Abstract
CD4+CD25+regulatory T cells (Treg) can inhibit a variety of autoimmune and inflammatory diseases, but their involvement in regulating virus-induced immunopathology is not known. We have evaluated the role of Tregin viral immunopathological lesion stromal keratitis. This frequent cause of human blindness results from a T cell-mediated immunoinflammatory response to HSV in the corneal stroma. The results show that lesions were significantly more severe if mice were depleted of Treg之前感染。Tregwas also shown to modulate lesion expression induced by adoptive transfer of pathogenic CD4+T cells in infected SCID recipients. The mechanism of Tregcontrol of stromal keratitis involved suppressed antiviral immunity and impaired expression of the molecule required for T cell migration to lesion sites. Interestingly, Tregisolated from ocular lesions in nondepleted mice showed in vitro inhibitory effects involving IL-10, but were not very effective in established lesions. Our results decipher the in vivo role of Tregin a virus-induced immunopathology and imply that manipulation of regulatory cell function represents a useful approach to control viral-induced immunoinflammatory disease.
Ocular infection with HSV can result in an immunoinflammatory lesion in the cornea which represents the most common infectious cause of blindness in the western world (1).Animal model studies have revealed that the blinding inflammatory lesions are orchestrated by CD4+T cells that principally generate type 1 cytokines (2).然而,身份啊f target Ags which drive the immunopathology remains unclear (3).病变在一些动物解决without treatment and in such cases the cytokine IL-10 is up-regulated (4).In addition, expression of IL-10 or TGF-β early in the experimental disease may alleviate lesion severity (5,6).Whether or not dedicated cell types that produce anti-inflammatory cytokines enter the cornea and regulate responses has not been explored. Thus, conceivably this viral-induced immunopathology, like several autoimmune lesions, might be subject to control by regulatory T cells (Treg)3such as the CD4+CD25+Tregpopulation described initially by Sakaguchi et al. (7,8,9).To date, the role of CD4+CD25+Tregin the pathogenesis of viral immunopathological disease has not been reported.
Our results demonstrate a major role for CD4+CD25+Tregcells in the pathogenesis of murine herpetic stromal keratitis (HSK). We demonstrate that Tregdepletion before infection results in lesions of greater severity and the induction of disease with lower infecting doses of virus. However, the role of CD25+Tregin HSK pathogenesis was complex. One effect was to minimize the extent of immunopathogenic CD4+T cell induction, whereas another was to limit the migration of pathogenic T cells to the tissue inflammatory site. The CD25+Tregwere present in the ocular inflammatory sites, could be recovered from the site, and were shown to express IL-10-dependent inhibitory effects on activated CD4+T cells in vitro. Our results indicate that CD25+Tregplay a beneficial role to minimize viral immunological lesions and imply that procedures that enhance their function may prove therapeutically beneficial.
Materials and Methods
Mice and virus
Female 5- to 6-wk-old C57BL/6, Thy1.2+BALB/c, and BALB/c SCID (H-2d) mice were purchased from Harlan Sprague Dawley (Indianapolis, IN) and Thy1.1+BALB/c mice were a kind gift from Dr. D. Woodland (Trudeau Institute, Saranac Lake, NY) and housed in the animal facilities at the University of Tennessee. SCID mice were kept in our special pathogen-free facility where all food, water, bedding, and instruments were autoclaved and all manipulations were done in a laminar flow hood. All experimental procedures were in complete agreement with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. HSV-1 RE was propagated and titrated on Vero cells (ATCC CCL81) using standard protocols. The virus was stored in aliquots at −80°C until use.
Abs and reagents
PC61 (anti-CD25) hybridoma was purchased from American Type Culture Collection (Manassas, VA) and Ab was grown in tissue culture roller bottles. For all injections, an ammonium sulfate cut of PC61 mAb (1.0 mg/mice) was used. Abs purchased from BD PharMingen (San Diego, CA) were FITC, PE, and biotin anti-CD4, FITC anti-CD25 (7D4), PE-anti-CD25 (PC61), CD62 ligand, CD44, CD69, CD49d, CTLA-4, streptavidin-PerCP-Cy5.5, and ELISA Abs such as anti-IFN-γ, anti-IL-2, anti-IL-10, and their biotinylated counterparts.
