September 2008
Volume 49, Issue 9
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Immunology and Microbiology  |   September 2008
The B Subunit of Escherichia coli Heat-Labile Enterotoxin Inhibits Th1 but Not Th17 Cell Responses in Established Experimental Autoimmune Uveoretinitis
Author Affiliations
  • Ben J. E. Raveney
    From the Department of Cellular and Molecular Medicine, School of Medical Sciences and the
  • Claire Richards
    From the Department of Cellular and Molecular Medicine, School of Medical Sciences and the
  • Marie-Laure Aknin
    Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
  • David A. Copland
    Unit of Ophthalmology, Department of Clinical Sciences at South Bristol, University of Bristol, Bristol, United Kingdom; and the
  • Bronwen R. Burton
    From the Department of Cellular and Molecular Medicine, School of Medical Sciences and the
  • Emma Kerr
    Unit of Ophthalmology, Department of Clinical Sciences at South Bristol, University of Bristol, Bristol, United Kingdom; and the
  • Lindsay B. Nicholson
    From the Department of Cellular and Molecular Medicine, School of Medical Sciences and the
    Unit of Ophthalmology, Department of Clinical Sciences at South Bristol, University of Bristol, Bristol, United Kingdom; and the
  • Neil A. Williams
    From the Department of Cellular and Molecular Medicine, School of Medical Sciences and the
  • Andrew D. Dick
    From the Department of Cellular and Molecular Medicine, School of Medical Sciences and the
    Unit of Ophthalmology, Department of Clinical Sciences at South Bristol, University of Bristol, Bristol, United Kingdom; and the
Investigative Ophthalmology & Visual Science September 2008, Vol.49, 4008-4017. doi:10.1167/iovs.08-1848
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      Ben J. E. Raveney, Claire Richards, Marie-Laure Aknin, David A. Copland, Bronwen R. Burton, Emma Kerr, Lindsay B. Nicholson, Neil A. Williams, Andrew D. Dick; The B Subunit of Escherichia coli Heat-Labile Enterotoxin Inhibits Th1 but Not Th17 Cell Responses in Established Experimental Autoimmune Uveoretinitis. Invest. Ophthalmol. Vis. Sci. 2008;49(9):4008-4017. doi: 10.1167/iovs.08-1848.

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

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Abstract

purpose. To investigate the efficacy of the B subunit of Escherichia coli heat-labile enterotoxin (EtxB) in the treatment of ocular autoimmune disease. Murine experimental autoimmune uveoretinitis (EAU) is an animal model of autoimmune posterior uveitis initiated by retinal antigen-specific Th1 and Th17 CD4+ T cells, which activate myeloid cells, inducing retinal damage. EtxB is a potent immune modulator that ameliorates other Th1-mediated autoimmune diseases, enhancing regulatory T-cell activity.

methods. EAU was induced in B10.RIII mice by immunization with peptide hIRBP161-180. Disease severity was measured by clinical and histologic assessment, and functional responses of macrophages (Mφs) and T cells were assessed, both in vivo and in cocultures in vitro. EtxB was administered intranasally daily for 4 days, starting either 3 days before or 3 days after EAU induction.

results. Preimmunization treatment with EtxB protected mice from EAU, limiting both the number and the activation status of retinal infiltrating immune cells. Treatment after EAU induction did not alter the disease course, despite suppression of IFN-γ. Although EtxB treatment of in vitro cocultures of T cells and Mφs increased IL-10 production, EtxB treatment in vivo increased the proportion and number of IL-17-producing CD4+ cells infiltrating the eye.

conclusions. EtxB preimmunization protects mice from EAU induction by inhibiting Th1 responses, but the resultant reduction in IFN-γ responses by EtxB does not effect infiltration or structural damage in established EAU, where Th17 responses predominate. These data highlight the critical importance of the dynamics of T-cell phenotype and infiltration during EAU when considering immunomodulatory therapy.

