Tissue-Resident Exhausted Effector Memory CD8+ T Cells Accumulate in the Retina during Chronic Experimental Autoimmune Uveoretinitis

Experimental autoimmune uveoretinitis is a model for noninfectious posterior segment intraocular inflammation in humans. Although this disease is CD4+ T cell dependent, in the persistent phase of disease CD8+ T cells accumulate. We show that these are effector memory CD8+ T cells that differ from their splenic counterparts with respect to surface expression of CD69, CD103, and Ly6C. These retinal effector memory CD8+ T cells have limited cytotoxic effector function, are impaired in their ability to proliferate in response to Ag-specific stimulation, and upregulate programmed death 1 receptor. Treatment with fingolimod (FTY720) during the late phase of disease revealed that retinal CD8+ T cells were tissue resident. Despite signs of exhaustion, these cells were functional, as their depletion resulted in an expansion of retinal CD4+ T cells and CD11b+ macrophages. These results demonstrate that, during chronic autoimmune inflammation, exhausted CD8+ T cells become established in the local tissue. They are phenotypically distinct from peripheral CD8+ T cells and provide local signals within the tissue by expression of inhibitory receptors such as programmed death 1 that limit persistent inflammation.

Experimental autoimmune uveoretinitis is a model for noninfectious posterior segment intraocular inflammation in humans. Although this disease is CD4 + T cell dependent, in the persistent phase of disease CD8 + T cells accumulate. We show that these are effector memory CD8 + T cells that differ from their splenic counterparts with respect to surface expression of CD69, CD103, and Ly6C. These retinal effector memory CD8 + T cells have limited cytotoxic effector function, are impaired in their ability to proliferate in response to Ag-specific stimulation, and upregulate programmed death 1 receptor. Treatment with fingolimod (FTY720) during the late phase of disease revealed that retinal CD8 + T cells were tissue resident. Despite signs of exhaustion, these cells were functional, as their depletion resulted in an expansion of retinal CD4 + T cells and CD11b + macrophages. These results demonstrate that, during chronic autoimmune inflammation, exhausted CD8 + T cells become established in the local tissue. They are phenotypically distinct from peripheral CD8 + T cells and provide local signals within the tissue by expression of inhibitory receptors such as programmed death 1 that limit persistent inflammation. The Journal of Immunology, 2014, 192: 4541-4550. E xperimental autoimmune uveoretinitis (EAU) is an Agspecific CD4 + T cell-dependent model of noninfectious intraocular inflammation, paralleling clinicopathological features of human uveitis. Animal models have proven useful in probing cellular mechanisms of disease and as a preclinical model for future treatments of human uveitis (1). EAU can be elicited in rodents by immunization with retinal autoantigens, such as retinolbinding protein (RBP)-3, previously known as interphotoreceptor retinoid-binding protein and S-Ag (also known as arrestin). In the C57BL/6 (H-2 b ) mouse model, immunization with the 1-20 RBP-3 peptide and adjuvants provokes persistent disease principally involving the posterior segment of the eye (2).
In murine models of EAU, it is possible to distinguish three phases of disease, the subclinical prodrome, a primary peak, and a period of secondary regulation (3). Secondary regulation is characterized by longstanding changes in the character of immunosurveillance as assessed by the level of immune cell infiltration. It manifests aspects of chronically disordered retinal regeneration, features that are also commonly found in human disease, specifically the development of intraretinal neovascular membranes (4).
Clinical disease in EAU depends on both CD4 + T cells and macrophages; depleting either during the prodromal phase prevents progression (5,6). However, other immune cells also play an important role in regulating disease, including CD8 + T cells (7)(8)(9). Recently, transcriptional profiling of CD8 + T cells from patients with severe autoimmune disease revealed them as a potential biomarker for patients with poor prognosis (10,11). In EAU and other models of organ-specific autoimmune disease, in which CD8 + T cells have been studied, they have been ascribed a variety of roles (12)(13)(14)(15)(16). CD8 + T cells have been reported to accumulate in late uveitis in rat models of disease, but depletion of these cells from the time of disease induction had little effect, and it remains unclear as to whether the cells regulate or contribute to the persistence of disease (17)(18)(19). Recently, there has been a growing awareness of heterogeneity among CD8 + T cells that are expanded as part of an acute immune response. The responding population is comprised of a mixture of different subsets that can be classified using cell surface markers, of which effector memory CD8 + T cells (T EM ) cells are the predominant subset that enters peripheral tissues (20,21). It has been of recent interest to determine the conditions that dictate whether T EM are retained in the target tissue or recirculate from the blood and continually repopulate the peripheral tissues. One outcome of acute viral infection is the generation of a subset of tissue-resident effector memory CD8 + T cells (T RM ) that populate normal and immune privileged peripheral organs such as the gut and the brain following the resolution of infection (22)(23)(24)(25). Further studies have revealed subsets of T RM residing in the skin, lung, and salivary glands (26)(27)(28)(29). This distinct population of cells has not only been identified in mouse models of infection but also in human mucosal tissue, and, importantly, expression patterns of key markers such as CD103 and CD69 are consistent in humans with those demonstrated in murine models (30,31). These T RM have been shown to provide protection against infection within the local tissue and limit secondary infection (27,32). This form of immunological memory has mainly been studied in viral models such as lymphocytic choriomeningitis virus (LCMV) or HSV infection and has not yet been characterized in autoimmune models. With this in mind, we set out to analyze the resident CD8 + T cells in the tissue of an autoimmune model, during the persistent phase of disease, in which secondary regulation may be in effect, and assess their role by depleting them from the tissue.