In vivo depletion, corneal HSV-1 infections, and clinical observations
BALB/c mice were given 1.0 mg PC61 mAb i.p. 3 days before corneal infection. Initial kinetics of depletion was studied in uninfected mice. Corneal infections of PC61-treated or nontreated BALB/c mice were conducted under deep anesthesia. Mice were scarified on their corneas with a 27-gauge needle, and a 3-μl drop containing the required viral dose was applied to the eye. The eyes were examined on different days postinfection (p.i.) with a slit-lamp biomicroscope (Kowa, Nagoya, Japan), and the clinical severity of keratitis of individually scored mice was recorded. The scoring system was as follows: +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity, but iris visible; +4, opaque cornea, iris invisible; and +5, necrotizing SK.
Immunofluorescence and immunohistochemical staining
Eyes were frozen in OCT compound (Miles, Elkart, IN) at different days p.i. Six-micrometer-thick sections were cut, air dried, and fixed in cold acetone for 5 min. The sections were then blocked with 3% BSA and stained for the presence of CD4+T cells and neutrophils with their respective mAbs. For immunohistochemistry, the biotinylated primary Abs were used followed by HRP-conjugated streptavidin, (1/1000; Jackson ImmunoResearch Laboratories, West Grove, PA) and 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA). In the case of immunofluorescence, the frozen sections were fixed in 4% paraformaldehyde and the primary Ab was labeled with FITC. Finally, sections were washed with PBS and mounted with Vectashield mounting medium with propidium iodide (Vector Laboratories).
Flow cytometric analysis
Cell preparation.
Single-cell suspensions were prepared from the cornea, draining lymph nodes (DLN), and spleen of mice at different time points p.i. Corneas were digested with Liberase (Roche Diagnostics). Briefly, a small incision was made at the junction of the limbus and the corneal cap was carefully removed. The pooled corneas were incubated with 60 U/ml Liberase for 60 min at 37°C in a humidified atmosphere of 5% CO2。After incubation, the corneas were disrupted by grinding with a syringe plunger on a stainless steel mesh and a single-cell suspension was made in complete RPMI 1640 medium. Cells were counted with trypan blue exclusion with high viability.
Staining for flow cytometry.
The single-cell suspension obtained from DLN, and corneal samples were stained for different cell surface molecules for FACS. Briefly, a total of 1 × 106cells were first blocked with an unconjugated anti-CD32/CD16 mAb for 30 min. in FACS buffer followed by addition of fluorochrome-labeled respective Abs for an additional 45 min. For intracellular staining, surface-labeled cells were permeabilized with permeabilization buffer (BD Biosciences, Mountain View, CA) and fluorochrome-labeled mAb was added. Finally, the cells were washed three times and samples were acquired on a FACScan (BD Biosciences). The data were analyzed using the CellQuest 3.1 software (BD Biosciences).
Cytokine ELISA
The culture supernatants from the bulk test cultures without addition of any exogenous cytokines were screened for the presence of IL-10 and IL-2 and IFN-γ. Briefly, ELISA plates were coated with capture Abs (2.0 μg/ml) in 0.1 M Na2HPO4(pH 9.0) for the respective cytokines and incubated at 4°C overnight. The plates were washed with PBS-Tween 20 and blocked with 3% BSA for 2 h at room temperature. After washing, serially diluted samples and standards were added to the plate and incubated at 4°C overnight. The plates were washed followed by the addition of cytokine-specific detection Abs for 2 h. Finally, peroxidase-conjugated streptavidin (Jackson ImmunoResearch) was added at 1/1000 dilutions. The color was developed by adding the substrate (ABTS) solution (Sigma-Aldrich, St. Louis, MO) and the concentration was calculated with an automated ELISA reader (SpectraMAX 340; Molecular Devices, Sunnyvale, CA).
