Parabiosis reveals the correlation between the recruitment of circulating antigen presenting cells to the retina and the induction of spontaneous autoimmune uveoretinitis.

BACKGROUND: Characterizing immune cells and conditions that govern their recruitment and function in autoimmune diseases of the nervous system or in neurodegenerative processes is an area of active investigation. We sought to analyze the origin of antigen presenting cells associated with the induction of retinal autoimmunity using a system that relies on spontaneous autoimmunity, thus avoiding uncertainties associated with immunization with adjuvants at remotes sites or adoptive transfer of in vitro activated T cells. METHODS: R161H mice (B10.RIII background), which spontaneously and rapidly develop severe spontaneous autoimmune uveoretinitis (SAU), were crossed to CD11cDTR/GFP mice (B6/J) allowing us to track the recruitment to and/or expansion within the retina of activated, antigen presenting cells (GFPhi cells) in R161H+/- × CD11cDTR/GFP F1 mice relative to the course of SAU. Parabiosis between R161H+/- × CD11cDTR/GFP F1 mice and B10.RIII × B6/J F1 (wild-type recipient) mice was done to explore the origin and phenotype of antigen presenting cells crucial for the induction of autoimmunity. Analysis was done by retinal imaging, flow cytometry, and histology. RESULTS: Onset of SAU in R161H+/- × CD11cDTR/GFP F1 mice was delayed relative to B10.RIII-R161H+/- mice revealing a disease prophase prior to frank autoimmunity that was characterized by expansion of GFPhi cells within the retina prior to any clinical or histological evidence of autoimmunity. Parabiosis between mice carrying the R161H and CD11cDTR/GFP transgenes and transgene negative recipients showed that recruitment of circulating GFPhi cells into retinas was highly correlative with the occurrence of SAU. CONCLUSIONS: Our results here contrast with our previous findings showing that retinal antigen presenting cells expanding in response to either sterile mechanical injury or neurodegeneration were derived from myeloid cells within the retina or optic nerve, thus highlighting a unique facet of retinal autoimmunity.

The retinal environment induces microglia-like properties in recruited myeloid cells.

BACKGROUND: Microglia are essential to the development of the CNS and its homeostasis. Our prior findings suggested a niche model to describe the behaviors of retinal microglia. Here, we ask whether new myeloid cells recruited to the retina are constrained to resemble endogenous microglia morphologically and functionally. METHODS: Use of CD11cDTR/GFP transgenic mouse allowed identification of two niches of retinal microglia distinguished by being GFPlo or GFPhi. We also used transgenic mice in which CX3CR1+ cells expressed YFP and were depletable following tamoxifen-induced expression of diphtheria toxin subunit A. We employed several ablation and injury stimulation protocols to examine the origin and fate of myeloid cells repopulating the retina. Analysis of retinal myeloid cells was done by microscopy, flow cytometry, and qRT-PCR. RESULTS: We found that the origin of new GFPhi and GFPlo myeloid cells in the retina of CD11cDTR/GFP mice, whether recruited or local, depended on the ablation and stimulation protocols. Regardless of origin, new GFPlo and GFPhi retinal myeloid cells were CD45medCD11b+Ly6G-Ly6CloIba1+F4/80+, similar to endogenous microglia. Following tamoxifen-induced diphtheria toxin ablation, myeloid cell repopulation differed in the retina compared to the brain and optic nerve. Stimulation of replacement GFPhi cells was substantially attenuated in repopulating retinas after tamoxifen-induced diphtheria toxin ablation compared to control or radiation-ablated mice. In radiation bone marrow chimeric mice, replacement GFPhi myeloid cells from the circulation were slow to repopulate the retina unless stimulated by an optic nerve crush injury. However, once stimulated, recruited GFPhi cells were found to concentrate on injured retinal ganglion cells and were morphologically similar to GFPhi cells in non-ablated control CD11cDTR/GFP mice. CONCLUSIONS: The results support the idea that GFPhi cells in the CD11cDTR/GFP mouse, whether recruited or from resident microglia, mark a unique niche of activated retinal myeloid cells. We conclude that the retinal environment has a potent influence on the function, morphology, and proliferative capacity of new myeloid cells regardless of their origin, compelling them to be equivalent to the endogenous microglia.

