveillance functions. 2A.Z., J.L.H., and S.N.C. contributed equally to this work. These cells do so by migrating constantly within the nonlymphoid ORCIDs: 0000-0002-2528-2783 (A.Z.); 0000-0001-9670-259X (W.R.H.); 0000- tissue environment, increasing the probability of contact with in- 0002-3838-3989 (S.N.M.). + fected cells or APCs (8, 9). In the skin, CD8 TRM cells are re- Received for publication April 21, 2017. Accepted for publication August 3, 2017. stricted to the epidermis and hair follicle epithelium (10). In This work was supported by the National Health and Medical Research Council of contrast to T cells within the dermis that have an amoeboid shape Australia. and relatively fast speed of migration (∼6 mm/min), TRM cells A.Z., J.L.H., and S.N.C. performed experiments and analyzed data; J.R.G. provided reagents; W.R.H. and L.K.M. contributed to experimental design; and S.N.M. de- within the epidermis adapt a striking dendritic morphology and signed the study, prepared ﬁgures, wrote the manuscript, and supervised the research. slow mode of migration (1–2 mm/min). This slow random mi-

Address correspondence and reprint requests to Assoc. Prof. Scott N. Mueller, De- gration promotes the long-term persistence of TRM cells at sites of partment of Microbiology and Immunology, The University of Melbourne, at the prior infection in the skin and may enhance local immuno- Peter Doherty Institute for Infection and Immunity, 792 Elizabeth Street, Melbourne, Victoria 3000, Australia. E-mail address: [email protected] surveillance by enhancing the ability of cells to scan the envi- The online version of this article contains supplemental material. ronment (8, 9). Although the epidermal environment appears to enforce a Abbreviations used in this article: bLN, brachial LN; DNFB, 2,4-dinitroﬂuoroben- zene; KO, knockout; LN, lymph node; PTx, pertussis toxin; ROCK, Rho-associated constrained mode of migration in skin TRM cells, what signals coiled-coil kinase; SHG, second harmonic generation; TRM, tissue-resident memory control the morphology and motility of these cells are not known. T; WT, wild-type. The range of signals that inﬂuence TRM cell formation and per- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 sistence within tissues, including the skin, are just beginning to be