Quantification of IL-2- and IFN-γ-secreting cells by ELISPOT
The ELISPOT assay was used for quantification of cytokine-producing cells as reported earlier (11).Briefly, ELISPOT plates (Millipore, Molseheim, France) were coated overnight at 4°C with anti-mouse IL-2 and IFN-γ Ab in 0.1 M Na2HPO4(pH 9.0). CD4+从virus-infe获得的T细胞(应答细胞)cted mice were mixed with syngeneic splenocytes (stimulator cells) pulsed earlier with UV-inactivated HSV (multiplicity of infection, 5.0) for 3 h along with 10 U/well human IL-2. Coincubation of the responder and stimulator cells was continued for 48 h at 37°C. The ELISPOT plates were washed three times with PBS and three times with PBS-Tween 20 (PBST), followed by addition of biotinylated IL-2 and IFN-γ Ab (1 μg/ml) for 1 h at 37°C. The spots were developed using nitroblue tetrazolium (Sigma-Aldrich) and 5-bromo-4-chloro-3-indolylphosphate (Sigma-Aldrich) as a substrate following incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch) for 1 h and counted 24 h later under a stereomicroscope.
CD4+CD25+Tregand CD4+CD25negT cell purification and adoptive transfer into BALB/c SCID mice
CD4+CD25+Treg
+T细胞。CD4+CD25+Tregwere then purified by incubating the enriched CD4+T cells obtained from the spleen and lymph node with biotin-conjugated anti-CD25 (15 μg/108cells) mAb in PBS-2% FCS for 15 min at 4°C. Positive magnetic separation was then performed with LS+columns (Miltenyi Biotec, Auburn, CA) according to the suggested protocol. The purity of cells ranged from 85–95%. The purified naive CD4+CD25−T细胞过继转移到SCID小鼠either alone or with CD25+Treg24 h before virus infection. Immune responses were analyzed 12 days p.i. Primed CD4+CD25−T cells were also purified by the same procedure 4 wk after HSV (i.p) infection. In some experiments, corneal CD4+CD25+T cells were purified by FACS sorting. Briefly, 32 corneal samples were collagenase digested and MACS-purified CD4+T cells were sorted into CD25+and CD25−fractions. The purity of sorted fractions was >95%.Proliferation assay
The purified CD4+CD25+Tregor CD4+CD25negT cells (5 × 103/well) were cultured with soluble anti-CD3 mAb (1.0 μg/ml) along with irradiated spot-forming cells (SPC) in the presence or absence of IL-2 (20 U/ml) for 72 h in 96-well round-bottom plates. To assess suppression by CD25+Treg, a graded number of Tregwere added into the anti-CD3 mAb-stimulated culture of CD4+CD25negT cells (104cells/well) in the presence of irradiated SPC. In one particular experiment, anti-IL-10R mAb (10 μg/ml) was added to a CD25+Treg(104/well) and CD25negT cell (104/well) coculture in the presence of anti-CD3 and irradiated SPC. Finally, [3H]thymidine (ICN Radiochemicals, Irvine, CA) was added 1 μCi/well for the last 18 h. The plates were harvested and were read using a beta scintillation counter (Trace 96; Inotech, Wohlen, Switzerland). The results were expressed as mean cpm ± SD.