Materials and Methods
Mice C57BL/6J mice were originally obtained from Harlan UK Limited (Oxford, U.K.), and breeding colonies were established within the Animal Services Unit at Bristol University (Bristol, U.K.). Mice were housed in specific pathogen-free conditions with continuously available water and food. Female mice immunized for disease induction were aged between 6 and 8 wk. All mice were kept in the animal house facilities of the University of Bristol, according to the Home Office Regulations. Treatment of animals conformed to United Kingdom legislation and to the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research.

Topical endoscopic fundal imaging
Photographs were taken of the retina using a method developed previously (33). A 5-cm-long tele-otoscope (1218AA; Karl Storz, Tuttlington, Germany) was attached to a digital camera (Nikon, Tokyo, Japan). A xenon lamp (201315-20; Karl Storz) connected through a flexible optic fiber to the endoscope was used as a light source. The pupils of the mice were dilated using 1% topical tropicamide and 2.5% phenylephrine (Chauvin Pharmaceuticals, Romford, U.K.), and then 0.4% oxybuprocaine (Chauvin Pharmaceuticals) and viscotears were applied (Novartis Pharmaceuticals). The camera was fixed to a bench, and the mouse was positioned at the end of the endoscope and orientated to the correct angle. All pictures were analyzed and cropped using Photoshop CS software (Adobe, Mountain View, CA). Fundal images were scored for inflammatory changes of the optic disc, retinal vessels, retinal lesions, and structural damage; all scores were added together to make a final disease score. This clinical grading system was adapted [from (34)] and has been previously published (35). Scoring was carried out independently by two trained assessors masked to the origin of the data.

Cell preparations
Spleens were disrupted mechanically through a 40-mm cell strainer, and RBCs were lysed. Eyes were enucleated and carefully cleaned to remove all extraneous connective and vascular tissue. The retinas (including the ciliary body) were microscopically dissected in HBSS media supplemented with 5% FCS. Retinas were then cut into small pieces and digested with 1 ml media containing 0.5 mg/ml collagenase D (Roche, Welwyn Garden City, U.K.) and 750 U/ml DNase I (Sigma-Aldrich) for 20 min at 37˚C. Retinal tissue was homogenized and forced through a 70-mm cell strainer with a syringe plunger, to obtain a single-cell suspension, and stained for flow cytometry analysis.
Quantification of the number of cells per eye has been described previously (3).

Fluorescent immunohistochemistry
Eyes were snap frozen in optimal cutting temperature medium and sectioned at 16 mm thickness. Following fixation with 1% paraformaldehyde and nonspecific goat serum block, slides were stained with monoclonal rat anti-mouse CD8a (clone 53-6.7; BD Biosciences). Secondary labeling was performed with an AlexaFluor 546-conjugated goat anti-rat Ab and then costained with DAPI and imaged by either fluorescent (Axio Observer.Z1; Carl Zeiss Microimaging, Jena, Germany) or confocal microscopy (Leica DM5500Q; Leica Microsystems). Images were exported and processed in Adobe Photoshop CS5.5 (Adobe Systems, San Jose, CA).