Optic nerve as a source of activated retinal microglia post-injury.

Using mice expressing green fluorescent protein (GFP) from a transgenic CD11c promoter we found that a controlled optic nerve crush (ONC) injury attracted GFPhi retinal myeloid cells to the dying retinal ganglion cells and their axons. However, the origin of these retinal myeloid cells was uncertain. In this study we use transgenic mice in conjunction with ONC, partial and full optic nerve transection (ONT), and parabiosis to determine the origin of injury induced retinal myeloid cells. Analysis of parabiotic mice and fate mapping showed that responding retinal myeloid cells were not derived from circulating macrophages and that GFPhi myeloid cells could be derived from GFPlo microglia. Comparison of optic nerve to retina following an ONC showed a much greater concentration of GFPhi cells and GFPlo microglia in the optic nerve. Optic nerve injury also induced Ki67+ cells in the optic nerve but not in the retina. Comparison of the retinal myeloid cell response after full versus partial ONT revealed fewer GFPhi cells and GFPlo microglia in the retina following a full ONT despite it being a more severe injury, suggesting that full transection of the optic nerve can block the migration of responding myeloid cells to the retina. Our results suggest that the optic nerve can be a reservoir for activated microglia and other retinal myeloid cells in the retina following optic nerve injury.

Enhanced Detection of Sub-Retinal Pigment Epithelial Cell Layer Deposits in Human and Murine Tissue: Imaging Zinc as a Biomarker for Age-Related Macular Degeneration (An American Ophthalmological Society Thesis).

PURPOSE: Understanding the apparent paradoxical role of zinc in the pathogenesis and prevention of age-related macular degeneration (AMD) has been limited by the lack of animal models for its detection in sub-retinal epithelial deposits (drusen), a definitive early hallmark of AMD. In-vitro studies using Zinpyr-1 showed drusen contained high levels of zinc, but the probe was not suitable for in-vivo studies. This study compares Zinpyr-1 to ZPP1, a new fluorescein-based probe for zinc, to assess the potential of ZPP1 for in-vivo detection of zinc in drusen. METHODS: Flat mounts of human sub-RPE tissue using the probes were analyzed by fluorescence and confocal microscopy. Flat mounts of sub-RPE tissue from mice deficient in superoxide dismutase isoform-1 (CuZn-SOD-KO) or isoform-2 (Mn-SOD-RPE-KO) were analyzed with sub-RPE deposits confirmed by histology. RESULTS: Drusen are detected in greater numbers and intensity with ZPP1 compared to Zinpyr-1. Using ZPP1, drusen was detected in a sample from a 46-year old human donor without ocular history, suggesting that ZPP1 might be sensitive enough to detect drusen at an early stage. With CuZn-SOD KO mice, ZPP1 detected sub-RPE deposits at 10 months of age, whereas Zinpyr-1 required 14 months. CONCLUSION: Detection of sub-RPE deposits by ZPP1 was greatly enhanced compared to Zinpyr-1. This enhanced sensitivity will allow for more insightful analysis of zinc in AMD using human specimens and mouse models. This could result in the development of a sensitive in-vivo probe to enhance research on the role zinc in drusen formation and the early clinical diagnosis of AMD.

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells.

BACKGROUND: We previously reported that the peripheral regulatory T cells (pTregs) generated 'on-demand' in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo. METHODS: Transgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (ßgal) as retinal neo-self antigen and ßgal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination. RESULTS: Local depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in ßgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal ßgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local ßgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after ßgal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation. CONCLUSIONS: Our results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.

Retinal dendritic cell recruitment, but not function, was inhibited in MyD88 and TRIF deficient mice.