Real-time quantitative PCR
The total cellular RNA was isolated form corneas by using an RNeasy protect mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction. All samples were treated with RNase-free DNase (Qiagen). The extracted RNA was reverse transcribed using oligo(dt) primers and reverse transcriptase enzyme (Promega, Madison, WI) according to standard protocol (4).The cDNA obtained was used as a template for real-time quantitative PCR and real-time PCR was performed using a Quantitect SYBR green PCR kit (Qiagen). Initially, mRNA levels were normalized to the GAPDH mRNA level. PCR primer pairs used for GAPDH were 5′-GCC TGC TTC ACC ACC TTC TTG ATG-3′ and 5′-CAT CCT GCA CCA CCA ACT GCT TAG-3′. Foxp3 expression was measured by using primers 5′-GGC CCT TCT CCA GGA CAG A-3′ and 5′-GCT GAT CAT GGC TGG GTT GT-3′. Briefly, for real-time PCR, a standard curve was generated using the PCR product of Foxp3 cloned into a pCR-XL-TOPO vector (Invitrogen, San Diego, CA). After spectrophotometric determination of the plasmid DNA concentration, the copy number was calculated using the following formula: (Xg/μl DNA/(plasmid length in bp × 660]) × 6.022 × 1023=Ymolecules/μl. Test samples were run in triplicate with a dilution series and the mean value of a particular dilution was used. Data are expressed as copy number per sample.
Statistical analysis
All analysis for statistically significant differences were performed with a Student’s pairedttest. Values ofp< 0.01 andp< 0.05 were considered to be statistically significant. Results are expressed as mean ± SD.
Results
CD4+CD25+Tregdepletion increases lesion severity
BALB/c mice were treated with the PC61 mAb using conditions in which >90% of CD4+CD25+cells were depleted by day 3 and ∼40–50% depleted at 18 days after depletion (data not depicted). Three days posttreatment, mice were infected on the ocular surface with different doses of HSV-1 and the severity of their stromal keratitis (SK) lesions were compared with age-matched control-infected mice treated with isotype control rat Ig. The results of a representative experiment are shown in Fig. 1⇓a。As is evident, CD25-depleted animals developed more severe lesions, an effect most apparent in animals infected with a lower dose (104PFU/eye) of virus. In this instance, 60% of eyes from depleted animals had lesion severity scores of 3.0 or greater on day 15 p.i. compared with none in the nondepleted animals (p< 0.01). A similar pattern of results was noted when comparing lesion severity in the HSV-resistant C57BL/6 mouse strain (Fig. 1⇓b).In these experiments, CD25+Tregdepletion resulted in approximately a 10-fold greater susceptibility to ocular HSV infection with an infecting dose of virus (105PFU/eye) producing lesions in depleted, but not in undepleted animals.
Lesions in Treg-depleted mice demonstrate enhanced CD4+T cell infiltration and an altered phenotype
SK is well known to be orchestrated by CD4+T cells (1).As shown in Table I⇓, the percentage of CD4+T cells recovered by collagenase digestion of corneas was greater in Treg-depleted animals at all time points tested after ocular HSV infection. Sample animals evaluated by immunocytochemistry at the time of peak lesions (day 15) are shown in Fig. 2⇓a。Increased T cell infiltration was notably more evident in sections from Treg-depleted animals.
In other experiments, the phenotype of CD4+T cells isolated from the DLN as well as from corneal lesions after collagenase digestion was evaluated on both days 9 and 15 p.i. No significant difference in the average phenotype between Tregdepleted and controls was noted for the following markers: CD62 ligand, CD69, and CD44 (data not depicted). However, a significant increase of DLN CD4+T cells from Treg-depleted mice expressed the integrin CD49d (very late Ag 4 (VLA-4)) at both time points (Fig. 2⇑b).Moreover, on day 15, the ocular CD4+T cell population of depleted animals also expressed an ∼2-fold increase in CD49d+CD4+T细胞。Additional experiments were conducted to compare the effector functions of infiltrated ocular CD4+T cells in the nondepleted and Treg-depleted mice. On day 15 p.i., cells isolated from collagenase-digested corneal tissue were stimulated with anti-CD3 mAb in the presence of IL-2 for 16 h. As shown in Fig. 2⇑c, the percentage of ocular CD4+T cells express more IFN-γ in the CD25-depleted group compared with the nondepleted group. Taken together, we interpret these observations to mean that after viral infection, Tregmay control the expression of molecules such as CD49d that are involved in migration of inflammatory cells to the ocular lesion site (10).Furthermore, once effector cells reach the cornea they function more effectively if not hampered by the presence of Treg。
Tregdepletion results in enhanced immune responsiveness to HSV
Another explanation for heightened lesion severity of Treg-depleted animals could be that these animals developed enhanced HSV-specific immune responses. As demonstrated in Fig. 3⇓, specific responses of both splenic and DLN CD4+T cell were ∼2-fold higher in Treg-depleted animals compared with controls as measured by ELISA and ELISPOT analysis for both IL-2 and IFN-γ. Thus, as noted previously with the specific CD8+T cell response to HSV (11), Tregdepletion also results in elevated CD4+T cell immunity to HSV.