Experimental autoimmune uveoretinitis (EAU) serves as a preclinical model of human uveitis for assessing efficacy of immunotherapies and has generated successful translation of such into clinical practice. Moreover, the close clinicopathologic correlation between EAU and human uveitis allows us to dissect immunopathologic mechanisms of autoimmune inflammation and tissue damage while interrogating pathways to facilitate the development of immunotherapies. 1 2 3 4 EAU in the mouse is initiated by activation of CD4+ T cells specific for ocular antigens, most frequently located within or around photoreceptor segments. 5 6 7 8 9 In particular, we use a model system in which EAU is induced by administration of dominant peptides from interphotoreceptor retinoid binding protein (IRBP) in an appropriate adjuvant. 10 Such infiltration recruits and activates macrophages (Mφs) in the eye that generate structural damage via mechanisms that include secretion of nitric oxide (NO). 11  
The ability of bacterial toxins from Vibrio cholerae (Ctx) and the related heat-labile enterotoxin from Escherichia coli (Etx) to modulate immune responses to several antigens including autoantigens after mucosal or systemic administration has been of interest in the development of potential vaccines and immunotherapy. 12 However, the inherent harm of such toxins has limited their use as an adjuvant for human use. The discovery that the nontoxic B subunit of these toxins could act as an adjuvant and immune modulator has led to several studies being performed on a range of disease models. Although the mode of action has yet to be fully determined, EtxB can induce a great range of effects including apoptosis in CD8+ T cells, 13 activation of B cells, 14 suppression of CD4+ Th1 responses, induction of CD4+ Th2 responses, 15 16 and induction of regulatory T cells. 17 The modulatory effect of EtxB is dependent on its ability to bind to receptors, the principle one being GM1 ganglioside, found on the surface of all mammalian cells. A non-receptor-binding mutant, EtxB(G33D) failed to induce modulatory responses and hence protection in both in vivo and in assays in vitro. 13 18 In the case of collagen-induced arthritis (CIA) in DBA/1 mice, intranasal administration of EtxB reduced the incidence and severity of arthritis when given either at the time or 25 days after disease induction. 17 Reduction in disease was associated with reduced levels of collagen-specific IgG2a antibodies and interferon (IFN)-γ, whereas IgG1, interleukin (IL)-4, and IL-10 were similar in both protected and unprotected animals. Disease protection was attributed to the reduction in Th1 cell reactivity, but in this disease model, it was not associated with a shift to Th2 responses. Transfer of CD4+ T cells from treated mice also protected recipient mice from disease induction, but such protection was markedly reduced if the transferred cells were depleted of CD25+ cells. This finding suggests that EtxB may enhance populations of CD4+ T regulatory cells to reduce autoimmunity. Differences in the pattern of disease observed after a transfer of whole spleen cell populations from protected mice, compared with splenocytes depleted of CD25+ cells, suggested that other cell populations may also have a role to play. 17 In a murine model of ocular HSV-1 infection, the infiltration of immune cells into the cornea leads to opacity, edema, and eventual scarring of this normally transparent tissue, resulting in herpes stromal keratitis. Coadministration of HSV-1 antigens with EtxB intranasally, before infection or after the establishment of latency, prevents the infiltration of tissue damaging immune cells, thus reducing disease severity. 16 19 In the absence of EtxB-mediated immunization, the initial influx of cells observed soon after disease induction was dominated by neutrophils (Gr-1+), as determined by immunohistochemistry, followed by a second influx of neutrophils together with CD4+ T cells. 20 The cytokine profile indicated that Th1 cells predominated. As an influx of cells similar to those observed in ocular HSV-1 infection is associated with EAU, and previous studies have demonstrated that cholera toxin B subunit (CtxB), which is structurally similar to EtxB, modulates EAU, 21 we wanted to test whether EtxB could also be used to prevent or treat ocular autoimmunity. 
Materials and Methods
Mice, EAU Induction, and Scoring
C57BL/6 and B10.RIII mice were originally obtained from Harlan UK, Ltd. (Oxford, UK). C57BL/6 OT-II transgenic mice expressing the TcR specific for chicken ovalbumin 323-339 (OVA) and I-Ab were the kind gift of Steve Anderton (Department of Biological Sciences, University of Edinburgh, UK). Breeding colonies were established within the Animal Services Unit at Bristol University. All mice were housed in specific pathogen-free conditions with food and water continuously available. Female B10.RIII mice were between 6 and 8 weeks of age at the time of disease induction. The treatment of animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
B10.RIII mice were immunized SC with 50 μg/mouse IRBP161-180 peptide in PBS (2% vol/vol DMSO) in emulsion with complete Freund’s adjuvant (CFA; 1 mg/mL; 1:1 vol/vol) supplemented with 1.5 mg/mL Mycobacterium tuberculosis complete H37 Ra (BD Biosciences, Oxford, UK). Mice also received 1 μg Bordetella pertussis toxin (Sigma-Aldrich, Poole, UK) IP at the time of immunization. Some mice received 50 μg EtxB in 20 μL PBS by the intranasal route according to the treatment regimens specified. From day 7, the mice were clinically scored by using slit lamp biomicroscopy after pupil dilation with cyclopentolate hydrochloride 1.0% (Chauvin, Montpellier, France); retinas were visualized using a microscope coverslip placed over the eye. The disease was scored as previously described. 9 Briefly, the retina, anterior chamber, and pupil were scored for disease, each using a scale of 0 to 3, giving a total disease score of 0 to 9: 0, normal; 1, early inflammation (partial disruption to pupil, some opacity in the vitreous, and retinal swelling and vascular sheathing, with white focal lesions visible); 2, increased inflammation (pupil disruption and fluid retention, dense vitritis with fundal view obscured, and increased sheathing, retinal swelling, and hemorrhage); and 3, full disease (anterior segment inflammation, 22 posterior synechiae and dense vitritis). 
Preparation of Tissue for Histology
At various time points after immunization, the eyes were snap frozen in optimal cutting temperature (OCT) compound (R. Lamb Ltd., East Sussex, UK). Serial 12-μm cryosections were cut and fixed in acetone for 10 minutes. They were stained with rat anti-mouse monoclonal anti-CD45 antibody (Serotec, Oxford, UK) and counterstained with hematoxylin (ThermoShandon, Pittsburgh, PA), before scoring for inflammatory infiltrate (presence of CD45-positive cells) and structural disease (disruption of morphology), as previously described. 23 24  
Reagents
