May 2008
Volume 49, Issue 5
Free
Retinal Cell Biology  |   May 2008
Suppression of Alkali Burn-Induced Corneal Neovascularization by Dendritic Cell Vaccination Targeting VEGF Receptor 2
Author Affiliations
  • Hiroshi Mochimaru
    From the Laboratory of Retinal Cell Biology, the
    Department of Ophthalmology, and the
    Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; the
  • Tomohiko Usui
    Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan; and the
  • Tomonori Yaguchi
    Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; the
  • Yasuharu Nagahama
    Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; the
  • Go Hasegawa
    Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; the
  • Yoshihiko Usui
    Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan.
  • Shigeto Shimmura
    Department of Ophthalmology, and the
  • Kazuo Tsubota
    Department of Ophthalmology, and the
  • Shiro Amano
    Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan; and the
  • Yutaka Kawakami
    Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; the
  • Susumu Ishida
    From the Laboratory of Retinal Cell Biology, the
    Department of Ophthalmology, and the
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2172-2177. doi:10.1167/iovs.07-1396
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      Hiroshi Mochimaru, Tomohiko Usui, Tomonori Yaguchi, Yasuharu Nagahama, Go Hasegawa, Yoshihiko Usui, Shigeto Shimmura, Kazuo Tsubota, Shiro Amano, Yutaka Kawakami, Susumu Ishida; Suppression of Alkali Burn-Induced Corneal Neovascularization by Dendritic Cell Vaccination Targeting VEGF Receptor 2. Invest. Ophthalmol. Vis. Sci. 2008;49(5):2172-2177. doi: 10.1167/iovs.07-1396.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by alkali injury.

methods. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2400–408) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of VEGFR2400–408- or gp70-pulsed mature DCs three times at 6-day intervals. After the third immunization, corneal neovascularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8+ T cells from immunized mice were subjected to ELISA for interferon (IFN)-γ and tumor necrosis factor (TNF)-α production and 51Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody.

results. Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2400–408 compared with those not immunized or immunized with gp70. VEGFR2400–408 or gp70, but not β-gal96–103, application led to dose-dependent induction of IFN-γ and TNF-α in the CD8+ T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not β-gal96–103. In vivo depletion of CD8+, but not CD4+, T cells significantly reversed the suppressive effect of VEGFR2400–408 immunization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals.

conclusions. These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.

