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of February 14, 2018. This information is current as Infection Response to Cutaneous Vaccinia Virus T Cell δ γ Dermal-Resident versus Recruited Delaney and Michael J. Bevan Amanda S. Woodward Davis, Tessa Bergsbaken, Martha A. doi: 10.4049/jimmunol.1402438 January 2015; 2015; 194:2260-2267; Prepublished online 21 J Immunol Material Supplementary 8.DCSupplemental average * 4 weeks from acceptance to publication Speedy Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* ? The JI Why References , 20 of which you can access for free at: cites 55 articles This article Subscription is online at: The Journal of Immunology Information about subscribing to Permissions Submit copyright permission requests at: Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright © 2015 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on February 14, 2018 Downloaded from by guest on February 14, 2018 Downloaded from

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    InfectionResponse to Cutaneous Vaccinia Virus

    T CellDermal-Resident versus Recruited

    Delaney and Michael J. BevanAmanda S. Woodward Davis, Tessa Bergsbaken, Martha A. 10.4049/jimmunol.1402438January 2015;

    2015; 194:2260-2267; Prepublished online 21J Immunol




    4 weeks from acceptance to publicationSpeedy Publication!

    Every submission reviewed by practicing scientistsNo Triage!

    from submission to initial decisionRapid Reviews! 30 days*

    ?The JIWhy


    , 20 of which you can access for free at: cites 55 articlesThis article


    is online at: The Journal of ImmunologyInformation about subscribing to

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    Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright 2015 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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  • The Journal of Immunology

    Dermal-Resident versus Recruited gd T Cell Response toCutaneous Vaccinia Virus Infection

    Amanda S. Woodward Davis,* Tessa Bergsbaken,*, Martha A. Delaney, and

    Michael J. Bevan*,

    The study of T cell immunity at barrier surfaces has largely focused on T cells bearing the ab TCR. However, T cells that express

    the gd TCR are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an

    important role responding to external stimuli. In this article, we report that, in a murine model of cutaneous infection with

    vaccinia virus, dermal gd T cell numbers increased 10-fold in the infected ear and resulted in a novel gd T cell population not

    found in naive skin. Circulating gd T cells were specifically recruited to the site of inflammation and differentially contributed to

    dermal populations based on their CD27 expression. Recruited gd T cells, the majority of which were CD27+, were granzyme B+

    and made up about half of the dermal population at the peak of the response. In contrast, recruited and resident gd T cell

    populations that made IL-17 were CD272. Using a double-chimera model that can discriminate between the resident dermal and

    recruited gd T cell populations, we demonstrated their divergent functions and contributions to early stages of tissue inflamma-

    tion. Specifically, the loss of the perinatal thymus-derived resident dermal population resulted in decreased cellularity and

    collateral damage in the tissue during viral infection. These findings have important implications for our understanding of

    immune coordination at barrier surfaces and the contribution of innate-like lymphocytes on the front lines of immune

    defense. The Journal of Immunology, 2015, 194: 22602267.

    The skin is host to a network of T lymphocytes, some ofwhich take up permanent residence within the tissue,having been recruited there throughout life, whereas others

    are only transiently present (1, 2). Following Ag-specific activationin central lymphoid organs, TCRab effector cells traffic to pe-ripheral sites, including the skin, and some become residentmemory T cells. These cells are recruited into the tissue as a resultof local infection or inflammatory signals and remain there toprotect against future external insults (38). In contrast to TCRabresident memory T cells, TCRgd T cells were shown to home totissues and acquire specific functions as a result of developmentalprogramming, and their response via TCR recognition of foreignAgs is poorly defined (911). This hardwiring, which drives themparticularly to barrier tissues, such as the intestine, reproductivetract, and skin, seems ideally suited to ensure that gd T cellsparticipate in innate host defense.Within the murine skin, dendritic epidermal T cells (DETC) are

    resident in the epidermis. They seed the skin during fetal devel-opment, express an invariant Vg5 TCR, have dendritic morphol-ogy, and are relatively immobile (12). They have been shown toparticipate in keratinocyte maintenance, to respond to wounding,

    and to assist in the healing process (1315). A different subset ofTCRgd T cells is resident in the dermis. Dermal gd T cells ac-

    count for up to half of the dermal T cells in mice (16) and 29% in

    humans (17). This population was shown to participate in host

    defense against bacterial infection of the skin (16, 18), as well as

    to contribute to psoriasis in the mouse (19, 20) and humans (21). It

    is now appreciated that, in mice, dermal gd T cells exit the thymus

    and seed the dermis near the time of birth (11, 22). Character-

    ization of dermal gd T cells by several groups revealed their

    memory phenotype and functions related to those of CD4+ Th17

    cells, being capable of producing IL-17 upon stimulation in vitro.