BACKGROUND: Immune system cells are known to affect loss of neurons due to injury or disease. Recruitment of immune cells following retinal/CNS injury has been shown to affect the health and survival of neurons in several models. We detected close, physical contact between dendritic cells and retinal ganglion cells following an optic nerve crush, and sought to understand the underlying mechanisms. METHODS: CD11c-DTR/GFP mice producing a chimeric protein of diphtheria toxin receptor (DTR) and GFP from a transgenic CD11c promoter were used in conjunction with mice deficient in MyD88 and/or TRIF. Retinal ganglion cell injury was induced by an optic nerve crush, and the resulting interactions of the GFPhi cells and retinal ganglion cells were examined. RESULTS: Recruitment of GFPhi dendritic cells to the retina was significantly compromised in MyD88 and TRIF knockout mice. GFPhi dendritic cells played a significant role in clearing fluorescent-labeled retinal ganglion cells post-injury in the CD11c-DTR/GFP mice. In the TRIF and MyD88 deficient mice, the resting level of GFPhi dendritic cells was lower, and their influx was reduced following the optic nerve crush injury. The reduction in GFPhi dendritic cell numbers led to their replacement in the uptake of fluorescent-labeled debris by GFPlo microglia/macrophages. Depletion of GFPhi dendritic cells by treatment with diphtheria toxin also led to their displacement by GFPlo microglia/macrophages, which then assumed close contact with the injured neurons. CONCLUSIONS: The contribution of recruited cells to the injury response was substantial, and regulated by MyD88 and TRIF. However, the presence of these adaptor proteins was not required for interaction with neurons, or the phagocytosis of debris. The data suggested a two-niche model in which resident microglia were maintained at a constant level post-optic nerve crush, while the injury-stimulated recruitment of dendritic cells and macrophages led to their transient appearance in numbers equivalent to or greater than the resident microglia.

Local "on-demand" generation and function of antigen-specific Foxp3+ regulatory T cells.

Extrathymically derived regulatory T cells (iTregs) protect against autoimmunity to tissue-specific Ags. However, whether Ag-specific iTreg generation and function is limited to secondary lymphoid tissue or whether it can occur within the tissue-specific local environment of the cognate Ag remains unresolved. Mice expressing ß-galactosidase (ßgal) on a retina-specific promoter (ßgal mice) in conjunction with mice expressing GFP and diphtheria toxin (DTx) receptor (DTR) under control of the Foxp3 promoter, and ßgal-specific TCR transgenic (BG2) mice were used to examine this question. Local depletion (ocular DTx), but not systemic depletion (i.p. DTx), of ßgal-specific iTregs enhanced experimental autoimmune uveoretinitis induced by activated ßgal-specific effector T cells. Injections of small amounts of ßgal into the anterior chamber of the eye produced similar numbers of ßgal-specific iTregs in the retina whether the mouse was depleted of pre-existing, circulating Tregs. Taken together, these results suggest that protection from tissue-specific autoimmunity depends on the function of local Ag-specific iTregs and that the retina is capable of local, "on-demand" iTreg generation that is independent of circulating Tregs.

Regulation of CD8(+) T Cell Responses to Retinal Antigen by Local FoxP3(+) Regulatory T Cells.

While pathogenic CD4 T cells are well known mediators of autoimmune uveoretinitis, CD8 T cells can also be uveitogenic. Since preliminary studies indicated that C57BL/6 mice were minimally susceptible to autoimmune uveoretinitis induction by CD8 T cells, the basis of the retinal disease resistance was sought. Mice that express ß-galactosidase (ßgal) on a retina-specific promoter (arrßgal mice) were backcrossed to mice expressing green fluorescent protein (GFP) and diphtheria toxin (DTx) receptor (DTR) under control of the Foxp3 promoter (Foxp3-DTR/GFP mice), and to T cell receptor transgenic mice that produce ßgal-specific CD8 T cells (BG1 mice). These mice were used to explore the role of regulatory T cells in the resistance to retinal autoimmune disease. Experiments with T cells from double transgenic BG1 × Foxp3-DTR/GFP mice transferred into Foxp3-DTR/GFP × arrßgal mice confirmed that the retina was well protected from attempts to induce disease by adoptive transfer of activated BG1 T cells. The successful induction of retinal disease following unilateral intraocular administration of DTx to deplete regulatory T cells showed that the protective activity was dependent on local, toxin-sensitive regulatory T cells; the opposite, untreated eye remained disease-free. Although there were very few Foxp3(+) regulatory T cells in the parenchyma of quiescent retina, and they did not accumulate in retina, their depletion by local toxin administration led to disease susceptibility. We propose that these regulatory T cells modulate the pathogenic activity of ßgal-specific CD8 T cells in the retinas of arrßgal mice on a local basis, allowing immuno regulation to be responsive to local conditions.

