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The Journal of Immunology, 2003, 171: 576-583.
Copyright © 2003 by The American Association of Immunologists

Blocking 4-1BB/4-1BB Ligand Interactions Prevents Herpetic Stromal Keratitis1

Su K. Seo*, Hye Y. Park*, Jae H. Choi*, Won Y. Kim*, Young H. Kim*, Hyo W. Jung*, Byungsuk Kwon*, Hyeon W. Lee* and Byoung S. Kwon2,*,{dagger}

* Immunomodulation Research Center, University of Ulsan, Ulsan, Korea; and {dagger} Louisiana State University Eye Center, Louisiana State University Health Sciences Center School of Medicine, New Orleans, LA 70112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Herpetic stromal keratitis (HSK) is a chronic inflammatory process in corneal stroma that results from recurrent HSV type 1 infection. We used the murine model of HSK to demonstrate the importance of the interaction between an inducible T cell costimulatory receptor, 4-1BB, and its ligand, 4-1BB ligand (4-1BBL), in the development of this disease. In BALB/c mice, HSK ordinarily induced by infection with the RE strain of herpes was prevented by blocking 4-1BB/4-1BBL interaction, either by deleting 4-1BB (in mutant 4-1BB-/- mice) or by introducing mAbs against 4-1BBL. The majority of T cells infiltrating the infected corneas were 4-1BB+ activated effector cells that expressed cell surface markers CD44, CD25, and/or CD62L, as well as chemokine receptors CCR1, CCR2, and CCR5, and a limited number of TCR V{beta} chains (V{beta}8.1/8.2, V{beta}8.3, V{beta}10b, and V{beta}5.1/5.2, in order of abundance). Analysis of cell surface phenotypes showed that the failure to develop HSK in the 4-1BB-/- mice was associated with a reduced expression of CD62L at the time of T cell migration into the corneal stroma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Herpetic stromal keratitis (HSK)3 is an inflammatory disorder induced by HSV-1 infection and characterized by T cell-dependent destruction of corneal tissues. Complications of HSK include corneal melting, glaucoma, and irreversible corneal scarring that often leads to blindness (1).

Several mechanisms have been proposed for the pathogenesis of HSK in mice. In corneas of BALB/c mice infected with the RE strain of HSV-1, Russell et al. (2) showed that HSK resulted from CD4+ T cells secreting IFN-{gamma}, IL-12, and other lymphokines typically produced by Th1 cells. Deshpande et al. (3, 4) showed that activated CD4+ T cells were required for the development of HSK, which occurred as a non-TCR-mediated event. On the basis of these studies, the authors proposed a bystander activation model of HSK. Other investigators found that mice carrying the IgHd/e alleles (i.e., C.AL-20) were susceptible to HSK when their corneas were infected with the KOS strain of HSV-1, whereas strains carrying the IgHb allele (i.e., C.B.-17 and C57BL/6) were resistant (5). However, when soluble IgG2ab was injected into C.AL-20 mice, they became tolerant and did not develop HSK. Zhao et al. (6) found that the IgG2ab-specific T cell clones also recognized a peptide sequence embedded in the HSV virion-associated protein UL-6. Infection with a mutant HSV-1 strain that was unable to express the UL-6 protein or had an altered UL-6 T cell epitope did not induce HSK, whereas wild-type control or gB-mutated KOS did produce the corneal disease (7). The results of these studies led to the proposal of an autoimmune mechanism in the pathogenesis of HSK.

HSK induced in mice by infection with the RE or KOS strain of HSV is marked by intense stromal inflammation that becomes evident on postinfection (PI) days 7–8, reaches a peak on PI days 14–21, and decreases thereafter (8). We demonstrate in this study that blocking 4-1BB/4-1BB ligand (4-1BBL) (CD137/CD137 ligand) interactions abrogates clinical symptoms, leukocyte infiltration, and induction of proinflammatory cytokines and chemokines in the HSV-infected cornea. These results suggest that the 4-1BB/4-1BBL pathway plays a critical role in the pathogenesis of HSK. Elucidation of the underlying mechanism that enhances T cell infiltration into the corneal stroma and the discovery of strategies to block T cell entry may lead to a new therapy for this leading infectious cause of human blindness.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

4-1BB-deficient mice were established as previously described (9) and backcrossed with BALB/c mice for more than nine generations. Wild-type BALB/c mice were obtained from Hyochang Bioscience (Tae-Ku, Korea). All mice were maintained under specific pathogen-free conditions in the animal facility of the Immunomodulation Research Center (University of Ulsan) and were used at 7–10 wk of age.

Antibodies

Anti-m4-1BBL mAb was used for in vivo neutralization. The hybridoma cells (TKS-1) were a kind gift from Drs. H. Yagita and K. Okumura (Juntendo University, Tokyo, Japan). The mAb was produced from ascites, purified by protein G column (Sigma-Aldrich, St. Louis, MO), and then tested on P815-m4-1BBL transfectants. Anti-m4-1BB (3E1), a kind gift from Dr. R. Mittler (Emory University, Atlanta, GA), was conjugated with FITC for detection of 4-1BB. The following Abs were purchased from BD PharMingen (San Diego, CA): PE-conjugated anti-mouse CD3 (145.2C11), PE-conjugated anti-mouse CD44 (IM7), PE-conjugated anti-mouse CD62L (MEL-14), biotin-conjugated anti-mouse CD25 (7D4), biotin-conjugated anti-mouse CD11b (M1/70), purified anti-mouse CD16/CD32 (Fc{gamma}III/IIR), streptavidin-PE, PE-conjugated hamster IgG, FITC-conjugated rat IgG2a, PE-conjugated anti-mouse {gamma}{delta} TCR (GL3) mAb, and a mouse V{beta} TCR screening panel that contained FITC-conjugated mAb against V{beta}2, -3, -5, -5.1 and -5.2, -6, -7, -8.1 and -8.2, -8.3, -9, -10b, -11, -12, -13, -14, and -17a TCRs.