    Dermal gd T cells express CCR6, CXCR6, CD103, CD44, IL-

    23R, and IL-7R (2123); however, it is unclear whether IL-17

    producing dermal gd T cell populations observed during infection

    or inflammation are derived from the resident population having

    been seeded there at birth or whether unique populations are

    recruited from the circulation.A recent study identified a population of gd T cells that is

    enriched in skin draining lymph nodes (dLN) of mice with

    properties similar to those of dermal gd T cells (22). This dis-

    covery demonstrated a potential link between dermal gd T cells

    and those found in the circulation. Although several models of

    systemic viral infection (24, 25) elicited a gd T cell response in

    the spleen and lymph nodes (LN), these studies did not address

    questions concerning tissue trafficking or provide a relevant con-

    text for natural modes of infection. Importantly, studies with hu-

    man PBMC found that gd T cells in the circulation are impacted in

    patients with inflammatory bowel disease (26), melanoma (27), or

    psoriasis (28), suggesting that local inflammation can affect global

    gd T cell homeostasis. These findings point to the need to un-

    derstand the trafficking patterns of gd T cells during steady-state

    conditions and in the context of disease.Emerging data from studies of cells in central lymphoid organs

    suggest that CD27 delineates gd T cell functional subsets, leading

    to a paradigm shift for how gd T cells are classified (11, 29).

    *Department of Immunology, University of Washington, Seattle, WA 98109;Howard Hughes Medical Institute, University of Washington, Seattle, WA 98109;and Department of Comparative Medicine, University of Washington, Seattle, WA98109

    Received for publication September 24, 2014. Accepted for publication December21, 2014.

    Address correspondence and reprint requests to Dr. Michael J. Bevan, Department ofImmunology, University of Washington, Box 358059, 750 Republican Street, Seattle,WA 98109. E-mail address: [email protected]

    The online version of this article contains supplemental material.

    Abbreviations used in this article: BM, bone marrow; DETC, dendritic epidermalT cell; dLN, draining lymph node; DN, double negative; dpi, d postinfection LN,lymph node; pi, postinfection; pThy, perinatal thymocyte; VV, vaccinia virus, WT,wild-type.

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  • Expression of CD27 by mouse gd T cells is tightly correlated withtheir ability to produce IFN-g, where failure to express CD27 cor-relates with IL-17 production (30). Importantly, analogous gd T cellpopulations were found in human PBMC (3133), making this arelevant axis from which to orient experiments in model organisms.In the current study, we analyzed the gd T cell response in

    mouse skin to local infection with vaccinia virus (VV). We soughtto dissect the contribution of resident versus immigrant gd T cellsfollowing infection and to determine whether gd T cells recruitedfrom the circulation give rise to long-term residents in the skinafter the cutaneous infection has cleared. Many previous reportsstudied the gd T cell response in TCRa2/2 or TCRb2/2 mice,which lack TCRab T cells (24, 25), or performed transfers of gdT cells isolated from TCRb2/2 mice (34) to investigate the an-tiviral response. In such scenarios, the gd T cell makeup and re-sponse are likely abnormal (35). Analyzing gd T cells in a wild-type (WT) setting allowed us to better appreciate their role in askin infection. Our experiments revealed that CD27+ and CD272

    gd T cells are specifically recruited from the circulation to the siteof infection; surprisingly, however, they do not become resident inthe tissue, even when encountering a relatively empty niche. Theimmigrant gd T cells have functional attributes distinct from theresident dermal gd T cells, with the latter being primarily re-sponsible for enhancing the inflammatory response in the tissue.

    Materials and MethodsMice

    C57BL/6, B6.SJL-Ptprca Pepcb/BoyJ, and TCRd2/2 mice were obtainedfrom the Jackson Laboratory, and TCRd-H2BeGFP (TCRd-GFP) micewere obtained from I. Prinz (Hannover Medical School, Hannover, Ger-many); all were described previously (36). Mice were housed in specificpathogenfree conditions in the animal facilities at the University ofWashington and, unless otherwise specified, were used at 610 wk of age.All experiments were done in accordance with the Institutional AnimalCare and Use Committee guidelines of the University of Washington.


    A total of 2 3 106 PFU recombinant VV expressing full-length chickenOVA was used for epicutaneous infection by skin scarification, as de-scribed previously (37). Mice were anesthetized with isoflurane, and 5 mldiluted virus was applied to the dorsal side of the ear. The skin area wasgently scratched 20 times with a 28-gauge needle. For flow cytometryexperiments, both ears of mice were infected and combined to make upone sample, and the numbers were divided by two to obtain the number ofcells/ear.