Extensive corneal injury from chemical burns develops conjunctivalization of the corneal epithelial surface with massive neovascularization, leading to severe reductions in corneal transparency and visual acuity. 1 Previous studies demonstrated the involvement of inflammation with corneal conjunctivalization and neovascularization. 2 Of several growth factors and inflammatory cytokines, vascular endothelial growth factor (VEGF) was shown to play a central role in corneal neovascularization, 3 suggesting the potential validity of targeting the VEGF-VEGF receptor (VEGFR) system. Aptamer- and antibody-based VEGF blockers are now clinically used for ocular neovascular diseases, including age-related macular degeneration, 4 5 6 whereas the inhibitory effect of these agents on corneal neovascularization has only recently been confirmed with animal models. 7 Although corneal transplantation surgery is conventionally applied for the treatment of corneal opacity resulting from neovascularization, it is well known that the vascularized cornea substantially decreases the success rate of penetrating keratoplasty. 8 The relatively poor outcome of the current modality has been arousing further interest for the establishment of a novel therapeutic strategy for corneal neovascularization. 
As an immunologic approach to combat angiogenesis-dependent solid tumor, the regression of murine renal carcinoma was achieved by interleukin (IL)-12/pulse IL-2 combination therapy eliciting CD8+ cytotoxic T lymphocyte (CTL)-mediated apoptosis of endothelial cells. 9 In the eye as well, we previously demonstrated CD8+ CTL-mediated regression of physiologic and pathologic retinal new vessels. 10 11 In addition, our recent data using dendritic cell (DC) vaccination targeting VEGFR2 has shown the induction of CD8+ CTLs led to significant suppression of laser-induced choroidal neovascularization. 12 VEGFR2, which plays a pivotal role in endothelial cell proliferation and migration, is upregulated in pathologic corneal vessels. 13 Indeed, oral immunization with recombinant Salmonella typhimurium harboring VEGFR2 proved useful to reduce herpetic keratitis-associated corneal neovascularization. 14 In tumor models, immunization with full-length cDNA or recombinant protein of VEGFR2 successfully inhibited tumor growth and angiogenesis. 15 16 In particular, specific immunization with the major histocompatibility (MHC) class I-restricted epitope peptides of VEGFR2, which were recently identified in separate studies, 17 18 led to significant suppression of tumor angiogenesis. In the present study, we investigated for the first time the potential usefulness and efficacy of DC vaccination against a specific VEGFR2 peptide to suppress alkali burn-induced corneal neovascularization. 
Materials and Methods
Animals
Male C57BL/6 mice (CLEA, Tokyo, Japan) at the age of 6 weeks were purchased and maintained in the specific pathogen-free Animal Facility of the Research Park, Keio University School of Medicine. Animals were allowed free access to food and water. A 12-hour light-dark cycle was maintained. All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Induction of Corneal Neovascularization
Corneal neovascularization was induced by alkali injury, as described previously. 19 Briefly, after general anesthesia with xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (35 mg/kg), 2 μL of 0.15 M NaOH was applied onto the corneal surface. Subsequently, total corneal limbus and epithelium were scraped off with a surgical blade under a microscope. Erythromycin ophthalmic ointment was instilled immediately after the operation. 
Quantification of Corneal Neovascularization
Corneal neovascularization was imaged by lectin angiography. 19 Mice received intravenous injections of BS-1 lectin conjugated with FITC (10 μg/g; Vector, Burlingame, CA) and were killed 30 minutes later. The eyes were enucleated and fixed with 1% paraformaldehyde for 15 minutes. After fixation, the corneas were placed on glass slides and studied by a fluorescence microscopy (Leica, Deerfield, IL), as described elsewhere. 20 Briefly, NIH Image was used for the image analysis. Neovascularization was quantified by setting a threshold level of fluorescence, above which only vessels were depicted. The vascularized area was outlined using the innermost vessel of the limbal arcade as the border. 
Epitope Peptides
The MHC class I H-2Db-restricted peptide corresponding to amino acids 400 to 408 of murine VEGFR2 (VILTNPISM; VEGFR2400–408) 18 was used for an epitope peptide to induce cellular immunity specific for VEGFR2 in C57BL/6 mice. The gp70 peptide, the epitope sequence of p15E (KSPWFTTL; p15E604–611), 21 served as a negative control for VEGFR2-specific immunotherapy and a positive control for cytokine assays, because the gp70 peptide has a potent immunogenicity in C57/BL6 mice. The p15E is the envelope protein of an endogenous murine retrovirus of the Akv family found in the germ line of C57BL/6 mice. We also used the epitope sequence of β-galactosidase (DAPIYTNV; β-gal96–103) 22 as a negative control for cytokine assays and cytotoxicity assays to confirm the peptide specificity of T-cell responses. Peptides were synthesized and purified with high-performance liquid chromatography (Sigma-Aldrich, St. Louis, MO). 
Preparation of Mature DCs
