DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Invariant natural killer T cells play dual roles in the development of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) represents an experimental model for human endogenous uveitis, which is caused by Th1/Th17 cell-mediated inflammation. Natural killer T (NKT) cells recognize lipid antigens and produce large amounts of cytokines upon activation. To examine the role of NKT cells in the development of uveitis, EAU was elicited by immunization with a peptide from the human interphotoreceptor retinoid-binding protein (hIRBP1-20) in complete Freund's adjuvant and histopathology scores were evaluated in C57BL/6 (WT) and NKT cell-deficient mice. NKT cell-deficient mice developed more severe EAU pathology than WT mice. When WT mice were treated with ligands of the invariant subset of NKT cells (α-GalCer or RCAI-56), EAU was ameliorated in mice treated with RCAI-56 but not α-GalCer. IRBP-specific Th1/Th17 cytokines were reduced in RCAI-56-treated compared with vehicle-treated mice. Although the numbers of IRBP-specific T cells detected by hIRBP3-13/I-Ab tetramers in the spleen and the draining lymph node were the same for vehicle and RCAI-56 treatment groups, RORγt expression by tetramer-positive cells in RCAI-56-treated mice was lower than in control mice. Moreover, the eyes of RCAI-56-treated mice contained fewer IRBP-specific T cells compared with control mice. These results suggest that invariant NKT (iNKT) cells suppress the induction of Th17 cells and infiltration of IRBP-specific T cells into the eyes, thereby reducing ocular inflammation. However, in sharp contrast to the ameliorating effects of iNKT cell activation during the initiation phase of EAU, iNKT cell activation during the effector phase exacerbated disease pathology. Thus, we conclude that iNKT cells exhibit dual roles in the development of EAU.

Retinal laser burn-induced neuropathy leads to substance P-dependent loss of ocular immune privilege.

Inflammation in the eye is tightly regulated by multiple mechanisms that together contribute to ocular immune privilege. Many studies have shown that it is very difficult to abrogate the immune privileged mechanism called anterior chamber-associated immune deviation (ACAID). Previously, we showed that retinal laser burn (RLB) to one eye abrogated immune privilege (ACAID) bilaterally for an extended period of time. In an effort to explain the inflammation in the nonburned eye, we postulated that neuronal signals initiated inflammation in the contralateral eye. In this study, we test the role of substance P, a neuroinflamatory peptide, in RLB-induced loss of ACAID. Histological examination of the retina with and without RLB revealed an increase of the substance P-inducible neurokinin 1 receptor (NK1-R) in the retina of first, the burned eye, and then the contralateral eye. Specific antagonists for NK1-R, given locally with Ag within 24 h, but not 3, 5, or 7 d post-RLB treatment, prevented the bilateral loss of ACAID. Substance P knockout (KO) mice retained their ability to develop ACAID post-RLB. These data support the postulate that substance P transmits early inflammatory signals from the RLB eye to the contralateral eye to induce changes to ocular immune privilege and has a central role in the bilateral loss of ACAID. The possibility is raised that blocking of the substance P pathway with NK1-R antagonists postocular trauma may prevent unwanted and perhaps extended consequences of trauma-induced inflammation in the eye.

The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)-associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80(-/-) mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80(-/-) APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80(-/-) mice generated an efferent CD8(+) T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80(-/-) mice by adoptive transfer of F4/80(+) APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8(+) T reg cells.

Retinal laser burn disrupts immune privilege in the eye.

Immune privilege allows for the immune protection of the eye in the absence of inflammation. Very few events are capable of overcoming the immune-privileged mechanisms in the eye. In this study, we report that retinal laser burn (RLB) abrogates immune privilege in both the burned and nonburned eye. As early as 6 hours after RLB, and as late as 56 days after RLB, antigen inoculation into the anterior chamber of the burned eye failed to induce peripheral tolerance. After RLB, aqueous humor samples harvested from nontreated eyes but not from either the burned or the contralateral eye, down-regulated the expression of CD40 and up-regulated interleukin-10 mRNA in peritoneal exudate cells, and converted peritoneal exudate cells into tolerogenic antigen-presenting cells (APCs). Unlike F4/80(+) APCs from nontreated mice, F4/80(+) APCs from RLB mice were unable to transfer tolerance after anterior chamber inoculation of antigen into naïve mice. The increased use of lasers in both the industrial and medical fields raises the risk of RLB-associated loss of immune regulation and an increased risk of immune inflammation in the eye.

