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Background Studies of antigen presentation in retina using mice that expressed green fluorescent protein (GFP) from a transgenic CD11c promoter found that retinal GFP hi cells possessed antigen presentation function. Subsequent studies found that these high GFP hi cells preferentially localized to sites of retinal injury, consistent with their APC function. Interest in the roles of macrophages in degenerative CNS diseases led us to study the GFP hi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65 −/− knockout mice induced the GFP hi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival. Methods Rpe65 −/− mice were bred to CD11c GFP mice on the B6/J background. CD11c GFP Rpe65 −/− mice were also backcrossed to CX3CR1 YFP-creER ROSA DTA mice so that CX3CR1 + mononuclear cells could be depleted by Tamoxifen. Retinas were analyzed by immunohistochemistry, confocal microscopy, fluorescence fundoscopy and flow cytometry. Results Elevated numbers of GFP hi cells were concentrated in photoreceptor cell layers of CD11c GFP Rpe65 −/− mice coinciding with the peak of cone death at 2 to 4 weeks of age, and persisted for at least 14 months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFP hi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFP hi cells and MG in Rpe65 −/− mice from day 13 to day 41, and from day 390 to day 420 promoted a small increase in cone survival. We found no evidence that the GFP hi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFP hi cells were well segregated in the dystrophic retina; GFP hi cells were foremost in the photoreceptor cell layer, while MG were concentrated in the inner retina. Conclusions The expression of GFP on a subset of retinal mononuclear cells in CD11c GFP mice identified a distinct population of cells performing functions previously attributed to MG. Although GFP hi cells dominated the macrophage response to cone death in the photoreceptor cell layer, their ablation led to only an incremental increase in cone survival. The ability to identify, ablate, and isolate these cells will facilitate analysis of this activated, antigen-presenting subset of MG.

Purpose We previously demonstrated that passive transfer of lymphocytes from glaucomatous mice induces retinal ganglion cell (RGC) damage in recipient animals, suggesting a role for immune responses in the multifactorial pathophysiology of glaucoma. Here we evaluate whether absence of an adaptive immune response reduces RGC loss in glaucoma. Methods Elevated intraocular pressure (IOP) was induced in one eye of C57BL/6J (B6) or T- and B-cell-deficient Rag1-/- knockout mice. After 16 weeks RGC density was determined in both the induced and the normotensive contralateral eyes. Data were compared to mice having received injections of "empty" vector (controls). The number of extravascular CD3+ cells in the retinas was determined using FACS. Results Retinas of eyes with elevated IOP contain significantly more extravasated CD3+ cells than control retinas (46.0 vs. 27.1, P = 0.025). After 16 weeks of elevated IOP the average RGC density in B6 mice decreased by 20.7% (P = 1.9 × 10-4). In contrast, RGC loss in Rag1-/- eyes with elevated IOP was significantly lower (10.3%, P = 0.006 vs. B6). RGC loss was also observed in the contralateral eyes of B6 mice, despite the absence of elevated IOP in those eyes (10.1%; P = 0.008). In RAG1-/- loss in the contralateral eyes was minimal (3.1%) and significantly below that detected in B6 (P = 0.02). Conclusions Our findings demonstrate that T Rag1-/- mice are significantly protected from glaucomatous RGC loss. In this model, lymphocyte activity contributes to approximately half of all RGC loss in eyes with elevated IOP and to essentially all loss observed in normotensive contralateral eyes.

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. Electronic supplementary material The online version of this article (10.1186/s40478-018-0571-8) contains supplementary material, which is available to authorized users.

Extrathymically derived regulatory T cells (iTregs) protect against autoimmunity to tissue-specific antigens (Ag). 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 beta-galactosidase (βgal) on a retina specific promoter (βgal mice) in conjunction with mice expressing green fluorescent protein (GFP) and diphtheria toxin (DTx) receptor (DTR) under control of the FoxP3 promoter, and βgal-specific T cell receptor transgenic (BG2) mice were used to examine this question. Local depletion (ocular DTx), but not systemic depletion (intra-peritoneal DTx), of βgal-specific iTregs enhanced experimental autoimmune uveoretinitis induced by activated βgal-specific effector T cells. Injections of small amounts of βgal into the anterior chamber of the eye produced similar numbers of βgal-specific iTregs in the retina whether or not the mouse was depleted of pre-existing, circulating Tregs. 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.

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 β-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.

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.

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 β-galactosidase (βgal) on the retinal photoreceptor cell arrestin promoter, in conjunction with TCR Tg mice producing CD4+ T cells specific for βgal (βgalTCR). Several strategies were used to test the hypothesis that βgal 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 βgal following systemic immunization with βgal. This regulation was transferable to naive mice by CD3+4+25+ T cells from naive retinal βgal+ donors. Experiments that removed the βgal+ retina by enucleation showed that subsequent development of a regulatory response was lost. Adoptive transfer of CD25− βgalTCR T cells into retinal βgal Tg mice on the Rag−/− background led to regulatory activity that limited lymphopenia-induced proliferation of βgalTCR T cells in mice with retinal expression of βgal 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.