Local activation of dendritic cells alters the pathogenesis of autoimmune disease in the retina.

Interest in the identities, properties, functions, and origins of local APC in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of APC activity of DC from quiescent retina showed that they promoted generation of Foxp3(+) T cells and inhibited activation of naive T cells by splenic DC and Ag. Conversely, in vitro assays of DC purified from injured retina demonstrated an enhanced ability to activate T cells and reduced induction of Foxp3(+) T cells. These findings were supported by the observation that in situ activation of DC before adoptive transfer of ß-galactosidase-specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of Ag in vivo showed that quiescent retina promoted development of parenchymal Foxp3(+) T cells, but assays of preinjured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function and affected the pathogenesis of EAU by both CD4 and CD8 T cells.

Generation of Regulatory T Cells to Antigen Expressed in the Retina.

Regulatory T cells (Tregs) are generated to antigens (Ag) found in the retina. Some Tregs are the result of ectopic expression of the retinal Ags in the thymus, where developing T cells are committed to enter the regulatory lineage. However, the generation of retinal Ag-specific Tregs independent of the thymus was uncertain. Our studies show that Tregs can be generated from mature, peripheral T cells based on exposure to retinal Ags. These peripherally induced Tregs limited immune responses and experimental autoimmune disease induced by retinal Ags and thus constitute a crucial component of retinal immune privilege.

Dendritic cells are early responders to retinal injury.

The presence and activity of dendritic cells (DC) in retina is controversial, as these cells are difficult to identify in retina due to limited markers and sparse numbers. Transgenic mice that express green fluorescent protein (GFP) on the CD11c promoter to label DC allowed the visualization and quantification of retinal DC. Two retina injury models, the optic nerve crush (ONC) and light injury, were used to study their injury response. Many GFP(+) DC were tightly associated with retinal ganglion cell nerve fibers following ONC, while very few microglia (GFP(-)CD11b(+) cells) were found in close contact. The GFP(+) cells were greatly elevated in the outer plexiform layer following photic injury. All of the GFP(+) DC were CD11b(+), suggesting a myeloid origin. In addition, the GFP(+) DC upregulated expression of MHC class II after injury, while the GFP(-)CD11b(+) microglia did not. This study shows that DC were found in the retina and that they rapidly responded to neural injuries. We propose that they are a previously overlooked population, distinct from microglia, and may be important in the injury response.

Peripheral induction of tolerance by retinal antigen expression.

The contribution of peripheral expression of tissue-specific CNS Ags to the generation of tolerance is uncertain. To study this question, we examined mice transgenic (Tg) for expression of beta-galactosidase (beta gal) on the retinal photoreceptor cell arrestin promoter, in conjunction with TCR Tg mice producing CD4(+) T cells specific for beta gal (beta galTCR). Several strategies were used to test the hypothesis that betagal expressed in the retina supported thymus-independent tolerance and regulatory T cell development. Retinal expression generated an immunoregulatory response that depressed development of immune responses to beta gal following systemic immunization with beta gal. This regulation was transferable to naive mice by CD3(+)4(+)25(+) T cells from naive retinal beta gal(+) donors. Experiments that removed the beta gal(+) retina by enucleation showed that subsequent development of a regulatory response was lost. Adoptive transfer of CD25(-) beta galTCR T cells into retinal beta gal Tg mice on the Rag(-/-) background led to regulatory activity that limited lymphopenia-induced proliferation of beta galTCR T cells in mice with retinal expression of beta gal and inhibited the ear-swelling assay for delayed type hypersensitivity. These results show that retinal expression of very small amounts of a tissue-specific Ag can generate tolerance that includes regulatory T cells.