    Adoptive transfer and sorting

    C57BL/6 or CD45.2+ TCRd-GFP mice received 7 3 105 gd T cellsenriched from spleen and skin dLN of CD45.1+ TCRd-GFP mice by i.v.injection 1 d before infection. Enrichment of gd T cells was performed byincubating single-cell suspensions with biotin-labeled anti-TCRb and anti-CD19 mAb for 30 min on ice, followed by passage through the Stem CellBiotin negative selection kit. Mice were infected on one ear, and theinfected (or uninfected) ears from two mice were combined for eachsample. In some experiments, the enriched population was further purifiedas follows: cells were stained with fluorochrome-conjugated anti-CD27and sorted on a FACSAria for the GFP+TCRb2CD192CD27+ or GFP+

    TCRb2CD192CD272 population, of which .97% were TCRgd+. A totalof 6 3 105 CD27+ or 2.7 3 105 CD272 sorted gd T cells from B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice was transferred into separate C57BL/6(CD45.2) hosts. Mice that received sorted cells were infected on both ears,and ears from a single mouse were combined for a sample.

    Flow cytometry

    Single-cell suspensions were prepared from spleens and LN by mechanicaldisruption. Dorsal and ventral halves of ears were separated, minced, andenzymatically digested for 2 h at 37C in Liberase TM (Roche). Both earsfrom each mouse were combined for processing to obtain adequate cellnumbers. All tissues were passaged through a 100-mm nylon sieve (BDBioscience). In some experiments, to avoid the Liberase digestion (that

    cleaved CD27), dorsal and ventral halves of the ears were separated andfloated dermal-side down in complete media overnight at 37C. Superna-tant, into which cells had migrated, was collected the following day andstained. Cells were stained for 30 min on ice with the appropriate mix-ture of mAb and washed with PBS containing 1% BSA. The followingconjugated mAb were obtained from BD Pharmingen, eBioscience, orBioLegend: anti-TCRb (H57-597), CCR6 (140706), CD27 (LG.3A10),CD103 (2E7), Vg5 (BioLegend Vg3 clone 536), CD44 (IM7), CD45.1(A20), and CD45.2 (104). In the ear, gd T cells from TCRd-GFP wereidentified as GFP+Vg52 (dermal) or GFP+Vg5+ (DETC), according to thenomenclature described by Heilig and Tonegawa (38). For the analysis ofintracellular IFN-g, IL-17, and granzyme B, single-cell suspensions wereincubated in the presence or absence of 50 ng/ml PMA and 500 ng/mlionomycin for 4 h at 37C in the presence of brefeldin A. Cells werestained for surface markers and then processed using the BD PharmingenCytofix/Cytoperm kit. Samples were analyzed on a FACSCanto II (BD)using FlowJo software (Tree Star).

    In situ proliferation

    Infected mice were injected i.p. with 2 mg BrdU in 200 ml PBS1 h beforebeing euthanized. Single-cell suspensions were obtained from each tissueand stained using the BD Biosciences APC BrdU Kit. Cells were fixedwith paraformaldehyde before Cytofix/Cytoperm to ensure retention of theTCRd-GFP signal.

    Bone marrow chimeras

    Chimeras were made as previously described (22). Briefly, perinatal thy-mocytes (pThy) were harvested 048 h after birth, and 5 3 106 cells weretransferred i.v. to congenic recipients that had been lethally irradiated with1000 rad in a cesium irradiator. The next day, 12 3 106 congenic bonemarrow (BM) cells were transferred. These mice are referred to as BMpThychimeras, whereas controls without pThy are referred to as BM chimeras.Chimeras were analyzed or infected $8 wk after reconstitution.

    Virus titration

    The viral load in organs was determined by plaque assays on 143B cells,with dilutions of a 40% tissue homogenate added to confluent wells. Titersreported are log10 PFU per ear.


    Ears from infected mice were fixed in 10% buffered formalin for 3 d beforebeing embedded vertically in paraffin. Sections were stained with H&E, andthe severity of inflammation and necrosis was determined on a scale of 04(with 4 being the most severe). Scoring of all samples was blinded, anduninfected mice were used as controls.

    Statistical analysis

    Prism software (GraphPad) was used for all statistical analysis. The two-tailed, unpaired Student t test was used for comparisons of gd T cellfrequencies and histological scores.