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700571 2 SIGNALS FOR SKIN TRM CELL FORMATION AND MIGRATION determined. Signaling through the TGF-b receptor and aryl hy- prior to acquisition by flow cytometry (BD FACSCanto or LSRFortessa). drocarbon receptor, IL-15 signals, as well as CD103 and CD69 Data were analyzed using FlowJo software (Tree Star). The following expression, are each required for skin T cell development or Abs were purchased: BD Pharmingen: Va2-PE-Cy7 (B20.1) and CD69- RM PE ( 1H.2F3); eBioscience: CD3e-PerCP-Cy5.5 (145-2C11), CD44– persistence (5, 11, 12). Furthermore, downregulation of key mole- Alexa Fluor 700 (IM7), CD8a–allophycocyanin–eFluor 780 (53-6.7), cules required for T cell trafficking, including CCR7, S1PR1, and CD62L-PerCP-Cy5.5 (MEL-14), CD45.1-FITC (A20), CD45.1-PE KLF2, as well as regulation of T-bet and Hobit are central to tissue (A20), and CD45.2–Alexa Fluor 700 (104); and BioLegend: CD103- residence by enforcing a gene expression program that prevents allophycocyanin (2E7). migration of TRM cells out of the tissues (4, 5, 11, 13). Whether Cell labeling and transfers other chemokine or cytokine receptors also contribute to skin T RM gBT-I T cells were incubated with 5 mM CellTrace Violet at 37˚C for 10 cell formation, migration, or persistence needs to be determined. min and washed in complete media. T cells were then adoptively trans- We have examined signals controlling TRM cell morphology and ferred i.v. into recipient mice prior to infection as described above. For motility in the skin of mice, using intravital two-photon micros- in vitro–generated effector CD8+ T cells, splenocyte samples enriched for + copy to track T cells in real time. Via the topical application of CD8 T cells were activated by anti-CD3ε (5 mg/ml; clone 145-2C11; eBioscience) and anti-CD28 (5 mg/ml; clone 37.51). Activated CD8+ various inhibitors to the skin, we could directly alter T cell 6 RM T cells (5 3 10 ) were transferred at a 1:1 ratio of wild-type (WT) and behavior. This revealed key roles for actin polymerization and CXCR32/2 or CXCR62/2 cells into recipients by intradermal injection myosin-II, as well as the Rho-associated coiled-coil kinases (five 20-ml injections over an area of skin 1 3 1.5 cm2) with a 30-gauge (ROCK) for T cell dendritic morphology, and a critical role for needle. Mice were shaved and depilated before the application of 15 ml RM 2 microtubules for maintaining T cell shape. Skin T cells re- 2,4-dinitrofluorobenzene (DNFB) in acetone/oil (4:1) to a 1-1.5 cm area RM RM of skin 24 h prior to T cell transfer. quired pertussis toxin (PTx)–sensitive signals for motility, yet the Downloaded from chemokine receptors CXCR3, CCR8, CXCR6, and CCR10 were Intravital skin imaging + not required. However, CD8 T cells lacking expression of Intravital imaging of the skin flank was performed as described elsewhere. CXCR6 or CCR10 formed reduced numbers of skin TRM cells, Briefly, mice were anesthetized with isoflurane (Cenvet; 2.5% for induction, and lack of CCR10 also impaired numbers of circulating memory 1–1.5% for maintenance, vaporized at 80:20 mixture of O2 and air), shaved T cells, demonstrating a role for CCR10 for T cell priming and on the left flank, and hair was depilated. Two incisions (∼15 mm apart) were made longitudinally along the left flank to contain the herpes lesion memory formation after skin infection. http://www.jimmunol.org/ site. Subcutaneous connective tissue below the dermis was carefully cut away from the dermis and an 18-mm-wide 3 1-mm-thick stainless steel platform was inserted under the exposed dermis, upon which the underside Materials and Methods of the dermis was attached using Vetbond tissue adhesive (3M). The edges Mice of the skin were lined with vacuum grease (Dow Corning), warm PBS was placed on top of the skin, and a glass coverslip was carefully positioned. a b C57BL/6J, B6.SJL-Ptprc Pep3 /BoyJ (B6.CD45.1), gBT-I.CD45.1, gBT- Imaging was performed with an upright LSM 710 NLO multiphoton mi- 2/2 2/2 2/2 I.EGFP, gBT-I.dsRed, gBT-I.EGFP Itgae (CD103 ), gBT-I.CCR10 , croscope (Carl Zeiss) with a 320/1.0 numerical aperture water immersion 2/2 2/2 2/2 gBT-I.EGFP CCR8 , OT-I.EGFP CXCR3 , and CXCR6 mice objective enclosed in an environmental chamber (Precision Plastics) were used in this study. gBT-I mice encode transgenes expressing TCR maintained at 35˚C with heated air. Fluorescence excitation was provided recognizing the HSV glycoprotein B–derived epitope gB . CCR10- 498–505 by a Chameleon Vision II Ti:Sapphire laser (Coherent) with dispersion by guest on September 25, 2021 deficient (EGFP knock-in) mice were from Lu and colleagues and correction and fluorescence emission detected using external non- C. Gerard (14). Animal experiments were approved by The University of descanned photomultiplier tubes. EGFP and second harmonic generation Melbourne Animal Ethics Committee. (SHG) were excited at 920 nm, whereas samples with dsRed and EGFP were excited at 900 nm. For four-dimensional data sets, three-dimensional Virus z-stacks (40 mm thick) were acquired every 60 s for 45–60 min. Raw Epicutaneous infection with HSV-1 KOS was performed as described imaging data were processed using Imaris (Bitplane). Cells were surface- elsewhere (15). Briefly, mice were anesthetized with a 1:1 mixture of rendered using Imaris surface module to generate sphericity data. Videos ketamine and Xylazil (10 ml/g body weight) by i.p. injection. Hair was generated in Imaris were composed in After Effects (Adobe). removed from the left skin flank and depilated (Veet; Reckitt Benckiser). Statistical analysis Scarification was performed by lightly abrading the skin using a grindstone 6 tip of a rotary tool (Dremel) and infected with 10 PFUs of HSV. Comparison of data sets was performed using Kruskal–Wallis tests with a Dunn posttest or Mann–Whitney U test where appropriate. Inhibitor treatment Formulations of inhibitors were prepared for transcutaneous delivery. Cytochalasin D (20 mg), blebbistatin (50 mg), or Y27632 (50 mg) were Results mixed with acetone (in an 80 ml vol). Colchicine (50 mg) or PTx (100 ng) Cytoskeletal control of skin T cell migration and was added to 100 mg of Sorbolene cream and mixed thoroughly. Com- RM pounds mixed in acetone were applied to the skin using a 200-ml micro- morphology pipette. Compounds mixed in Sorbolene were applied and the mixture was T cells that enter the epidermis acquire a dendritic morphology and contained on the skin using a bandage consisting of an adhesive film low migrational velocity that become more pronounced after the (OpSite Flexigrid; Smith & Nephew) and surgical tape (Micropore tape, + 3M). The mixture was allowed to absorb through the skin for 3 h prior to resolution of infection (8–10). The shape of skin CD8 TRM cells removal of the bandage. Mice were imaged 8–10 h after application. is highly dynamic during the process of migration within the constrained epidermal layer, and it appears unique from the mode of Tissue processing and flow cytometry migration used by T cells in other tissues (16). We performed 2 Mice were euthanized by CO2. Mice were perfused with PBS and 1 cm intravital two-photon microscopy on the flank skin of anesthetized skin regions were excised. Skin samples were placed into dispase (2.5 mg/ml mice to visualize HSV-specific gBT-I CD8+ T cells in the epi- in PBS) and incubated at 37˚C for 90 min, then placed into collagenase RM type III (3 mg/ml in RPMI 1640 plus 10% FCS and 5 mg/ml DNase I), dermis. We observed elongated cell bodies and persistent extension finely chopped and further incubated at 37˚C for 30 min. Samples were and retraction of dendritic protrusions from the TRM cells as they transferred into RPMI 1640 plus 10% FCS, and vigorously mixed. Cells navigated the epidermis (Fig. 1A). Many of the smaller protru- were successively filtered through 70- and 30-mm nylon meshes and sions appeared to resemble lamellipodia, suggesting that the cells resuspended in FACS buffer (PBS with 2% FCS, 5 mM EDTA, 0.1% probed their environment to move and make directional decisions. NaN3), prior to Ab staining. LN and/or spleens were harvested and pushed through a 70-mm sieve to obtain single-cell suspensions and resuspended To best understand the molecular mechanisms controlling the + in FACS buffer for Ab staining. Propidium iodide was added to samples behavior of skin CD8 TRM cells, in situ analysis of cell movement The Journal of Immunology 3