Flow cytometry
Cell suspensions were incubated with 24G2 cell supernatant (Fc block) for 10 min at 4˚C. Cells were then stained with fluorochrome-conjugated mAb against cell surface markers for 20 min at 4˚C. Multiparameter flow cytometry was carried out using different fluorochromes all purchased from BD Biosciences, except for CD107a (gift of D. Morgan, University of Bristol). Granzyme B and programmed death 1 (PD-1) were purchased from eBioscience (Hatfield, U.K.). Cells were resuspended in 7-aminoactinomycin D (7AAD), and dead cells were excluded from analysis by gating on 7AAD 2 cells. TNF-a, granzyme B, and CD107a were detected after 4 h of activation with PMA (20 ng/ml) and ionomycin (1 mM) with 1 ml/ml Golgiplug (BD Biosciences). IFN-g production was detected either after PMA/ionomycin stimulation as above or from cells cultured with 10 mg/ml RBP-3 1-20 or OVA 323-339 peptides, and with 100 U/ml human rIL-2 for 30 min before the addition of 1 ml/ml Golgiplug for an additional 3 h. Cells were incubated with supernatant 24G2 for 10 min and then stained for cell surface markers for 20 min at 4˚C. The cells were then permeabilized with Perm/Fix kit (BD Biosciences), according to the manufacturer's instructions, and subsequently stained for mAb against intracellular cytokines or appropriate isotype controls. Cell suspensions were then washed and acquired on a flow cytometer (LSR II, FACSDiva software; BD Cytometry Systems). All analyses were performed using FlowJo software (Tree Star, Ashland, OR). The numbers of cells were calculated by reference to known standard curve (3).

FTY720 treatment
Immunized C57BL/6J mice were treated with 0.3 mg/kg FTY720 or control analog AAL149 in a maximum volume of 150 ml in PBS, by oral gavage. A dose previously titrated to reduce cell infiltrate by 75% (35).
In vivo CD8 depletion mAbs were purified from supernatant by growing hybridomas YTS 169.4 and YTS 156.7 (a gift of A. Gallimore, Cardiff University) in complete media with 2.5% FCS. Supernatant was purified using G protein column (GE Healthcare) on AKTAprime chromatography system (GE Healthcare), and concentration of protein was determined by NanoDrop (Thermoscientific). Protein was dialyzed into PBS before injection. Immunized mice were given 250 mg of either both depleting Abs or IgG isotype control by i.p. injection.

Statistical analysis
All data were analyzed using a Mann-Whitney U test, except for retinal CFSE and IFN-g assays. Data were analyzed using Wilcoxon signed rank test and considered significant when results had a p value # 0.05. All graphs and statistical tests were performed using GraphPad Prism (GraphPad Software).
To characterize the underlying disease process in more mechanistic detail, we carried out extensive phenotyping of the cellular infiltrate. We quantified the leukocyte content of the retinas of animals with EAU at two time points, during the primary peak (days 24-26) and the persistent phase (days 36-43), with methodology established in the B10.RIII model (3) (Fig. 1C). Total average CD4 + T cell numbers were not different at these two time points (Fig. 1D), whereas CD8 + T cell numbers did increase significantly ( Fig. 1E) (p , 0.05). This accumulation was specific to the inflamed retina, as there was no difference in the percentage of CD8 + T cells in the spleen at these two time points (Fig. 1H). Normalizing the CD8 + T cells as a fraction of the total CD45 + leukocyte population confirmed the selective accumulation of CD8 + T cells in the retina (Fig. 1G) compared with CD4 + T cells, which showed no selective accumulation (Fig. 1F). The CD8 + T cells increased from 10 to 16% of the total CD45 + CD11b 2 cell population (Fig. 1I). We concluded that, in the C57BL/6J mouse, persistent uveitis was accompanied by a selective increase of CD8 + T cells localized within the retina, the function of which is poorly understood.