    Resultsgd T cells accumulate at the site of infection with theemergence of a novel dermal population

    Although it is known that gd T cells reside at barrier surfaces inmice and humans (16, 17), few studies have addressed the role thatthey play during infection of the skin, particularly in response toviral infection. To address this, we used a model of VV scarifi-cation, in which virus localizes primarily to the site of inoculation(39). Following infection of the ear pinna, we observed that thenumber of dermal gd T cells increased 10-fold in the infected earand dLN (Fig. 1A, 1B). The response kinetics were slightly un-usual, because the numbers in the skin and dLN remained elevatedfor a protracted period of time. Furthermore, the number of dermalgd T cells at late time points was significantly higher than in naiveears, suggesting that infection could result in prolonged alterationsin the dynamics of the dermal gd T cell population. The number ofDETC also increased 3-fold following infection and returned tothe number found in control ears at late time points (Fig. 1A).Dermal gd T cells were reported to express CCR6 and CD103

    (22). We confirmed that, in resting skin, all dermal gd T cells are

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  • CD103+, and more than half are CCR6+ (Fig. 1C, 1D). Skinscarification with VV resulted in dramatic changes in the dermalgd T cell population. By 3 d postinfection (dpi), a novel subset

    appeared in the dermis that was CCR6 and CD103 double nega-tive (DN). This novel dermal gd T cell subset accounted for al-most half of the gd T cell population at the peak of the responseand then disappeared entirely from the dermis by 30 dpi (Fig. 1C,1D). We also assessed TCR Vg-chain expression using commer-cially available Ab on the three dermal populations and found thatall were similarly heterogeneous (data not shown).

    Circulating gd T cells are rapidly recruited to the VV-infectedear

    We performed an adoptive transfer to investigate the origin of thedermal gd T cell populations following infection and to determinethe contribution of circulating gd T cells to the expanded dermalpopulation. Accordingly, 7 3 105 gd T cells isolated from thespleen and skin dLN of naive TCRd-GFP mice were transferred i.v.into normal C57BL/6 recipients before VV scarification. Infectionof the recipients showed that donor gd T cells were specificallyrecruited to the skin of the infected ear, and their numbers peaked7 dpi (Fig. 2A). Although these adoptively transferred, circulatingcells were capable of entering the dermis during active infection,very few remained permanently integrated into the dermis at a latetime point.In the peripheral lymphoid organs and the circulation, it was

    established that gd T cells can be functionally segregated basedon CD27 expression (11, 29). We found that CD27 and CCR6staining were mutually exclusive in peripheral lymphoid organsand identified three gd T cell populations with distinct surfaceand transcription factor staining patterns (Fig. 2B, SupplementalFig. 1A). In line with previous reports that CD272 gd T cells pro-duce IL-17 upon stimulation, we found that baseline expressionof the transcription factor RORgt was higher in CD272 than inCD27+ gd T cells. Conversely, the transcription factor Tbet washighest in the CD27+ gd T cells. CCR6+CD272 gd T cells ex-pressed the highest levels of CD44 and CD103, whereas the smallnumber of CD272CCR62 cells and the CD27+ cell subset hadlower levels of CD44, and only half expressed CD103.Transfer experiments revealed that the donor gd T cells con-

    tributed to all three dermal gd T cell populations defined byCD103 and CCR6 staining (Fig. 2C), but it was not clear howthese populations related to those in the circulation. Our initialattempts to stain for CD27 on gd T cells isolated from the ear werenegative. Further analysis revealed that CD27 was cleaved duringthe digestion process used to isolate cells from the ear. Floatingears (dorsal and ventral sides separated) overnight in completemedia without enzymatic digestion released some gd T cells fromthe tissue and permitted analysis of CD27 expression. This tech-nique revealed that, although CD27 was not found on dermal gdT cells in naive animals, it was expressed on some dermal gdT cells following infection, all of which belonged to the CD1032

    CCR62 DN population (Supplemental Fig. 1B). This suggestedthat the CD1032CCR62 DN subset appearing in infected ears wasan immigrant population of circulating CD27+ gd T cells. Becauseyields and subset composition from the floating technique werevariable, we opted to use CD103 and CCR6 for further analyses ofskin samples.To determine whether CD27+ or CD272 gd T cells from the

    circulating pool differentially contributed to the three dermal gdT cell subsets, we sorted and transferred them into separate hostsbefore ear scarification with VV. The CD272 donor gd T cellscontributed to all three dermal populations defined by CD103and CCR6, although few were DN, and most expressed CD103(Fig. 2D). In contrast, immigrant gd T cells from the CD27+ do-nor population were exclusively CD1032CCR62 DN, eventhough 50% of cells in the donor population expressed CD103

    FIGURE 1. Following skin scarification with VV, gd T cells accumulate

    in dLN and dermis. TCRd-GFP mice were infected with VVon the ear by

    scarification. gd T cells were gated as TCRb2GFP+ in the LN and as

    TCRb2GFP+Vg5+ (DETC) or TCRb2GFP+Vg52 (dermal gd T cells) in

    the ear. (A) Number of DETC and dermal gd T cells in the ear following

    infection. Open circle represents the number of dermal gd T cells in the

    contralateral uninfected ear at 7 dpi. (B) Number of gd T cells in the dLN

    following infection. Open circle represents the number of gd T cells in

    the contralateral non-dLN (ndLN) at 7 dpi. (C) Representative flow plots

    show CCR6 and CD103 profile of dermal gd T cells following infection.