Human IRBP161-180 SGIPYIISYLHPGNTILHVD peptide and OVA323-339 ISQAVHAAHAEINEAGR were synthesized by Sigma-Aldrich to at least 95% purity, as determined by HPLC. Culture medium (DMEM) was supplemented with 10% vol/vol fetal calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine, 1 mM sodium pyruvate, and 5 μM 2-mercaptoethanol (all Invitrogen, Paisley, UK), unless otherwise stated. rEtxB was purified from Vibrio sp. 60 (pMMB68), as previously described. 25 26 Briefly, rEtxB expression was induced by IPTG addition to Vibrio sp. 60 (pMMB68) grown in Luria Bertini medium supplemented with 1% (wt/vol) NaCl. After 16 hours, the culture medium was recovered by dia-ultrafiltration and then subjected to ammonium sulfate precipitation (30% saturation) and hydrophobic and anion exchange chromatography. The single peak eluting from the anion exchange column (Resource Q; GE Healthcare, Waukesha, WI) was desalted by dialysis against PBS and stored at −80°C before use. Levels of endotoxin were <30 EU/mg of rEtxB, as determined by a chromogenic endpoint Limulus amebocyte lysate assay (Lonza, Berks, UK). 
Isolation of Single-Cell Suspensions
For isolation of retinal-infiltrating cells, the eyes were enucleated and the retinas of each animal were dissected microscopically and washed in wash medium (complete RPMI supplemented with 10% vol/vol FCS and 1 mM HEPES; all from Invitrogen). The retinas were then cut into small pieces and digested in 1 mL wash medium, supplemented with 0.5 mg/mL collagenase D (Roche UK, Welwyn Garden City, UK) and 750 U/mL DNase I (Sigma-Aldrich) for 20 minutes at 37°C. An additional 0.5 mg collagenase D and 750 U DNase was then added, before the specimens were incubated for a further 10 minutes at 37°C. The cell suspensions were forced through a 40-μm cell strainer (BD Falcon, Bedford, MA) by using a syringe plunger and then stained for flow cytometry. 
Splenocyte and lymph node cell suspensions were generated by excising spleens and cervical lymph nodes (draining lymph nodes for the eye) and disassociating tissue, by passing through a 40-μm cell strainer with a syringe plunger. 
Flow Cytometry
Cell suspensions were incubated with 24G2 cell supernatant for 5 minutes at 4°C. For cell counting, retinal cell suspensions were stained with PE-Cy5,5-conjugated anti-mouse CD4 monoclonal antibody (mAb), APC-Cy7-conjugated anti-mouse CD11b mAb, and PE-Cy7-conjugated anti-mouse CD45 mAb (all BD PharMingen, Oxford, UK), at 4°C for 20 minutes. For assessment of Mφ phenotype, cell suspensions were additionally stained with PE-conjugated anti-mouse CD206 mAb (Serotec) and FITC-conjugated mAb against the murine inducible nitric oxide synthase enzyme (iNOS; BD PharMingen). For intracellular cytokine analysis, cell suspensions were stained by using a kit according to manufacturer’s instructions (Cytofix/Cytoperm; BD PharMingen) and FITC-conjugated anti-mouse IFN-γ mAb and PE-conjugated anti-mouse IL-17 (both BD PharMingen). Cell suspensions were acquired with an LSR-II flow cytometer (BD Cytometry Systems, Oxford, UK), and analysis was performed (FlowJo software; TreeStar, San Carlos, CA). The number of cells was calculated by reference to a known standard. 
Generation of Bone Marrow-Derived Macrophages
Bone marrow-derived macrophages (BM-Mφs) were generated with an adaptation of a method described by Munder et al. 27 Briefly, BM cells were resuspended at 5 × 105 cells/mL in complete media supplemented with 5% vol/vol horse serum (Invitrogen) and 50 pg/mL macrophage-colony stimulating factor. The cell suspension was transferred to hydrophobic PTFE-coated tissue culture bags (supplied by Markus Munder, University of Heidelberg, Heidelberg, Germany) and incubated for 8 days at 37°C in 5% CO2 (vol/vol). BM-Mφs were harvested and cocultured with OT-II CD4+ T cells, isolated from OT-II spleens by CD4+ MACS enrichment (Miltenyi Biotech, Surry, UK). 
Assessment of Cocultures
To measure proliferation, cultures were pulsed with 18.5 kBq triturated thymidine (GE Healthcare, Bucks, UK) per well for the final 8 hours of a 72-hour incubation. Cells were harvested with a 96-well harvester, and thymidine uptake (measured in cpm) was determined by liquid scintillation with a liquid scintillation counter (Microbeta 1450; Wallac Oy, Turku, Finland). Accumulated NO production was measured after 36 hours in culture by assaying culture supernatants using Griess reagent (Sigma-Aldrich), as previously described. 28 Cytokine production (IFN-γ, IL-4, and IL-10) in culture supernatants was assayed by standard capture enzyme-linked immunosorbent assay (ELISA), as previously described. 28 In some experiments, cytokine concentrations in tissue culture supernatants were also assessed using a murine Th1/Th2 multiplex bead array (Flow Cytomix; Bender Medsystems, Vienna, Austria) used according to the manufacturer’s instructions. Beads were visualized on a flow cytometer (FACSCalibur; BD Cytometry Systems), and data were analyzed on computer (FlowCytomix Pro 2.2 Software; Bender Medsystems). 
Statistical Analyses
Commercial software (Prism 4; GraphPad Software Inc., San Diego, CA) was used, and comparisons of statistical significance between groups were assessed by the Mann-Whitney U test. P < 0.05 was regarded as significant. 
Results
Protection from EAU after Pretreatment with EtxB
Administration of the immunomodulatory agent EtxB via a mucosal route can prevent the onset of Th1-mediated autoimmune disease. 29 30 As the murine correlate of human uveitis, EAU, has been described as a Th1-mediated autoimmune disease, 31 we wanted to test whether EtxB could be used to prevent onset of EAU. To this end, groups of B10.RIII mice, a strain highly susceptible to EAU, were treated with four daily intranasal doses of EtxB in PBS before induction of disease by immunization with a uveitogenic peptide (IRBP161-180) in CFA. Control mice received only PBS intranasally before immunization. The progression of the disease was monitored by both clinical and histologic scoring, according to previously published methods. 9 24 Clinical funduscopic scoring showed that control mice developed EAU with an onset at day 13 (Fig. 1A) , with peak disease at day 15, and disease was maintained until day 21 after immunization. In contrast, EtxB-treated mice demonstrated suppression of clinical disease throughout the time course assessed. Histologic examination at day 21 after immunization revealed fewer infiltrating CD45+ cells in EtxB-treated mice, and tissue morphology showed very little sign of disease (Figs. 1B 1C) . There was a statistically significant reduction in both infiltrative and structural disease scores in EtxB-treated mice (Fig. 1D)
Effect of EtxB on Both Number and Activation of Mφ Infiltrating the Eye
We have previously shown that tissue damage in EAU is, in part, dependent on Mφ-derived nitric oxide (NO) production in the eye 32 and that such activation results from stimulation by infiltrating CD4+ T cells. 6 It is clear that EAU can be modulated both by reducing CD4+ T cell infiltration 6 and by reducing Mφ activation. 33 EtxB has been shown to prevent T-cell proliferation through the induction of regulatory T cells. 17 We wanted to examine whether the reduction in the number of ocular infiltrating CD45+ cells (Figs. 1B 1C 1D)generated by EtxB treatment was due to a reduction in Mφ activation resulting from an altered cytokine environment or to a reduction in the number of T cells. Thus, using flow cytometry, we assessed the number of CD45+ cells infiltrating the eye during peak EAU. Concordant with our histologic data, flow cytometry confirmed a marked reduction in CD45+ cells in the eyes of EtxB-treated B10.RIII mice immunized for EAU compared with untreated immunized mice (Fig. 2A) . There was a reduction in both myeloid (CD11b+) and CD4+ T cell infiltrate, whereas the number of other CD45+ T cells (CD11bCD4) was not altered by EtxB treatment. However, the number of CD11b+ and CD4+ cell number in EtxB-treated mice was significantly greater than those isolated from age-matched unmanipulated control mice (data not shown); thus, despite treatment, immunization still allowed some immune cell infiltration. 