Lymphopenia-induced proliferation is a potent activator for CD4+ T cell-mediated autoimmune disease in the retina.

To study retinal immunity in a defined system, a CD4+ TCR transgenic mouse line (betagalTCR) specific for beta-galactosidase (betagal) was created and used with transgenic mice that expressed betagal in retinal photoreceptor cells (arrbetagal mice). Adoptive transfer of resting betagalTCR T cells, whether naive or Ag-experienced, into arrbetagal mice did not induce retinal autoimmune disease (experimental autoimmune uveoretinitis, EAU) and gave no evidence of Ag recognition. Generation of betagalTCR T cells in arrbetagal mice by use of bone marrow grafts, or double-transgenic mice, also gave no retinal disease or signs of Ag recognition. Arrbetagal mice were also resistant to EAU induction by adoptive transfer of in vitro-activated betagalTCR T cells, even though the T cells were pathogenic if the betagal was expressed elsewhere. In vitro manipulations to increase T cell pathogenicity before transfer did not result in EAU. The only strategy that induced a high frequency of severe EAU was transfer of naive, CD25-depleted, betagalTCR T cells into lymphopenic arrbetagal recipients, implicating regulatory T cells in the T cell inoculum, as well as in the recipients, in the resistance to EAU. Surprisingly, activation of the CD25-depleted betagalTCR T cells before transfer into the lymphopenic recipients reduced EAU. Taken together, the results suggest that endogenous regulatory mechanisms, as well as peripheral induction of regulatory T cells, play a role in the protection from EAU.

Evidence for extrathymic generation of regulatory T cells specific for a retinal antigen.

BACKGROUND: Thymic expression of a photoreceptor cell antigen, interphotoreceptor retinoid-binding protein, is known to generate regulatory T cells (T(reg)) that prevent spontaneous autoimmune disease of the retina. However, the contribution of other endogenous, tissue-specific antigens (Ags) expressed in the retina to the generation of T(reg) is uncertain. METHODS: Transgenic mice that express beta-galactosidase (beta-gal) in photoreceptor cells, together with beta-gal-specific T cell receptor transgenic mice, were used to study the induction of T(reg) in vivo. RESULTS: Transgenic expression of beta-gal on the arrestin promoter led to a spontaneous immunoregulatory response that inhibited the development of immune responses to beta-gal. The regulation was transferred by CD3+4+25+ T(reg). Several strategies were then used to show that beta-gal expressed in the retina supported spontaneous, thymus-independent T(reg) development. The endogenous T(reg) also differed from the T(reg) induced by Ag inoculation into the anterior chamber of the eye. CONCLUSION: These results demonstrate that retinal expression of very small amounts of a tissue-specific Ag can generate T(reg) in the periphery.

Interaction of retinal pigmented epithelial cells and CD4 T cells leads to T-cell anergy.

PURPOSE: Retinal pigmented epithelial (RPE) cells may contribute to retinal immune privilege. Daily phagocytosis and degradation of photoreceptor cell outer segment tips by RPE provide substantial amounts of retinal autoantigens for potential MHC occupancy. RPE are well placed to modulate antigen (Ag)-specific activation of T cells in the outer retina under conditions in which inflammatory mediators may upregulate major histocompatibility complex (MHC) on RPE cells. The Ag-presenting ability of RPE cells was examined to determine whether they induce Ag-dependent modulation of CD4 T-cell activity. METHODS: The effects of RPE on Ag-specific activation of naive, Ag-specific CD4 T cells were tested in cultures with immortalized, syngeneic murine RPE cells. Flow cytometry, proliferation, and cytokine production were used to assess T-cell activation and phenotype. RESULTS: Naive CD4 T cells exposed to peptide-pulsed RPE upregulated expression of CD25, CD69, and CD44, showing receptor occupancy. However, T-cell proliferation and production of IL-2, IL-17, and IFN-gamma were severely depressed. Provision of whole beta-gal, as opposed to beta-gal peptide, gave no evidence of T-cell activation. T cells recovered from RPE cocultures were hyporesponsive to restimulation with splenic APC and Ag, but did not exhibit significant regulatory activity. Although CD25 was upregulated on RPE-activated T cells, expression of FoxP3 was similar to that found after activation with splenic APC and Ag. The inhibitory activity of RPE was dominant, since T-cell activation remained inhibited if splenic APCs were included in the cocultures. CONCLUSIONS: RPE cells directly presented extracellular peptides through MHC class II to naive CD4 T cells, leading to an anergic state in the T cells. The anergic T cells survived, but were not immunoregulatory. The ability to modulate T-cell responsiveness in this manner may underlie the contribution of the RPE to immune privilege.