Purified DCs were obtained using previously described methods 23 24 25 with slight modification. Briefly, marrow from tibias and femurs of C57BL/6 mice were harvested and then followed by DC enrichment with a magnetic cell sorting (MACS) kit (BD Biosciences PharMingen, San Jose, CA). Isolated precursors were cultured in the presence of granulocyte/macrophage-colony stimulating factor (GM-CSF; 10 ng/mL) to induce differentiation into DCs. After 1 week, OK432 and either of the epitope peptides (VEGFR2400–408 or gp70) were added to the culture to induce DC maturation. OK432, a penicillin- and H2O2-killed lyophilized preparation of the Su strain of Streptococcus pyogenes, 26 was kindly provided by Chugai Pharmaceutical Co., Ltd (Tokyo, Japan). After 6 hours, generated mature DCs were harvested and used for subsequent vaccination. The maturation of DCs as CD11c+ CD40+ cells or CD11c+ CD86+ cells was confirmed by flow cytometry using a fluorescence-activated cell sorter (FACSCalibur; Becton Dickinson, Mountain View, CA) with CellQuest (Becton Dickinson) software (data not shown). 
Vaccination with Peptide-Pulsed DCs
Mice were immunized as previously described, 17 18 27 with slight modification. In brief, C57BL/6 mice received subcutaneous inoculation of 5 × 105 mature DCs and 200 μL vaccine mixture containing 100 μg epitope peptides (VEGFR2400–408 or gp70) and 100 μL incomplete Freund adjuvant (IFA; Difco Laboratories, Detroit, MI), every 7 days for 3 weeks. Immediately after the third immunization, alkali injury was made to induce corneal neovascularization. Two weeks after the injury, immunized mice were killed to harvest the corneas and splenocytes. 
Cytokine Assays
To confirm the peptide-specific T-cell responses, we examined the concentration of interferon (IFN)-γ and tumor necrosis factor (TNF)-α that CD8+ T cells released in the medium. TNF-α and IFN-γ are regarded as indicators for CTL responses. Naive splenocytes irradiated with 40 Gy served as stimulator cells. Using a MACS kit for CD8+ T cell isolation (Miltenyi Biotech, Auburn, CA), CD8+ T cells were isolated from the splenocytes excised from VEGFR2400–408 or gp70-immunized mice 2 weeks after the third vaccination. Isolated CD8+ T cells, subjected to 1-week in vitro restimulation with 10 μg/mL epitope peptides (VEGFR2400–408 or gp70) and stimulator cells plus 10 U/mL IL-2, were used as effector cells. 18 After 2 × 105 effector cells were cocultured overnight with 1 × 106 stimulator cells at 37°C in a 95% air/5% CO2 atmosphere in 200 μL medium containing epitope peptides (VEGFR2400–408, gp70 or β-gal96–103) at the dose of 0, 0.1, 1, or 10 μg/mL, the supernatant was collected and the protein levels of IFN-γ and TNF-α were measured with mouse IFN-γ and TNF-α ELISA kits (BioSource, Camarillo, CA), respectively. 
Cytotoxicity Assay
CD8+ T cells isolated from VEGFR2400–408-immunized mice were restimulated in vitro, as described, and served as effector cells to kill VEGFR2400–408-presenting target cells. As for target cells, the murine endothelial cell lines H5V and bEND3 were used as MHC-matched (H-2Db+) and MHC-unmatched (H-2Db) VEGFR2-expressing cells, respectively. In addition, the EL4 murine lymphoma cell line was used as MHC-matched nonendothelial cells not expressing VEGFR2. H5V cells were kindly provided by Annunciata Vecchi (Department of Immunology and Cell Biology, Mario Negri Institute for Pharmacological Research, Milan, Italy). H5V (H-2Db+) and bEND3 (H-2Db) endothelial cells were maintained in DMEM supplemented with 10% fetal calf serum, whereas EL4 lymphoma cells (H-2Db+) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and pulsed with 10 μg/mL epitope peptides (VEGFR2400–408 or β-gal96–103). Cytotoxic activities were tested in a 4-hour Na51CrO4 (51Cr) release assay, as previously described. 18 28 Briefly, target cells were incubated with 100 μCi of 51Cr for 60 minutes. Target cells (5 × 103) were then mixed with effector cells for 4 hours at the effector-to-target ratio of 10:1, 20:1, or 40:1. The amount of 51Cr release was determined by gamma counting, and the percentage of lysis was calculated with the following formula: (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100. 
In Vivo Depletion of T-Cell Subsets
Immune cell subsets were depleted in vivo, as previously described. 29 30 Mice were injected intraperitoneally with 5 mg/kg body weight of either an anti-CD4 or -CD8 depletion antibody (clone GK1.5 or clone 53-6.72, respectively; eBioscience, San Diego, CA) or an isotype control antibody (BD Biosciences PharMingen) 1 day before and 1 week after alkali injury. The depletion of T-cell subsets was confirmed by flow cytometry using splenocytes from immunized mice 1 week after the second injection. The percentage of CD4 T cells (CD3+ CD4+) or CD8 T cells (CD3+ CD8+) was compared between anti-CD4 or -CD8 antibody-treated mice and control animals. 
Results
Suppression of Corneal Neovascularization by Vaccination with VEGFR2400–408-Pulsed DCs
The vascularized area after alkali injury was quantified to evaluate the effect of vaccination with VEGFR2400–408-pulsed or gp70-pulsed DCs on the development of corneal vascularization (Fig. 1) . Interestingly, the VEGFR2400–408-immunized mice exhibited significant (P < 0.05) suppression of corneal neovascularization compared with nonimmunized or gp70-immunized controls on day 14 after alkali injury. Importantly, there was no significant (P > 0.05) difference between nonimmunized and gp70-immunized mice, suggesting that the observed suppression of corneal neovasculature was attributable to the VEGFR2400–408 peptide-specific immunologic responses. 
