Tissue-resident memory CD8⁺ T cells continuously patrol skin epithelia to quickly recognize local antigen

Model to Dissect the Function of Skin-Resident Memory T Cells.

To understand how tissue-resident CD8⁺ memory T cells guard against viral reinfections, we first set out to develop an experimental model in which the activity of skin-resident versus systemic CD8⁺ Tmem can be analyzed without potential confounding effects of virus-specific CD4⁺ memory T cells or antibodies. To this purpose, we transferred naive CD8⁺ herpes simplex virus-1 (HSV)–specific (10), GFP⁺ T cells (GFP⁺gB) into C57BL/6j recipients and subsequently vaccinated the mice by skin DNA injection, using a DNA vaccine encoding the gB_498–505 epitope that is recognized by gB T cells (Fig. 1A). The magnitude of vaccine-induced gB_498–505-specific CD8⁺ T-cell responses in these mice (up to 5% of circulating CD8⁺ T cells at days 11–15 postvaccination; Fig. 1B) was comparable to that induced in mice that had not received adoptive transfer. However, the gB_498–505-specific CD8⁺ T-cell response in recipients of GFP⁺ gB T cells fully consisted of GFP⁺ cells (Fig. S1A), thus ensuring that the fluorescent cells observed during imaging represented the entire population of gB_498–505-responsive T cells. Analysis of the vaccination site several weeks after treatment revealed the presence of large numbers of GFP⁺ cells that displayed a dendritic morphology (Fig. 1C). In agreement with previous literature (2), multiphoton analysis of the skin established that the GFP⁺ cells were exclusively restricted to the epidermal layers, including hair shafts (Fig. 1 D and E). Flow cytometry demonstrated that the GFP⁺ cells extracted from the skin were uniformly CD8⁺TCRβ⁺ and K^bgB_498–505tetramer⁺, thereby qualifying them as bona fide HSV-specific CD8⁺ Tmem (Fig. 1F); furthermore, they expressed a high level of CD103 (integrin α_Eβ₇; Fig. 1G), an adhesion molecule implicated in T-cell retention within the epithelia (6). Consistent with published literature (6), the skin-resident memory T-cell pool that was induced by skin vaccination displayed a morphology and phenotype similar to that found after local skin infection with HSV, demonstrating that long-term tissue retention of CD8⁺ Tmem is not dependent on the presence of virus. To determine the contribution of the vaccine-induced CD8⁺ skin-resident memory T-cell pool to the control of secondary infections, we vaccinated recipients of GFP⁺gB T cells on one flank with the gB_498–505 DNA vaccine, to create a localized CD8⁺ memory T-cell pool. Three months after vaccination, mice were challenged with an HSV strain that expresses the td-tomato fluorescent protein (HSV_TOM) at both the ipsilateral (i.e., previously vaccinated) and contralateral site, and infection growth was monitored over time (Fig. S1B). Intravital imaging of the infected skin revealed the presence of similarly sized HSV-infected lesions early after infection (P = 0.4402). In contrast, whereas infections at the contralateral site continued to grow rapidly, HSV foci at memory CD8⁺ T-cell sites were in large part contained (Fig. S1 C and D; comparison contra – ipsi, P < 0.001), establishing that skin-resident CD8⁺ T cells play an important role in the control of local secondary HSV infections.

Skin-Resident Memory T Cells Display a Continuous Migratory Behavior.