To study retinal immunity in a defined system, a CD4+ TCR transgenic mouse line (βgalTCR) specific for β-galactosidase (βgal) was created and used with transgenic mice that expressed βgal in retinal photoreceptor cells (arrβgal mice). Adoptive transfer of resting βgalTCR T cells, whether naive or Ag-experienced, into arrβgal mice did not induce retinal autoimmune disease (experimental autoimmune uveoretinitis, EAU) and gave no evidence of Ag recognition. Generation of βgalTCR T cells in arrβgal mice by use of bone marrow grafts, or double-transgenic mice, also gave no retinal disease or signs of Ag recognition. Arrβgal mice were also resistant to EAU induction by adoptive transfer of in vitro-activated βgalTCR T cells, even though the T cells were pathogenic if the βgal 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, βgalTCR T cells into lymphopenic arrβgal 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 βgalTCR 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.

It is well known that immunoproteasome generates peptides for MHC Class I occupancy and recognition by cytotoxic T lymphocytes (CTL). The present study focused on evidence for alternative roles for immunoproteasome. Retina and brain were analyzed for expression of immunoproteasome subunits using immunohistochemistry and western blotting under normal conditions and after injury/stress induced by CTL attack on glia (brain) or neurons (retina). Normal retina expressed substantial levels of immunoproteasome in glia, neurons, and retinal pigment epithelium. The basal level of immunoproteasome in retina was two-fold higher than in brain; CTL-induced retinal injury further up-regulated immunoproteasome expression. Immunoproteasome up-regulation was also observed in injured brain and corresponded with expression in Purkinje cells, microglia, astrocytes, and oligodendrocytes. These results suggest that the normal environment of the retina is sufficiently challenging to require on-going expression of immunoproteasome. Further, immunoproteasome up-regulation with retinal and brain injury implies a role in neuronal protection and/or repair of damage.

Background: Thymic expression of a photoreceptor cell antigen, interphotoreceptor retinoid-binding protein, is known to generate regulatory T cells (Treg) 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 Treg is uncertain. Methods: Transgenic mice that express β-galactosidase (β-gal) in photoreceptor cells, together with β-gal-specific T cell receptor transgenic mice, were used to study the induction of Treg in vivo. Results: Transgenic expression of β-gal on the arrestin promoter led to a spontaneous immunoregulatory response that inhibited the development of immune responses to β-gal. The regulation was transferred by CD3+4+25+ Treg. Several strategies were then used to show that β-gal expressed in the retina supported spontaneous, thymus-independent Treg development. The endogenous Treg also differed from the Treg 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 Treg in the periphery.

Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus-specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death.

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.

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 (βgal) as retinal neo-self antigen and βgal-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 βgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal βgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local βgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after βgal 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.

T cell adoptive transfer models of autoimmune disease have been used in conjunction with radiation/bone marrow chimeras to define the minimal requirements for antigen (Ag) recognition. In models with central nervous system Ags, major histocompatibility complex (MHC) class II compatibility achieved by grafting F1 bone marrow into parental recipients was reported to be necessary and sufficient for transfer of CD4 T cell-mediated experimental autoimmune encephalomyelitis. Bone marrow-derived, perivascular microglia are now widely regarded to play a critical role in the expression of experimental autoimmune diseases of the nervous system. Similar results might be expected in the experimental autoimmune uveoretinitis model, as retina is an extension of the brain. Using an allogeneic Ag-presenting cell (APC) adoptive transfer strategy, it was found that resident APC were not essential and that their replacement with MHC-compatible cells by bone marrow-grafting techniques was not necessary. Instead, APC were recruited from the circulation.

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 β-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-γ, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-β 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.

The retina is a well-known immune-privileged tissue in the eye. Gene therapy and transgenic strategies have been taken to explore the relationship between the immune system and retinal antigens. Retroviruses were used to express retina-specific antigens or fragments systemically, leading to an antigen-specific loss of susceptibility to autoimmune disease. Transgenic strategies used a neo self-antigen, beta-galactosidase, or a known retinal antigen, interphotoreceptor retinoid-binding protein, to show that immune recognition of antigen by mice, which express solely in the retina, is not detectably different than that of mice that don't express this antigen. Together, these studies show that antigens expressed solely in the retina do not appear to be seen by the immune system, demonstrating that sequestration contributes to the lack of antigen recognition and absence of tolerance. Provision of these antigens outside of the retina provides the opportunity for development of peripheral tolerance, protection from autoimmunity, and potential therapies.