    Numbers denote the percentage of the total dermal gd T cell population

    within the indicated quadrant. (D) Cell numbers within the indicated

    dermal gd T cell subsets/ear over the course of infection (inset, day 0).

    Error bars signify SEM. Data are compiled from .5 (A and B) or .3 (Cand D) independent experiments (n = 313 mice/group). *p , 0.001, two-tailed, unpaired t test.


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  • (Supplemental Fig. 1A). On a per-cell basis, the CD272 gd T cellswere recruited more efficiently to the infected skin (data notshown). These experiments additionally demonstrate that in vivoin the presence of inflammatory signals, CD27 expression remainsstable in the LN and spleen as a marker of distinct gd T celllineages. Based on these experiments and the fact that we ob-served very limited proliferation within the tissue (SupplementalFig. 2), it appears that the majority of gd T cell accumulation isdue to recruitment.

    Adult BM- and perinatal thymusderived gd T cells occupydiscrete niches

    To tease apart the distinct roles of the resident dermal and cir-culating gd T cell populations, we generated mice with andwithout resident dermal gd T cells (22). We found that gd T cellsin the circulation and dermis were sensitive to whole-body irra-diation and that donor BM failed to reconstitute the dermal(Fig. 3A) or the circulating CCR6+ gd T cell populations(Supplemental Fig. 3). The majority of the gd T cells in the spleenand LN derived from the BM were CD27+ with a minority ofCD272CCR62 gd T cells. Although small numbers of host gdT cells remained in the dermis following irradiation (Fig. 3B),their phenotype was abnormal (data not shown), and their numberswere significantly lower than in unirradiated T cell populations lost as a result of irradiation were replaced

    by transferring pThy into irradiated hosts along with congenicallymarked BM cells to generate mice referred to in this article asBMpThy chimeras. This resulted in the restoration of both dermalgd T cells (Fig. 3A, 3B) and the circulating CCR6+ population bypThy (Fig. 3C). Total gd T cell numbers in the dermis returned tonormal levels, and the phenotype of the pThy-derived dermal gdT cells resembled that of unirradiated mice. Although pThy-derivedgd T cells contributed to all three circulating populations definedby CD27 and CCR6 staining, CD27+ cells were a relatively minorpopulation, allowing us to generally discriminate between the res-ident dermal and recruited gd T cell populations.Using BMpThy chimeras, we confirmed that, following VV

    infection, adult BM-derived gd T cells (largely CD27+ in the

    circulation) were transiently recruited from the circulation andcontributed primarily to the CCR62CD1032 DN dermal subset(Fig. 3D). In contrast, pThy-derived gd T cells contributed mainlyto the two CD103+ subsets in the dermis and their expansionduring infection. As early as day 3 following infection, pThy-derived gd T cells had expanded in the dermis, and their num-bers remained elevated for a protracted period of time. In contrast,the number of BM-derived gd T cells peaked at 7 dpi and thendeclined dramatically. Importantly, at a late time point the BM-derived gd T cells were absent from the ear, whereas the pThy-derived gd T cells plateaued at a level similar to WT mice fol-lowing infection.

    Adult BM- and pThy-derived gd T cells are functionallydistinct

    Several groups have established that dermal gd T cells are fated toproduce IL-17 (16, 21, 40). We found that a fraction of dermal gdT cells in uninfected ears produced this cytokine following in vitrostimulation with PMA/ionomycin. On day 3 after VV scarifica-tion, CD103+ gd T cells in the dermis were capable of producingIL-17 at a level similar to that of naive tissue (Fig. 4A). Althoughat any given time point the majority of IL-17 production was byCD103+ gd T cells (Supplemental Fig. 4A), it was surprising thatCD1032 gd T cells also produced IL-17 in the dermis. Using thefloat method to preserve CD27 expression, we found that all of theIL-17 production was by CD272 gd T cells (Supplemental Fig.4B), which we predict are derived from the circulating CCR62

    CD272 population (Fig. 2C). Notably, there were no granzyme B+

    gd T cells in the naive dermis at day 3 pi, but by day 7 (which isthe peak for the immigrant population), the CD1032 gd T cellsproduced granzyme B. None of the gd T cells in the dermisstained positive for IFN-g at any time following VV scarification(Supplemental Fig. 4C).We repeated these intracellular staining experiments with

    BMpThy chimeras to determine whether the BM- and pThy-derived populations had distinct functions (Fig. 4B). At day 3pi, there were too few BM-derived cells to be analyzed, but thepThy-derived gd T cells readily produced IL-17. By day 7 pi,

