Immune-privileged Tissue Research Articles

Adhesion protein Fibulin-7 (Fbln7) regulate differentiation and polarisation of human monocytes and macrophages.

Abstract Fibulin-7 (Fbln7) is a member of the fibulin family of secreted glycoproteins, majorly found in developing teeth, bone and cartilage and shows interaction with other ECM proteins. Recently, it was shown that its C-terminal fragment (Fbln7C) and its peptides show anti-angiogenic effects. Fbln7 is also expressed in immune privileged tissues such as eye and placenta but to date very little is known about its functional significance. The results from this study show that human monocytes adhered to Fbln7 full-length (Fbln7-FL) and Fbln7C via integrin α5β1. Interestingly, actin stress fiber formation of monocytes was promoted by Fbln7-FL and not by Fbln7C. Morphological studies and surface expression analysis of CD14, mannose receptor (CD206), MHC-II and CD11b receptors revealed that both Fbln7-FL and Fbln7C inhibited MCSF induced monocyte differentiation with more significant effect with Fbln7C as compared to Fbln7-FL. Similarly, both protein fragments inhibited IFNγ and IL-4 induced M1 and M2 polarization of human blood monocyte derived macrophages. Collectively, these data suggests that Fbln7 and its fragments could negatively regulate immune cell in the expressed tissues.

Abstract A021: Development of an ALK vaccine to treat ALK-rearranged non-small cell lung cancers

Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) expressed during development in the nervous system. Aberrant expression of ALK drives several tumor malignancies. About 3-7% of non-small cell lung cancers (NSCLC) harbor ALK rearrangements. Treatment with the Tyrosine Kinase inhibitor (TKI) crizotinib results in a high objective rate response (ORR) of ∼60%, but median progression-free survival (mPFS) is only 8-10 months, owing to the rapid emergence of acquired drug resistance. Alectinib and ceritinib, a second generation of TKIs developed to overcome crizotinib resistance, induce a transient response followed by tumor relapses. Currently, there are at least 7 additional ALK TKIs in clinical development. Emergence of mechanisms of resistance to the new TKI is expected. Thus, innovative therapeutic approaches are needed to complement the partial success obtained by TKIs in ALK-positive NSCLC patients. Three properties make of ALK a rather unique shared antigen: 1) ALK is a driver oncogene that is expressed in all the tumoral cells; 2) ALK is not expressed by normal cells with only marginal expression in few cells in the nervous system of adults, an immuneprivileged tissue, anticipating limited if any autoimmune side effects by an ALK-specific immunotherapy; 3) ALK is immunogenic in humans because ALK-specific antibodies and T cell responses are detected in ALK-positive lymphoma and lung cancer patients. As an additional advantage, the availability of ALK TKI to reduce tumor might facilitate immunotherapeutic approaches. Therefore, we aim to develop a strategy of vaccination in human patients to treat ALK-positive tumors in combination with TKIs and/or immunocheckpoints blockade. We recently developed an ALK vaccine based on a DNA plasmid that generates an efficacious anti-ALK immune response in pre-clinical mouse models. This vaccine potentiates the effects of ALK TKI and can be combined with immune checkpoint blockade therapy. However, a consistent theme in human DNA vaccine trials has been their suboptimal immunogenicity when compared with protein-based vaccine approaches. To move towards a peptide-based vaccine, we sought to identify antigenic peptides responsible for these immune responses by in vitro screening with ALK overlapping peptides in splenocytes from ALK vaccinated mice and by MHC-I binding predictive algorithms. Four peptides induced IFN-γ production in splenocytes and one of these peptides contained a predicted CD8+ T-cell epitope. To validate these results in vivo, mice were vaccinated with these peptides. The peptide containing the predicted CD8+ 9mer was confirmed to induce CD8+ T-cell responses while the others induced CD4+ T-cell responses. To increase immunogenicity, peptides were modified with an amphiphilic tail. As we recently showed, this conjugation promotes binding to endogenous albumin, increasing lymph node accumulation and T-cell priming. In prophylactic experiments, mice vaccinated with the CD8+ T-cell epitope, but not the CD4+ T-cell epitopes, almost completely rejected ALK-expressing lung orthotropic tumors. To validate the efficacy of the vaccine in a therapeutic protocol, EML4-ALK transgenic mice that spontaneously develop ALK-positive lung tumors were vaccinated with a combination of these peptides. The vaccine significantly delayed the progression of primary lung tumors. We propose that a peptide-based ALK vaccine, combined with ALK TKI treatment and/or immune checkpoint blockade therapy, could represent a novel therapeutic option for ALK-positive lung cancer patients. A Phase I clinical trial with a peptide-based ALK vaccine in NSCLC patients is currently under preparation. Citation Format: Rafael B. Blasco. Development of an ALK vaccine to treat ALK-rearranged non-small cell lung cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A021.

Is cancer a severe delayed hypersensitivity reaction and histamine a blueprint?