Activated T cells recognize Ag in the retina, an immune privileged tissue, and may mediate autoimmune disease. In contrast, this report asks if resting, Ag-specific CD4(+) CD44(+) T cells can recognize Ag expressed in the retina. As a probe for Ag, 3E9 T cells specific for an immunodominant epitope of beta-galactosidase (beta-gal) were transferred to transgenic (Tg) mice expressing beta-gal in retinal photoreceptor cells, or to ROSA26 mice which express beta-gal widely. The survival, phenotype, and responsiveness of transferred 3E9 T cells were unaffected by the presence of retinal beta-gal, but altered by recognition of beta-gal in the ROSA26 mice. Inoculation or induction of activated T cells with specificity for this epitope produced autoimmune uveoretinitis, showing that the retinal beta-gal is expressed at immunologically significant levels. We conclude that sequestration provides a substantial barrier to recognition of Ag in quiet retina, and that insufficient Ag leaves the retina for detectable immune recognition outside of the retina.

To assess the role of sequestration in the maintenance of the immune privilege of the retina, retrovirally mediated gene transfer was used to express a defined, specific retinal autoantigen, rat soluble retinal Ag (S-Ag), in a systemic, nonsequestered manner. In this study we report the stable, long term transduction of rat retinal S-Ag into PBMC. Tolerance to S-Ag was assayed by challenging the S-Ag chimeric animals with S-Ag peptides in CFA and monitoring the time course and severity of experimental autoimmune uveoretinitis (EAU). The resulting data showed a correlation between the incidence of S-Ag chimerism and the loss of susceptibility to EAU. The development of resistance to EAU induction supports the hypothesis that Ag sequestration contributes to retinal immune privilege.

Recent studies revealing active mechanisms of immune privilege in neural tissues have diminished the putative role of passive tolerance. To examine the significance of Ag localization in the retina on immune privilege, the immune responses of transgenic mice expressing high and low levels of β-galactosidase (β-gal) in the photoreceptor cells of the retina were compared with those of normal mice and those of mice expressing moderate levels of β-gal systemically. Immunization with β-gal induced experimental autoimmune uveoretinitis indistinguishable from that induced by known photoreceptor cell autoantigens, including destruction of photoreceptor cells, in transgenic mice with high level retinal expression. Retinal expression had no apparent effect on the immune responses to β-gal, showing that tolerance was not elicited by levels of retinal β-gal sufficient to serve as a target for autoimmune disease. Mice with systemic expression exhibited reduced lymphoproliferative responses following immunization with β-gal and did not develop autoimmune disease. T cells prepared from normal mice immunized with β-gal transferred experimental autoimmune uveoretinitis to the transgenic mice with high level retinal β-gal expression, but no disease was found in mice with systemic transgene expression under these conditions. The results of our experiments are most consistent with sequestration being the primary mechanism of retinal immune privilege. The results also show that β-gal can serve as an immunopathogenic neural autoantigen, and that T cells raised by immunization of normal mice with a foreign Ag can be immunopathogenic in certain transgenic recipients.

Oral administration of antigen modulates subsequent immune responses raised by conventional subcutaneous priming. If experimental autoantigens are administered, subsequent induction of autoimmune diseases may be inhibited. However, feeding autoantigens after priming or disease induction is more clinically relevant, but the trials have been less successful. Using therapeutic feeding of peptides to inhibit experimental autoimmune uveoretinitis (EAU) induced in LEW rats by bovine S-Ag peptides, we found that only mild disease could be inhibited if feeding was delayed until after immunization, and relatively high feeding doses were required. In recipients with more severe EAU, the clinical efficacy of therapeutic feeding was minimal despite concurrent down-regulation of in vitro antigen-specific lymphocyte proliferation and serum antibody responses. No further inhibition of EAU was found by increasing the feeding dose. Feeding the same peptides prior to immunization produced resistance to moderate to severe disease induction. Unlike prophylactic feeding protocols, conditions were found such that feeding after immunization with low doses of antigen led to worsening of mild disease, raising a note of caution.

The inoculation of antigens into the anterior chamber (AC) of the eye induces an antigen-specific immune response that inhibits delayed-type hypersensitivity (DTH). This regulatory response is known as anterior chamber-associated immune deviation (ACAID). The ACAID response appears to be complex, as it can be elicited by a wide variety of soluble and cell-associated antigens, including foreign, self, tumor, and alloantigens. To evaluate the contribution of Fas/Fas ligand (FasL) interaction to the induction of ACAID to alloantigens, gld and lpr mutant mice were used in conjunction with normal C3H, MRL, and BALB/c mice. ACAID was induced by inoculation of non-irradiated splenocytes from donor mice into the AC of various recipients. After 1 week, recipients were primed intradermally with donor splenocytes. One week later DTH was measured by ear swelling. C3Hgld mutants lacking functional FasL did not develop ACAID after the AC inoculation of BALB/c splenocytes. Conversely, the AC inoculation sensitized these mutants. MRL/pr mutants, which lack Fas, developed ACAID following inoculation of BALB/c cells. AC inoculation of lpr splenocytes did not induce ACAID, but sensitized C3H recipients. Treatment of the AC inoculum with an anti-Fas antibody blocked ACAID induction in a transient manner, as the recipients developed ACAID later. These results show that interaction of the Fas and FasL is required to induce ACAID to allogeneic cells. In the absence of Fas expression on donor splenocytes, or FasL expression by the recipient, AC inoculation primes for a DTH response rather than inducing ACAID.