Specific Cytokine Responses by Peptide-Induced CD8+ T Cells
To confirm the induction of T cells specific for the immunized peptides, we used ELISA to examine the peptide-induced secretion of IFN-γ and TNF-α in the culture medium (Fig. 2) . VEGFR2400–408 application to CD8+ T cells harvested from mice immunized with VEGFR2400–408 led to significant (P < 0.01) production of both IFN-γ and TNF-α in a dose-dependent manner (Figs. 2A 2B) . Similarly, a significant (P < 0.01) increase in the production of these cytokines in response to gp70 stimulation was detected in a dose-dependent manner with CD8+ T cells from the gp70-immunized mice (Figs. 2C 2D) , in which the vascularized area was not reduced (Fig. 1) . In contrast, irrelevant β-gal96–103 application at the maximal dose in the assay (10 μg/mL) did not significantly (P > 0.05) induce the production of these cytokines by CD8+ T cells isolated from mice that received VEGFR2400–408 or gp70 immunization (Figs. 2A 2B 2C 2D)
Specific Cell Lysis by VEGFR2400–408-Induced CD8+ T Cells
51Cr release assay was conducted to determine whether T cells could recognize the presentation of H-2Db (MHC class I)-VEGFR2400–408 complex, thus leading to lysis or killing of target cells (Fig. 3) . VEGFR2-positive syngeneic endothelial line H5V cells (H-2Db+, VEGFR2+) served as target cells, whereas the VEGFR2-positive allogeneic endothelial line bEND3 (H-2Db; VEGFR2+) and the VEGFR2-negative syngeneic line EL4 (H-2Db+; VEGFR2) functioned as controls. The ability of VEGFR2400–408-induced CD8+ T cells (effector cells) to lyse MHC-matched H5V endothelial cells was significantly more potent than that of MHC-unmatched bEND3 endothelial cells at all the effector/target ratios examined (Fig. 3A) . Importantly, the effector cells significantly killed MHC-matched, VEGFR2-negative, EL4 nonendothelial cells pulsed with VEGFR2400–408 but not with the irrelevant peptide β-gal96–103 (Fig. 3B)
CD8+ T Cells as In Vivo Effector Cells to Inhibit Corneal Neovascularization
To confirm the role of CD8+ T cells as in vivo effectors in the vaccination-induced suppression of corneal neovascularization (Fig. 1) , in vivo depletion of immune cell subsets using an anti-CD4 or -CD8 antibody was performed to mice immunized with VEGFR2400–408 (Fig. 4) . Compared with treatment with an isotype-matched control antibody, antibody-based depletion of CD8+, but not of CD4+, T cells significantly (P < 0.05) reversed the VEGFR2400–408-induced reduction of the vascularized area to the level in nonimmunized mice (Fig. 4A) . The depletion of T cells in immunized mice was confirmed by flow cytometry 1 week after the second treatment with each deletion antibody (Fig. 4B) . The percentage of CD4+ T cells out of splenocytes was 22% with the isotype-matched control compared with 3% with the anti-CD4 antibody. Similarly, the percentage of CD8+ T cells was 12.3% with the isotype-matched control compared with 4.9% with the anti-CD8 antibody. 
Discussion
The present study reveals, for the first time to our knowledge, that the induction of VEGFR2 peptide-specific CTLs targeting endothelial cells inhibits corneal neovascularization caused by alkali injury (Fig. 1) . Vaccination of DCs pulsed with the epitope peptide VEGFR2400–408 elicited T-cell responses specific for the peptide (Fig. 2) . CD8+ T cells from VEGFR2400–408-immunized mice also exhibited potent cytotoxicity against MHC-matched endothelial cells (Fig. 3) . Additionally, CD8+ T cells were shown as the in vivo major effectors in the immunologic treatment of corneal neovascularization (Fig. 4) . These emerging data indicate the new concept of CTL-mediated specific immunotherapy for corneal neovascularization. 
In our peptide-pulsed DC vaccination, VEGFR2400–408 immunization led to significant suppression of corneal neovascularization compared with no immunization in mice with corneal alkali injury (Fig. 1) . Although peptide-specific CTL responses were induced by VEGFR2400–408 and gp70 (Fig. 2) , gp70 immunization did not reduce the area of corneal neovascularization (Fig. 1) , suggesting that the suppressive effect observed in the present study resulted from VEGFR2400–408-specific induction of T-cell response. In the recent report 18 showing that VEGFR2400–408 immunization inhibited tumor growth and angiogenesis, the adjuvant mixture of IFA, GM-CSF, and an anti-CD40 activating antibody was applied to enhance the specific immunoreaction. Because GM-CSF signaling and CD40 ligation proved to be proangiogenic, 31 32 33 34 these were not used as the vaccine adjuvant in the present study. Instead, we determined the use of DCs, the most potent antigen-presenting cells, 24 25 26 27 validated as effective adjuvant therapy in our recent report showing that the vaccination of VEGFR2400–408-pulsed DCs led to significant suppression of laser-induced choroidal neovascularization. 12  