To test whether or not EtxB affects Mφ activation, in addition to reducing the number of infiltrating Mφs, we stained retinal digests intracellularly for the presence of the enzyme iNOS. This enzyme is upregulated in Mφs classically activated in a Th1 environment 34 35 and is responsible for the production of NO during EAU. 11 Expression was compared with that in CD4+ cells, which do not express iNOS. CD11b+ cells infiltrating the eye in EAU from control mice did upregulate iNOS, as demonstrated by flow cytometry (Fig. 2B) , whereas EtxB treatment led to lower expression of iNOS. As Mφs can also be activated in an alternative manner in a Th2 environment 36 leading to expression of the mannose receptor (CD206), 37 we examined retinal digests for the expression of CD206. Lack of CD206 upregulation at peak disease in both EtxB-treated and control mice indicated that infiltrating CD11b+ cells were not alternatively activated (data not shown). 
EtxB and T-Cell Priming in EAU
As described in Figure 2 , EtxB treatment not only reduced the number of infiltrating cells into the eye during EAU, but also limited the activation of Mφs in the tissue. However, as there was still a significant number of infiltrating CD11b+ cells and CD4+ T cells in the eyes of EtxB-treated mice, we wanted to elucidate the activity of such cells. To this end, splenocytes and cells from cervical lymph nodes (which drain the site of activation and reflect the cytokine environment of the eye 38 ) of immunized mice, either EtxB- or PBS-treated control specimens were restimulated with the immunizing peptide. After 72 hours, proliferation and cytokine production was measured. EtxB treatment had very little effect on the priming of IRBP-specific responses among splenocytes in terms of proliferative capacity and their ability to produce IFN-γ, IL-4, and IL-10 on restimulation (Fig. 3) . On the other hand, EtxB treatment dramatically affected T-cell responses in the draining lymph nodes, inhibiting proliferation and IFN-γ and IL-10 production (Fig. 3)
Effect on EAU of EtxB Treatment of Mice Subsequent to Immunization
Although EtxB treatment prevented EAU induction when administered before immunization, to consider EtxB as a therapeutic agent in human uveitis, EtxB must be effective in suppressing disease once initiated. To test any possible therapeutic potential, mice were immunized, and EtxB was administered intranasally daily for 4 days beginning 3 days after immunization for EAU. Control mice were similarly treated with PBS. Disease severity was assessed by clinical and histologic scoring, as well as by flow cytometry at day 13. Surprisingly, EtxB treatment when given after disease induction appeared to have no effect on disease outcome, as shown by clinical scoring, histology, and flow cytometry (Fig. 4) . Overall, there was equivalent structural damage and a similar number of infiltrating CD11b+ and CD4+ T cells between treated and control animals. 
Effect of EtxB on Mφ-T-Cell Cocultures
We have demonstrated that pretreatment with EtxB modulates immune responses and thus prevents EAU (Fig. 1) , but EtxB treatment did not suppress disease when administered after immunization (Fig. 4) . To address this apparent discrepancy, we examined the mechanism by which EtxB may modulate immune responses in vitro. EAU is an autoimmune disease mediated by ocular antigen-specific CD4+ T cells, that activate Mφs to generate localized damage. 2 Thus, we used a model system where naïve BM-Mφs were cocultured with OVA-specific CD4+ T cells and cognate peptide in the presence or absence of EtxB, and examined T-cell and BM-Mφ responses by assessing proliferation, as well as the production of cytokines and NO. These studies were performed on a C7BL/6 background owing to the availability of a suitable in vitro system. 
BM-Mφs were able to activate OT-II T cells to produce IFN-γ in a peptide-dependent manner; however, they did not stimulate T-cell proliferation (Figs. 5A 5C) . This result was probably due to the large amounts of NO produced by BM-Mφ in these cultures (Fig. 5B) , which have been linked with suppression of T-cell proliferation. 39 Treatment of BM-Mφs markedly inhibited the production of NO; however, it did not, lead to a restoration of T-cell proliferation (Figs. 5A 5B) . Analysis of the supernatants from cultures indicated that EtxB inhibited IFN-γ production (Fig. 5C)but enhanced production of IL-10, IL-6, and TNF-α (Figs. 5D 5E 5F) . Furthermore, EtxB stimulated the production of high levels of PGE2, even in the absence of peptide (Fig. 5G) . These findings are in keeping with observations that have shown that EtxB upregulates COX-2 mRNA in vivo (Tong KK, Williams NA, unpublished data, 2007) and may explain the suppression of T-cell responses in these cultures, even in the absence of NO. 40 In the absence of EtxB, BM-Mφs cocultured with OT-II T cells produced PGE2 in a peptide-dependent manner, which was abrogated by treatment with a COX inhibitor, indomethacin (Fig. 5G)
Treatment with EtxB and Differentiation of Th17 Cells
EAU has customarily been defined as a Th1-mediated disease 31 ; however, more recent data suggest that other similar models of organ-specific autoimmune disease may be mediated by IL-17-producing CD4+ T cells—for example EAE 41 42 —and several new studies also indicate that Th17-mediated effector responses may occur in EAU as well. 43 44 45 This paradigm may explain why EtxB, which reduces IFN-γ responses both in vitro and in vivo (Figs. 3 5C , respectively), does not ameliorate EAU. With this is mind, it is interesting to note that our in vitro data show that EtxB promotes macrophage IL-6 production, and if this occurs in the eye in the context of the constitutive presence of TGF-β 46 and inhibition of IFN-γ secretion, then this may provide optimal differentiation stimuli for autopathogenic Th17 cells. 47 To determine whether or not EtxB treatment enhances Th17 responses in EAU, groups of IRBP161-180-immunized mice were treated with EtxB, as before, on days 3 to 7 after immunization; control immunized mice received only PBS. At various time points, retinal cells were restimulated and then stained intracellularly for IFN-γ, IL-4, and IL-17. Virtually no IL-4 was detected at any time point in any sample (data not shown). A high proportion of CD4+ T cells infiltrating the retinas of diseased animals produced either IL-17 or IFN-γ on restimulation. In both groups, a higher proportion of cells produced IL-17 than IFN-γ at the peak of infiltration, day 13 (Fig. 6) . Over time, the ratio of IL-17-secreting cells to IFN-γ-producing cells was reduced in both groups. Critically, at day 13, the ratio of IL-17- to IFN-γ-producing cells was higher in EtxB-treated mice than in control mice (Fig. 6)
Discussion
Modulation of immune responses by EtxB administration has alleviated clinical disease in a range of animal models of autoimmune disease. 15 17 29 48 We proposed that similar EtxB administration may provide a useful therapeutic approach in the treatment of ocular autoimmune inflammatory disease. Thus, we examined how EtxB modulates the immune responses that occur during EAU and the efficacy of this treatment strategy. 
In this study, treating mice with EtxB immediately before EAU induction prevented the initiation of autoimmunity, inhibited immune cell infiltration into the eye, and limited activation of both T cells and Mφs in the target organ. Conversely, EtxB treatment after EAU induction did not abrogate disease progression and instead may enhance Th17 responses in the eye at peak infiltrative disease. 