Background. The expression of Fas ligand (FasL) in the eye has been proposed to be an important component of ocular immune privilege. Since the unusually favorable outcome of corneal transplantation is thought to result from the immune privilege of the eye, examination of the function of FasL on corneal allografts would be a test of that hypothesis. Methods. To investigate the role of Fas-FasL interaction in corneal allografts, orthotopic corneal transplantation was performed using C57BL/6 (B6, FasL + ) and B6-gld (FasL - ) mice as cornea donors and BALB/c mice as recipients. The rejection rate of B6-gld grafts (FasL - group) was compared with that of normal B6 control corneas. Results. The rejection rate at the final observation (8 weeks) in the FasL - group (89%) was significantly higher than in the FasL + control group (47%). FasL expression was found on the corneal endothelium by staining with anti-FasL monoclonal antibodies. The TdT-mediated dUTP nick-end labeling assay revealed that apoptotic cells were attached to the endothelium in the control group but not in the FasL - groups. Conclusions. Apoptosis of infiltrating cells on the corneal endothelium resulting from Fas-FasL interaction plays an important role in the high success rate of corneal transplantation.

Immunoproteasome is a protease abundant in immune cells and also present, albeit at lower concentrations, in cells outside the immune system. Recent evidence supports a novel role for the immunoproteasome in the cellular stress response potentially through regulation of NFκB signaling, which is the primary response to multiple stressors. The current study tests whether the Classical or Alternative Pathways are regulated by immunoproteasome following chronic TNFα exposure in cultured retinal pigment epithelial cells isolated from wild-type mice and mice deficient in one (LMP2, L2) or two (LMP7 and MECL-1, L7M1) immunoproteasome subunits. Assays were performed to assess the expression of NFκB responsive genes, the content and activity of NFκB transcription factors (p65, p50, p52, cRel, RelB), and expression and content of regulatory proteins (IκBα, A20, RPS3). Major findings include distinct differences in expression of NFκB responsive genes in both KO cells. The mechanism responsible for the altered gene expression could not be established for L7M1 since no major differences in NFκB transcription factor content or activation were observed. However, L2 cells exhibited substantially higher content and diminished activation of NFκB transcription factors associated with the Alternative Pathway and delayed termination of the Classical Pathway. These results provide strong experimental evidence supporting a role for immunoproteasome in modulating NFκB signaling.

Recent studies have revealed roles for immunoproteasome in regulating cell processes essential for maintaining homeostasis and in responding to stress and injury. The current study investigates how the absence of immunoproteasome affects the corneal epithelium under normal and stressed conditions by comparing corneas from wildtype (WT) mice and those deficient in two immunoproteasome catalytic subunits (lmp7(-/-)/mecl-1(-/-), L7M1). Immunoproteasome expression was confirmed in WT epithelial cells and in cells of the immune system that were present in the cornea. More apoptotic cells were found in both corneal explant cultures and uninjured corneas of L7M1 compared to WT mice. Following mechanical debridement, L7M1 corneas displayed delayed wound healing, including delayed re-epithelialization and re-establishment of the epithelial barrier, as well as altered inflammatory cytokine production compared to WT mice. These results suggest that immunoproteasome plays an important role in corneal homeostasis and wound healing.

Immunoproteasomes contain replacements for the three catalytic subunits of standard proteasomes. In most cells, oxidative stress and proinflammatory cytokines are stimuli that lead to elevated production of immunoproteasomes. Immune system cells, especially antigen-presenting cells, express a higher basal level of immunoproteasomes. A well-described function of immunoprotea-somes is to generate peptides with a hydrophobic C terminus that can be processed to fit in the groove of MHC class I molecules. This display of peptides on the cell surface allows surveillance by CD8 T cells of the adaptive immune system for pathogen-infected cells. Functions of immunoprotea-somes, other than generating peptides for antigen presentation, are emerging from studies in immunoproteasome-deficient mice, and are complemented by recently described diseases linked to mutations or single-nucleotide polymorphisms in immunoproteasome subunits. Thus, this growing body of literature suggests a more pleiotropic role in cell function for the immunoproteasome.