F4/80: the macrophage-specific adhesion-GPCR and its role in immunoregulation.

As a macrophage-restricted reagent, the generation and application of the F4/80 mAb has greatly benefited the phenotypic characterization of mouse tissue macrophages for three decades. Following the molecular identification of the F4/80 antigen as an EGF-TM7 member of the adhesion-GPCR family, great interest was ignited to understand its cell type-specific expression pattern as well as its functional role in macrophage biology. Recent studies have shown that the F4/80 gene is regulated by a novel set of transcription factors that recognized a unique promoter sequence. Gene targeting experiments have produced two F4/80 knock out animal models and showed that F4/80 is not required for normal macrophage development. Nevertheless, the F4/80 receptor was found to be necessary for the induction of efferent CD8+ regulatory T cells responsible for peripheral immune tolerance. The identification of cellular ligands for F4/80 and delineation of its signaling pathway remain elusive but are critical to understand the in vivo role of this macrophage-specific adhesion-GPCR.

Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.

In the inflamed cornea, there is a parallel outgrowth of blood and lymphatic vessels into the normally avascular cornea. We tested whether adaptive and/or innate immune cells were actively involved in the genesis of new lymphatic vessels. Our results indicate that innate immune cells (CD11b+ macrophages, but not CD11c+ dendritic cells) physically contributed to lymphangiogenesis under pathological conditions and that bone marrow-derived CD11b+ macrophages expressed lymphatic endothelial markers such as LYVE-1 and Prox-1 under inflamed conditions in the corneal stromata of mice. Furthermore, blood vascular endothelial cells that expressed the Tie2 promoter did not contribute to newly formed lymphatic vessels under inflamed conditions. Our in vitro experiments demonstrated that CD11b+ macrophages alone were capable of forming tube-like structures that expressed markers of lymphatic endothelium such as LYVE-1 and podoplanin. The novel finding that CD11b+ macrophages are critical for the development of inflammation-dependent lymphangiogenesis in the eye suggests a new mechanism of lymphangiogenesis.

An eye's view of T regulatory cells.

T regulatory (Treg) cells have been studied for more than 30 years. Recently, changing technology and attitudes have led to new interest in T cell regulation of the immune responses. The eye is an immune-privileged site with unique mechanisms for the prevention of damaging immune inflammation. The eye fashions its Treg cells in novel ways to prevent immune inflammation locally and systemically. The purpose of this mini-review is to condense and summarize reports of Treg cells dependent on the eye in the context of the Treg literature in general.

Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma.

Glaucoma is the most prevalent neurodegenerative disease and a leading cause of blindness worldwide. The mechanisms causing glaucomatous neurodegeneration are not fully understood. Here we show, using mice deficient in T and/or B cells and adoptive cell transfer, that transient elevation of intraocular pressure (IOP) is sufficient to induce T-cell infiltration into the retina. This T-cell infiltration leads to a prolonged phase of retinal ganglion cell degeneration that persists after IOP returns to a normal level. Heat shock proteins (HSP) are identified as target antigens of T-cell responses in glaucomatous mice and human glaucoma patients. Furthermore, retina-infiltrating T cells cross-react with human and bacterial HSPs; mice raised in the absence of commensal microflora do not develop glaucomatous T-cell responses or the associated neurodegeneration. These results provide compelling evidence that glaucomatous neurodegeneration is mediated in part by T cells that are pre-sensitized by exposure to commensal microflora.

Author Correction: Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma.