Previous studies have reported multiple mechanisms by which EtxB modulates immune responses. In particular, when EtxB is given alone, it has been shown to ameliorate autoimmunity by inhibiting Th1 responses and promoting T regulatory responses. 17 29 When given in combination with antigen, EtxB appears to promote Th2- and T-regulatory responses, 15 48 and this process has been shown to reduce chronic ocular inflammation generated by HSV-1 infection. 16 Modulation of the immune response in favor of Th2 responses can prevent EAU induction. 31 In keeping with the fact that we used EtxB in the absence of antigen in these studies, EtxB-mediated protection from induction of autoimmune ocular inflammation was not associated with a skewing of responses to a Th2 phenotype. There was no evidence of enhanced IL-4 production in the eyes and no increase in CD206 expression on the Mφs found within the tissue (CD206+ alternatively activated Mφs are generated in the presence of high levels of IL-4 37 ). EtxB pretreatment reduced the number of Mφs infiltrating the eye and suppressed the levels of IFN-γ, in keeping with a reduction in the Th1 response. This result is in keeping with observations of infiltration of the pancreas in NOD mice that have been treated with EtxB as a means of inhibiting the development of type 1 diabetes, 29 in which a reduced number of CD11b+ cells were noted and with other systems in which EtxB has been shown to modulate immunity. 
In our experiments, EtxB administration did not alter the proliferation and cytokine responses of splenocytes when restimulated with the immunizing peptide ex vivo. EtxB pretreatment entirely inhibited peptide-specific responses by cells from the cervical lymph nodes that drain the target organ. A small but significant T-cell infiltrate was observed in the eyes of immunized mice that were pretreated with EtxB; and, combined with the fact that immunization of such mice generates activation of IRBP-specific T cells in the spleen, it is unlikely that this lack of response by lymph node cells is due to an absence of activated IRBP-specific T cells. We have demonstrated that EtxB can also modulate immune responses by enhancing the generation of regulatory T cells, 17 and we suggest that this may be its mechanism of action in preventing EAU, whereby EtxB causes differentiation of regulatory T cells in the target organ, inhibits autoimmune T-cell responses, and thus prevents Mφ activation in situ. The trafficking of such regulatory T cells to the draining lymph nodes would also explain the reduction in these peptide-specific responses, although both theories have yet to be tested directly. 
Despite its clear effects on the immune response to IRBP when given before challenge, EtxB treatment was insufficient to abrogate established EAU. To dissect possible reasons for the lack of effect after immunization, we examined the response to EtxB treatment in cocultures of T cells and BM-Mφs in the presence of cognate peptide. In the absence of EtxB, Th1 responses predominated in these cocultures, with high levels of IFN-γ that led to classic Mφ activation and production of high concentration of NO. The lack of T-cell proliferation observed under these in vitro conditions concurs with previous studies demonstrating inhibition of T-cell proliferation by NO production. 39 Taken together, two lines of evidence support possible NO-mediated mechanisms of control of T-cell proliferation, despite ongoing tissue damage during EAU: first, our previous observations of retained T cells within protected retina of EAU animals when treated with l-NAME 49 and second, NO and NOS2 expression in infiltrating Mφs during the evolution of EAU. 50  
In keeping with our observations in vivo, the addition of EtxB to this coculture system inhibited Th1-associated responses as evidenced by reduced levels of IFN-γ and NO, but did not enhance Th2 responses, as evidenced by a lack of IL-4 production. Of interest, in T-cell-BM-Mφ cocultures, EtxB enhanced production of IL-10, which is associated with inhibition T-cell proliferation by regulatory T cells. 51 Previously, EtxB has been shown to promote regulatory T-cell populations and thus inhibit autoimmunity. 17 Therefore, it is possible that EtxB treatment of cocultures also enhances the generation regulatory T cells and IL-10 production. Thus, the inhibition of T-cell proliferation in these cocultures is maintained, despite the reduction in NO. 
Myeloid-derived cells in tumor infiltrates have also been found to have the ability to suppress T-cell proliferation 52 ; also, such myeloid suppressor cells may be generated after treatment with immunosuppressive agents. 53 Myeloid-derived suppressor cells have been shown to inhibit T-cell proliferation by a number of mechanisms, including NO production 39 and PG production. 40 EtxB treatment dramatically stimulated PGE2 production in cocultures; thus, it is possible that EtxB maintains inhibition of T-cell proliferation through PGE2 production, rather than by generating regulatory T cells. EtxB also promotes PG production in vivo as indicated by increased levels of the PG synthase enzyme COX-2 in EtxB-treated DBA-1 mice (Tong KK, Williams NA, unpublished data, 2007); thus, PG may be involved in the immunomodulation by EtxB that suppresses autoimmunity, potentially via its known role in promoting the generation of certain types of T-regulatory cells. 54  
EtxB-mediated modulation of Mφ activation was associated with enhanced levels of IL-6 and TNF-α production. It is possible that this provides a clue to the reasons that EtxB may fail to control EAU after induction. Evidence clearly points toward a role for IL-6 in T-cell differentiation into Th17 cells 55 ; however, the presence of IL-10 can downregulate the pathogenic activity of such cells. 56 Th17 cells thrive in the absence of IFN-γ, 57 and it has been shown that EAU can be mediated by autoreactive Th17 cells. 45 Therefore, it is possible that the ability of EtxB to suppress Th1 responses but at the same time to promote IL-6 production creates an environment in which Th17 responses become more dominant. Our data support this hypothesis, showing increased proportions of IL-17-producing CD4+ T cells in the eyes of EtxB-treated mice at peak disease. However, despite the data, there was no increase in clinical or histologic signs of inflammatory disease. We suggest that, this enhanced Th17 activity is controlled either directly as a result of the production of IL-10 or as a result of increased levels of T-regulatory cell activity. 
Although EtxB is a potent immunomodulator and can prevent the initiation of ocular inflammation, treatment after EAU induction does not reduce clinical disease. This may be related to the differential activity of the components of the immune system which are active and which are modulated by EtxB at different stages of ocular inflammation. For example, PG has been shown to prevent ocular inflammation, but administration at later stages enhances disease. In other autoimmune models, PGE2 has also been shown to drive pathogenic Th17 responses, 58 which may explain the increased proportion of IL-17-producing cells in the eyes of EtxB-treated mice. Modulating the interplay between IL-17 and PG may allow therapeutic intervention to treat ocular autoimmunity. 
 