Immunoproteasomes contain replacements for the three catalytic subunits of standard proteasomes. In most cells, oxidative stress and proinflammatory cytokines are stimuli that lead to elevated production of immunoproteasomes. Immune system cells, especially antigen-presenting cells, express a higher basal level of immunoproteasomes. A well-described function of immunoproteasomes is to generate peptides with a hydrophobic C terminus that can be processed to fit in the groove of MHC class I molecules. This display of peptides on the cell surface allows surveillance by CD8 T cells of the adaptive immune system for pathogen-infected cells. Functions of immunoproteasomes, other than generating peptides for antigen presentation, are emerging from studies in immunoproteasome-deficient mice, and are complemented by recently described diseases linked to mutations or single-nucleotide polymorphisms in immunoproteasome subunits. Thus, this growing body of literature suggests a more pleiotropic role in cell function for the immunoproteasome.

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 β-galactosidase (βgal) on a retina-specific promoter (arrβgal 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 βgal-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 × arrβgal 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 βgal-specific CD8 T cells in the retinas of arrβgal mice on a local basis, allowing immuno regulation to be responsive to local conditions.

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.

Cells and tissues of the anterior uvea and aqueous humor express activities which inhibit immune responses. These activities include soluble factors such as TGF-β and uncharacterized cell surface interactions. Relatively little is known regarding the immunologic activities of corneal endothelium, despite its potentially important role in contributing to the immune privilege of the anterior chamber and the high success rate of corneal transplantation. In this report, in vitro studies of cultured rat corneal endothelial (CE) cells were done using S-antigen-specific LEW rat T cell lines, or S-antigen-specific T cell hybridomas, to examine the immunologic capabilities of CE cells. Monolayers of LEW rat CE cells were unable to present antigen or a mitogen, Con A, to T cell lines or hybridomas as assessed by the lack of a proliferative response or IL-2 secretion. Furthermore, the CE cells exerted a potent inhibitory effect when added to in vitro proliferation assays of T cell lines stimulated with antigen or Con A. When T cells were preactivated on conventional antigen presenting cells and then transferred to wells containing CE cells, their proliferation was not inhibited. Although CE cells inhibited activation of T cell lines and hybridomas, they did not inhibit the growth of T cell hybridomas or CTLL cells, nor did the CE cells adversely affect the viability of resting T cells cultured on CE monolayers. The inhibitory effect was reversible as preincubation of T cells on CE cells for up to 6 days followed by washes restored T cell responsiveness when assayed on splenocytes. The inability to stimulate proliferative responses was not affected by preincubation of the CE cells with lymphokines which increase MHC antigen expression. The inhibition observed in these assays was not MHC-restricted as CE cells from both LEW and BN rats were equally inhibitory. CE cells from rabbits and cats were also potent inhibitors of T cell activation, suggesting that the mechanism is evolutionarily conserved. The mechanism of inhibition by CE cells is unknown at this time.

Experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are CD4+ T cell-mediated inflammatory diseases of the uveal tract and retina of the eye and of the pineal gland. EAU and EAP can be induced by several retinal autoantigens including S-antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP). In this study we investigated the effect of intravenous administration of S-Ag and IRBP coupled to syngeneic spleen cells on the development of EAU and EAP. Injection of S-Ag or IRBP coupled to spleen cells 5 days prior to immunization with native S-Ag or IRBP, respectively, was effective in preventing the induction of EAU and EAP in LEW rats. Conversely, LEW rats receiving S-Ag-coupled spleen cells and challenged with IRBP or LEW rats receiving IRBP-coupled spleen cells and challenged with S-Ag developed a severe EAU within 10 days to 2 weeks following immunization, as did all control animals receiving sham-coupled spleen cells and challenged with the two retinal antigens. The results show that the administration of retinal autoantigens coupled to spleen cells effectively protects against the development of EAU when animals are subsequently challenged with the tolerizing antigen but not when challenged with another unrelated pathogenic retinal autoantigen.

S-Antigen, a photoreceptor cell protein, is highly efficient in inducing experimental autoimmune uveoretinitis (EAU), a severe inflammation of the uveal tract and retina of the eye. S-Antigen and six synthetic peptides, all of which correspond to known T-cell or B-cell recognition sites, were tested for their ability to induce oral tolerance to EAU in LEW rats. Feeding three 1-mg doses of native S-Antigen or three doses of one synthetic peptide, designated BSA(343-362) (200 micrograms per dose), reduced the incidence and severity of EAU induced by immunization with 50 micrograms of S-Antigen. Another peptide, BSA(270-289), was able to inhibit EAU only when a low dose (10 micrograms) of the uveitogenic S-Antigen was used to induce EAU. Animals which received 200 micrograms doses of four other immunologically active peptides, BSA(31-51), BSA(143-162), BSA(303-327) and BSA(333-352), were not significantly protected. Furthermore, animals fed BSA(343-362) were significantly less susceptible to EAU induced by adoptive transfer (tEAU) of the uveitogenic R9 T-cell lines. Con A-activated lymphocytes purified from spleens of rats fed peptide BSA(343-362) transferred partial resistance to tEAU induced by adoptive transfer of R9 line cells. The resistance of orally tolerized rats to induction of EAU by adoptive transfer of an activated, pathogenic T-cell line, and the ability of lymphocytes from orally-tolerized animals to transfer resistance to tEAU shows that effector mechanisms can be inhibited by oral feeding as well as the afferent mechanisms reported here and elsewhere. Circulating levels of IgG specific for S-Antigen were not affected by feeding any of the peptides.