    FIGURE 2. Circulating gd T cells migrate specifically to VV-infected skin and contribute to all three dermal subsets. C57BL/6 mice (CD45.2) or

    CD45.2+ TCRd-GFP mice received 7 3 105 gd T cells isolated from spleen and skin dLN of CD45.1+ TCRd-GFP mice. The following day, mice wereinfected with VV on the ear. (A) Graphs show numbers of transferred TCRd-GFP+ cells recovered from the LN or ear (data are mean 6 SEM). (B)Representative flow plot shows the CCR6 and CD27 profile of the donor gd T cell population prior to transfer. (C) Representative flow plots show CCR6

    and CD103 profile of donor and recipient dermal cells at 7 dpi. (D) gd T cells from spleen and skin dLN of TCRd-GFP mice were sorted based on CD27

    expression, and 6 3 105 CD27+ or 2.7 3 105 CD272 gd T cells were transferred into separate C57BL/6 hosts. Recipients were infected with VVon the earthe following day. Flow plots show the phenotype of CD272 and CD27+ at the time of transfer and 7 dpi in the spleen and ear. Numbers denote the

    percentage of cells within the indicated quadrant. Data are representative of at least two independent experiments (n = 37 mice/group).

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  • a clear distinction between the two populations could be seen,demonstrating that, in this infection model, the function of adultBM-derived gd T cells from the circulation was to make granzymeB, whereas the dermal pThy-derived gd T cells gave rise to apopulation capable of making either IL-17 or, to a lesser extent,granzyme B.

    Resident dermal gd T cells enhance the early immune responseto infection

    To determine whether gd T cells contributed to inflammation andresolution of infection, we assessed tissue pathology and viraltiters in WT and TCRd2/2 mice, which lack all gd T cells. By3 dpi, WT ears had evidence of both epidermal and cartilage ne-crosis, often with entire sections of the epidermis replaced byserocellular crusts (Fig. 5A). The dermis of these mice predomi-nantly contained accumulation of intact and degenerate neu-trophils along with some macrophages and fewer lymphocytes. Incontrast, there was less infiltration of neutrophils and macrophagesin the ears of TCRd2/2 mice, and the cartilage and epidermisremained largely intact. Comparing the histological analysis fornecrosis (combined for epidermis and cartilage) and cellularity,WT mice scored significantly higher than TCRd2/2 mice (Fig.5B). By day 7 pi, the two groups had similar pathology (data notshown), and there was no difference in viral load between the earsof WT and TCRd2/2 mice over the course of infection either byPFU assay (Fig. 5C) or by quantitative PCR (data not shown).We next compared histologic changes of BMpThy chimeras and

    BM chimeras to determine whether the differences between WTand TCRd2/2 mice could be assigned to the presence or absenceof resident dermal gd T cells. By and large, the BMpThy chimerasrecreated the picture seen in WT mice, with high scores fornecrosis and cellularity. However, the BM chimeras had mildneutrophil infiltration, little to no cartilage necrosis, and rareepidermal necrosis (Fig. 5D, 5E). It was striking to see that re-placement of dermal gd T cells in BMpThy mice recapitulatedresults obtained with WT mice and, similarly, that BM micemirrored gd T cellnull mice (Fig. 5F). Thus, the loss of thedermal CD103+ gd T cell population appears sufficient to reduceear inflammation and damage.

    DiscussionScarification of the skin with VV was used as a vaccine to eradicatesmall pox infection, and the virus is now appreciated as an im-portant vector for vaccine development and, potentially, cancertreatment (41). We used VV scarification of mouse ears to inter-rogate the distinct roles of recruited and resident dermal gd T cellsand to better understand their diversity. In this study, we describethe expansion, recruitment, and contraction of gd T cells in thedermis following localized VV infection. About half of the ex-panded dermal gd T cells at day 7 were composed of a uniquepopulation of CD1032CCR62 gd T cells that rapidly appeared inthe dermis in response to infection. This novel population wasderived primarily from circulating CD27+ gd T cells. A portionof the immigrant gd T cells was phenotypically similar to theCD103+ IL-17producing dermal-resident gd T cell populationand derived from circulating CD272 gd T cells, although somealso were found in the CD1032 fraction. These immigrant

    FIGURE 3. Contribution of adult BM-derived and perinatal thymus-

    derived gd T cells to the dermal response. CD45.2+ TCRd-GFP mice were

    irradiated and reconstituted with BM or with BM plus pThy (BMpThy).