Tregsuppress pathology by CD4+CD25negT cells
Mice lacking T cells fail to express SK upon ocular infection with HSV, but readily do so if given adoptive transfers of CD4+T cells (12).To assess the influence of Tregon this form of immunopathology, CD25+T cells were isolated from naive BALB/c mice and transferred into SCID recipients along with a population of CD4+CD25negT cells from either naive or animals primed 5 wk previously by HSV infection. Mice were then infected ocularly with HSV. The results expressed in Fig. 4⇓aindicate that the lesions induced by both naive and primed CD4+CD25negT cells were reduced in severity if animals additionally received Treg。The effect was greater against the naive cells than against the primed population, but in both instances the differences were statistically significant (p< 0.01). In these experiments, the transfer of Tregalone failed to cause SK and animals in this group died earlier from HSV encephalitis than those in the other groups. Curiously, the adoptive transfer recipients of both Tregand CD25negT cells also had less neutrophil infiltration in their inflammatory lesions (Fig. 4⇓b).This might mean that the Tregadditionally function in the eye to limit innate aspects of immunity.
Mechanism of lesion suppression by Treg
The mechanism by which the adoptive transfer of CD25+Tregsuppressed lesion induction by CD25negprimed and naive CD4+T cells was further investigated and seen to have at least two explanations. First, as shown in Fig. 5⇓a, cotransfer of Tregwith CD25negT cells resulted in significantly diminished Th1-type immune responses in viral Ag-stimulated splenocytes. This effect was more evident against the response of naive CD25negcells (∼4-fold reduction) than against primed cells (∼2-fold reduction). In neither group could Ag-induced IL-10 production be demonstrated in splenocytes (data not depicted). Such results indicate that the Tregserved to inhibit the induction of HSV immunopathogenic CD4+T cells involved in lesion expression.
A second effect observed in the recipients of CD25+Tregwith suppressed lesions was a change in the average phenotype of the CD4+CD25negpopulation in lymphoid tissues compared with controls not receiving Treg。In such experiments, recipient SCID mice were given adoptive transfers of Thy1.1 Tregalong with Thy1.2 HSV-primed CD4+CD25negT cells isolated 5 wk p.i. Animals were ocularly HSV infected 1 day later and animals were sacrificed after an additional 12 days to measure the phenotype of CD4+T cells in spleens.
In these experiments, lesion severity was greater in control animals given only CD25negT cells compared with those that additionally received Treg(data not depicted). FACS analysis of splenocytes from animals in the two groups revealed that the adoptively transferred Thy1.2 cells showed phenotypic differences. Thus, whereas 49.0% of Thy1.2 cells in recipients of only CD25negT cells were positive for CD49d, those that additionally received Treghad significantly fewer CD49d-positive Thy1.2 cells (19.0%; Fig. 5⇑b).In addition, random corneal samples were taken from both groups and either stained by immunohistochemistry (Fig. 5⇑c) or collagenase digested to quantitate the influx of CD4+T cells (Fig. 5⇑d).The presence of Tregstrongly diminished the number of CD4+T cells in virus-infected corneas. Similar results were observed when naive CD4+CD25negT cells were adoptively transferred with Treginto SCID mice (data not depicted). Since ∼70% of ocular CD4+T cells express CD49d, at least in the early phase of SK lesions (Fig. 2⇑b), we interpret these experiments to indicate that Tregmay down-regulate CD49d in lymphoid organs and that in consequence fewer cells were able to migrate to the ocular damage site. Interestingly, we also observed that adoptively transferred CD4+CD25+regulatory T cells expressed very high levels of CD49d (data not shown), a molecule involved in the migration of cells to the eye (10).