Virus

The RE strain of HSV-1 was grown on CV-1 cells in MEM supplemented with 2% heat-inactivated FBS (HyClone, Logan, UT), penicillin (100 U/ml), and streptomycin (100 µg/ml). Virus was titered on CV-1 cells by plaque assay. Virus was aliquoted in 1-ml vials and stored at -70°C.

HSV-1 infection, HSK scoring, and mAb treatment

Mice were anesthetized with ketamine hydrochloride (1 mg/kg) and xylazine (0.5 mg/kg). Corneas were scarified with a 30-gauge needle in a criss-cross pattern and infected with 1 x 105 PFU of HSV-1 RE strain. Stromal keratitis was scored with a slit lamp biomicroscope or a surgical microscope (Olympus, Melville, NY) as follows: 0, normal cornea; 1.0+, edema and mild opacity and neovascularization; 2.0+, edema and moderate opacity and neovascularization; 3.0+, severe edema and opacity and neovascularization, iris not visible; and 4.0+, corneal perforation. Mice were injected i.p. with 200 µg of anti-mouse 4-1BBL mAb or rat IgG on PI days 0, 3, 6, and 8.

Immunohistochemistry

Inflamed eyes were excised at different times after infection and immediately frozen in optimum cutting temperature medium. Sections (7-µm thick) were cut at -20°C, stained with H&E, and observed for corneal thickness and numbers of infiltrating cells. For immunoperoxidase staining, sections were blocked with serum, incubated with biotin-conjugated anti-murine 4-1BB mAb (3E1) for 1 h, and washed in Tris-buffered saline (pH 7.4). Cells with bound Abs were detected with a peroxidase substrate kit (Vectastain ABC (avidin/biotin complex) kit; Vector Laboratories, Burlingame, CA). Sections were washed, counterstained with hematoxylin, and examined by light microscopy.

In situ TUNEL assay

In situ detection of DNA fragmentation was performed using terminal transferase (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions. Briefly, the sections were fixed in 4% paraformaldehyde in PBS (pH 7.4) and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 20 min at 4°C. The sections were then incubated with terminal transferase in reaction buffer for 1 h at 37°C and washed with PBS. Fluorescein-labeled DNA strands were detected by HRP-conjugated anti-fluorescein Ab. After substrate reaction (3-amino-9-ethylcarbazole; Vector Laboratories), stained cells were analyzed under light microscopy. For double fluorescence staining, frozen sections were fixed in 3.2% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature and blocked with Fc Block (2.4G2; BD PharMingen) containing 10% normal mouse serum for 30 min at room temperature. Sections were incubated with the biotin-conjugated anti-CD3 (145.2G11) for 1.5 h, washed in PBS, and incubated with PE-conjugated streptavidin for 30 min. To detect DNA fragmentation, the sections were incubated with terminal transferase for 1 h at 37°C and mounted in Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL). Staining was visualized with a laser scanning confocal microscope (FV500; Olympus).

RNase protection assay (RPA)

Total RNA was isolated from mouse corneas (n = 5) using Fenozol reagent (Active Motif, Carlsbad, CA). Cytokine and chemokine mRNA levels were quantified by RPA according to the manufacturer’s instructions (Riboquant; BD PharMingen). Briefly, 8 µg of total RNA was hybridized with [{alpha}-32P]UTP-labeled riboprobes overnight at 56°C. After hybridization, unhybridized ssRNA was digested by RNase treatment. dsRNA was then purified by phenol/chloroform extraction and ethanol precipitation. The samples were subjected to electrophoresis on a 6% polyacrylamide/7 M urea gel. The gel was dried and subjected to autoradiographic analysis.

Isolation of infiltrating cells in the corneal stroma and flow cytometry

The corneas were aseptically removed, cut into small fragments, and incubated with 100 U/ml collagenase type IV (Sigma-Aldrich) for 60 min at 37°C in a humidified atmosphere with 5% CO2. Thereafter, the fragments were disrupted by grinding and passage through a cell strainer (BD Falcon, Bedford, MA). The cells (5 x 104) were washed with FACS buffer (Dulbecco’s PBS containing 1% FBS and 0.1% sodium azide), incubated with Fc Block (2.4G2) for 15 min on ice, then stained with Abs for CD3, CD11b, 4-1BB, 4-1BBL, CD44, CD25, CD62L, and a TCR panel, and analyzed by FACS (FACSCalibur; BD Biosciences, San Jose CA).

RT-PCR

RNA was extracted from normal and inflamed corneas. RNA was reverse transcribed into cDNA using a PCR cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA). PCR was performed using the sense/antisense primers. PCR primer sequences were as follows: mouse 4-1BB, forward, 5'-TGTGTGCAGGCTATTTCAGG-3', and reverse, 5'-GAGCTGCTCCAGTGGTCTTC-3' (expected size, 504 bp); mouse 4-1BBL, forward, 5'-ATTCACAAACACAGGCCACA-3', and reverse, 5'-GATAAGCCCTCAGACCCACA-3' (expected size, 203 bp); and mouse GAPDH, forward, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3', and reverse, 5'-CACCACCTGGAGTACCGGATGTAC-3' (expected size, 982 bp). PCR products were visualized using ethidium bromide after electrophoresis on 1% agarose gels.