    (A) Graphs show the total number of gd T cells .8 wk following recon-stitution in the dermis and skin dLN. Geometric mean is shown. (B)

    Contribution of BM-derived and pThy-derived gd T cells in the dermis and

    LN. Geometric mean is shown. (C and D) BMpThy chimeras were infected

    with VV on the ear .8 wk following reconstitution. (C) CCR6 and CD27profiles of gd T cells in the skin dLN at day 0 and CCR6 and CD103

    profiles of gd T cells in the dermis at day 0 and 7 dpi. Numbers denote

    the percentage of that population within the indicated quadrant. (D) The

    number of BM- and pThy-derived gd T cells contributing to subsets in the

    dermis following infection. Error bars signify SEM. Data are results from

    more than three (A and B) or more than two independent experiments

    (C and D) (n = 315 mice/group). *p , 0.02, **p , 0.0001, two-tailed,unpaired Student t test. N.D., not done.


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  • populations were functionally distinct and played a discrete rolefrom the resident dermal population, which was ultimately re-sponsible for driving increased cellularity and tissue damage at anearly time point.The number of dermal gd T cells increased 10-fold following

    VV infection, and our data suggest that the majority of this in-crease can be ascribed to immigration rather than expansion ofthe resident population. Thus, 7 3 105 adoptively transferred gdT cells made up 5% of the total gd T cell population in the spleenand LN before infection (data not shown), and this donor/host gdT cell ratio was maintained in the dermis at day 7 following in-fection. Furthermore, the CD103/CCR6 profile of the adoptivelytransferred immigrants resembled that of the host gd T cells in thedermis at day 7 pi (Fig. 2C), suggesting that not only is the entireCD1032 population recruited, but many of the CD103+ gd T cells

    in the dermis may be, as well. This, in combination with the factthat we found very little in situ proliferation (SupplementalFig. 2), leads us to conclude that the accumulation of gd T cellsin the dermis is primarily due to recruitment.The ability of immigrant cells to diversify into all of the dermal

    subsets led us to ask what the contribution was of the individualpopulations in the circulation. Emerging evidence confirms thatCD27 delineates functional gd T cell subsets in both mice (11, 29,30) and humans (3133). Transfer of CD27+ and CD272 circu-lating gd T cells into separate hosts, followed by VV scarification,demonstrated their unique contribution to CD1032 or CD103+

    dermal populations, respectively. These experiments also con-firmed the independent maintenance of these two populations,even in the presence of inflammation. The stability of these twosubsets and the constancy of the CCR6+/CCR62 ratio within the

    FIGURE 4. Adult BM-derived and pThy-derived gd T cells

    in the dermis are functionally distinct. TCRd-GFP mice (A)

    and BMpThy chimeras (B) were infected with VV on the ear

    and sacrificed on day 3 or 7. Ears were digested in brefeldin

    A, and cells were restimulated with PMA/ionomycin for 4 h

    at 37C, followed by intracellular staining for cytokines and

    granzyme B. Representative flow plots are shown, and gates

    are based on unstimulated naive samples. Numbers indicate

    the percentage of that population in each quadrant. Data are

    representative of more than two independent experiments.

    FIGURE 5. Dermal gd T cells accelerate early collateral damage and contribute to increased tissue cellularity during VV infection. Mice were infected

    with VVon the ear. (A) Representative histological sections of ears stained with H&E from C57BL/6 WT and TCRd2/2 mice 3 dpi (original magnification

    3200). (B) Graphs show scores for necrosis (epidermis and cartilage scored separately and then combined) and cellularity. Data are mean 6 SEM. (C) WTand TCRd2/2mice were sacrificed at the indicated time points following infection, and ears were taken for plaque assay. Error bars indicate SEM.

    Representative histological sections stained with H&E (D) and scores (E) for BM chimeras and BMpThy chimeras 3 dpi. Data are mean 6 SEM. (F)Comparison of necrosis scores for WT, TCRd2/2, BMpThy chimeras, and BM chimeras in relation to the presence (+) or absence (2) of gd T cellpopulations. In (A) and (D), arrows indicate necrotic cartilage, arrowheads indicate neutrophilic infiltrates, and asterisks indicate serocellular crusts. Data in

    (C) are representative of more than one independent experiment (n = 36 mice/group), and data in (A), (B), (D), and (E) are representative of more than two

    independent experiments (n = 56 mice/group). Scale bars, 100 mm. *p , 0.05, **p , 0.01, ***p , 0.005, two-tailed, unpaired Student t test. Circ.circulating gd T cells; Derm., dermal gd T cells.