CD8 + T cells are localized throughout the retina
To determine whether the CD8 + T cells accumulate in a specific location or are dispersed throughout the retina, fluorescent immunohistochemistry was performed on retinal sections from mice immunized for EAU (Fig. 2). Confocal images demonstrate the presence of CD8 + cells at days 26 and 40 EAU throughout the retinal layers. There were no striking differences in the pattern of accumulation at these two time points. Sections show staining that appeared within regions of vasculitis, retinal folds, and the vitreous. The histology also confirms that the architecture of the retina is relatively well preserved compared with some other models of uveitis (36), providing an ongoing source of retinal Ags to activate specific infiltrating T cells.

Retinal CD8 + T cells show Ag-specific activation
Next, we investigated the response of the CD8 + T cells to activation with the immunizing peptide. First, we used CFSE staining with Ag-specific stimulation to assess activation and proliferation. Not surprisingly, we detected Ag-specific CD8 + T cells in the spleen (37). Segregating splenic CD8 + cells into a dividing and nondividing population on the basis of CFSE dilution allowed quantification of CD3 downregulation as a consequence of TCR engagement by cognate peptide (Fig. 3A) (38). Similarly, retinal CD8 + T cells displayed a significant downregulation of CD3 when stimulated with 1-20 peptide (Fig. 3A), albeit no proliferation was observed, as we have previously noted with retinal CD4 + T cells (39) (Fig. 3B). To test the functional response of these cells, we analyzed retinal Ag-induced IFN-g intracellular cytokine expres-sion in the presence of IL-2, a method previously used to identify Ag-specific T cells (40). IFN-g was produced in response to Ag by a much higher percentage of retinal CD8 + T cells than their splenic counterparts, achieving levels seen in the spleen only following PMA/ionomycin stimulation (Fig. 3C). Activation of retinal T cells with IL-2 also induced IFN-g in the presence of nonspecific peptide stimulation, but stimulation with RBP-3 1-20 elicited significantly more IFN-g than seen in the control (p , 0.01) (Fig. 3D). We also noted that the IFN-g production from cells obtained from the retina segregates to the CD8 low population, consistent with coreceptor tuning of the cells in response to Ag engagement (41). Taken together, these results indicate that a significant population of retinal CD8 + T cells has undergone recent activation.

Phenotype of retinal T EM
T RM have a distinct phenotype based on cell surface markers that has been used to characterize their presence in a wide range of different tissues; we used this approach to determine the phenotype of CD8 + T cells in the retina. First, we assessed the expression of the well-known memory and activation markers CD44 and CD62L (20) (Fig. 4). Flow cytometry on live splenic CD8 + T cells on days 25 and 40 postimmunization revealed naive and memory, effector, and central CD8 + T cell populations. In contrast, in the retina, .95% of cells were CD44 high CD62L low . It should be noted that these cells lacked the markers CD25 and CD28 (data not shown). This indicates that, by the late phase of EAU, during CD8 + T cell accumulation, the majority of the CD8 + T cells have a phenotype consistent with Ag-activated T cells that have become T EM .
Further phenotyping using additional markers revealed that T EM found in the retina have a different phenotype to their splenic counterparts (Fig. 4A). The expression of the surface marker Ly6C on T EM in the spleen and the retina at day 25 EAU was similar; however, by day 40, Ly6C was significantly downregulated on retinal T EM (Fig. 4B). CD69 expression is well known as an early activation marker on T cells, but it is also expressed on T RM populations (31,42). In the retina, CD69 was upregulated on T EM compared with spleen at day 25 (Fig. 4A), and the percentage of cells expressing CD69 doubled by day 40 (Fig. 4B). Downregulation of Ly6C and increased expression of CD69 is a phenotype that has been reported in chronic LCMV infection, in which T EM undergo changes in phenotype upon migration to infected tissue (23,43). CD103 is a key marker found on T RM subsets (24). Naive

CD8 + T cells express intermediate amounts of CD103
, and this expression is downregulated upon activation. In this study, splenic T EM expressed minimal CD103 (Fig. 4A) compared with CD44 low cells (data not shown). Within the retina, upregulation of CD103 was limited; ∼25% of these cells expressed this surface marker (Fig. 4B).
Retinal T EM retain the potential to kill, as shown by the expression, after stimulation with PMA/ionomycin, of granzyme B (Fig. 4C). However, they have low levels of CD107a expression, which declines as disease progresses, indicating that they have not recently degranulated (44). To assess function further, we also tested the cytokine production by these cells. Between days 25 and 40, there was a significant decrease in the percentage of cells capable of producing IFN-g in response to PMA/ionomycin (p , 0.05) (Fig. 4C). There was also a trend toward reduced production of TNF-a that was not statistically significant. Therefore, there is no evidence that the selective accumulation of retinal T EM is accompanied by an increase in effector function. Collectively, the phenotype indicates that the retinal CD8 + T EM have experienced Ag stimulation in vivo, but within the retina effector function is held in check.