FIGURE 1. Cytoskeletal components required for TRM cell morphology and motility in the skin. (A) gBT-I.GFP CD8+ T cells (green) imaged by two-photon microscopy in the skin .30 d after HSV infection. SHG (blue) delineates the collagen- rich dermis. Scale bar, 5 mm. (B) In- travital images of gBT-I.GFP T cells in skin treated with the indicated compounds dissolved in acetone (vehicle). Two examples are shown for each condition, at different magniﬁ- cations. (C) Disruption of microtubules by treatment with colchicine results in pronounced elongation of skin TRM cells and fragmentation of some cells. Four examples from different regions of skin are shown. (D) Downloaded from Morphology of skin TRM cells after treatment with the indicated agents. Sphericity measurements for individual cells are plotted. Red lines are mean 6 SEM. Means: acetone, 0.62; cytochalasin D, 0.75; blebbistatin, 0.71; Y27632, 0.77; colchicine, 0.65. http://www.jimmunol.org/ (E) Mean velocity of TRM cells migrating within the epidermis after treatment with the indicated agents. Red lines are mean 6 SEM. Means (mm/min): acetone, 0.75; cytochalasin D, 0.52; blebbistatin, 0.63; Y27632, 0.57; colchicine, 0.84. Data are shown from two to three independent experiments. n = 5–6 mice per condition, one to two videos per mouse, 92–136 by guest on September 25, 2021 individual cells. *p , 0.05, ****p , 0.0001. ns, not signiﬁcant.