To investigate how tissue-resident memory T cells can provide such local protection we performed long-term imaging of skin-resident GFP⁺CD8⁺ Tmem at formerly infected sites. These experiments revealed that at all of the examined time points postinfection, these cells were not sessile, but migrated productively within the epidermis, with mean speeds of about 1.3 µm/min (Movie S1). Whereas the speed of Tmem migration was relatively low, Tmem migration was characterized by long directional persistence, with cells moving in a similar direction for an average of ∼10.5 min. Combined, these speed and directionality parameters result in a high motility coefficient of ∼9 µm²/min, close to that of lymph node B cells (11). To address whether this steady-state migration could reflect continued low-level T-cell receptor (TCR) triggering due to the presence of residual antigen, we generated an experimental setting in which tissue-resident memory T cells reside in skin areas in which their cognate antigen was never introduced. To this purpose, naive GFP⁺OTI T cells were transferred into C57BL/6j recipients and activated by application of a DNA vaccine encoding the OVA_257–264 epitope that is recognized by the OTI TCR to create a pool of effector GFP⁺OTI T cells. At this time point, mice were infected by epidermal HSV application, and entry and maintenance of the “bystander” GFP⁺OTI T cells at the site of HSV infection was evaluated. These analyses demonstrated that effector CD8⁺ T cells also form long-term local T-cell memory at sites of former bystander infection (Fig. 2A). Importantly, these bystander tissue-resident memory T cells displayed a dendritic morphology (Fig. 2B) and showed a productive migration through the epidermis (Movie S2), indicating that both of these Tmem cell properties are antigen-independent. Thus, regardless of the presence of cognate antigen, CD8⁺ T cells that enter the skin epithelium up-regulate the surface expression of CD103 and acquire dendritic morphology and constitutive motility.

Migratory Behavior of Memory T Cells Allows Contact with a Large Number of Surrounding Cells.

The continuous movement of the epithelium-resident memory pool (Fig. 2C) suggests that in time CD8⁺ T cells can cover long distances and as a consequence contact a large number of surrounding tissue cells. To visualize the cumulative skin area that can be patrolled by migrating populations of skin-resident CD8⁺ Tmem over time, all consecutive images from a 4-h imaging session were overlayed (Fig. 2D). This analysis revealed that at the cell densities found within previously infected areas, the migratory behavior of CD8⁺ Tmem is sufficient to allow the exploration of a large portion of skin within a period of hours. Constitutive steady-state migration of skin-resident Tmem was observed up to 18 mo after primary infection, the latest time point analyzed. As a control, and consistent with prior data (12), the cell body of skin-resident Langerhans cells (LCs) did not show detectable displacement (Fig. 2E). LCs, epidermal γδ T cells, and CD8⁺ Tmem within the epidermis all have a dendritic shape. It has been established that LCs project their dendrites upward, toward the stratum corneum (13), a property that allows them to capture antigens from the most superficial skin layers. Likewise, the dendrites of γδ T cells invariably project toward the stratum corneum (14). However, the observation that skin-resident CD8⁺ Tmem show a continuous migration through the epidermis, together with the fact that the dendrites continuously change size, length, and direction (Movies S1 and S2), suggests that these dendrites could serve a different function. To compare the morphology of dendrites from skin-resident γδ T cells, LC, and CD8⁺ Tmem we analyzed these cell types in whole-mount stains. CD8⁺ Tmem had fewer but thicker dendrites compared with γδ T cells; more importantly, whereas the dendritic extensions of Langerhans cells and epidermal γδ T cells pointed upward, the dendrites of CD8⁺ Tmem were almost all parallel to the epidermis, consistent with patrol along a 2D surface (Fig. 3 and Movie S3). Thus, memory T cells residing in the epidermis differ from other immune cells within the same environment with respect to their morphology and migration.

Dendritic Shape of Skin-Resident Memory T Cells Is Independent of Skin Inflammatory State.