Figure 1.
 
B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days; control mice received only PBS. The mice were then immunized with 50 μg IRBP161-180 in CFA. The mice’s fundi were examined daily for clinical disease with a slit lamp. (A) Individual clinical disease score and medians over time in control mice (PBS) and treated mice (EtxB). *EtxB-treated significantly lower than PBS control at an equivalent time point (P < 0.005). The eyes were excised on day 21 after immunization, frozen, and cryosectioned. The resultant 12-μm sections were stained with anti-CD45 and scored histologically for EAU. Representative photomicrographs are shown for control mice (B) and ExtB-treated mice (C). (D) Mean infiltrate compared with the structural damage disease score for mice treated with EtxB with control mice (PBS). *Significantly different from control mice (P < 0.05). Data are representative of three independent experiments, with n ≥ 6 animals in each group.
Figure 1.
 
B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days; control mice received only PBS. The mice were then immunized with 50 μg IRBP161-180 in CFA. The mice’s fundi were examined daily for clinical disease with a slit lamp. (A) Individual clinical disease score and medians over time in control mice (PBS) and treated mice (EtxB). *EtxB-treated significantly lower than PBS control at an equivalent time point (P < 0.005). The eyes were excised on day 21 after immunization, frozen, and cryosectioned. The resultant 12-μm sections were stained with anti-CD45 and scored histologically for EAU. Representative photomicrographs are shown for control mice (B) and ExtB-treated mice (C). (D) Mean infiltrate compared with the structural damage disease score for mice treated with EtxB with control mice (PBS). *Significantly different from control mice (P < 0.05). Data are representative of three independent experiments, with n ≥ 6 animals in each group.
Figure 2.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization; whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. Thirteen days later, the retinas were digested with collagenase, and the resultant cell suspensions were surface stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b and intracellularly stained with anti-iNOS mAb. The cells were analyzed by flow cytometry and the number of cells calculated by reference to a standard. (A) The mean number of cells isolated from retinas of EtxB-treated mice with those from control mice. (B) Increase in geometric mean fluorescence intensity of iNOS staining among CD11b+ cells from each group compared with geometric mean fluorescence among CD4+ cells. †*Significantly different from control mice (P < 0.05 and P < 0.005, respectively). Data are representative of two independent experiments; n = 6.
Figure 2.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization; whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. Thirteen days later, the retinas were digested with collagenase, and the resultant cell suspensions were surface stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b and intracellularly stained with anti-iNOS mAb. The cells were analyzed by flow cytometry and the number of cells calculated by reference to a standard. (A) The mean number of cells isolated from retinas of EtxB-treated mice with those from control mice. (B) Increase in geometric mean fluorescence intensity of iNOS staining among CD11b+ cells from each group compared with geometric mean fluorescence among CD4+ cells. †*Significantly different from control mice (P < 0.05 and P < 0.005, respectively). Data are representative of two independent experiments; n = 6.
Figure 3.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization, whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. On day 21 after immunization, single-cell suspensions from spleens and draining lymph nodes were cultured for 72 hours in the presence or absence of 10 μg/mL IRBP161-180. Proliferation was measured by 3H-thymidine incorporation over the final 8 hours of culture and supernatant concentration of IFN-γ, IL-4, and IL-10 was determined by ELISA. The data are representative of two independent experiments, each containing at least six individual animals per experimental group.
Figure 3.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization, whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. On day 21 after immunization, single-cell suspensions from spleens and draining lymph nodes were cultured for 72 hours in the presence or absence of 10 μg/mL IRBP161-180. Proliferation was measured by 3H-thymidine incorporation over the final 8 hours of culture and supernatant concentration of IFN-γ, IL-4, and IL-10 was determined by ELISA. The data are representative of two independent experiments, each containing at least six individual animals per experimental group.
Figure 4.
 