was required. To influence this behavior, we applied various dendrite formation (20). Treatment of skin with the ROCK in- compounds topically to the skin of mice. We developed a protocol hibitor Y27632 induced reduced formation of dendritic projec- to apply nonaqueous compounds dissolved in acetone topically to tions by the TRM cells and inhibited motility (Fig. 1). We also the shaved skin of mice. Skin TRM cells treated with vehicle alone treated mice with a Rac1 inhibitor, NSC1, to determine a role for (acetone) displayed no change in their morphology or motility branched actin formation and membrane protrusions; however, no (Fig. 1B) compared with untreated TRM cells (8). Because poly- difference was observed between treated and untreated mice (data merization of actin is crucial for cell migration and morphology, not shown). This could be due to compensation by Rac2, which is in particular to control cell shape and cytoplasmic projections also required for T cell migration (21). (17), we first examined the effect of disrupting the actin cyto- Next, we examined a role for microtubules for TRM cell mi- skeleton of skin TRM cells. Examination of the skin of the mice gration. Microtubules are important for organization of cytoskel- after treatment did not reveal obvious changes or disruption of the etal structures to stabilize cell uropods and lamellopodia, and epidermis or underlying SHG+ dermis. Compared to mice treated microtubule retraction may help cells to migrate through confined with vehicle alone, treatment with the actin depolymerization spaces (22, 23). Treatment of TRM cells in the skin with the mi- compound cytochalasin D induced marked rounding of the TRM crotubule destabilization agent colchicine resulted in a marked cells and a significant reduction in cell motility (Fig. 1B–E, change in the morphology and behavior of the cells (Fig. 1C–E, Supplemental Video 1). Because T cells also require myosin-II for Supplemental Video 1). We observed marked elongation of TRM motility, at least for amoeboid modes of migration (18), we treated cells, some extending dendrites for 30–40 mm, yet retaining a the skin of mice with the compound blebbistatin to inhibit myosin- rounded and sessile cell body. We also observed rounded cells and II function. TRM cells rounded up and reduced motility after substantial cell debris that was indicative of cell death. Although blebbistatin treatment, although some cells retained the ability to some cells appeared to migrate normally after treatment, we were extend some small dendritic projections, albeit with a reduced unable to confirm that the compound evenly penetrated across motility (Fig. 1B–E). We then examined a role for the Rho the skin. Notably, we saw similar effects on skin TRM cell mor- GTPase ROCK for TRM cell motility. ROCK has been shown to phology and migration after treatment with another microtubule- play an important role in T cell migration within constrained destabilizing agent nocodazole (Supplemental Fig. 1A). These environments by activating myosin-IIA–mediated actomyosin data show that skin TRM cells use actin-driven migration ma- contraction (19). In dendritic cells ROCK is also important for chinery, including the Rho GTPase ROCK and myosin-II, to adopt 4 SIGNALS FOR SKIN TRM CELL FORMATION AND MIGRATION a unique dendritic morphology and migrate within the epidermis. behavior of the cells was markedly altered, indicating that G protein– Notably, skin TRM cells also require an intact microtubule network coupled receptors are required for TRM cell morphology and motility. to migrate and maintain cellular integrity within the epidermis. Because PTx can inhibit chemokine receptor signaling, which may contribute to TRM cell behavior, we examined the roles of CD103 expression influences skin TRM cell migration but not chemokine receptors involved in CD8+ T cell responses and homing morphology to the skin. We recently demonstrated that expression of CXCR3 on T cells resident in the epidermis after infection or inflammation effector T cells was important for entry of TRM cell precursors into 2/2 express high levels of the integrin aE chain, CD103 (24), which the epidermis, and fewer TRM cells formed from CXCR3 Tcells + binds to the ligand E-cadherin. Although CD8 T cells that lack (5). We sought to determine whether the migration of skin TRM expression of CD103 can initially enter the epidermis and develop cells or their dendritic morphology required expression of CXCR3. into TRM cells, persistence of these cells over time is impaired (5). We transferred equal numbers of in vitro–activated WT and It was previously demonstrated that skin dendritic epidermal CXCR32/2 T cells into WT mice and then treated the flank skin gdT cells lacking CD103 had fewer dendrites in vivo and showed with the contact-sensitizing agent DNFB to recruit T cells into the + + slower motility in vitro (25). To investigate the role for CD103 skin where they formed populations of CD69 CD103 TRM cells + 2/2 expression by CD8 TRM cells, we cotransferred CD103 and (Supplemental Fig. 1B) (26). Intravital imaging revealed that TRM WT HSV-specific TCR transgenic CD8+ T cells from gBT-I mice cells lacking CXCR3 displayed equivalent cell morphologies and into WT recipients. Following infection with HSV, we imaged motility compared with WT cells (Fig. 3A, 3B, Supplemental 2/2 2/2 both WT and CD103 epidermal TRM cells. CD103 TRM cell Video 4), suggesting that CXCR3 signals are not required for morphology was equivalent to that of WT cells (Fig. 2A, Supple- TRM cell migration in the epidermis. Downloaded from mental Video 2). Tracking of the cells revealed greater motility by We next examined the role of CCR8, because we found that this 2/2 CD103 TRM cells compared with WT cells (Fig. 2B). These chemokine receptor was highly expressed specifically by skin TRM data suggest that CD103 expression may contribute to TRM cell cells (5). We examined whether the migration or morphology of adhesion in the epidermis and thereby influence local retention. skin TRM cells was altered in the absence of CCR8. However, we However, the unique cell morphology of skin TRM cells was not observed no difference in the morphology of the two populations,