Upregulation of PD-1 on retinal T EM correlates with lack of effector function
If the T EM that accumulate in the retina during the persistent phase of disease are responsive to local Ag, their lack of effector func-tion must have some cause. There are well-known instances in which CD8 + Ag-specific T cells do accumulate in tissues without initiating their cytotoxic program, for example, in the presence of tumors (45). In our experiments, the T EM in the retina could be chronically stimulated by autoantigens through their TCR. One outcome of chronic stimulation in CD8 + T cells is the development of an exhausted phenotype, which is described both in viral infections and tumor models (46). This is associated with a lack of effector function and upregulation of surface markers, including PD-1 receptor (47,48). In T EM recovered from the retinas of mice with EAU, we do find upregulation of PD-1, which significantly increased from days 25 to 40, consistent with continuous local activation (Fig. 5A, 5B) (p , 0.01). Dual staining of PD-1 with cytokines showed that PD-1 expression correlates with a lack of IFN-g and TNF-a production along with CD107a and granzyme B, albeit to a lesser extent (Fig. 5C). Studying this PD-1 + population further, the majority of PD-1-positive cells were also CD69 + (5:1), whereas in the PD-1-negative compartment this ratio was lower (3:1). A small population of the PD-1 + CD69 + subset was also positive for CD103 (Fig. 5D). Ly6C, which aids cytolytic activity (49), segregated into the PD-1-negative population (Fig. 5E).

FTY720 treatment during persistent EAU retains T EM in tissue
The phenotype of the T EM accumulated in the retina during persistent EAU has many similarities with populations of T RM that FIGURE 4. Phenotype of retinal CD8 + T cells. Mice were immunized for EAU, and, at different time points, CD8 + T cells were examined for the indicated markers. After dissection, retinas were pooled together before analysis, at least three retinas per experiment. (A) Representative flow cytometry plots of CD8 + T cells isolated from the spleen (gray filled histograms) and the retina (black histograms). CD44 and CD62L markers were gated on CD8 + CD3 + cells; CD69, Ly6C, and CD103 markers were gated on CD8 + CD3 + CD44 high CD62L low populations. (B and C) Average cell percentage for CD8 + T cells prepared from the retina for surface markers (B) and cytotoxic markers and cytokine expression (C). Data represent at least three independent experiments at each time point. Data expressed as mean 6 SEM. *p , 0.05. arise following infection and localize in tissues such as the CNS, gut, lung, and the skin, where they are thought to provide local immune memory (24,25,27). These cells remain within their target tissues for extended periods of time without recirculating. To determine whether the T EM that we have identified in persistent retinal inflammation are resident, we used FTY720 (Fingolimod) treatment, which arrests lymphocytes in the secondary lymphoid tissues and prevents trafficking back to tissues (50,51). This leads to a decrease in lymphocytes within peripheral tissues and sites of inflammation, such as the brain and the retina, following FTY720 treatment (35,52,53). Mice with EAU were treated from day 35 postimmunization with 0.3 mg/kg FTY720 or control analog administered daily for 8 d before the CD8 + T cells in the retina were quantified. Treatment was associated with a significant decrease in EAU disease score (Fig. 6A) (p , 0.001). As expected, the numbers of CD8 + and CD4 + T cells were significantly reduced in the blood and significantly increased in the spleen, confirming that FTY720 interrupts the circulation of both CD4 + and CD8 + T cell populations (Fig. 6B). Analyses of the retinas of mice posttreatment with FTY720 (Fig. 6C) revealed a significant decrease in the number of CD4 + T cells and CD11b + macrophages compared with the analog control AAL149 (p , 0.001). In contrast, no significant decrease was shown in the overall CD8 + T cell population, indicating that their t 1/2 within the tissue was much greater than the CD4 + T cells. A subphenotype analysis of the CD8 + T cells remaining in the retina indicated that at least some of the CD8 + CD69 2 T cells were recirculating with kinetics similar to the CD4 + cells but that the CD69 + CD8 + population was not (Fig. 6D).