Effector CD8⁺ T cells, which are present in both the epidermis and dermis of HSV-infected mice, exhibit an amoeboid shape, whereas skin-resident memory CD8⁺ T cells, which are located exclusively in the epidermis (2; Fig. 1 D and E), display a dendritic shape. To evaluate whether the shape difference between the two cell populations could be due to the skin layer in which they reside, we compared the shape of effector T cells (Teff) and Tmem in the same skin layer using LCs as an internal reference (Fig. S2). This analysis demonstrated that the shape difference between the two is not due to a simple difference in location. A second explanation for the “dendricity” acquired by memory T cells upon retention in the epidermis could be that this shape is imposed on CD8⁺ Tmem by the tight surrounding tissue after infection resolves. To investigate whether the dendritic shape displayed by the memory T-cell pool in the epidermis is solely imposed by the environment, we compared the shape of GFP⁺OTI T cells present at sites of ongoing HSV infections, when either present as recently primed effector T cells or as locally resident memory T cells (Fig. 4A). Effector OTI T cells migrating within HSV-infected skin displayed the amoeboid, round shape that is also seen for antigen-specific effector T cells within effector sites (Fig. 4B). In contrast, skin-resident memory OTI T cells that infiltrated an HSV_TOM-infected area retained their dendritic, crawling behavior (Fig. 4 B and C and Movie S4). Thus, the dendritic migration mode of CD8⁺ Tmem that are present in the epidermis does not merely stem from the physical constraints imposed by the environmental architecture, but at least partially reflects an adaptation of the memory T cells present at that site. The observation that skin-resident CD8⁺ Tmem that move within the epidermis continuously extend and contract dendrites along a 2D plane suggests that these cells migrate in between keratinocytes. To visualize the cellular environment of skin-resident CD8⁺ Tmem, we transferred naive Kaede⁺gB T cells (15) into Histone-2B-GFP mice (in which GFP marks the nuclei of all skin cells) and created a local skin-resident CD8⁺ Tmem pool. Several weeks postinfection, Kaede was locally photoconverted in vivo, to distinguish the transferred T cells from the GFP⁺ skin cells. Imaging of the photoconverted areas confirmed that skin-resident CD8⁺ Tmem extend and retract dendrites continuously in between skin cells while moving through the epidermis (Fig. 4D and Movie S5). To evaluate whether the differential behavior of Tmem and Teff correlated with phenotypic differences, the surface expression of a small number of T-cell markers was studied. CCR4 and NKG2D expression did not differ between Tmem and Teff (Fig. S3), making their contribution to the differential behavior of the two cell types unlikely. The chemokine receptor CXCR3, involved in T-cell migration to peripheral tissues (16), was detected on skin-resident memory T cells but was nearly absent on HSV-infected skin-infiltrating effector T cells (Fig. S3), conceivably reflecting down-regulation induced by ligand binding.

Quantification of the Efficiency of Target Cell Encounter by Computational Modeling.

The above data demonstrate that memory T cells continuously migrate through the epidermal surface for long periods after infection and in the absence of antigenic stimulation. These observations raise the hypothesis that the combination of migratory behavior and morphology could represent a type of tissue surveillance, in which CD8⁺ Tmem patrol the epidermis to search for skin cells that express cognate antigen. To explore to what extent the migration and morphology of Tmem would increase the contact and scanning of surrounding cells, we developed a 2D Cellular Potts Model (17–20), in which the density of keratinocytes (21) and T-cell migration and shape parameters closely match the experimentally determined values (SI Materials and Methods, Movie S6, Fig. 5A, and Fig. S4 A and B). Subsequently, simulated “target cells” and memory T cells were positioned randomly within an in silico field (size 0.54 mm²) and Tmem–target encounters were monitored over time. At the experimentally determined Tmem densities of about 430 cells per square millimeter, the majority of targets was detected within 1 h (Fig. 5B). Furthermore, even at T-cell densities that were up to fivefold below those determined experimentally, most targets were encountered within hours. If T-cell densities are reduced even further, the efficiency of steady-state target scanning no longer allows identification of each target cell with time periods of hours. However, even in this case viral outgrowth may potentially still be limited upon encounter of a small number of targets, through the “field effect” described by Müller et al. (22).

Comparisons of the number of target cell encounters by these simulated cells with that of simulated T cells that were either nonmotile or nondendritic indicate that T-cell motility is the primary factor that determines the efficiency of target cell encounter, with dendricity adding another 33% (Fig. S4C). As a side note, modeling also indicated that the dendritic (compared with amoeboid) morphology allowed migrating T cells to touch an 11% larger cell surface area of each contacted keratinocyte per time point (P < 0.001), a property that may conceivably increase the efficiency of scanning.

Skin Patrol by Tissue-Resident Memory T Cells Allows Rapid Recognition of Antigen-Expressing Cells in Vivo.