influenced by expression of CD103. and both displayed a similar rate of motility in the skin (Fig. 3C, http://www.jimmunol.org/ 3D, Supplemental Video 4). We then cotransferred WT and Influence of chemokine receptors on TRM cell formation and CCR82/2 gBT-I CD8+ T cells into mice prior to skin HSV in- motility fection and examined T cell responses in the draining brachial LN We next sought to determine other signals capable of influencing (bLN), spleen, and skin. Equivalent numbers of WT and CCR82/2 + TRM cell formation and behavior in the skin. We treated the flank CD8 T cells were found in all tissues 10 d after skin HSV in- skin of mice with PTx dissolved in Sorbolene cream to inhibit Gai fection (Fig. 3E), indicating that expression of CCR8 was not protein–coupled receptors. In contrast to vehicle treatment, PTx required for T cell responses or migration to the skin. Likewise, + induced rounding of skin CD8 TRM cells and a substantial loss of numbers of skin TRM cells that formed after infection were dendritic projections (Fig. 2C, 2D, Supplemental Video 3). Some equivalent between populations of WT and CCR82/2 T cells. by guest on September 25, 2021 TRM cells retained the ability to extend cytoplasmic projections Therefore, despite increased expression of CCR8 by skin TRM and “wiggle” their cell bodies back and forth. However, the overall cells, we did not find a role for expression of this chemokine

FIGURE 2. (A and B) CD103 restrains cell motility but does not inﬂuence TRM cell morphology in the skin. (A) Representative image of WT (green) and 2/2 + 2/2 CD103 (red) gBT-I CD8 TRM cells after HSV infection. Mice were cotransferred with equal numbers of WT and CD103 gBT-I cells prior to skin 2/2 + HSV infection. (B) Sphericity measurements and average velocity of WT and CD103 CD8 TRM cells in the skin .30 d postinfection. n = 6 mice, one to two videos per mouse, 42–78 individual cells. Black lines are mean 6 SEM. Mean sphericity: WT, 0.52; CD1032/2, 0.56. Mean velocity (mm/min): WT, 2/2 + 1.07; CD103 , 1.41. (C and D) PTx-sensitive signals control TRM cell migration in the skin. (C) Representative images of gBT-I CD8 TRM cells treated + with PTx or vehicle (control). (D) Sphericity measurements and average velocity of gBT-I CD8 TRM cells in the skin after PTx or control treatment. n =6 mice, one to two videos per mouse, 89–92 individual cells. Red lines are mean 6 SEM. Mean sphericity: WT, 0.61; PTx, 0.75. Mean velocity (mm/min): WT, 1.12; PTx, 0.76. Data are shown from two independent experiments. ****p , 0.0001. ns, not signiﬁcant. The Journal of Immunology 5

FIGURE 3. (A and B) CXCR3 does not inﬂuence skin TRM cell morphology or motility. (A) Repre- sentative image of WT (red) and 2/2 + CXCR3 (green) OT-I CD8 TRM cells .30 d after DNFB treatment. (B) Sphericity measurements and average velocity of WT and CXCR32/2

TRM cells in the skin. n = 7 mice, one to two videos per mouse, 76–163 individual cells. Black lines are mean 6 SEM. Mean sphericity: WT, 0.72; CXCR32/2, 0.73. Mean velocity (mm/min): WT, 1.52; CXCR32/2, 1.45. (C–E) CCR8 expression is not Downloaded from required for skin TRM cell formation or migration. (C) Representative image of WT (red) and CCR82/2

(green) gBT-I TRM cells 60 d after HSV infection. (D) Sphericity measurements and average velocity of 2/2 WT and CCR8 TRM cells in the http://www.jimmunol.org/ skin. n 5 7 mice, one video per mouse, 54–115 individual cells. Black lines are mean 6 SEM. Mean sphericity: WT, 0.68; CCR82/2, 0.70. Mean velocity (mm/min): WT, 1.07; CCR82/2, 1.00. (E) Mice were cotransferred with equal numbers of WT and CCR82/2 gBT-I cells prior to skin HSV infection. Cell numbers were enumerated in the spleen and by guest on September 25, 2021 skin 10 and 30 d postinfection. n 5 8–10 mice per time point. ns, not signiﬁcant.