In vivo migration assay

Cervical lymph node cells were isolated from 4-1BB+/+ and 4-1BB-/- mice on various days after HSV-1 infection. The CD4+ cells were suspended in PBS at a concentration of 1 x 107 cells/ml and incubated with CFSE (Molecular Probes, Eugene, OR) at a final concentration of 5 µM for 5 min at 37°C, followed by two washes in PBS. Equal numbers (8 x 106) of 4-1BB+/+ and 4-1BB-/- draining cervical lymph node (DLN) CD4+ T cells were resuspended in 400 µl of PBS, and injected i.v. into normal BALB/c mice. Three and one-half hours later, spleens and lymph nodes were removed, and the numbers of fluorescent cells were determined by FACS. At least 5 x 105 events were counted.

Statistical analysis

Severity score data were analyzed using generalized estimating equations for non-normally distributed longitudinal data that had a repeated measures term to account for the within-subject variation of mice that were repeatedly scored over the weeks of the study period (10). The models also included main effect terms for treatment and time, as well as the interaction of treatment and time. When an overall significance for the model was found, post hoc testing of differences between the time mean score levels for wild-type and mutant mice was performed using specific contrasts for each comparison. For all analyses, the severity score or other scores were analyzed as a binary outcome, either 0 or ≥1. Data manipulation and statistical testing were conducted using SAS software (Statistical Analysis System, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Deletion of 4-1BB prevents development of HSK

To evaluate the effect of the elimination of the 4-1BB costimulatory receptor, we infected the corneas of 4-1BB-/- and 4-1BB+/+ littermates on BALB/c background with the RE strain of HSV-1 and scored the severity of HSK in each eye on a scale of 0 to 4.0 over a 25-day period (Fig. 1a). In the 4-1BB+/+ mice (n = 19), HSK first appeared on PI days 7–8 (severity score, 0.32 ± 0.2; n = 6 of 38 eyes; mean ± SD). Severe inflammatory disease was evident by day 19 (severity score, 2.31 ± 0.4; n = 22 of 38 eyes) and persisted through PI day 22. Thereafter, the severity of HSK declined and the eyes were disease free by PI day 32. The incidence of HSK in the 4-1BB+/+ eyes peaked at 57.9% (22 of 38 eyes) (Fig. 1b). However, in the 4-1BB-/- mice (n = 25), only 8 of 50 eyes (16%) developed HSK, and the inflammation was mild (severity score, 0.21 ± 0.2) and lasted for just 1 or 2 days (Fig. 1b). The differences in incidence (p < 0.0001) and severity (p = 0.013 for PI day 11 and p < 0.0001 for PI days 12–25) were significant.



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  		FIGURE 1. 4-1BB-/- mice do not develop HSK. a, Herpes-infected 4-1BB+/+ BALB/c mouse eyes with HSK. HSK is visible on PI day 7 (1) and increases in severity through day 21 (2 and 3). Numbers in parentheses are severity scores. b, Incidence and severity of HSK in 4-1BB+/+ and 4-1BB-/- mice. Peak incidence (1) and severity (2) of HSK in 4-1BB+/+ mouse eyes (n = 38 eyes of 19 mice) were 57.9% and 2.19 ± 0.4, respectively. Peak incidence (1) and severity (2) of HSK in 4-1BB-/- mouse eyes (50 eyes of 25 mice) were 16% and 0.22 ± 0.22, respectively. Data on severity are means ± SD. Similar results were obtained in three independent experiments. c, Histologic examination of HSK corneas from 4-1BB+/+ and 4-1BB-/- mice on PI day 15. 4-1BB+/+ mouse cornea with severe HSK shows swelling and heavy infiltration of leukocytes (1). 4-1BB-/- mouse cornea with no evidence of HSK appears normal, with no leukocyte infiltration (2). H&E. Magnification, x250 (1); x400 (2).

 
Histological examination showed that the inflamed corneas of the 4-1BB+/+ mice were swollen and heavily infiltrated with leukocytes (Fig. 1c), whereas the corneas of the 4-1BB-/- mice appeared to be normal and showed no infiltration of leukocytes (Fig. 1c). The results indicate that 4-1BB expression may be required for the induction of HSK.

Blocking 4-1BBL inhibits the development of HSK

Because of the dramatic differences in the incidence and severity of HSK in the 4-1BB-/- and 4-1BB+/+ littermates, we considered the possibility that the 4-1BB-/- mice might have some other, unknown defects that contributed to the results. To rule out this possibility and examine another means of inhibiting the 4-1BB/4-1BBL interactions, we injected normal BALB/c mice with anti-4-1BBL mAb and compared the incidence and severity of HSK with that seen in control mice treated only with IgG (Fig. 2). The results were similar to those obtained with 4-1BB+/+ and 4-1BB-/- littermates; the control mice developed severe disease (PI day 15: incidence, 55.0%; mean severity score, 2.42 ± 0.7; n = 19 of 40 eyes), and the mice treated with anti-4-1BBL mAb did not (incidence, <5% at all times; peak mean severity score, 0.35 ± 0.7 on day 17; n = 2 of 40 eyes). These differences were also significant (p < 0.0001). The results suggest that blocking 4-1BB/4-1BBL interaction can prevent the development of HSK.



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  		FIGURE 2. Anti-4-1BBL mAb treatment prevents the development of HSK. The incidence (a) and severity (b) of HSK in the corneas of anti-4-1BBL mAb-treated and IgG-treated mice were recorded. Data on severity are means ± SD. Similar results were obtained in three independent experiments. Anti-4-1BB mAb-treated: n = 40 eyes of 20 mice; IgG-treated: n = 40 eyes of 20 mice.