CD4+CD25+Tregare present in SK lesions
In autoinflammatory lesions that are subject to control by CD25+Treg, it remains unclear whether Tregare present in lesions and exert their effects at such extralymphoid tissue sites or whether they act only in lymphoid tissues (13,14).Two approaches indicated that in the SK model, Tregwere present in tissue lesions. The first approach took advantage of the observation that CD25+Tregexpress higher levels of intracellular CTLA-4 and lower cell surface expression of CD45RB (15).At 15 days p.i., corneal inflammatory cells were recovered by collagenase digestion and analyzed by FACS for their CD4, CD25, CD45RB, and intracellular CTLA-4 phenotype. In populations from undepleted animals, an average of 35% of CD4+cells were CD25+and all expressed higher levels of CTLA-4. Additionally, they possessed lower levels of CD45RB (Fig. 6⇓a).However, in ocular cells of CD25-depleted animals, an average of 12% of CD4+cells were CD25+and they expressed significantly lower levels of CTLA-4 and higher levels of CD45RB than cells from intact animals (Fig. 6⇓a).These results indicate that Tregmay migrate to the site of inflammation in virus-infected undepleted animals.
A second approach to detect Tregin lesions involved isolation of RNA from the corneas of infected depleted and nondepleted mice and to quantify by real-time PCR the levels of mRNA of transcription factor Foxp3. This molecule was recently shown to be present in high levels in CD25+Treg(16,17).Foxp3 expression was readily detectable in samples from virus-infected undepleted mice, but were significantly less in material from depleted mice even though such samples were obtained from mice with significantly more severe lesions (Fig. 6⇑b).Furthermore, a comparative analysis of FoxP3 expression in the DLN and corneal tissue from the same HSV-infected mouse exhibited higher copy number of FoxP3 at the site of inflammation (Fig. 6⇑b).Such results add further evidence that Tregare present in lesions of undepleted mice.
Role of lesion CD4+CD25+Tregin controlling SK
To seek evidence that the lesion Tregin undepleted animals expressed immunomodulatory activity, cells were collected and fractionated into CD4+CD25negand CD4+CD25+T细胞。t细胞分数被检测heir ability to proliferate in response to a polyclonal stimulant, soluble anti-CD3 mAb. The CD25negpopulation underwent extensive proliferation but the CD25+cells proliferated only when exogenous IL-2 was added (Fig. 7⇓a).Furthermore, the addition of graded numbers of ocular CD25+cells to a fixed number of ocular CD25negresponders showed a dose-dependent inhibition of anti-CD3-driven proliferation of the CD25negcells (Fig. 7⇓b).
Additional experiments were also done in which anti-IL-10R mAb was added to the cell mixtures and the level of suppressed proliferation was compared with the CD25+and CD25negmixture without Ab. As shown in Fig. 7⇑d, this resulted in partial abrogation of the suppression, which likely means that the regulation proceeded via IL-10 secretion. Further support for this came from an experiment where anti-CD3 mAb stimulation of lesion derived CD4+CD25+, but not CD4+CD25negT cells, resulted in detectable IL-10 secretion measured by ELISA (Fig. 7⇑c).Taken together, these results indicate that the Tregpresent in SK lesions do act, at least in vitro, to modulate the function of CD25negT cells and that this may proceed by an IL-10-mediated mechanism.