receptor for formation, persistence, or migration of these memory CD8+ T cells into mice prior to DNFB treatment and examined T cells. T cell responses. Equivalent numbers of WT and KO CD8+ T cells were found in the spleen (Fig. 4C). However, numbers of CXCR62/2 CXCR6 is required for skin CD8+ T cell formation RM T cells were reduced in the skin by day 10 and significantly fewer + + T cells resident in several tissues, including the skin, express CD69 CD103 TRM cells formed in the skin. Intradermal injection the chemokine receptor CXCR6 (4). The ligand for CXCR6, of in vitro–activated CXCR62/2 CD8+ T cells to distinguish po- CXCL16, can be expressed by keratinocytes as well as dendritic tential defects in recruitment versus retention of these cells in the cells in various tissues. TRM cells in the liver express high levels of skin revealed decreased numbers of TRM cells compared with WT CXCR6, and expression of this receptor contributes to mainte- T cells (Fig. 4D). The CXCR62/2 and WT T cells were present in nance of memory cells in this site (27, 28). We examined a role for similar numbers in the spleen, indicating that poor survival of the GFP/GFP this chemokine receptor using homozygous CXCR6 CXCR62/2 T cells may have contributed to the reduced retention 2/2 knock-in mice (CXCR6 ), comparing T cells from these mice and formation of TRM populations in the skin. These data identify with WT cells. TRM cells generated following DNFB treatment an important role for the chemokine receptor CXCR6 for the were imaged in the skin, revealing that cells lacking CXCR6 formation of TRM cells in the skin. showed more rounded cell morphologies, whereas migration in the + epidermis was unaffected (Fig. 4A, 4B, Supplemental Video 4). A role for CCR10 for memory CD8 T cell programming after We also observed a marked reduction in numbers of CXCR62/2 skin infection 2 TRM cells in the epidermis (WT, 42.3 6 +7.5 cells/mm ; knockout We next examined a role for CCR10 in TRM cell formation and [KO], 6.8 6 1.6 cells/mm2 skin from six mice). migration in the skin. CCR10 is a chemokine receptor implicated To examine the influence of CXCR6 expression on memory in the homing of T cells to the skin (29), and CD8+ T cells have T cell formation, we cotransferred activated WT and CXCR62/2 been shown to be reduced in the skin of CCR10-deficient mice 6 SIGNALS FOR SKIN TRM CELL FORMATION AND MIGRATION

FIGURE 4. CXCR6 is required for skin TRM cell formation. (A and B) Inﬂuence of CXCR6 on TRM cell formation and morphology. (A) Rep- resentative image of WT (red) and 2/2 + CXCR6 (green) CD8 TRM cells .30 d after DNFB treatment. Low 2/2 numbers of CXCR6 TRM cells were observed in most regions of skin. (B) Sphericity measurements and average velocity of WT and 2/2 CXCR6 TRM cells in the skin. n =6 mice, one to two videos per mouse, 60–83 individual cells. Black lines are mean 6 SEM. Mean sphericity: WT, 0.72; CXCR62/2, 0.75. Mean Downloaded from velocity (mm/min): WT, 1.42; CXCR62/ 2,1.34.(C) Mice were cotransferred with equal numbers of WT and CXCR62/2 gBT-I cells. Cell numbers were enumerated in the spleen and skin 10 and 30 d after transfer and DNFB treatment. n =10mice http://www.jimmunol.org/ per time point. (D) Numbers of CXCR32/2 or WT CD8+ T cells in the spleen or skin (CD69+CD103+) from recipient mice given intradermal cotransfer of in vitro–activated cells, assessed at various times (horizontal axes) after transfer. n = 9micepertimepoint.Dataare shown from two independent ex- by guest on September 25, 2021 periments. **p , 0.005, ***p , 0.001, ****p , 0.0001. ns, not signiﬁcant.