 
Profile of cytokines and chemokines in HSK

Various cytokines and chemokines have been implicated in the pathogenesis of HSK (11, 12). We analyzed the expression of cytokines and chemokines by RPA using total corneal RNA from normal infected and uninfected mice. Infected corneas with HSK of 1.0+ severity demonstrated mRNA for RANTES, macrophage-inflammatory protein (MIP)-1{beta}, MIP-1{alpha}, MIP-2, INF-{gamma}-inducible protein (IP)-10, monocyte chemoattractant protein-1, and T cell activation 3, whereas uninfected corneas showed only a small amount of RANTES (Fig. 3a). Production of these chemokines is characteristic of activated T cells and monocytes. Corneas with HSK also had IL-15, IL-6, and IFN-{gamma} mRNA, whereas uninfected corneas showed only IL-15 mRNA (Fig. 3a).



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  		FIGURE 3. Expression of cytokine and chemokine mRNA in HSK. a, Cytokine and chemokine mRNA levels in infected (1.0+: severity score) and uninfected (C) corneas. b, Cytokine and chemokine mRNA levels in corneas from 4-1BB+/+ mice (HSK severity, 2.0), and 4-1BB-/- mice (HSK severity, 0.5). Results are representative of three experiments; n = 5 corneas with the same degree of HSK in each group on PI day 15.

 
Similarly, herpes-infected 4-1BB+/+ corneas with severe HSK (2.0 severity score) demonstrated high levels of chemokines, whereas herpes-infected but relatively disease-free corneas from 4-1BB-/- mice (severity score, 0.45) showed low to undetectable levels (Fig. 3b). Also, IL-6 and IFN-{gamma} mRNA were detected in the 4-1BB+/+ corneas, but not in the 4-1BB-/- corneas. IL-15 was present in both, but at a reduced level in the 4-1BB-/- corneas (Fig. 3b). In other studies (S. Seo, H. Park, Y. Kim, J. Choi, and B. Kwon, manuscript in preparation), we have found that cultured stromal keratocytes produce IL-15 constitutively; thus, some of the IL-15 mRNA detected in these corneas may have been produced by stromal keratocytes rather than T cells.

Expression of 4-1BB on infiltrating T cells

4-1BB is inducible on T cells that are activated through TCR signaling (9), and 4-1BB+ T cells presumably recognize Ags in corneal stroma, including herpes Ags. Therefore, we examined the time course of T cell infiltration and 4-1BB expression during the development of HSK. Infiltrating leukocytes were extracted from infected corneas at various time points (Fig. 4). CD3+ T cells constituted ~3% of the cells on PI day 8, when the mean severity score was 0.5; ~17% on PI day 13, when the mean score increased to 1.0; and ~23% on day 15, when the mean score peaked at 2.0 (Fig. 4, a and b). Immediately thereafter, the proportion of T cells decreased sharply (<7% on PI day 17), although the ocular disease remained severe (Fig. 4, a and b).



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  		FIGURE 4. 4-1BB expression on infiltrating T cells over time in corneas with HSK. a, Flow cytometry of infiltrating leukocytes stained with anti-CD3 and anti-4-1BB or anti-CD11b and anti-4-1BB. Plots were gated on leukocytes. Numbers in each quadrant indicate the percentage of cells of interest relative to the total number of infiltrating cells on various PI days. We normally recovered an average of 421 ± 102 (SD) CD3+ T cells per cornea on PI day 15. Upper left quadrant, CD3+ or CD11b+; upper right quadrant, CD3+ 4-1BB+ or CD11b+ 4-1BB+; numbers in parentheses are HSK severity scores. b, Graphic presentation of the data in a. Percentage of total infiltrating leukocytes that were CD3+ T cells and CD3+ 4-1BB+ T cells (1). Percentage of total infiltrating leukocytes that were CD11b+ cells (2). c, Section of HSK cornea stained to detect 4-1BB+ T cells (arrows); Vectastain ABC. d, mRNA for 4-1BB and 4-1BBL on various PI days detected by RT-PCR. Data shown are representative of the results of three separate experiments. Score indicates the severity of HSK.

 
CD11b+ cells constituted ~30% of the population on PI day 8, increased steadily as the disease worsened (67.4 ± 1.84% on PI day 15), remained abundant through day 17 when the disease scores were at their maximum, and then decreased as the disease resolved (27.7 ± 4.24% on PI day 25, when the severity score was 0.5). Thus, the peak population of CD11b+ cells coincided with the sharp decline in T cells (Fig. 4, a and b), suggesting that T cells played a role in the induction of HSK and CD11b+ cells in the maintenance of the disease.

Approximately 65% (65.6 ± 6.0% on PI day 15) of the infiltrating CD3+ T cells expressed 4-1BB (Fig. 4, b–d). In contrast, expression of 4-1BB and 4-1BBL was not detected in the peripheral blood T cells in mice with HSK (data not shown). The data again indicate that interaction between 4-1BB and 4-1BBL in the corneal stroma contributes to the pathogenesis of HSK.

T cell apoptosis in the inflamed corneal stroma

We sought to identify the mechanism that leads to the disappearance of T cells immediately after the peak of HSK severity (Fig. 5). Using the TUNEL assay, we observed massive T cell apoptosis on PI day 16 (Fig. 5, c, e–g), immediately before the precipitous drop in T cell numbers seen on day 17 (Fig. 4b). Fewer apoptotic cells were observed on PI day 17 (Fig. 5d), by which time the number of T cells had decreased to <7% of total infiltrating cells. Dual staining for CD3+ T cells (Fig. 5e) and apoptotic cell death in the corneal sections obtained on PI day 16 (Fig. 5, f and g) supports the idea that the reduction in T cell numbers seen on day 17 was the result of apoptosis.