Retinal CD8 + T cells control local infiltrate
FTY720 treatment reduces clinical disease, despite the persistence of CD8 + T cells within the retina; this is further evidence that these cells are not causing tissue damage. To determine other roles for these cells, we investigated whether the depletion of CD8 + T cells would affect the progression of persistent EAU and retinal cell in-filtrate. Mice with EAU were given one treatment of either 250 mg CD8-depleting mAbs or an isotype control on day 35 postimmunization; 4 d later, retinal cell infiltrate was quantified by flow cytometry. As expected after treatment with depleting Abs, few CD8 + T cells were seen in the periphery and the retina (Fig. 7B, 7C). Although depletion of CD8 + T cells revealed little change in disease scores (Fig. 7A), a significant increase in total retinal infiltrate was observed (p , 0.05) (Fig. 7D). Treatment was associated with an increase in total CD45 + leukocytes, comprising CD4 + T cells and CD11b + macrophages. We conclude that the removal of T EM in the retina provokes an influx of pathogenic CD4 + T cells and CD11b + cells, implying that exhausted tissue-resident T EM play a role in limiting local disease.

Discussion
In the current study, we report that CD8 + T cells that populate the inflamed retinal tissue late in the course of EAU are of T EM  phenotype, are Ag experienced, and exhibit features comparable with a chronically stimulated T RM phenotype. These cells accumulate as disease progresses and have a long t 1/2 in the tissue, as indicated by their retention following blockade of T cell circulation by FTY720. Depletion of this resident population from the retina leads to an influx of CD4 + T cells and CD11b + macrophages. These data support the proposal that, during the primary peak of EAU, CD8 + T EM migrate to the retina, where they encounter a specific microenvironment, which over time drives functional changes, including the upregulation of CD69 and PD-1, thereby allowing retention and accumulation of these Ag-specific cells in an exhausted state within the tissue, where they regulate local cell infiltrate.
We have established that there are changes in the levels of TCR and CD8 expression consistent with Ag-specific activation (40,41). Testing Ag-specific activation confirms that a proportion of retinal resident CD8 + T cells responds to autoantigens, but they do not proliferate and they survive poorly when removed from the retina and cultured in IL-2 (data not shown). This is consistent with previous work demonstrating that T RM do not survive well when removed from the brain (25). The selective accumulation of CD8 + T cells over time, despite similar levels of tissue inflammation and equivalent levels of CD4 + T cell infiltration, argues for a cognate recruitment process. Elegant studies in a diabetic NOD mouse model (54) demonstrate the absolute requirement of autoantigen recognition to the recruitment of CD8 + T cells to inflamed islet tissue. However, in other systems, only a very small number of autoantigen-specific cells is necessary for diabetes to develop (55). Taking the level of PD-1 activation as a sign of recent cognate interaction, we suggest that at least 40% of the T EM are likely to be specific for autoantigen, rather than retained by bystander recruitment to the retina. However, without the development of Agspecific class I tetramers and a complete understanding of epitope spreading, a precise accounting of cell numbers within the retina remains infeasible.
CD8 + T cell accumulation is a recognized feature of uveitis, but the role of these cells in the disease process is poorly defined (18). We now show that the CD8 + T cells that accumulate in the tissue during the persistent phase of disease ( Fig. 1) are related to the T RM cells that have been described in epithelial and neuronal tissues after viral infection (25,32). We exploited previous findings that showed effective reduction in CD4 + T cells and CD11b + macrophages in the retina within 24 h of treatment with FTY720 (35), revealing that most CD8 + T cells are retained. This is a novel observation that in a persistent autoimmune inflammatory setting CD8 + T cells have a prolonged tissue residency in contrast to their CD4 + T cell counterparts (Fig. 6). There is also a degree of heterogeneity in the CD8 + T cell populations within the retina; those that expressed CD69 were less affected by FTY720 treatment than CD69 2 CD8 + T cells. In the lymph node, upregulation of CD69 inhibits the exit of T cells mediated by the sphingosine 1-phosphate receptor 1 acting as a retention signal (56). Furthermore, expression of CD69 has a role in promoting the migration and retention of virus-infected CD8 + T cells to the lung, as cells lacking CD69 failed to traffic to the tissue efficiently (28). Therefore, the relative unresponsiveness of the CD69 + CD8 + T cell population to FTY720 treatment in late EAU may implicate CD69 as the mediator of retinal T EM persistence. Whether retinal T EM are replenished from circulating memory cells over longer periods of time is yet to be explored.