(30). The ligand for CCR10, CCL27, is highly expressed in the Notably, by day 30, numbers of CCR102/2 memory gBT-I T cells epidermis (29). To examine CCR10 in the context of skin infec- were significantly reduced in the spleen and skin, and this was 2/2 tion, WT and CCR10 gBT-I T cells were cotransferred into most pronounced among TRM cells in the skin, which contained CCR10+/2 mice prior to HSV infection and 30 d later, the memory ∼12-fold more CCR10+/2 gBT-I T cells. This was not due to a T cell populations that formed in the skin were imaged by intra- delay in the activation or expansion of the CCR102/2 T cells, as vital two-photon microscopy. The morphology and motility of proliferation was similar between WT and KO CD8+ T cells in the 2/2 + skin CCR10 and WT TRM cells was indistinguishable (Fig. 5A, draining bLN 3 d postinfection (Fig. 5E). Importantly, CD8 5B, Supplemental Video 4). These results indicated that expres- T cells responding to skin infection upregulated CCR10 expression of CCR10 by skin TRM cells was not required for migration. sion by the first division, with expression among virus-specific 2/2 However, we observed that numbers of CCR10 TRM cells were T cells highest at the peak of the response (day 7) (Fig. 5F, 5G). slightly reduced (WT, 71 6 7.5 cells/mm2;KO,24.76 3.8 cells/mm2 CCR10 expression was downregulated on most memory T cells in skin from seven mice). lymphoid organs and skin .30 d postinfection, indicating that To directly determine whether CCR10 contributed to memory CCR10 expression by CD8+ T cells controls effector and memory T cell formation in the skin after HSV infection, CCR10+/2 and cell responses during skin virus infection, possibly by promoting CCR102/2 gBT-I CD8+ T cells were cotransferred in equal num- their migration and survival. bers into CCR10+/2 mice and T cell responses were examined in the draining bLN, spleen, and skin. Ten days after infection, Discussion 2/2 +/2 numbers of CCR10 and CCR10 gBT-I T cells were not sig- TRM cells that form in the skin after infection play an important nificantly different in the spleen and skin, although based on mean role in rapid protection from reinfection (2). Integral to this role, +/2 values 2- to 3-fold more CCR10 gBT-I T cells were present in TRM cells must accomplish effective immunosurveillance of their these tissues compared with CCR10-deficient cells (Fig. 5C, 5D). tissue microenvironment in the search for invading pathogens. The The Journal of Immunology 7

FIGURE 5. CCR10 is required for memory T cell responses to skin HSV infection. (A) Representative image of WT (red) and CCR102/2 + (green) gBT-I CD8 TRM cells 60 d after HSV infection. (B) Sphericity measurements and average velocity 2/2 of WT and CCR10 TRM cells in the skin. n = 7 mice, one to two videos per mouse, 67–110 individual cells. Black lines are mean 6 SEM. Mean sphericity: WT, 0.68; CCR102/2,

0.70. Mean velocity (mm/min): WT, Downloaded from 0.86; CCR102/2,0.96.(C)Mice were cotransferred with equal numbers of CCR10+/2 and CCR102/2 gBT-I cells prior to skin HSV infection. Cell numbers were enumerated in the spleen and skin 10 and 30 d postinfection. (D) Ratios of CCR10+/ http://www.jimmunol.org/ 2 to CCR102/2 gBT-I in individual mice postinfection. n = 8–10 mice per time point. (E) Number of divided (CTV-low) gBT-I CCR10+/2 or CCR102/2 T cells in bLN 3 d after HSV infection. (F)Regulationof CCR10 (EGFP) on gBT-I CD8+ T cells in bLN, spleen, and skin at

different times after HSV infection. by guest on September 25, 2021 (G) Graph of CCR10 expression on gBT-I T cells after infection. n =5– 15 mice per time point. Data are shown from two to three independent experiments. *p , 0.05, **p , 0.005. ns, not signiﬁcant.

epidermis presents a highly constrained environment for the treatment on surrounding keratinocytes, although we predict this T cells to navigate, and we have shown that TRM cells adopt a would be secondary to the T cell–intrinsic roles of the cytoskeletal striking morphology that is accompanied by a slow mode of mi- components. Similar to T cells migrating in less constrained tis- gration (8). Interestingly, epidermal gdT cells and Langerhans sues such as LN, myosin-II was required for TRM cell migration in cells also adopt dendritic cell morphologies (31), although both of the epidermis. Myosin-II might therefore help TRM cells to navi- these cell subsets extend thinner and more deﬁned dendrites from gate the epidermis by preventing prolonged adhesion to the en- largely immotile cell bodies. In contrast, epidermal TRM cells vironment, much like it does in LN (18). Thus, T cell migration remain motile and extend and retract many cellular protrusions as within the conﬁned epidermis requires the actomyosin cyto- they move. skeleton to facilitate the probing dendritic morphology that is To facilitate dissection of the molecular mechanisms of TRM cell required for navigation in this constrained microenvironment. morphology, we treated the skin of mice with selected compounds Our experiments demonstrate a key requirement for microtu- to determine the effects on T cell behavior in situ. The actin bules for TRM cell migration in the skin. Memory T cells treated cytoskeleton was integral to skin TRM cell migration and for the with a microtubule-destabilizing drug continued to stretch and dynamic cell morphology that TRM cells presumably use to probe project dendrites, but were observed to often overextend and to their environment and generate adhesions to help propel the cells disintegrate or lose pieces of cytoplasm. These stretched TRM cells forward. We do not rule out potential effects of topical inhibitor maintained a sessile cell body, suggesting that microtubules were 8 SIGNALS FOR SKIN TRM CELL FORMATION AND MIGRATION