Peptide-specific T-cell responses were confirmed by cytokine assays (Fig. 2) , indicating that our peptide-pulsed DC vaccination broke immunotolerance against the self-antigen VEGFR2400–408. In these assays, IFN-γ and TNF-α were used as indicators for the peptide-specific induction of CTLs. 35 The direction of acquired immunity is regulated by the Th1/Th2 balance, in which Th1 and Th2 CD4+ T cells promote cellular and humoral immunity, respectively, through cytokines inhibitory to each other. 36 37 IFN-γ, produced by Th1 cells and CTLs, is one of the most important cytokines for the induction of cellular immunity. Moreover, IFN-γ was shown to induce endothelial cell apoptosis 38 and to contribute to CTL-mediated tumor rejection, 39 suggesting its role in the observed suppression of corneal neovascularization in this study. TNF-α is known to be capable of activating cell survival and death. 40 Recent data demonstrated that genetic ablation of TNF-α led to significant suppression of cautery-induced corneal neovascularization, suggesting the antiangiogenic role of TNF-α in the cornea. 41 TNF-α stimulates the nuclear factor-κB pathway leading to cell proliferation, whereas endothelial cells undergo apoptosis through the TNF-α-induced activation of caspase 8. 42 Accordingly, CTL-derived TNF-α is thought to contribute to cytotoxicity, together with perforin and Fas ligand (FasL), 43 each of which triggers key distinct pathways responsible for CTL-mediated apoptosis. 
We further confirmed the peptide-specific cytotoxic activity of CD8+ T cells induced by VEGFR2400–408-pulsed DC vaccination (Fig. 3) . The substantial ability of VEGFR2400–408-induced CD8+ T cells to kill H5V endothelial cells (H-2Db+, VEGFR2+), but not MHC-unmatched bEND3 endothelial cells (H-2Db; VEGFR2+), demonstrated that the VEGFR2400–408 epitope peptide was naturally processed and was presented with MHC class I H-2Db by endothelial cells. Moreover, the CTL-induced killing of MHC-matched EL4 nonendothelial cells (H-2Db+; VEGFR2) pulsed with VEGFR2400–408 but not with the irrelevant peptide β-gal96–103, indicated that CD8+ T cells of VEGFR2400–408-immunized C57BL/6 mice recognized the H-2Db-VEGFR2400–408 complex presented on the cell surface, thus leading to effective and selective cytotoxicity. In accordance with the in vitro killing assay (Fig. 3) , the in vivo depletion experiments (Fig. 4)indicated CD8+ T cells as the major effector cells for the suppression of corneal neovascularization in our immunotherapy. This is compatible with the previous data showing that CD8+ CTLs as negative regulators of tumoral, 9 15 16 retinal, 10 11 and choroidal 12 neovascularization. Given the in vitro results confirming the specific lysis of endothelial cells by VEGFR2400–408-induced CTLs (Fig. 3) , the suppressive effect on corneal neovascularization observed in the present study is thought to depend mainly on the specific CTL-endothelial cell interaction. 
In the present study, we used the murine model of conjunctivalization of the cornea from alkali burn, a relevant model to reflect chemical injury in human ocular surface leading to vision loss because of corneal scarring and neovascularization. 1 Considering immunotherapy in clinical practice, highly purified peptides for vaccination have several advantages over full-length proteins. Peptides are more easily synthesized, and they lack the potential dangers of infection by recombinant viruses or exposure to exogenous allergens. Clinically, DC vaccination is already established in the cancer field. 44 The sustained effect of immunotherapy may theoretically benefit patients with chemical burn-induced corneal neovascularization; this angiogenic activity lasts at least several months. Long-term attention should be paid, however, to systemic adverse events potentially caused by the antiangiogenic action of VEGFR2-targeting CTLs. Additionally, VEGF signaling through VEGFR2, weakly expressed on normal vascular and nonvascular cells in various organs including the eye, is suggested to play physiologic roles in cell survival and tissue maintenance. 45 Importantly, CTLs induced by active immunization against VEGFR2 in the present and previous studies 12 14 15 16 17 18 do not target functional VEGFR2 to block its downstream signaling but induce apoptosis exclusively in endothelial cells that present the epitope peptide(s) naturally processed from VEGFR2 protein (VEGFR2400–408 in the present study) with the MHC class I molecule. In previous data on mice receiving anti-VEGFR2 immunotherapy, vaccination of S. typhimurium transfected with a VEGFR2-containing plasmid led to delayed wound healing and negligible impact on fertility. 16 In contrast, vaccination of DCs pulsed with VEGFR2 full-length protein did not affect wound healing. 15 Similarly, mice immunized with VEGFR2400–408, which we used in the present study, exhibited no obvious side effects. 12 18 In the eye as well, VEGFR2400–408 immunization did not affect retinal vasculature or leukocyte recruitment in our study (data not shown). Minimal side effects observed in these series of immunotherapy suggest that the MHC-mediated presentation of the VEGFR2 epitope peptide(s) is preferentially limited to proliferating endothelial cells during tumor growth and corneal chemical injury. However, delayed wound healing, 16 conceivably resulting from cytotoxicity for proliferating endothelial cells, has raised safety concerns as a potential adverse event in future clinical settings. 
In summary, the present data are the first to show that VEGFR2-specific CTL induction leads to significant suppression of corneal neovascularization caused by chemical injury. These findings indicate the possibility of active immunization as a novel therapeutic strategy to inhibit corneal neovascularization. 
 