RPE cells resist bystander killing by CTLs, but are highly susceptible to antigen-dependent CTL killing.

PURPOSE: Retinal pigmented epithelial (RPE) cells maintain the blood-retinal barrier, sustain retinal photoreceptor cell health and function, and may play a role in ocular immune privilege. If RPE immunomodulatory activities were antigen specific, their expression would require antigen presentation. In a study of antigen processing and major histocompatibility complex (MHC) class I-restricted presentation by RPE cells, the cells' sensitivity to the activity of cytotoxic T lymphocytes (CTLs) was determined. METHODS: RPE was cultured, with and without proinflammatory cytokines and antigen, followed by the addition of beta-galactosidase (beta-gal)-specific CTLs. Cytotoxic activity was measured by the CTL-dependent activation of caspase-3 in the RPE. Sensitivity to the CTLs was used to evaluate the activity of pathways of antigen processing and presentation using an antigen (beta-gal) that was either applied to or expressed in RPE. RESULTS: RPE cells were sensitive targets for activated CTL-mediated killing in vitro only if prepulsed with cognate peptide, or if beta-gal-expressing RPE was pretreated to induce upregulation of immunoproteasome. Activated CTLs induced apoptosis in RPE within 3 hours of coculture with antigen-positive RPE monolayers. Application of CTLs in a resting state to antigen-positive RPE led to their activation in the absence of exogenous antigen-presenting cells (APCs). This antigen-dependent activation and killing required 24 hours of co-incubation of RPE with resting CD8 T cells specific for beta-gal. Although RPE cells are highly phagocytic, functional evidence for processing that allowed phagocytosed antigens to load into class I MHC was not detected. RPE was minimally sensitive to bystander killing by activated CTLs. CONCLUSIONS: Although there are many reports of T-cell inhibition by RPE, we found that CTLs efficiently killed RPE cells by induction of apoptosis in an antigen-dependent manner. The survival of RPE in the face of extensive CTL destruction of adjacent photoreceptor cells in vivo appears to be based on their insensitivity to injury via bystander mechanisms.

Bystander killing of neurons by cytotoxic T cells specific for a glial antigen.

To explore pathways to neuron loss in inflammatory diseases, transgenic mice expressing beta-galactosidase (beta-gal) in either astrocytes or photoreceptor cells, or both, were inoculated with activated, beta-gal-specific cytotoxic CD8 T lymphocytes (CTLs). beta-gal-positive astrocytes in brain were rapidly attacked, with particular damage in cerebellum. Substantial loss of cerebellar granule cells was found, even though these neurons did not express beta-gal. The small number of beta-gal-positive retinal astrocytes present in these mice was also rapidly destroyed by transferred CTLs, but without detectable consequences for retinal neurons. However, in mice with photoreceptor cell-specific beta-gal expression, near-total destruction of photoreceptor cells was produced by CTL transfer. Attack on photoreceptor cells displayed minimal inflammation, and onset was a week later than onset of astrocyte-directed disease. CTL transfer into F1 mice expressing beta-gal in both astrocytes and photoreceptor cells confirmed that pathogenesis directed against antigen expressed in glia versus neurons proceeded in two distinct, independent phases. beta-gal-positive retinal astrocytes were severely affected by 5 days post-transfer, followed by rapid resolution. Photoreceptor cells in the same retina were unaffected until 12 days post-transfer. The susceptibility of photoreceptor cells was not enhanced by the prior CTL attack on beta-gal-expressing retinal astrocytes. The results demonstrate that extensive bystander killing of neurons can occur in vivo as a result of direct CTL attack on surrounding astrocytes. Antigen-expressing retinal neurons were also efficiently killed by CTLs, but by a mechanism that was substantially delayed and dissociated from the killing of retinal astrocytes.