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  		FIGURE 5. T cell apoptosis in corneas with HSK. Apoptotic cells (arrows) detected by in situ cell death assay (TUNEL assay). a, Negative control, Section of HSK cornea processed without terminal transferase. b, Positive control, Section of HSK cornea treated with DNase I. c, Section of HSK cornea on PI day 16. d, Section of HSK cornea on PI day 17. e–g, Sections were stained with PE-anti-CD3 (145.2C11) and terminal transferase. Red, CD3+ T cells (e); green, apoptotic cells (f); and yellow, apoptotic CD3+ T cells (g).

 
Phenotype of 4-1BB+ T cells

The majority of infiltrating 4-1BB+ T cells also expressed CD44, CD25, and CD62L on their surfaces (Fig. 6, a and b). On day 15, ~12% of the total cells isolated from the HSK corneas were 4-1BB+ T cells (Fig. 6a). Of the 4-1BB+ T cells, 68.1 ± 7.1% were CD44+, 67 ± 2.1% were CD25+, and 76 ± 15.6% were CD62L+ (Fig. 6b). Taken together, the results indicate that the infiltrating T cells, especially the ones expressing 4-1BB, were effector T cells.



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  		FIGURE 6. Phenotypic analysis of 4-1BB+ T cells. Infiltrating lymphoid cells from HSK corneas were isolated on PI day 15 (when the HSK severity scores reached 1.0–2.0), stained with mAbs against 4-1BB and CD44, CD25, or CD62L, and analyzed by flow cytometry. a, Plots were gated on lymphocytes. Rat IgG2b, rat IgM, and rat IgG2a mAbs were used as isotype controls. Numbers in each quadrant indicate the percentage of cells of interest relative to the total number of infiltrating cells on various PI days. Upper left quadrant, CD44+, CD25+, or CD62L+; upper right quadrant, CD44+ 4-1BB+, CD25+ 4-1BB+, or CD62L+ 4-1BB+; lower right quadrant, 4-1BB+. b, Percentage of 4-1BB+ T cells positive for a second cell surface marker. Results in a and b are representative of three experiments. c, RPA-detected expression of CCRs in CD4+ T cells from the corneas of 4-1BB+/+ and 4-1BB-/- mice.

 
Using RPA, we found that the infiltrating leukocytes produced mRNA for the chemokine receptors CCR1, CCR2, and CCR5 (Fig. 6c). High levels of CCR5 expression were detected in the T cells in 4-1BB+/+ corneas but not in 4-1BB-/- corneas. If we assume that CCR5 is expressed on T cells, the detection of CCR5 in 4-1BB+/+ corneas suggests that most of the infiltrating T cells were Th1 cells and that 4-1BB has a role in promoting the Th1 response. This idea is further corroborated by the markedly reduced expression of IP-10 in 4-1BB-/- corneas (Fig. 3b), compared with 4-1BB+/+ corneas. IP-10 is linked to IFN-{gamma}, which also promotes the Th1 response.

We then determined the TCR V{beta} expression profile for the infiltrating T cells. We found that 14.5 ± 3.0% of the T cells carried V{beta}8.1/8.2, 10.8 ± 2.7% carried V{beta}8.3, 9.8 ± 1.5% carried V{beta}10b, and 8.9 ± 2.1% carried V{beta}5.1/5.2 (Fig. 7). No population expressed {gamma}{delta} TCR chains. This result indicates that the infiltrating T cells in HSK display a limited heterogeneity.



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  		FIGURE 7. TCR {beta}-chain expression on infiltrating T cells. Infiltrating CD3+ T cells were isolated from HSK corneas on PI day 15 (when the severity scores reached 1.0–2.0), stained with a V{beta} TCR mAb panel or {gamma}{delta} TCR mAbs, and analyzed by flow cytometry. Plots were gated on lymphocytes. Results are representative of three experiments.

 
Reduced expression of CD62L in 4-1BB-/- T lymphocytes associated with the absence of HSK

To further elucidate the mechanisms involved in the diminished T cell infiltration into the corneas of 4-1BB-/- mice, the abundance of various T cell surface markers was determined from infection through PI day 8, when the T cells begin to migrate into the corneal stroma from the cervical lymph nodes. Among the markers tested, only CD62L showed a significant difference in expression in herpes-infected 4-1BB+/+ and 4-1BB-/- mice.

The expression levels of CD62L were first measured in CD4+ T cells from the spleens and DLNs in normal HSV-1-infected BALB/c mice. We found that the expression of CD62L was reduced in the cells from both organs early in the period after infection. Specifically, CD62Llow/- was seen on ~3% of DLN CD4+ T cells on PI day 0, 35% on PI day 6, and 14% by PI days 8–12 (Fig. 8a). In splenic CD4+ T cells, the CD62Llow/- phenotype increased even more dramatically, from 3% on PI day 0 to 60% on days 6–12 (Fig. 8a).



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  		FIGURE 8. CD62L (L-selectin) expression in 4-1BB+/+ and 4-1BB-/- mice after HSV-1 infection. a, CD62L expression was measured for DLN and splenic CD4+ T cells from BALB/c mice at the indicated times after infection. b, CD62L expression was measured for DLN and splenic CD4+ T cells from 4-1BB+/+ and 4-1BB-/- mice on PI days 3, 6, and 8. Numbers within plots represent the percentage of CD62Llow/- cells. c and d, Percentages of T cells expressing CD62Llow/- (CFSE+) of the total T cells from the cervical lymph nodes and spleens of 4-1BB+/+ and 4-1BB-/- mice on PI days 3 and 8, counted by FACS. Results are representative of three experiments. *, Significant difference from 4-1BB+/+ mice (p < 0.001).