Noteworthy differences were observed in the retinal T EM compared with their peripheral counterparts (Fig. 4A, 4B), including the upregulation of CD69, which is consistent with reports following acute LCMV infection (23). CD103 expression on pe-ripheral T EM was minimal, but was expressed on ∼25% of the retinal T EM , but, unlike the increase established on CD8 + T RM in other tissues (23), the expression was not upregulated in situ as disease progressed, rather it declined slightly. A similar pattern was seen for Ly6C with downregulation of this receptor segregating predominantly into the PD-1 + compartment. Moreover, an increase of PD-1 expression was seen over the course of disease and was associated with cells that lacked evidence of cytokine production and cytotoxic effector function (Fig. 5C).
T RM can be identified by their expression of both CD69 and CD103 (24,25,27). CD103 interaction with its ligand, Ecadherin, has been shown to promote CD8 + T cell retention in the tissue (25,28), and E-cadherin is present in the retina (57), but our data show that in persistent EAU CD103 is not required for CD8 + T cells to be retained, as both CD103 + and CD103 2 cells remain in the retina after treatment with FTY720 (data not shown). However, it has been noted that persistent Ag stimulation prevents upregulation of CD103 expression in a chronic LCMV setting (24). Therefore, we conclude that chronic stimulation in the tissue leads to the difference in CD103 expression compared with T RM initiated by acute viral infection.
In tissues such as the skin or gut, Ag is not required for the retention of T RM and nonspecific inflammation is enough to produce these cells in other nonlymphoid tissues (24,32). This implies that some tissue-specific T RM are more influenced by their microenvironment than others; TGF-b can induce a T RM phenotype in peripheral effector memory cells in ex vivo culture, and in vivo loss of TGF-b reduces the number of CD69 + CD103 + cells in the small intestine (24). However, in studies of T RM in tissues such as the brain, which is isolated by a blood barrier, local Ag is essential (24,25). Hence, the differentiation or maintenance of CD8 + T RM in peripheral tissue seems to be dependent on location (31). Whether our retinal T EM require Ag to survive or are controlled by the microenvironment was not addressed directly in this study.
PD-1 is widely used as a marker of T cell exhaustion and its blockade has been successful in experimental and clinical therapy for tumors (58,59). T cell exhaustion due to chronic viral infections or tumor immunology leads to a hierarchical loss of function in effector CD8 + T cells (46,60). Reversal of this exhausted phenotype, by blocking PD-1 interactions, results in an increase in viral clearance and reduced tumor growth (48,(61)(62)(63). Some exhausted T cells also coexpress other inhibitory molecules such as TIM-3 and LAG-3, and blockade of these inhibitory receptors in unison with PD-1 can enhance the restoration of functionality (61,64). Furthermore, it has been shown that complex coexpression of these inhibitory receptors exists depending on the severity of viral infection (61). We found negligible expression of TIM-3 and LAG-3 on the T EM in the retina (data not shown), which indicates an intermediate level of exhaustion in this model.
Expression of PD-1 is found constitutively in retinal neurons, and with active retinal inflammation the ligand programmed cell death ligand 1 (PD-L1) is upregulated (65,66). Similarly, PD-L1 levels on astrocytes and microglia in the brain are elevated during disease (67). Interestingly, under inflammatory conditions, retinal pigment epithelium expresses PD-L1 and suppresses T cell activation in vitro (68).
Finally, there is the question of whether exhausted T EM tissueresident cells have a functional role. Depleting these cells leads to a significant increase in the number of retinal CD4 + T cells and macrophages, and the number of cells recruited is much greater than the number of CD8 + cells that are lost. This suggests that CD8 + T cells may play a part in regulating the level of cellular infiltrate. This might be through engagement of PD-1 by other cells locally. PD-1 signaling can limit IFN-g production and proliferation (69), as well as control regulatory T cells (66). Alternatively, other less well-defined mechanisms may be involved. Taken together, the data indicate that retinal resident T EM are a potential source of local regulation during persistent intraocular inflammation.