B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. A group of these mice received 20 μg EtxB in PBS intranasally daily for 4 days on days 3 to 7 after immunization; control mice received only PBS. Mice were clinically scored by funduscopy. On day 15 after immunization, the left eyes were frozen, cryosectioned, stained with anti-CD45, and scored histologically for EAU. (A) Individual clinical disease score and medians over time for control mice (PBS) and treated mice (EtxB). Data sets are not significantly different. Representative photomicrographs of eye sections stained with anti-CD45 mAb are shown for control mice (B) and EtxB-treated mice (C). (D) Mean histologic disease score for treated mice (EtxB) versus control mice (PBS). Retinas from right eyes were digested with collagenase, and the single cell suspensions were stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b. (D, E) Mean number of cells obtained from retinas of mice treated with EtxB or those from control mice. Data are representative of two independent experiments, with n ≥ 5 animals in each experimental group.
Figure 4.
 
B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. A group of these mice received 20 μg EtxB in PBS intranasally daily for 4 days on days 3 to 7 after immunization; control mice received only PBS. Mice were clinically scored by funduscopy. On day 15 after immunization, the left eyes were frozen, cryosectioned, stained with anti-CD45, and scored histologically for EAU. (A) Individual clinical disease score and medians over time for control mice (PBS) and treated mice (EtxB). Data sets are not significantly different. Representative photomicrographs of eye sections stained with anti-CD45 mAb are shown for control mice (B) and EtxB-treated mice (C). (D) Mean histologic disease score for treated mice (EtxB) versus control mice (PBS). Retinas from right eyes were digested with collagenase, and the single cell suspensions were stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b. (D, E) Mean number of cells obtained from retinas of mice treated with EtxB or those from control mice. Data are representative of two independent experiments, with n ≥ 5 animals in each experimental group.
Figure 5.
 
BM-Mφs were cocultured with CD4+ MACS-enriched OT-II splenocytes in the (A) presence or (B) absence of cognate OVA for 72 hours. To some cultures, EtxB or indomethacin was added at the indicated concentrations. Proliferation was measured by 3H thymidine incorporation over the final 8 hours of culture, supernatant concentration of (C) IFN-γ, (D) IL-10, (E) IL-6, and (F) TNF-α and was determined by flow cytometry, NO production was measured by Griess reaction, and PGE2 concentration was determined by ELISA (G). Results are representative of at least two independent experiments.
Figure 5.
 
BM-Mφs were cocultured with CD4+ MACS-enriched OT-II splenocytes in the (A) presence or (B) absence of cognate OVA for 72 hours. To some cultures, EtxB or indomethacin was added at the indicated concentrations. Proliferation was measured by 3H thymidine incorporation over the final 8 hours of culture, supernatant concentration of (C) IFN-γ, (D) IL-10, (E) IL-6, and (F) TNF-α and was determined by flow cytometry, NO production was measured by Griess reaction, and PGE2 concentration was determined by ELISA (G). Results are representative of at least two independent experiments.
Figure 6.
 
Groups of B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. The mice were then treated with 20 μg EtxB in PBS intranasally daily on days 3 to 7 after immunization; a group of immunized control mice received only PBS. Thirteen days later, the retinas were digested with collagenase to form single-cell suspensions. The cells were restimulated with PMA/ionomycin for 6 hours before surface staining with fluorochrome-conjugated mAb against CD45, CD4, CD11b, and 7-AAD, and intracellular staining with anti-IFN-γ, anti-IL-17, and anti-IL-4. The cells were analyzed by flow cytometry and a ratio of IL-17-producing CD4+ cells to IFN-γ-producing live CD4+ cells was calculated (n = 6).
Figure 6.
 
Groups of B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. The mice were then treated with 20 μg EtxB in PBS intranasally daily on days 3 to 7 after immunization; a group of immunized control mice received only PBS. Thirteen days later, the retinas were digested with collagenase to form single-cell suspensions. The cells were restimulated with PMA/ionomycin for 6 hours before surface staining with fluorochrome-conjugated mAb against CD45, CD4, CD11b, and 7-AAD, and intracellular staining with anti-IFN-γ, anti-IL-17, and anti-IL-4. The cells were analyzed by flow cytometry and a ratio of IL-17-producing CD4+ cells to IFN-γ-producing live CD4+ cells was calculated (n = 6).
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Figure 1.
 
B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days; control mice received only PBS. The mice were then immunized with 50 μg IRBP161-180 in CFA. The mice’s fundi were examined daily for clinical disease with a slit lamp. (A) Individual clinical disease score and medians over time in control mice (PBS) and treated mice (EtxB). *EtxB-treated significantly lower than PBS control at an equivalent time point (P < 0.005). The eyes were excised on day 21 after immunization, frozen, and cryosectioned. The resultant 12-μm sections were stained with anti-CD45 and scored histologically for EAU. Representative photomicrographs are shown for control mice (B) and ExtB-treated mice (C). (D) Mean infiltrate compared with the structural damage disease score for mice treated with EtxB with control mice (PBS). *Significantly different from control mice (P < 0.05). Data are representative of three independent experiments, with n ≥ 6 animals in each group.
Figure 1.
 
B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days; control mice received only PBS. The mice were then immunized with 50 μg IRBP161-180 in CFA. The mice’s fundi were examined daily for clinical disease with a slit lamp. (A) Individual clinical disease score and medians over time in control mice (PBS) and treated mice (EtxB). *EtxB-treated significantly lower than PBS control at an equivalent time point (P < 0.005). The eyes were excised on day 21 after immunization, frozen, and cryosectioned. The resultant 12-μm sections were stained with anti-CD45 and scored histologically for EAU. Representative photomicrographs are shown for control mice (B) and ExtB-treated mice (C). (D) Mean infiltrate compared with the structural damage disease score for mice treated with EtxB with control mice (PBS). *Significantly different from control mice (P < 0.05). Data are representative of three independent experiments, with n ≥ 6 animals in each group.
Figure 2.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization; whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. Thirteen days later, the retinas were digested with collagenase, and the resultant cell suspensions were surface stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b and intracellularly stained with anti-iNOS mAb. The cells were analyzed by flow cytometry and the number of cells calculated by reference to a standard. (A) The mean number of cells isolated from retinas of EtxB-treated mice with those from control mice. (B) Increase in geometric mean fluorescence intensity of iNOS staining among CD11b+ cells from each group compared with geometric mean fluorescence among CD4+ cells. †*Significantly different from control mice (P < 0.05 and P < 0.005, respectively). Data are representative of two independent experiments; n = 6.
Figure 2.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization; whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. Thirteen days later, the retinas were digested with collagenase, and the resultant cell suspensions were surface stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b and intracellularly stained with anti-iNOS mAb. The cells were analyzed by flow cytometry and the number of cells calculated by reference to a standard. (A) The mean number of cells isolated from retinas of EtxB-treated mice with those from control mice. (B) Increase in geometric mean fluorescence intensity of iNOS staining among CD11b+ cells from each group compared with geometric mean fluorescence among CD4+ cells. †*Significantly different from control mice (P < 0.05 and P < 0.005, respectively). Data are representative of two independent experiments; n = 6.
Figure 3.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization, whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. On day 21 after immunization, single-cell suspensions from spleens and draining lymph nodes were cultured for 72 hours in the presence or absence of 10 μg/mL IRBP161-180. Proliferation was measured by 3H-thymidine incorporation over the final 8 hours of culture and supernatant concentration of IFN-γ, IL-4, and IL-10 was determined by ELISA. The data are representative of two independent experiments, each containing at least six individual animals per experimental group.
Figure 3.
 