Figure 1.
 
Suppression of corneal neovascularization by vaccination with VEGFR2400–408-pulsed, but not gp70-pulsed, DCs. Representative photographs of the corneas from nonimmunized, VEGFR2400–408-immunized, and gp70-immunized mice (A). Graph shows the vascularized area (in pixels) of the cornea (B). Corneal neovascularization was significantly reduced by immunization with VEGFR2400–408. Results represent mean ± SD; n = 8 for all. *P < 0.05 by Mann-Whitney U test.
Figure 1.
 
Suppression of corneal neovascularization by vaccination with VEGFR2400–408-pulsed, but not gp70-pulsed, DCs. Representative photographs of the corneas from nonimmunized, VEGFR2400–408-immunized, and gp70-immunized mice (A). Graph shows the vascularized area (in pixels) of the cornea (B). Corneal neovascularization was significantly reduced by immunization with VEGFR2400–408. Results represent mean ± SD; n = 8 for all. *P < 0.05 by Mann-Whitney U test.
Figure 2.
 
Specific cytokine responses by peptide-induced CD8+ T cells. CD8+ T cells from VEGFR2400–408-immunized (A, B) or gp70-immunized (C, D) mice released IFN-γ (A, C) and TNF-α (B, D) in a dose-dependent manner by stimulation with each corresponding peptide but not with β-gal96–103. Results represent the mean ± SD; n = 12 each (A, B), and n = 6 each (C, D). *P < 0.05, **P < 0.01 by Mann-Whitney U test.
Figure 2.
 