The originally published version of this Article contained an error in Figure 4. The bar chart in panel f was inadvertently replaced with a duplicate of the bar chart in panel e. This error has now corrected in both the PDF and HTML versions of the Article.

Invariant NKT cells and tolerance.

Invariant natural killer T (iNKT) cells are innate cells that can bias an immune response toward inflammation or toward a negative regulatory response. iNKT cells can produce cytokines immediately on exposure to activating signals, but the role of iNKT cells in the differentiation of T regulatory (Treg) cells and peripheral tolerance was elucidated only within the past decade. The purpose of this review is to outline the current knowledge of how iNKT cells function in various tolerance paradigms. The roles of iNKT cell in anterior chamber-associated immune deviation (ACAID), oral tolerance, other tolerance systems, and autoimmune diseases is discussed.

Cross talk among cells promoting anterior chamber-associated immune deviation.

The visual axis of the eye focuses light images precisely on the retina. The retina is intolerant of distortion that might be induced by innate or immune inflammation. In addition, the corneal endothelium and the neurosensory retina are unable to regenerate if injured by trauma or inflammation. Within the environment of this visual organ a phenomenon called ocular immune privilege provides the eye with the necessary immune protection against infectious agents by allowing the expression of the least deleterious immune effector mechanisms. Moreover, the mechanisms of immune privilege are multiple, overlapping, and include both active and passive suppression of innate and immune inflammation. At the very basis of an effective immune response are cellular interactions and their cross talk. Central to the ability of cells to communicate are the intercellular channels that are established to isolate signals and movement of proteins between cells. Within this secure nano-environment, cells signal each other and even exchange proteins. Studies reviewed here are centered on knowledge and exploration of the tolerogenic synapse rather than the immunogenic synapse. The unique cells (invariant natural killer T cells, F4/80+ antigen-presenting cells, and T and B lymphocytes) that cluster within the marginal zone following injection of antigen in the anterior chamber (AC) express a phenotype of cell surface molecules that that seem to be uniquely critical for the development of AC-associated immune deviation. How these cell surface molecules behave during the cellular interactions that result in the development of regulatory T cells and peripheral tolerance induced through the eye is discussed.

Therapies based on principles of ocular immune privilege.

Anterior chamber (AC)-associated immune deviation (ACAID) is a form of ocularderived peripheral tolerance that helps to maintain the immune privilege of the eye by suppressing both the priming and elicitation of adaptive immune responses. ACAID is known to facilitate the survival of corneal grafts and suppression autoimmune uveitis in the eye. Intravenous inoculation of in vitro generated ACAID tolerance-inducing antigen presenting cells (APCs) treated with transforming growth factor-Beta2 (tolerogenic APCs) generates the kind of T regulatory cells found in in vivo ACAID when antigen is inoculated into the AC of the eye. Here, we review the application of peripheral tolerance induction by ACAID with either AC inoculation or in vitro generated tolerogenic ACAID-APCs in suppressing ongoing Th1- and Th2-mediated immune pathogenesis in naive and presensitized hosts. Transfer of tolerogenic APCs has suppressed antigen-specific immune inflammation in animal models of experimental autoimmune encephalomyelitis, hapten immune pulmonary interstitial fibrosis, and ovalbumin-induced allergic pulmonary inflammation. The possibility of immune therapy by in vitro generated ACAID-like tolerogenic APCs in humans is discussed.

CD8+ T regulatory cells use a novel genetic program that includes CD103 to suppress Th1 immunity in eye-derived tolerance.