 
Next, we compared the level of CD62L expression on T cells from spleens and DLNs from herpes-infected 4-1BB+/+ and 4-1BB-/- mice. In the DLN samples obtained on PI day 3, there was no difference: ~85% of the T cells were CD62L+ and 15% were CD62Llow/- in both groups of mice (Fig. 8b). However, on PI days 6 and 8, T cells expressing CD62L were less numerous in the 4-1BB-/- mice relative to the 4-1BB+/+ mice: on day 6, 44.3% of the T cells from the 4-1BB-/- mice were CD62Llow/-, compared with 34.4% in the 4-1BB+/+ mice (Fig. 8b). By day 8, the corresponding numbers were 37.5% in the 4-1BB-/- mice and 14.4% in the 4-1BB+/+ mice (p = 0.008) (Fig. 8b). No differences in levels of CD62L were observed in the splenic CD4+ T cells from 4-1BB+/+ and 4-1BB-/- mice (Fig. 8b).

Finally, to evaluate migratory potential, DLN CD4+ T cells purified from 4-1BB+/+ and 4-1BB-/- littermates on PI days 3 and 8 were labeled with CFSE, injected i.v. into normal mice, and counted in specimens from cervical lymph nodes and spleens. When the T cells obtained on PI day 3 were used, the numbers of labeled cells in the two groups of mice were similar for both spleens and lymph nodes (Fig. 8c). However, when the PI day 8 T cells were injected, the 4-1BB-/- mice showed significantly fewer labeled T cells in the DLN and spleen, compared with the 4-1BB+/+ littermates (spleen, p = 0.013; DLN, p = 0.0094) (Fig. 8d).

In summary, CD62L expression in 4-1BB+/+ and 4-1BB-/- littermates was similar during the acute phase of infection (PI day 3). By PI day 6, when the acute infection had usually resolved, somewhat higher numbers of T cells expressing CD62Llow/- were seen in the 4-1BB-/- mice, compared with their 4-1BB+/+ littermates, although the difference was not significant. However, by day 8, when T cells were beginning to infiltrate the corneas, CD62L expression was significantly less in the 4-1BB-/- mice, compared with their 4-1BB+/+ littermates. These results suggest the possibility that reduced infiltration of T cells in herpes-infected 4-1BB-/- mice is related to the lower expression of CD62L in a subset of T cells, potentially those T cells that would have been expected to migrate into the corneal stroma. This notion is supported by the finding that CD4+ T cells isolated from DLNs of 4-1BB-/- mice on PI day 8, but not those isolated on PI day 3, had reduced migratory potential. If so, CD62L expression may be regulated by signals through 4-1BB. The absence of 4-1BB/4-1BBL interactions may result in reduced expression of CD62L on a subset of T cells. If the 4-1BB-/-CD62Llow/- T cells have a reduced migratory potential as a result of the absence of CD62L, the ultimate result could be fewer T cells available to stimulate inflammation and the development of HSK in the cornea.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A variety of costimulatory receptors on T cells and their ligands on dendritic cells are required for full activation of T cells, which ultimately leads to the generation of effector T cells. Generally, costimulatory receptors can be classified into two families: 1) the CD28/inducible costimulator family and 2) the TNFR family, to which 4-1BB belongs.

4-1BB is expressed on activated CD4+ T cells, CD8+ T cells, NK cells, and NK T cells (reviewed in Ref.12). CD4+CD25+ regulatory T cells appear to express 4-1BB constitutively (13, 14). Recent studies have shown that 4-1BB expression is not restricted to subpopulations of lymphoid cells, but rather is distributed across a variety of blood cells. For example, myeloid cells, including monocytes, neutrophils, and dendritic cells, express 4-1BB constitutively (15, 16). 4-1BBL is expressed on activated APCs, such as dendritic cells, B cells, and macrophages (12), and on keratinocytes (8). This expression pattern raises the possibility that 4-1BB/4-1BBL interactions may be involved in multiple steps in various innate and adaptive immune responses. Studies that block or stimulate the 4-1BB costimulatory pathway demonstrate the involvement of 4-1BB in a variety of CD4+ T cell-mediated responses in vivo. These responses include induction of a Th1 cell anergy (17), an alloimmune response (18), an acute inflammation (19), an autoimmune disease (20), and a Th1 cell response to tumor cells (21). A critical role for 4-1BB in the Th1-mediated immune response suggests that intervening in the 4-1BB costimulatory pathway could provide an immunotherapeutic approach to the treatment of inflammatory diseases.

Present studies demonstrate that interference in the interactions between 4-1BB and 4-1BBL can abolish the development of HSK in the murine model. 4-1BB/4-1BBL interactions can be blocked by deletion of 4-1BB or by mAbs to 4-1BBL (Figs. 1 and 2).

Absence of the 4-1BB signal appears to produce a dual effect: blocking T cell migration from the regional lymph nodes to the corneal stroma and inhibiting the inflammatory responses in the corneal stroma. Blocking B7-1/CD28 interactions, as well as CD154/CD40 interactions, was also observed to reduce the incidence of HSK in mice (22).

The most intriguing aspect of the present investigation is the potential effect of the 4-1BB signal on CD62L expression (Fig. 8). CD62L is expressed constitutively on all classes of leukocytes. It mediates the binding of lymphocyte subpopulations to specialized high endothelial cells present in postcapillary venules of peripheral lymph nodes, and also facilitates the rolling of leukocytes on the endothelium at sites of tissue injury or inflammation (23, 24). The migration of lymphocytes into peripheral lymph nodes and the recruitment of leukocytes to sites of inflammation are reduced dramatically in CD62L-deficient mice (25, 26). In fact, Grewal et al. (27) showed that CD62L-deficient mice did not develop experimental allergic encephalomyelitis. These studies indicate a key role for CD62L in inflammation and leukocyte trafficking.