Specific cytokine responses by peptide-induced CD8+ T cells. CD8+ T cells from VEGFR2400–408-immunized (A, B) or gp70-immunized (C, D) mice released IFN-γ (A, C) and TNF-α (B, D) in a dose-dependent manner by stimulation with each corresponding peptide but not with β-gal96–103. Results represent the mean ± SD; n = 12 each (A, B), and n = 6 each (C, D). *P < 0.05, **P < 0.01 by Mann-Whitney U test.
Figure 3.
 
Specific cell lysis by VEGFR2400–408-induced CD8+ T cells. VEGFR2-positive H5V (H-2Db+; black circle) and bEND3 (H-2Db; white circle) endothelial cells served as targets (A). VEGFR2-negative EL4 (H-2Db+) nonendothelial cells pulsed with VEGFR2400–408 (black circle) or β-gal96–103 (white circle) served as targets (B). VEGFR2400–408-induced CD8+ T cells (effector cells) effectively killed MHC-matched H5V endothelial cells, but not MHC-unmatched bEND3 cells, at all the effector/target ratios examined (A). Effector cells exhibited the substantial ability to lyse EL4 cells pulsed with VEGFR2400–408 but not those pulsed with β-gal96–103 (B).
Figure 3.
 
Specific cell lysis by VEGFR2400–408-induced CD8+ T cells. VEGFR2-positive H5V (H-2Db+; black circle) and bEND3 (H-2Db; white circle) endothelial cells served as targets (A). VEGFR2-negative EL4 (H-2Db+) nonendothelial cells pulsed with VEGFR2400–408 (black circle) or β-gal96–103 (white circle) served as targets (B). VEGFR2400–408-induced CD8+ T cells (effector cells) effectively killed MHC-matched H5V endothelial cells, but not MHC-unmatched bEND3 cells, at all the effector/target ratios examined (A). Effector cells exhibited the substantial ability to lyse EL4 cells pulsed with VEGFR2400–408 but not those pulsed with β-gal96–103 (B).
Figure 4.
 
CD8+, but not CD4+, T cells as in vivo effector cells in the vaccination-induced suppression of corneal neovascularization. Compared with treatment with an isotype control, antibody-based depletion of CD8+, but not CD4+, T cells significantly reversed the VEGFR2400–408-induced reduction of the vascularized area to the level in nonimmunized mice (A). Flow cytometric analyses showing the depletion of T cells in immunized mice by anti-CD4 and anti-CD8 antibodies (B). Representative data on the decreased ratio of the CD3+CD4+ cells (22%–3%) or CD3+CD8+ cells (12.3%–4.9%) after antibody treatment. Results represent the mean ± SD; n = 7 for each. *P < 0.05 by Mann-Whitney U test.
Figure 4.
 
CD8+, but not CD4+, T cells as in vivo effector cells in the vaccination-induced suppression of corneal neovascularization. Compared with treatment with an isotype control, antibody-based depletion of CD8+, but not CD4+, T cells significantly reversed the VEGFR2400–408-induced reduction of the vascularized area to the level in nonimmunized mice (A). Flow cytometric analyses showing the depletion of T cells in immunized mice by anti-CD4 and anti-CD8 antibodies (B). Representative data on the decreased ratio of the CD3+CD4+ cells (22%–3%) or CD3+CD8+ cells (12.3%–4.9%) after antibody treatment. Results represent the mean ± SD; n = 7 for each. *P < 0.05 by Mann-Whitney U test.
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Figure 1.
 
Suppression of corneal neovascularization by vaccination with VEGFR2400–408-pulsed, but not gp70-pulsed, DCs. Representative photographs of the corneas from nonimmunized, VEGFR2400–408-immunized, and gp70-immunized mice (A). Graph shows the vascularized area (in pixels) of the cornea (B). Corneal neovascularization was significantly reduced by immunization with VEGFR2400–408. Results represent mean ± SD; n = 8 for all. *P < 0.05 by Mann-Whitney U test.
Figure 1.
 
Suppression of corneal neovascularization by vaccination with VEGFR2400–408-pulsed, but not gp70-pulsed, DCs. Representative photographs of the corneas from nonimmunized, VEGFR2400–408-immunized, and gp70-immunized mice (A). Graph shows the vascularized area (in pixels) of the cornea (B). Corneal neovascularization was significantly reduced by immunization with VEGFR2400–408. Results represent mean ± SD; n = 8 for all. *P < 0.05 by Mann-Whitney U test.
Figure 2.
 