Retinal S-antigen (SAg) and interphotoreceptor retinol-binding protein (IRBP) induced experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) are good models for studying the mechanisms involved in autoimmune diseases. Many immunogenetically active epitopes have been identified in these proteins but immunodominance of one or more epitopes in vivo, has not yet been established. In this paper we present and discuss some experiments that led to the discovery of a dominant "tolerogenic" epitope in SAg. We also demonstrate the presence of cross reactive epitopes in the two potent retinal antigens, SAg and IRBP and finally introduce early data on a unique anti S2.4.c5 idiotypic (Id) monoclonal antibody (MAb) which appears to be a site non associated antibody that binds not only to s2.4.c5 but also to SAg.

Virus-specific CD8+ T cells (TCD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector TCD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated TCD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the TCD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines., Author Summary Understanding the pathways by which protective immunity is mediated against viral pathogens is essential to allow the design of effective vaccines. No effective vaccine has been designed to activate killer cells of the immune system expressing CD8, although CD8+ T cells are the most effective cells at modulating anti-viral immunity. We have studied the process that activates the CD8+ T cell to better understand how the cells are triggered so future vaccines might readily activate these cells. CD8+ T cells are activated following recognition of small peptides derived from a virus that binds to a cell surface MHC molecule. Many viruses have evolved to prevent the presentation of these peptide-MHC complexes to CD8+ T cells. However, the immune system avoids these viral “evasion” mechanisms by allowing virus-derived peptides to be generated from viral proteins that are taken up by uninfected cells, a process termed “cross presentation”. We have shown that a poxvirus can specifically prevent the presentation of its proteins by uninfected cells, the first demonstration of evasion of cross presentation. This knowledge is vital in the use of certain viral vectors during vaccine design and adds to the numerous ways in which viruses can evade the immune system.

Retinal S-antigen is widely used to study the LEW rat model of experimental autoimmune uveoretinitis (EAU). In this report, we have examined the T cell receptor V gene usage of several T cell lines recognizing either pathogenic or nonpathogenic sites on S-antigen to determine whether the V alpha 510 and V beta 510 rat homologues of the murine V alpha 2 and V beta 8 families, respectively, are used by uveitogenic T cells. Using cDNA probes for a LEW rat T cell receptor specific for the encephalitogenic determinant of myelin basic protein, we have found that in the retinal S-antigen/EAU model for autoimmune disease, pathogenicity correlates with usage of those rat V genes. Thus, all of the pathogenic lines were found to express T cell receptors of the V beta 510 and V alpha 510 families; conversely, V beta 510 usage was not detected in any of the nonpathogenic lines. Usage of these V regions has been associated with pathogenicity in the murine and rat models of experimental autoimmune encephalomyelitis, and now with S-antigen-induced EAU.

Retinal S-antigen induced experimental autoimmune uveitis (EAU) is a severe, predominantly T-cell mediated inflammatory disease of the uveal tract and retina of the eye. Pretreatment of LEW rats with the monoclonal antibody, MAbS2.4.C5, which defines an epitope in S-antigen, has been shown to effectively inhibit the subsequent induction of EAU with S-antigen. Using synthetic peptides and cyanogen bromide fragments of S-antigen we found the binding site of MAbS2.4.C5 to be located at the carboxy terminus of the molecule corresponding to amino acid positions 375 to 380. Limited Staphylococcus aureus V8 protease digestion yielded several polypeptide fragments including one large 43 kD fragment which retained antibody binding to a variety of both polyclonal and monoclonal antibodies which identify epitopes that span the length of the S-antigen. This treatment, however, completely destroys the MAbS2.4.C5 binding site and dramatically reduces uveitopathogenicity. Limited trypsin and papain digestion, on the other hand, had little effect on pathogenicity or on MAbS2.4.C5 binding to S-antigen or its peptide fragments. These results indicate that the carboxy-terminus of S-antigen plays a predominant role in the pathogenesis of EAU.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of the eye which can be induced in LEW rats by immunization with either human or bovine S-antigen (S-Ag). In previous reports, two nonimmunodominant pathogenic sites were found using synthetic peptides corresponding to conserved sequences at amino acid residues 303–314 and 286–297 of the bovine sequence. In this report, a 20-residue synthetic peptide encompassing amino acids 343–362 located near the C-terminus was found to be highly immunopathogenic in LEW rats. The onset of EAU was observed at as early as 8 days when high doses of a peptideencompassing residues 343–362 were used. EAU was elicited with as little as 0.5 μg of peptide per animal. Smaller peptides from this region were also tested for uveitogenicity, further refining the site to 13 amino acids. Uveitogenic T cell lines were made to this site in two ways; first, by the in vitro selection of a bulk T cell line raised to human S-Ag with peptide 343–362. Second, by the in vitro selection of a peptide-specific line from an animal immunized with peptide 352–364, which corresponds to the minimal uveitogenic site. Both of these lines adoptively transferred EAU to LEW rats, further establishing the pathogenicity of this site. A proliferative site distinct from, but overlapping, the uveitogenic site was also found. The potent uveitopathogenicity of peptides from this region indicates that it is a major pathogenic site responsible for EAU induced in LEW rats by immunization with human S-Ag.