As shown in the present investigation (Fig. 8), it is possible that 4-1BB signaling regulates the expression of CD62L. We have previously noticed that 4-1BB stimulation induces ICAM-1, a molecule that is involved in cell adhesion in monocyte cell lines (H. Lee, W. Kang, and B. Kwon, manuscript in preparation). It may be important to determine the role of 4-1BB-mediated signals in CD62L expression. Additionally, a defect in the APCs in 4-1BB-deficient mice cannot be ruled out.

The phenotypes of the stroma-infiltrating T cells were analyzed. As shown in Fig. 3, the infiltrating T cells produced chemokines that are normally induced when T cells are activated. In the absence of 4-1BB, levels of CCR5 (Fig. 3) and IP-10 (Fig. 1) were markedly reduced, suggesting that without 4-1BB, Th1 responses may be diminished. Although the abundance of chemokines and IL-6 mRNA may reflect the severity of HSK, the markedly elevated levels of MIP-2, IP-10, monocyte chemoattractant protein-1, and IL-6 are also noteworthy. Lausch and colleagues (28, 29) reported that MIP-2 and IL-6 are important in the development of HSK. How much the cytokine network contributes to the initiation and maintenance of HSK is not known, and the significance of the unusually high expression of the chemokines noted above, as well as IL-6, remains to be determined.

The majority of the infiltrating T cells (CD44+, CD25+, and CD62L+) were effector cells, with possibly some memory T cells, and most appeared to express 4-1BB on their cell surfaces (Fig. 6). It appeared that HSK was induced by 4-1BB+ T cells, and then, at the peak of disease, the T cells were suddenly eliminated by apoptosis (Fig. 5). The mechanism controlling this phenomenon is unknown, although it is possible that CD95 is induced at this stage of inflammation and interacts with CD95 ligand on stromal tissue (30). The course of HSK was prolonged, probably by the presence of CD11b+ monocytes, but the disease eventually resolved without treatment. The disappearance of the T cells might be a precursor to resolution of the disease, but how these two phenomena are related remains to be determined.

In summary, blocking 4-1BB/4-1BBL interactions can abolish the development of HSK. The mechanism appears to act at the effector level of the immune response. Ultimately, it may be possible to develop an agent to block 4-1BB/4-1BBL activity for treatment of HSK in the clinical setting.


   Acknowledgments
 
We thank Dr. Hilary W. Thompson at the Clinical Trials and Biometry Unit (Louisiana State University Eye Center) for the statistical analysis and Carole Hoth for typing the manuscript.


   Footnotes
 
1 This work was supported by the Science Research Center Fund (to the Immunomodulation Research Center, University of Ulsan) from the Korea Science Engineering Foundation and the Korean Ministry of Science and Technology, by U.S. Public Health Service Grants R01EY013325 (to B.S.K.) and P30EY02377 (core grant to Louisiana State University Eye Center) from the National Eye Institute, National Institutes of Health (Bethesda, MD), and by an unrestricted departmental grant (to Louisiana State University Eye Center) from Research to Prevent Blindness, Inc. (New York, NY). Back

2 Address correspondence and reprint requests to Dr. Byoung S. Kwon, Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 680-749. E-mail address: bskwon{at}mail.ulsan.ac.kr Back

3 Abbreviations used in this paper: HSK, herpetic stromal keratitis; PI, postinfection; 4-1BBL, 4-1BB ligand; DLN, draining cervical lymph node; RPA, RNase protection assay; MIP, macrophage-inflammatory protein; IP, IFN-{gamma}-inducible protein. Back