required to propel the cells forward. We recently found that the and support from microtubules. Skin TRM cells also required signals guanine nucleotide exchange factor DOCK8, through CDC42 and from PTx-sensitive receptors for migration and cellular mor- p21-activated kinase, is required to coordinate cytoskeletal phology. Although the integrin CD103 and chemokine receptors structures (32). T cells lacking DOCK8 stretched and died CCR8, CXCR3, CXCR6, and CCR10 were not critical for epi- (cytothripsis) within constrained environments and were unable to dermal TRM cell migration, formation of skin memory T cells was + form TRM cell populations in the skin. This disturbed cytoskeletal impaired when CD8 T cells lacked expression of CXCR6 or behavior was accompanied by abnormal positioning of microtu- CCR10. Further work will be needed to define the roles of these bules. We also show in the present study that TRM cells required chemokine receptors in T cell migration, survival, and immune ROCK for migration in the epidermis. It has been shown that responses in peripheral tissues. ROCK can influence actomyosin contractility as well as microtubule stability (22). Microtubules have also been proposed to Acknowledgments contribute to the deformability of T cells in confined spaces (23). We thank C. Jones, G. Davey, and M. Damtsis for technical assistance. Together with our data in the present study, this suggests that microtubules are critical for the proper coordination of T cell Disclosures migration in tight cellular environments such as the epidermis. The authors have no financial conflicts of interest. Whether T cells also require microtubules for migration in other tissues is less clear, although we would predict that this would be highly dependent on the tissue microenvironment. References

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CD103

CXCR6 -/- 20um WT CXCR6 9 6 D C

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CCR10 WT CCR10-/- 2 α V

CD103

Supplemental Figure 1 (A) Disruption of microtubules by treatment with nocodozole results in elongation and + fragmentation of skin TRM cells. Intravital 2-photon image of gBT-I.GFP CD8 T cells (green) in skin. SHG (blue) delineates the collagen-rich dermis. (B) Normal expression

of the canonical TRM markers CD69 and CD103 on wild-type (WT) and the indicated chemokine receptor knockout CD8+ T cells in skin, analysed 21-30 days after transfer, Video legends Video 1

Cytoskeletal components required for TRM cell morphology and motility in the skin. Mice + containing skin gBT-I.EGFP CD8 TRM cells were treated with vehicle (acetone) or the compounds cytochalasin D (inhibitor of actin polymerization), blebbistatin (inhibitor of myosin II), Y-27632

(ROCK inhibitor) or colchicine (inhibitor of microtubule polymerization). Epidermal TRM cells were imaged in the skin by intravital 2-photon microscopy. A maximum intensity projection time series of gBT-I.GFP T cells (green) is shown. The collagen-rich dermis was visualized by second harmonic generation (SHG; blue). Frames were acquired at 1 min intervals. Display rate: 10 frames per second (fps).

Video 2

-/- CD103 does not influence TRM morphology or motility in the skin. WT (green) and CD103 (red) gBT-I CD8 TRM cells in the skin 32 days after HSV infection. SHG, blue. Frames were acquired at 1 min intervals. Display rate: 10 fps.

Video 3

Skin TRM migration and morphology is pertussis toxin sensitive. Mice containing skin gBT- + I.EGFP CD8 TRM cells (green) were treated with vehicle (sorbolene cream) or pertussis toxin

(inhibitor of G protein-coupled receptor signaling). Epidermis TRM cells were imaged in the skin by intravital 2-photon microscopy. SHG, blue. Frames were acquired at 1 min intervals. Display rate: 10 fps.

Video 4

Role of the chemokine receptors CXCR3, CCR8, CXCR6 and CCR10 in skin TRM cell morphology + and motility. Mice containing WT (red) or chemokine receptor deficient CD8 TRM cells (green) were imaged in the skin by intravital 2-photon microscopy. SHG, blue. Frames were acquired at 1 min intervals. Display rate: 10 fps.