Peripheral expression of rod photoreceptor arrestin induces an epitope-specific, protective response against experimental autoimmune uveoretinitis.

PURPOSE: To examine the immunological basis for reduced susceptibility to experimental autoimmune uveoretinitis (EAU) in rats expressing retinal photoreceptor cell arrestin in the periphery. METHODS: Peripheral expression of arrestin in Lewis rats was achieved by engraftment of syngeneic bone marrow (BM) transduced with retroviruses encoding wild-type arrestin or a mutant arrestin lacking the immunodominant epitope Arr(273 - 289) (Delta273-Arr). EAU was induced by immunization with arrestin peptides Arr(273-289) or Arr(343-362). Cultured splenocytes and/or lymphocytes from immunized rats were assayed for antigen-induced proliferation, antibody production, and cytokines. RESULTS: Rats expressing Delta273-Arr were not protected from Arr(273 - 289)-induced EAU, showing that protection was epitope specific. Proliferation assays found little difference in the ability of draining lymph node cells from arrestin-transduced rats to proliferate in response to the antigen, indicating that antigen-responsive T cells were not deleted in BM recipients. Only rats immunized with Arr(343 - 362) elicited antibodies, but no difference in titer was found between transduced and control animals. Higher levels of IFN-gamma mRNA were made by Arr(273 - 289)-immunized rats than Arr(343 - 366)-immunized rats, but in either case, the levels did not correlate with chimeric status or EAU susceptibility. Arr(273 - 289)-immunized rats had higher levels of IL-10 mRNA than Arr(343 - 362)-immunized rats, and those levels were decreased in arrestin chimeric rats. Overall, immunization with the more potently uveitogenic Arr(343 - 362) induced lower levels of IL-10 and IFN-gamma than the less uveitogenic Arr(273 - 289). A strong correlation was found between the ability of lymphocytes to make IL-4 in the arrestin-chimeric animals and inhibition of EAU. CONCLUSIONS: Peripheral expression of arrestin in a regenerating immune system induces an epitope-specific protective response to EAU induced by arrestin peptides. Although IL-4 and IL-10 levels were altered in arrestin-chimeric mice, the outcome was not consistently T(H)2-like. Only IL-4 production was clearly associated with reduced susceptibility to EAU.

The antigen-presenting activity of fresh, adult parenchymal microglia and perivascular cells from retina.

Although several observations show local T cell recognition of retinal Ag, there has been no direct demonstration that the APC were retinal derived, rather than recruited. In this study, CD45(+) cells isolated from immunologically quiescent murine retina were tested in vitro for functional evidence of Ag presentation to naive and Ag-experienced CD4 T cells specific for beta-galactosidase. Because CD45(+) cells from brain have been reported to be efficient APC, they were included for comparison. Measures of activation included changes in CD4, CD25, CD44, CD45RB, CD62L, CD69, caspase-3 activation, CFSE dilution, size, number of cells recovered, and cytokine production. Retinal CD45(+) cells gave no evidence of Ag-dependent TCR ligation in naive T cells, unlike splenic APC and CD45(+) cells from brain, which supported potent responses. Instead, addition of retinal CD45(+) cells to cocultures of naive 3E9 T cells plus splenic APC reduced the yield of activated T cells and cytokine production by limiting T cell activation at early time points. Ag-experienced T cells responded weakly to Ag presented by retinal CD45(+) cells. Activating the retinal cells with IFN-gamma, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-beta did not reveal suppressed retinal APC activity. Because retina lacks tissue equivalents of meninges and choroid plexus, rich sources of dendritic cells in brain, cells from retina may better represent the APC activity of fresh, adult CNS parenchymal and perivascular cells. The activity of the retinal CD45(+) cells appears to be directed to limiting T cell responses.