Previous attempts to prepare monoclonal antibodies (MAbs) against S-antigen, a photoreceptor cell protein involved in the visual process and a potent autoantigen for the induction of experimental autoimmune uveitis (EAU), have yielded MAbs which define only carboxyl terminal epitopes. In this study we devised alternate strategies to prepare five MAbs directed to other regions of the molecule. MAbC10C10 and MAbH11-A2 were prepared against synthetic peptides known to be uveitopathogenic and they were selected for more detailed studies. MAbC10C10 was generated against synthetic peptide BSA281-302 which contains a predictive consensus sequence for defined T cell epitopes (GIALD) as well as a consensus sequence for GTP-binding proteins. One human adenosine deaminase synthetic peptide containing an extensive amino acid sequence homology to BSA281-302 was a potent inhibitor of MAbC10C10 binding in a competitive inhibition radioimmunoassay. MAbH11-A2 was generated against peptide BSA303-332 which also contains a uveitopathogenic site. The binding site of MAbH11-A2 was determined to be within amino acid positions 305 to 314 (NLASSTIIKE) in S-antigen. This binding site corresponded closely to the binding site of an affinity-purified rat polyclonal antibody raised to human S-antigen. MAb5C6.47 was isolated from a mouse hyperimmunized with bovine S-antigen and was specific for a highly conserved sequence near the amino terminus, amino acid residues 42 to 48 (DGVVLVD). Both MAbC10C10 and MAb5C.47 were useful in screening gt11 cDNA libraries expressing S-antigen polypeptides as fusion proteins. Our results demonstrate the feasibility of producing site-specific MAbs potentially useful in the study of T cell-mediated immune mechanisms in EAU as well as in the phototransduction of vision.

S-antigen is a highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis (EAU). EAU is predominantly a T cell mediated autoimmune disease of the uveal tract and retina of the eye and the pinealocytes of the pineal gland. Using synthetic peptides it has been possible to identify several B cell and T cell epitopes in the molecule. In addition, synthetic peptides derived from proteins of diverse origin with amino acid sequence homology to pathogenic regions of S-antigen induce an EAU which is indistinguishable from the disease induced by native S-antigen. These studies aid in the understanding of immune mechanisms in EAU and provide a basis for the pathogenesis of uveitis in humans.

The immunoproteasome is upregulated by disease, oxidative stress, and inflammatory cytokines, suggesting an expanded role for the immunoproteasome in stress signaling that goes beyond its canonical role in generating peptides for antigen presentation. The signaling pathways that are regulated by the immunoproteasome remain elusive. However, previous studies suggest a role for the immunoproteasome in the regulation of PTEN and NF-κB signaling. One well-known pathway upstream of NF-κB and downstream of PTEN is the Akt signaling pathway, which is responsible for mediating cellular survival and is modulated after optic nerve crush (ONC). This study investigated the role of retinal immunoproteasome after injury induced by ONC, focusing on the Akt cell survival pathway. Retinas or retinal pigment epithelial (RPE) cells from wild type (WT) and knockout (KO) mice lacking either one (LMP2) or two (LMP7 and MECL-1) catalytic subunits of the immunoproteasome were utilized in this study. We show that mRNA and protein levels of the immunoproteasome subunits are significantly upregulated in WT retinas following ONC. Mice lacking the immunoproteasome subunits show either a delayed or dampened apoptotic response as well as altered Akt signaling, compared to WT mice after ONC. Treatment of the RPE cells with insulin growth factor-1 (IGF-1) to stimulate Akt signaling confirmed that the immunoproteasome modulates this pathway, and most likely modulates parallel pathways as well. This study links the inducible expression of the immunoproteasome following retinal injury to Akt signaling, which is important in many disease pathways.