Received for publication November 8, 2002. Accepted for publication May 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Metcalf, J. F., H. E. Kaufman. 1976. Herpetic stromal keratitis: evidence for cell-mediated immunopathogenesis. Am. J. Ophthalmol. 82:827.[Medline]
  2. Russell, R. G., M. P. Nasisse, H. S. Larsen, B. T. Rouse. 1984. Role of T-lymphocytes in the pathogenesis of herpetic stromal keratitis. Invest. Ophthalmol. Visual Sci. 25:938.[Abstract/Free Full Text]
  3. Deshpande, S. P., S. Lee, M. Zheng, B. Song, D. Knipe, J. S. Kapp, B. T. Rouse. 2001. Herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis. J. Virol. 75:3077.[Abstract/Free Full Text]
  4. Deshpande, S., M. Zheng, S. Lee, K. Banerjee, S. Gangappa, U. Kumaraguru, B. T. Rouse. 2001. Bystander activation involving T lymphocytes in herpetic stromal keratitis. J. Immunol. 167:2902.[Abstract/Free Full Text]
  5. Avery, A. C., Z. S. Zhao, A. Rodriguez, E. K. Bikoff, M. Soheilian, C. S. Foster, H. Cantor. 1995. Resistance to herpes stromal keratitis conferred by an IgG2a-derived peptide. Nature 376:431.[Medline]
  6. Zhao, Z. S., F. Granucci, L. Yeh, P. A. Schaffer, H. Cantor. 1998. Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection. Science 279:1344.[Abstract/Free Full Text]
  7. Panoutsakopoulou, V., M. S. Sanchirico, K. M. Huster, M. Jansson, F. Granucci, D. J. Shim, K. W. Wucherpfennig, H. Cantor. 2001. Analysis of the relationship between viral infection and autoimmune disease. Immunity 15:137.[Medline]
  8. Seo, S. K., B. M. Gebhardt, H. Y. Lim, S. W. Kang, S. Higaki, E. D. Varnell, J. M. Hill, H. E. Kaufman, B. S. Kwon. 2001. Murine keratocytes function as antigen-presenting cells. Eur. J. Immunol. 31:3318.[Medline]
  9. Kwon, B. S., J. C. Hurtado, Z. H. Lee, K. B. Kwack, S. K. Seo, B. K. Choi, B. H. Koller, G. Wolisi, H. E. Broxmeyer, D. S. Vinay. 2002. Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol. 168:5483.[Abstract/Free Full Text]
  10. Liang, K. Y., S. L. Zeger. 1986. Longitudinal data analysis using generalized linear models. Biometrika 73:13.[Abstract/Free Full Text]
  11. Tumpey, T. M., H. Cheng, D. N. Cook, O. Smithies, J. E. Oakes, R. N. Lausch. 1998. Absence of macrophage inflammatory protein-1{alpha} prevents the development of blinding herpes stromal keratitis. J. Virol. 72:3705.[Abstract/Free Full Text]
  12. Tang, Q., W. Chen, R. L. Hendricks. 1997. Proinflammatory functions of IL-2 in herpes simplex virus corneal infection. J. Immunol. 158:1275.[Abstract]
  13. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, A. Rudensky. 2002. Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo. Nat. Immunol. 3:33.[Medline]
  14. McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. Collins, M. C. Byrne. 2002. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity 16:311.[Medline]
  15. Heinisch, I. V., I. Daigle, B. Knopfli, H. U. Simon. 2000. CD137 activation abrogates granulocyte-macrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils. Eur. J. Immunol. 30:3441.[Medline]
  16. Wilcox, R. A., A. I. Chapoval, K. S. Gorski, M. Otsuji, T. Shin, D. B. Flies, K. Tamada, R. S. Mittler, H. Tsuchiya, D. M. Pardoll, L. Chen. 2002. Cutting edge: expression of functional CD137 receptor by dendritic cells. J. Immunol. 168:4262.[Abstract/Free Full Text]
  17. Mittler, R. S., T. S. Bailey, K. Klussman, M. D. Trailsmith, M. K. Hoffmann. 1999. Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy. J. Exp. Med. 190:1535.[Abstract/Free Full Text]
  18. Blazar, B. R., B. S. Kwon, A. Panoskaltsis-Mortari, K. B. Kwak, J. J. Peschon, P. A. Taylor. 2001. Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients. J. Immunol. 166:3174.[Abstract/Free Full Text]
  19. Seko, Y., S. Ishiyama, T. Nishikawa, T. Kasajima, M. Hiroe, S. Suzuki, S. Ishiwata, S. Kawai, Y. Tanaka, M. Azuma, et al 2001. Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3. J. Pathol. 195:593.[Medline]
  20. Sun, Y., X. Lin, H. M. Chen, Q. Wu, S. K. Subudhi, L. Chen, Y. X. Fu. 2002. Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis. J. Immunol. 168:1457.[Abstract/Free Full Text]
  21. Ye, Z., I. Hellstrom, M. Hayden-Ledbetter, A. Dahlin, J. A. Ledbetter, K. E. Hellstrom. 2002. Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nat. Med. 8:343.[Medline]
  22. Xu, M., A. J. Lepisto, R. L. Hendricks. 2002. Co-stimulatory requirements of effector T cells at inflammatory sites. DNA Cell Biol. 21:461.[Medline]
  23. Tedder, T. F., D. A. Steeber, A. Chen, P. Engel. 1995. The selectins: vascular adhesion molecules. FASEB J. 9:866.[Abstract]
  24. Ley, K., T. F. Tedder. 1995. Leukocyte interactions with vascular endothelium: new insights into selectin-mediated attachment and rolling. J. Immunol. 155:525.[Abstract]
  25. Arbones, M. L., D. C. Ord, K. Ley, H. Ratech, C. Maynard-Curry, G. Otten, D. J. Capon, T. F. Tedder. 1994. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity 1:247.[Medline]
  26. Tedder, T. F., D. A. Steeber, P. Pizcueta. 1995. L-selectin deficient mice have impaired leukocyte recruitment into inflammatory sites. J. Exp. Med. 181:2259.[Abstract/Free Full Text]
  27. Grewal, I. S., H. G. Foellmer, K. D. Grewal, H. Wang, W. P. Lee, D. Tumas, C. A. Janeway, Jr, R. A. Flavell. 2001. CD62L is required on effector cells for local interactions in the CNS to cause myelin damage in experimental allergic encephalomyelitis. Immunity 14:291.[Medline]
  28. Tumpey, T. M., H. Cheng, X. T. Xan, J. E. Oakes, R. N. Lausch. 1998. Chemokine synthesis in the HSV-1-infected cornea and its suppression by interleukin-10. J. Leukocyte Biol. 63:486.[Abstract]
  29. Fenton, R. R., S. Molesworth-Kenyon, J. E. Oakes, R. N. Lausch. 2002. Linkage of IL-6 with neutrophil chemoattractant expression in virus-induced ocular inflammation. Invest. Ophthalmol. Visual Sci. 43:737.[Abstract/Free Full Text]
  30. Raftery, M. J., C. K. Behrens, A. Müller, P. H. Krammer, H. Walczak, G. Schönrich. Herpes simplex virus type 1 infection of activated cytotoxic T cells: induction of fratricide as a mechanism of viral immune evasion. J. Exp. Med. 190:1103.



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