Discussion
This investigation demonstrates that CD25+CD4+Tregcells notably influence the expression of a viral-induced immunopathological disease. Using a mouse model of the blinding immunoinflammatory disease, SK, our results show that lesions are significantly more severe and animals become more susceptible to infection if depleted of CD25+Treg之前感染。Increased lesion severity was associated with a greater influx of CD4+T cells into the cornea. In an adoptive transfer model of disease, the cotransfer of Tregwith primed or naive pathology inducing CD4+CD25negT cells reduced lesion severity and diminished the Ag-specific cytokine response of splenic CD4+T细胞。In addition, Tregrecipients had significantly fewer splenic CD4+T cells that expressed CD49d (VLA-4), an integrin likely involved in T cell migration to the ocular inflammatory site. Interestingly, in undepleted animals, Tregcould be demonstrated in the inflamed corneas and CD4+CD25+cells isolated from such corneas inhibited the proliferation of polyclonal stimulated CD25negT cells, an effect impaired by anti-IL-10R mAb. These results are the first to indicate a role for Tregin viral-induced immunopathology and imply that manipulation of regulatory cell function might represent a useful approach to control viral-induced immunoinflammatory disease.
The surge of recent interest in regulatory cells has primarily focused on disease models that involve autoreactivity (18,19).However, increasing reports document that Tregalso influence immunity to several pathogens (11,20,21).Thus, we and others have shown that the absence of a Tregresponse may result in heightened immunity which includes a more durable memory response (11,22).然而,提高免疫reacti的缺点vity might be immunopathogenesis. Indeed, the initial description of CD25+Tregcell function emerged from studies showing that their absence resulted in organ-specific autoimmune diseases in an adoptive transfer system (7).The SK model represents a situation where normally immunoprotective CD4+T cells exert an immunopathological function in the special environment of the eye’s cornea (23).Thus, for reasons still unclear, corneal infection with HSV sets off an inflammatory reaction that usually fails to fully resolve, even long after viral replication has ceased and viral Ags are no longer detectable (24).Such observations have led to the suggestion that SK ultimately becomes an autoinflammatory lesion (25), but this attractive hypothesis remains to be substantiated.
Our observation that the severity of SK lesions was influenced by Tregwas supported by the observation of more severe lesions in Treg-depleted animals, as well as by noting a modulatory effect of Tregon lesions induced by pathogenic CD4+T cells in an adoptive transfer system. Our results suggest that Tregmay play a pivotal role in microbial pathogenesis, especially in situations that involve chronic inflammation and persistent infection. Other examples have also been recently reported, especially with parasitic infections (26,27).Thus, withLeishmaniainfection in the susceptible BALB/c mouse strain, Tregappeared to limit the extent of T cell-mediated inflammatory disease (28).This is similar to the circumstances we described for SK as well as forPneumocystis carnii-induced lung pathology in mice (21).However, in resistant mice the Tregplays a crucial role in maintaining concomitant immunity toLeishmania(20).Accordingly, Treglimit the protective effect of CD4+Th1 cells, resulting in a balance that permits parasite persistence, immunity to reinfection, and an absence of lesions. Removal of Tregresults in parasite clearance but susceptibility to infection. Clearly, manipulating Tregfunction in chronic microbial infections could represent a valuable approach to disease management.
Although our results substantiate that Tregmodulate the severity of SK lesions, we can only speculate how this process is accomplished. We anticipate that the effects are multiple. They include inhibition of virus-specific CD4+T cell induction, the cells that help orchestrate SK lesions (3).Accordingly, the presence of Treglimited the extent of CD4+T cell immunity, as was shown previously to occur with CD8+T cell responses (29).In immunocompetent mice, these inhibitory effects were modest. Greater effects were evident in an adoptive transfer model in which Tregwere transferred along with naive or primed CD4+CD25negresponders and HSV-specific immunity was compared with recipients that received responder cells only. In such experiments, the HSV-specific CD4+T cell response was inhibited by up to 4-fold in animals that received Treg。Thus, one explanation for the Tregeffect on SK lesion expression was immunosuppression, an effect likely mediated at the lymphoid sites of immune induction.