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 -galactosidase (-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 (-gal) that was either applied to or expressed in RPE. RESULTS. RPE cells were sensitive targets for activated CTLmediated killing in vitro only if prepulsed with cognate peptide, or if -gal-expressing RPE was pretreated to induce upregulation of immunoproteasome. Activated CTLs induced apoptosis in RPE within 3 hours of coculture with antigenpositive 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 -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. (Invest Ophthalmol Vis Sci. 2006;47:5385–5394) DOI:10.1167/ iovs.06-0636

Neural progenitor cells (NPCs) have shown ability to repair injured CNS, and might provide precursors to retinal neurons. NPCs were isolated from the brains of 14 day murine embryos of transgenic mice that express beta-galactosidase (beta-gal) on the arrestin promoter, which specifically directs expression to retinal photoreceptor cells. NPCs were transferred to adult, syngeneic mice via inoculation into the anterior chamber of the eye, the peritoneal cavity, or the brain. At 14 weeks postgrafting, tissues were collected and examined to determine if differentiated NPC progeny were present in retina based on histochemical detection of beta-gal. Four of six anterior chamber-inoculated recipients showed Bluo-gal-stained cells in retina, indicating the presence of transferred NPCs or their progeny. Because the progenitor cells do not express beta-gal, positive staining indicates differentiation leading to activation of the arrestin promoter. Two recipients inoculated by the intraperitoneal route also exhibited Bluo-gal staining in retina. The NPCs did not express beta-gal if inoculated into brain, but survived and dispersed. Most recipients, regardless of inoculation route, were PCR positive for beta-gal DNA in extraocular tissues, but no Bluo-gal staining was found outside of the retina. Injury to the retina promoted, but was not required, for progenitor cell engraftment. beta-Gal-positive cells were concentrated in the outer layers of the retina. In summary, a reporter gene specifically expressed in differentiated retinal photoreceptor cells due to the activity of the arrestin promoter was expressed in recipient mouse retina following transfer of NPCs prepared from the beta-gal transgenic mice. The presence of beta-gal DNA, but not Bluo-gal staining, in spleen and other tissues revealed that the cells also migrated elsewhere and took up residence in other organs, but did not undergo differentiation that led to beta-gal expression.

The type III variant of the epidermal growth factor receptor (EGFRvIII) mutation is present in 20-25% of patients with glioblastoma multiforme (GBM). EGFRvIII is not expressed in normal tissue and is therefore a suitable candidate antigen for dendritic cell (DC) based immunotherapy of GBM. To identify the antigenic epitope(s) that may serve as targets for EGFRvIII-specific cytotoxic T lymphocytes (CTLs), the peptide sequence of EGFRvIII was screened with two software programs to predict candidate epitopes restricted by the major histocompatibility complex class I subtype HLA-A0201, which is the predominant subtype in most ethnic groups. Three predicted peptides were constructed and loaded to mature human DCs generated from peripheral blood monocytes. Autologous CD8+ T cells were stimulated in vitro with the EGFRvIII peptide-pulsed DCs. One of the three peptides was found to induce EGFRvIII-specific CTLs as demonstrated by IFN-gamma production and cytotoxicity against HLA-A0201+ EGFRvIII transfected U87 glioma cells. These results suggest that vaccination with EGFRvIII peptide-pulsed DCs or adoptive transfer of in vitro elicited EGFRvIII-specific CTLs by EGFRvIII peptide-pulsed DCs are potential approaches to the treatment of glioma patients.

To examine the immunological basis for reduced susceptibility to experimental autoimmune uveoretinitis (EAU) in rats expressing retinal photoreceptor cell arrestin in the periphery.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.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.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.

Purpose The ability to mount antigen-specific immune reactions in the retina demonstrates local recognition of retinal antigens. However, properties of antigen-presenting cells (APCs) of the retina are uncertain. The current study was undertaken to look for evidence of CD45(+) cells with APC potential in the retina and to examine their in situ and in vitro responses to IFN-gamma and anti-CD40, two stimuli known to upregulate activities associated with antigen presentation. Methods Mice were pretreated with systemic or intracameral (IC) inoculations of IFN-gamma or anti-CD40. Retinas were harvested, enzymatically dissociated, positively selected with anti-CD45, and analyzed by flow cytometry with antibodies known to identify APCs. Results The most common CD45(+) retinal cells were CD11b(+), F4/80(+), CD8 alpha(+), CD80(+), and major histocompatibility complex (MHC) class II(lo), a phenotype characteristic of central nervous system (CNS) microglia (MG). There was also a small population of DEC-205(+) cells and a smaller number of CD11c(+) cells, both markers of dendritic cells (DCs). IC inoculation of IFN-gamma led to an increase in the number of CD45(+) cells and a modest upregulation of MHC class II on CD11b(+) cells. IC inoculation of anti-CD40 also increased the total number of CD45(+) cells and the number of CD11b(+) cells, but increases in CD80 and MHC class II expression on CD11b(+) cells were insignificant. After anti-CD40 treatment, CD45(hi)11c(+) cells increased in number and altered their expression of CD11b. Conclusions Retinal MG were readily identified as the most numerous population. A small population of cells with perivascular cell (PVC)-like properties was found. They were CD45(hi)11c(+), some had elevated MHC class II, and they were affected by anti-CD40 treatment in vivo. No conventional DCs were found, although there was a distinct DEC-205(+) population. Overall, the effects of IFN-gamma and anti-CD40 treatment were attenuated in the retina in vivo and also on CD45(+) cells in culture, compared with the control.