Specific cytokine responses by peptide-induced CD8+ T cells. CD8+ T cells from VEGFR2400–408-immunized (A, B) or gp70-immunized (C, D) mice released IFN-γ (A, C) and TNF-α (B, D) in a dose-dependent manner by stimulation with each corresponding peptide but not with β-gal96–103. Results represent the mean ± SD; n = 12 each (A, B), and n = 6 each (C, D). *P < 0.05, **P < 0.01 by Mann-Whitney U test.
Figure 2.
 
Specific cytokine responses by peptide-induced CD8+ T cells. CD8+ T cells from VEGFR2400–408-immunized (A, B) or gp70-immunized (C, D) mice released IFN-γ (A, C) and TNF-α (B, D) in a dose-dependent manner by stimulation with each corresponding peptide but not with β-gal96–103. Results represent the mean ± SD; n = 12 each (A, B), and n = 6 each (C, D). *P < 0.05, **P < 0.01 by Mann-Whitney U test.
Figure 3.
 
Specific cell lysis by VEGFR2400–408-induced CD8+ T cells. VEGFR2-positive H5V (H-2Db+; black circle) and bEND3 (H-2Db; white circle) endothelial cells served as targets (A). VEGFR2-negative EL4 (H-2Db+) nonendothelial cells pulsed with VEGFR2400–408 (black circle) or β-gal96–103 (white circle) served as targets (B). VEGFR2400–408-induced CD8+ T cells (effector cells) effectively killed MHC-matched H5V endothelial cells, but not MHC-unmatched bEND3 cells, at all the effector/target ratios examined (A). Effector cells exhibited the substantial ability to lyse EL4 cells pulsed with VEGFR2400–408 but not those pulsed with β-gal96–103 (B).
Figure 3.
 
Specific cell lysis by VEGFR2400–408-induced CD8+ T cells. VEGFR2-positive H5V (H-2Db+; black circle) and bEND3 (H-2Db; white circle) endothelial cells served as targets (A). VEGFR2-negative EL4 (H-2Db+) nonendothelial cells pulsed with VEGFR2400–408 (black circle) or β-gal96–103 (white circle) served as targets (B). VEGFR2400–408-induced CD8+ T cells (effector cells) effectively killed MHC-matched H5V endothelial cells, but not MHC-unmatched bEND3 cells, at all the effector/target ratios examined (A). Effector cells exhibited the substantial ability to lyse EL4 cells pulsed with VEGFR2400–408 but not those pulsed with β-gal96–103 (B).
Figure 4.
 
CD8+, but not CD4+, T cells as in vivo effector cells in the vaccination-induced suppression of corneal neovascularization. Compared with treatment with an isotype control, antibody-based depletion of CD8+, but not CD4+, T cells significantly reversed the VEGFR2400–408-induced reduction of the vascularized area to the level in nonimmunized mice (A). Flow cytometric analyses showing the depletion of T cells in immunized mice by anti-CD4 and anti-CD8 antibodies (B). Representative data on the decreased ratio of the CD3+CD4+ cells (22%–3%) or CD3+CD8+ cells (12.3%–4.9%) after antibody treatment. Results represent the mean ± SD; n = 7 for each. *P < 0.05 by Mann-Whitney U test.
Figure 4.
 
CD8+, but not CD4+, T cells as in vivo effector cells in the vaccination-induced suppression of corneal neovascularization. Compared with treatment with an isotype control, antibody-based depletion of CD8+, but not CD4+, T cells significantly reversed the VEGFR2400–408-induced reduction of the vascularized area to the level in nonimmunized mice (A). Flow cytometric analyses showing the depletion of T cells in immunized mice by anti-CD4 and anti-CD8 antibodies (B). Representative data on the decreased ratio of the CD3+CD4+ cells (22%–3%) or CD3+CD8+ cells (12.3%–4.9%) after antibody treatment. Results represent the mean ± SD; n = 7 for each. *P < 0.05 by Mann-Whitney U test.
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