To directly test the hypothesis that the epidermal migration by long-term resident memory T cells leads to antigen scanning and thereby allows the detection of antigen-expressing cells, we first established how antigen recognition by skin-resident memory T cells can be visualized in vivo. To this purpose, sites containing a GFP⁺gB memory T-cell pool were either left untreated or injected with the gB_498–505 peptide, with control peptides (CMV pp65_495–503 and Ova_257–264), or with vehicle. In all cases, skin-resident Tmem displayed a transient increase in circularity and a decrease in migration speed following injection, likely reflecting skin trauma (Fig. 5 C and D and Fig. S5A). However, 3 h postinoculation, GFP⁺gB Tmem that had either been exposed to vehicle or to control peptides had recovered their dendritic shape and migratory behavior (Fig. 5 C–E and Movie S7). In sharp contrast, gB Tmem that had been exposed to gB_498–505 peptide remained roundish and arrested for up to 9 h after peptide delivery (Fig. 5 C–E and Movie S8). This change in shape and motility was strictly antigen-dependent, because skin-resident OTI Tmem underwent similar changes upon Ova_257–264 delivery, but not in the presence of pp65_495–503 or gB_498–505 control peptides (Fig. S5B). In an independent set of experiments, s.c. peptide delivery was also shown to result in T-cell rounding up and stalling in an antigen-specific manner (Fig. S6). These data demonstrate that antigen recognition by skin-resident Tmem can be visualized by alterations in both motility and morphology; the loss of motility is reminiscent of prior data on antigen recognition by naïve and effector T cells (23), and the large change in morphology is a characteristic that is unique to skin-resident memory T cells.

Having established both loss in dendricity and migration as experimental readouts to determine whether migrating Tmem can identify rare antigen-expressing cells, we created a situation in which only a small fraction of skin cells underwent de novo expression of an antigen recognized by the skin-resident memory T-cell population. To this purpose, we injected epidermis hosting a local GFP⁺gB memory T-cell pool with DNA encoding the Katushka fluorescent protein as a genetic fusion to the gB_498–505 peptide. Following DNA delivery, Katushka-gB_498–505–expressing cells were readily detectable amid a large number of antigen-negative cells (Movie S9). Remarkably, GFP⁺gB Tmem could be shown to continue migration and dendrite protrusion through the skin until they contacted an antigen-expressing target cell, after which they immediately stalled and rounded up (Fig. 6 A and B). Furthermore, stalling and rounding could also be observed for migrating Tmem even before antigen expression by the neighboring cells became detectable by microscopy (Fig. 6C and Movie S9), suggesting that recognition of antigen expression during natural infection may occur before substantial pathogen replication can take place. Both stalling and rounding were fully antigen-dependent; Katushka-expressing skin cells that lacked the gB_498–505 epitope were completely ignored (Movie S9).

Adoptive Transfer, HSV Infection, Peptide, and DNA Injection.

Spleens from GFP⁺gBT I.1 or GFP⁺OTI mice were isolated and homogenized; after red blood cell lysis, CD8⁺ T cells were isolated and C57BL/6j mice were injected i.v. with 2.0 × 10⁵ sorted naïve CD8⁺ T cells. Intraepithelial HSV infection was carried out on the shaved flank of anesthetized mice by tattooing a droplet containing ∼2.5 × 10⁵ pfu HSV_TOM onto the skin using a sterile disposable 11-needle bar mounted on a rotary tattoo device. For peptide delivery, 750 ng of peptide in PBS/10% (vol/vol) DMSO solution was tattooed in the flank of anesthetized mice. For skin cell transfection, 30 µg of the indicated DNA in PBS was applied onto the skin and injected using a sterile disposable 11-needle bar mounted on a rotary tattoo device (25).

Confocal Microscopy.

Anesthetized mice were placed in a custom-built chamber with the infected flank gently placed against a coverslip at the bottom side of the chamber. Images were acquired using an inverted Leica TCS SP2 confocal scanning microscope equipped with diode and argon lasers and enclosed in a custom-built environmental chamber that was maintained at 37 °C using heated air. Images were acquired using a 20×/0.7 N.A. dry objective. GFP was excited at 488 nm wavelength and td-tomato at 561 nm. Typical voxel dimensions were 0.7–1.5 μm laterally × 1.5–2.5 μm axially. Three-dimensional stacks (typical size ∼500 µm × ∼500 µm × ∼30 µm) were captured every 2 min for a period of up to 4 h.