PURPOSE: The peripheral tolerance that arises after injection of antigen into the anterior chamber (anterior chamber-associated immune deviation; ACAID) is associated in part with CD8+ T cells that suppress the expression of Th1 and Th2 immunity. The purpose of these studies was to determine the genes and molecules that are critical for CD8+ T regulatory cell (T reg) functions in ACAID. METHODS: Ovalbumin (OVA)-specific CD8+ T cells from T-cell receptor (TCR) transgenic OT-1 mice acquire efferent regulatory properties similar to in vivo-generated CD8+ T regs after stimulation with OVA-pulsed TGF-beta2-treated APCs. Changes in the genetic program associated with acquisition of efferent regulatory function in OT-1 CD8+ T cells in vitro were determined by DNA microarray analyses and confirmed by RT-PCR analyses and biological assays. RESULTS: T regulatory OT-1 T cells acquired a novel transcriptional pattern indicative of their function. Genes for molecules associated with TGF-beta function, resistance to TCR-triggered apoptosis, and localization of cells to antigen deposition in peripheral tissues were upregulated, and genes related to cytolytic function were downregulated. Further study showed that CD103, a cell-adhesion molecule that binds E-cadherin, was highly upregulated in in vivo-generated ACAID T regs and was necessary for their suppression of T-cell activation in vitro. CONCLUSIONS: OT-1 CD8 T cells modulated in vitro by exposure to antigen-pulsed, TGF-beta2-treated APCs expressed genes related to immune suppression. Thus, the necessity for CD103 emerges in the efferent CD8+ T-cell regulatory mechanisms in eye-derived tolerance.

B7+ iris pigment epithelial cells convert T cells into CTLA-4+, B7-expressing CD8+ regulatory T cells.

PURPOSE: To determine whether iris PE (IPE) promotes the generation of regulatory T-cells (Tregs) with cell contact via B7-2/CTLA-4 interactions. METHODS: T cells were cocultured with IPE cells obtained from eyes of normal and B7-deficient mice, x-irradiated, and used as regulators. IPE T regulator cells (IPE Tregs) of normal and CD28- or CTLA-4-deficient mice were established. Target bystander T cells were established from normal splenic T cells with anti-CD3 antibodies. T-cell activation was assessed for proliferation by [3H]-thymidine incorporation. Neutralizing anti-B7-1 and/or B7-2 antibodies, anti-CTLA-4 antibodies, CTLA-4-Ig fusion proteins were used to abolish regulatory function. IPE-exposed CD8+ T cells were evaluated for expression of B7, CTLA-4, and Foxp3 by using RT-PCR and flow cytometry. CD8+ IPE Tregs were depleted of B7-2+ and CTLA-4+ T cells and assayed for suppressive activity by adding them to bystander T cells. RESULTS: T cells acquired T regulatory activity when exposed to cultured IPE. Ciliary body PE cells did not promote conversion of T cells into Tregs. IPE converted CD8+, but not CD4+, T cells into Tregs by direct cell contact. In the conversion, IPE and responding T cells must both express endogenously synthesized B7-1 and B7-2, and the T cells must also express CTLA-4. Expression of CD28 molecules was not necessary for Treg generation. In addition, the CD8+ Tregs that fully suppress activation of bystander T cells expressed Foxp3. CONCLUSIONS: IPE cells promote conversion of T cells into Tregs solely through a contact-dependent mechanism. T cells exposed to IPE cells acquire full regulatory capacity.

Induction of eye-derived tolerance does not depend on naturally occurring CD4+CD25+ T regulatory cells.

PURPOSE: Regulatory CD4+ T cells (T regs) arise in the spleens of mice with anterior chamber-associated immune deviation (ACAID), an eye-derived tolerance evoked by injection of antigen into the ocular anterior chamber (AC). The current study was conducted to investigate the possibility that these T regs express CD25 and are derived from natural CD4+CD25+ T cells. METHODS: Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGFbeta-treated antigen-presenting cells (APCs), and the expression of CD25 assayed by flow cytometry. OVA-specific ACAID T regs were obtained from the spleens of DO11.10 mice with ACAID to OVA. Immunomagnetic enrichment was used to sort out CD4+CD25+, and CD4+CD25- ACAID T cells before they were injected into OVA-immunized mice or examined for mRNA expression of the regulatory T-cell transcription factor Foxp3. In addition, before AC injection of OVA, systemic depletion of CD25+ T cells was performed with injections of anti-IL-2 receptor antibody into the mice. RESULTS: OVA-specific T cells from DO11.10 mice expressed CD25 when exposed to OVA-pulsed, TGFbeta-treated APCs, even when the DO11.10 T cells were depleted of CD25+ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4+CD25+ or CD4+CD25- ACAID T cells. The CD4+CD25+, but not the CD4+CD25-, ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25+ cells. CONCLUSIONS: Some of the CD4+ T regs of ACAID arise from CD25- precursors, and the induction of ACAID is not dependent on the presence of natural CD4+CD25+ T regs.