A second effect accounting for reduced lesion severity could be that Treg, perhaps as a consequence of activation by HSV as was shown to occur previously (11), could serve to modulate the expression of one or more homing molecules involved in T cell migration to the ocular inflammatory site. Although the signature of homing molecules involved in ocular migration remains unknown, there is evidence that the integrin VLA-4 is involved in the process (10).In addition, in SK 70–80% of corneal T cells in disease express VLA-4 early in lesion development. Thus, it was intriguing to observe that the VLA-4 integrin was expressed on a significantly lower percentage of lymphoid CD4+T cells when these were activated in the presence of Treg。Our observations could mean that Tregalso act to control lesion development by limiting the migration of pathogenic T cells to the extralymphoid inflammatory site. Such concepts are now under further investigation.
A final effect of Tregon SK may be that such cells also function at tissue sites to limit inflammatory events. Whether or not Tregact at nonlymphoid or lymphoid sites (or both) remains an unresolved issue. In some systems where Tregeffects have been noted, the regulatory cells cannot be demonstrated in lesions (14).In others they are present, but it was not clear whether this was the exclusive site for their activity. For example, in the inflammatory bowel disease model, Tregcould be demonstrated both in colon lesions as well as in the mesenteric lymph nodes, but whether they act at both sites was not evaluated (13).In the SK model, two lines of evidence indicated that Tregwere present in tissues. First, a high percentage of CD4+CD25+T cells isolated from lesions of undepleted mice also expressed high intracellular CTLA-4 and low cell surface expression of CD45RB, accepted markers for Treg(15).Additionally, taking advantage of the recent observation that the transcription factor Foxp3 is a marker product for Treg(16), we showed by real-time PCR abundant Foxp3 message only in the samples from nondepleted mouse lesions. Together, these approaches indirectly establish the presence of Tregin lesions, but do not indicate that they act to control immunopathology at the site.
Two lines of evidence indicated that such lesional Tregwere functioning in the SK system. First, the homing/activation marker VLA-4 was expressed on a significantly lower percentage of lesional CD4+T cells late after infection (15 days p.i.) in control compared with undepleted animals. This could mean that the Tregwere responsible for down-regulation. Second, CD25+T cells isolated from lesions at this late stage were shown to inhibit in vitro proliferation of polyclonal stimulated CD4+CD25negT cells isolated from the same lesions. This modulatory effect was partially inhibited by anti-IL-10R mAb, indicating that the Tregacted via IL-10 secretion as noted in some other systems (30,31).
Further studies are underway to directly demonstrate Treg在SK病变和决定是否和如何y control the immunopathology. We anticipate that the efficacy of such a function might vary as lesions progress. Conceivably, regulation may not occur in the early lesions when virus is still present because HSV infection is a potent stimulus for IL-6 production (32,33), and this cytokine was elegantly shown to render pathogenic T cells refractory to the effects of Treg(34).Furthermore, the corneal IL-6 concentrations have also been reported to decline during the later phase of HSK (35), the time when Tregmight help to resolve the inflammatory events. The kinetics of Tregingress and their control of SK lesions are under further investigations.
Acknowledgments
We thank Dr. Mark Sangster and Christopher D. Pack for helpful suggestions and Nancy Neilsen for technical support with flow cytometry.
Footnotes
↵1This work was supported by National Institutes of Health and National Eye Institute Research Grants AI-14981 and EYO5093.
↵2Address correspondence and reprint requests to Dr. Barry T. Rouse, Department of Microbiology, M409, Walters Life Sciences Building, University of Tennessee, Knoxville, TN 37996-0845. E-mail address:btr{at}utk.edu
↵3Abbreviations used in this paper: Treg, T regulatory cell; SK, stromal keratitis; p.i., postinfection; HSK, herpetic stromal keratitis; DLN, draining lymph node; SPC, spot-forming cell; VLA, very late Ag.
- ReceivedOctober 10, 2003.
- AcceptedJanuary 30, 2004.
- Copyright © 2004 by The American Association of Immunologists