Groups of B10.RIII mice received 20 μg EtxB in PBS intranasally daily for 4 days before immunization, whereas a group of control mice received only PBS. Mice were immunized with 50 μg IRBP161-180 in CFA. On day 21 after immunization, single-cell suspensions from spleens and draining lymph nodes were cultured for 72 hours in the presence or absence of 10 μg/mL IRBP161-180. Proliferation was measured by 3H-thymidine incorporation over the final 8 hours of culture and supernatant concentration of IFN-γ, IL-4, and IL-10 was determined by ELISA. The data are representative of two independent experiments, each containing at least six individual animals per experimental group.
Figure 4.
 
B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. A group of these mice received 20 μg EtxB in PBS intranasally daily for 4 days on days 3 to 7 after immunization; control mice received only PBS. Mice were clinically scored by funduscopy. On day 15 after immunization, the left eyes were frozen, cryosectioned, stained with anti-CD45, and scored histologically for EAU. (A) Individual clinical disease score and medians over time for control mice (PBS) and treated mice (EtxB). Data sets are not significantly different. Representative photomicrographs of eye sections stained with anti-CD45 mAb are shown for control mice (B) and EtxB-treated mice (C). (D) Mean histologic disease score for treated mice (EtxB) versus control mice (PBS). Retinas from right eyes were digested with collagenase, and the single cell suspensions were stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b. (D, E) Mean number of cells obtained from retinas of mice treated with EtxB or those from control mice. Data are representative of two independent experiments, with n ≥ 5 animals in each experimental group.
Figure 4.
 
B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. A group of these mice received 20 μg EtxB in PBS intranasally daily for 4 days on days 3 to 7 after immunization; control mice received only PBS. Mice were clinically scored by funduscopy. On day 15 after immunization, the left eyes were frozen, cryosectioned, stained with anti-CD45, and scored histologically for EAU. (A) Individual clinical disease score and medians over time for control mice (PBS) and treated mice (EtxB). Data sets are not significantly different. Representative photomicrographs of eye sections stained with anti-CD45 mAb are shown for control mice (B) and EtxB-treated mice (C). (D) Mean histologic disease score for treated mice (EtxB) versus control mice (PBS). Retinas from right eyes were digested with collagenase, and the single cell suspensions were stained with fluorochrome-conjugated mAb against CD45, CD4, and CD11b. (D, E) Mean number of cells obtained from retinas of mice treated with EtxB or those from control mice. Data are representative of two independent experiments, with n ≥ 5 animals in each experimental group.
Figure 5.
 
BM-Mφs were cocultured with CD4+ MACS-enriched OT-II splenocytes in the (A) presence or (B) absence of cognate OVA for 72 hours. To some cultures, EtxB or indomethacin was added at the indicated concentrations. Proliferation was measured by 3H thymidine incorporation over the final 8 hours of culture, supernatant concentration of (C) IFN-γ, (D) IL-10, (E) IL-6, and (F) TNF-α and was determined by flow cytometry, NO production was measured by Griess reaction, and PGE2 concentration was determined by ELISA (G). Results are representative of at least two independent experiments.
Figure 5.
 
BM-Mφs were cocultured with CD4+ MACS-enriched OT-II splenocytes in the (A) presence or (B) absence of cognate OVA for 72 hours. To some cultures, EtxB or indomethacin was added at the indicated concentrations. Proliferation was measured by 3H thymidine incorporation over the final 8 hours of culture, supernatant concentration of (C) IFN-γ, (D) IL-10, (E) IL-6, and (F) TNF-α and was determined by flow cytometry, NO production was measured by Griess reaction, and PGE2 concentration was determined by ELISA (G). Results are representative of at least two independent experiments.
Figure 6.
 
Groups of B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. The mice were then treated with 20 μg EtxB in PBS intranasally daily on days 3 to 7 after immunization; a group of immunized control mice received only PBS. Thirteen days later, the retinas were digested with collagenase to form single-cell suspensions. The cells were restimulated with PMA/ionomycin for 6 hours before surface staining with fluorochrome-conjugated mAb against CD45, CD4, CD11b, and 7-AAD, and intracellular staining with anti-IFN-γ, anti-IL-17, and anti-IL-4. The cells were analyzed by flow cytometry and a ratio of IL-17-producing CD4+ cells to IFN-γ-producing live CD4+ cells was calculated (n = 6).
Figure 6.
 
Groups of B10.RIII mice were immunized with 50 μg IRBP161-180 in CFA. The mice were then treated with 20 μg EtxB in PBS intranasally daily on days 3 to 7 after immunization; a group of immunized control mice received only PBS. Thirteen days later, the retinas were digested with collagenase to form single-cell suspensions. The cells were restimulated with PMA/ionomycin for 6 hours before surface staining with fluorochrome-conjugated mAb against CD45, CD4, CD11b, and 7-AAD, and intracellular staining with anti-IFN-γ, anti-IL-17, and anti-IL-4. The cells were analyzed by flow cytometry and a ratio of IL-17-producing CD4+ cells to IFN-γ-producing live CD4+ cells was calculated (n = 6).
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