A privileged view of NKT cells and peripheral tolerance through the eye.

The eye is an immune-privileged site that uses specialized mechanisms to protect itself from damage and to preserve its visual acuity. Among the mechanisms that contribute to the eye's privileged existence is its ability to induce both local and peripheral tolerance to antigens that may transgress its chambers. Experimentally, antigens that are inoculated into the anterior chamber induce an associated immune deviation of immune lymphocytes from their potential T-helper inflammatory responses to T regulatory cells. The prominent role of a somewhat rare cell called invariant (i)NKT cell in the process of tolerance induced through the anterior chamber-associated immune deviation model, ACAID, has revealed novel biological characteristics for the iNKT cell, as well as novel mechanisms for the induction of tolerance that have not been previously known or considered. The role of the iNKT cell in ACAID and its novel story is discussed in this mini-review.

Anterior chamber associated immune deviation (ACAID): regulation, biological relevance, and implications for therapy.

Immune privilege was first explored in the late 1800s by van Dooremaal, and was then extended by Medawar in the mid 1900s to fit in with emerging concepts of transplantation immunology. Modern concepts and understanding of immune privilege come from subsequent studies produced by Medawar, Billingham, and Streilein. The exploitation of the model of anterior chamber immune deviation (ACAID) in mice has allowed us to look at both cellular and molecular mechanisms involved in the prevention of potentially damaging immune responses in such privileged sites. This review gives a historical perspective of the immune privilege research and provides up-to-date information of molecules, cells, and concepts newly recognized as contributing to tolerance induction induced in such specialized areas of the body. Evidence is given to support the idea that application of such information may lead to potential for therapeutic applications of ACAID mechanisms in prevention of progression of immune-inflammatory diseases in humans.

Ocular immune privilege and CD1d-reactive natural killer T cells.

PURPOSE: Immune privilege in the eye is, in part, associated with the development of an antigen-specific systemic tolerance termed anterior chamber-associated immune deviation (ACAID). Natural killer T (NKT) cells express T-cell receptor and natural killer (NK) markers and are classified as innate immune cells partly because they produce cytokines within minutes of signals. The aim of this study was to elucidate the role of murine NKT cells in the induction of T regulatory cells in anterior chamber-associated immune deviation. METHODS: Anterior chamber-associated immune deviation T regulatory cell generation ability was examined in the following NKT cell-deficient mice: SJL mice, CDld or Jalpha281 knockout (KO) mice on C57BL/6 (B6) background, and NKT cell-depleted mice. To detect T regulatory cells, splenic T cells were harvested 7 days after anterior chamber inoculation of ovalbumin (50 microg/2 microL in Hanks balanced salt solution [HBSS]), mixed with ovalbumin-primed T cells (effector) and ovalbumin-pulsed antigen-presenting cells (stimulator), and then cotransferred into the ear pinnae of a syngeneic naive mouse (local adoptive transfer assay). Ear swelling was measured 24 hours later. RESULTS: Anterior chamber-inoculated B6 mice developed T regulatory cells, but all natural killer T cell-deficient mice did not generate T regulatory cells unless they were reconstituted with natural killer T cells. We also found that the number of splenic natural killer T cells were increased in anterior chamber-inoculated B6 mice and those natural killer T cells produced IL-10. CONCLUSIONS: CD1d-reactive natural killer T cells are essential for the induction of T regulatory cells in anterior chamber-associated immune deviation through their IL-10 production and are involved in the maintenance of immune privilege of the eye.