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  • CD272 population offer an important tool for studying gd T cellsduring inflammation. Although other groups compared CD27+ andCCR6+ gd T cells (11, 29), the circulating CCR62CD272 subsethas received less attention. This latter population is uniformlyRORgt+ and is distinguished from the CCR6+ population by lowerlevels of CD103 and CD44 (Supplemental Fig. 1A). Future studieswill be needed to explore the nuances between these populations.Although there is some disagreement concerning the radio-

    sensitivity of gd T cells and their reconstitution in an irradiatedadult mouse (11, 16, 22), our chimera experiments revealed that,unlike the DETC population, gd T cells in the circulation anddermis are radiosensitive. Our results confirm that transfer of pThyinto an irradiated host generates bona fide CCR6+CD272 gdT cells in the circulation and residents in the dermis. Differentialkinetics and functional profiles of the BM (immigrant) and pThy(resident) gd T cell populations in the BMpThy chimeras allowedus to ask whether they had distinct roles following infection.Determining the role of dermal gd T cells by comparing WT andTCRd2/2 mice is complicated by the absence of normal DETC inthe null mice (14), cells that are known to play a role in kerati-nocyte growth and survival, perhaps impacting tissue damage orrecovery. Whole-body irradiation allowed us to specifically de-plete the resident dermal gd T cells, creating mice that lack thispopulation while retaining healthy BM-derived circulating gdT cell populations. Comparing mice specifically with or withoutdermal gd T cells revealed that the dermal-resident population wasresponsible for the increased inflammation observed in infectedWT animals (Fig. 5D, 5E).The ability of pThy-derived dermal gd T cells to recruit in-

    flammatory cells, such as neutrophils, is likely due to their pro-duction of IL-17 (16, 20, 42), and recruitment of neutrophils bygd T cells directly results in increased tissue damage (43). Wedemonstrated increased necrosis and cellularity of infected ears3 d after VV infection, indicating that the resident IL-17+ gdT cells are early responders to viral infection. In contrast, theimmigrating population of CD1032CCR62 DN gd T cells, whichappeared concurrently, only upregulated granzyme B expression ata later time point. Although classically associated with cytotox-icity, which was shown for gd T cells (44), granzyme B has directantiviral effects within cells and can cleave structural proteinswhen released into the extracellular matrix (45, 46). Importantlyfor our study, extracellular granzyme B release was shown to haveremodeling activity (47), potentially facilitating migration of cellsinto the tissue and impacting wound repair. Considering that theabsence of gd T cells had no effect on viral load (Fig. 5C) and thenumber of gd T cells within the dermis remained elevated for anextended period of time, these granzyme-producing cells may playa nontraditional role during inflammation and in the recoveryprocess after viral clearance.Little is known about whether gd T cells can form memory.

    The majority of studies have examined total gd T cell populationre-expansion (48, 49) or relied on CD44 expression (33), whichis now appreciated to be regulated differently in gd T cells thanin classical ab T cells (50). When we transferred congenicallymarked gd T cells, none of the immigrant gd T cells remained inthe dermis as residents after the infection was cleared (Fig. 2B).This was true for the CD27+ circulating gd T cells characterizedas CD1032CCR62 DN cells in the dermis and for the CD272

    circulating cells that give rise to all three populations in the dermisat day 7 pi. Similarly, in the BMpThy chimeras, BM-derivedcirculating gd T cells that entered during the infection did notremain there long term (Fig. 3D). This indicates that inflammationis not sufficient to drive the long-term skin residence of gd T cellsthat enter the skin from the circulation. In contrast, it was ob-

    served that, after oral Listeria monocytogenes infection, a pop-ulation of gd T cells with memory-like function develops andpersists in intestinal tissues (51). However, generating residentdermal gd T cells requires a neonatal population (or their pre-cursors), derived in our case from the transfer of pThy into irra-diated hosts.The presence of pThy-derived gd T cells in the dermis was

    required to increase cellularity near the site of infection and toamplify the overall potency of the immune response, as demon-strated by increased tissue damage in WT and BMpThy mice.Although the complete absence of gd T cells did not translate intoincreased viral load in this or related systems (52, 53), othermodels showed that mice lacking gd T cells are more susceptibleto bacterial infection (54) and have a decreased capacity forwound healing (13). Together, the data indicate that gd T cells area dynamic population with a range of functions but that they mightnot respond equally to all inflammatory cues. Importantly, becausethere is no DETC counterpart in human skin (55), it has becomeincreasingly important to isolate the relevance of gd T cells in thedermis in models relevant to human disease. Our study using theadoptive transfer of circulating gd T cells into normal hosts andthe creation of chimeras specifically lacking a dermal-residentpopulation will be useful for interrogating gd T cell subsets andfurthering our understanding of the dermal immune network.

    AcknowledgmentsWe thank the University of Washington Histology and Imaging Core for

    preparation of histological sections, Dr. E. Gray for critical discussion

    and technical advice, and Dr. P. Fink for review of the manuscript. We also

    thank Dr. J. Turner (University of Chicago, Chicago, IL) for the TCRd-

    H2BeGFP mice originally derived by I. Prinz.

    DisclosuresThe authors have no financial conflicts of interest.

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