Longevity and accumulation of multiple context-dependent signaling pathways of long-standing inflammation (antigen-load or oxidative stress) are the results of decreased/altered regulation of immunity and loss of control switch mechanisms that we defined as Yin and Yang of acute inflammation or immune surveillance. Chronic inflammation is initiated by immune disruptors-induced progressive changes in physiology and function of susceptible host tissues that lead to increased immune suppression and multistep disease processes including carcinogenesis. The interrelated multiple hypotheses that are presented for the first time in this article are extension of author’s earlier series of ‘accidental’ discoveries on the role of inflammation in developmental stages of immune dysfunction toward tumorigenesis and angiogenesis. Detailed analyses of data on chronic diseases suggest that nearly all age-associated illnesses, generally categorized as ‘mild’ (e.g., increased allergies), ‘moderate’ (e.g., hypertension, colitis, gastritis, pancreatitis, emphysema) or ‘severe’ (e.g., accelerated neurodegenerative and autoimmune diseases or site-specific cancers and metastasis) are variations of hypersensitivity responses of tissues that are manifested as different diseases in immune-responsive or immune-privileged tissues. Continuous release/presence of low level histamine (subclinical) in circulation could contribute to sustained oxidative stress and induction of ‘mild’ or ‘moderate’ or ‘severe’ (immune tsunami) immune disorders in susceptible tissues. Site-specific cancers are proposed to be ‘severe’ (irreversible) forms of cumulative delayed hypersensitivity responses that would induce immunological chaos in favor of tissue growth in target tissues. Shared or special features of growth from fetus development into adulthood and aging processes and carcinogenesis are briefly compared with regard to energy requirements of highly complex function of Yin and Yang. Features of Yang (growth-promoting) arm of acute inflammation during fetus and cancer growth will be compared for consuming low energy from glycolysis (Warburg effect). Growth of fetus and cancer cells under hypoxic conditions and impaired mitochondrial energy requirements of tissues including metabolism of essential branched amino acids (e.g., val, leu, isoleu) will be compared for proposing a working model for future systematic research on cancer biology, prevention and therapy. Presentation of a working model provides insightful clues into bioenergetics that are required for fetus growth (absence of external threat and lack of high energy-demands of Yin events and parasite-like survival in host), normal growth in adulthood (balance in Yin and Yang processes) or disease processes and carcinogenesis (loss of balance in Yin–Yang). Future studies require focusing on dynamics and promotion of natural/inherent balance between Yin (tumoricidal) and Yang (tumorigenic) of effective immunity that develop after birth. Lawless growth of cancerous cells and loss of cell contact inhibition could partially be due to impaired mitochondria (mitophagy) that influence metabolism of branched chain amino acids for biosynthesis of structural proteins. The author invites interested scientists with diverse expertise to provide comments, confirm, dispute and question and/or expand and collaborate on many components of the proposed working model with the goal to better understand cancer biology for future designs of cost-effective research and clinical trials and prevention of cancer. Initial events during oxidative stress-induced damages to DNA/RNA repair mechanisms and inappropriate expression of inflammatory mediators are potentially correctable, preventable or druggable, if future studies were to focus on systematic understanding of early altered immune response dynamics toward multistep chronic diseases and carcinogenesis.

Soluble monomers, dimers and HLA-G-expressing extracellular vesicles: the three dimensions of structural complexity to use HLA-G as a clinical biomarker.

The HLA-G molecule belongs to the family of nonclassical human leukocyte antigen (HLA) class I. At variance to classical HLA class I, HLA-G displays (i) a low number of nucleotide variations within the coding region, (ii) a high structural diversity, (iii) a restricted peptide repertoire, (iv) a limited tissue distribution and (v) strong immune-suppressive properties. The physiological HLA-G surface expression is restricted to the maternal-fetal interface and to immune-privileged adult tissues. Soluble forms of HLA-G (sHLA-G) are detectable in various body fluids. Cellular activation and pathological processes are associated with an aberrant or a neo-expression of HLA-G/sHLA-G. Functionally, HLA-G and its secreted forms are considered to be key players in the induction of short- and long-term tolerance. Thus, its unique expression profile and tolerance-inducing functions render HLA-G/sHLA-G an attractive biomarker to monitor the systemic health/disease status and disease activity/progression for clinical approaches in disease management and treatments. Here, we place emphasis on (i) the current status of the tolerance-inducing functions by HLA-G/sHLA-G, (ii) the current complexity to implement this molecule as a meaningful clinical biomarker regarding the three dimensions of structural diversity (monomers, dimers and HLA-G-expressing extracellular vesicles) with its functional implications, and (iii) novel and future approaches to detect and quantify sHLA-G structures and functions.

The Potential of HLA-G-Bearing Extracellular Vesicles as a Future Element in HLA-G Immune Biology.

The HLA-G molecule is a member of the non-classical HLA class I family. Its surface expression is physiologically restricted to the maternal–fetal interface and to immune privileged adult tissues. Despite the restricted tissue expression, HLA-G is detectable in body fluids as secreted soluble molecules. A unique feature of HLA-G is the structural diversity as surface expressed and as secreted molecules. Secreted HLA-G can be found in various body fluids either as free soluble HLA-G or as part of extracellular vesicles (EVs), which are composed of various antigens/ligands/receptors, bioactive lipids, cytokines, growth factors, and genetic information, such as mRNA and microRNA. Functionally, HLA-G and its secreted forms are considered to play a crucial role in the network of immune-regulatory tolerance mechanisms, preferentially interacting with the cognate inhibitory receptors LILRB1 and LILRB2. The HLA-G mediated tolerance is described in processes of pregnancy, inflammation, and cancer. However, almost all functional and clinical implications of HLA-G in vivo and in vitro have been established based on simple single ligand/receptor interactions at the cell surface, whereas HLA-G-bearing EVs were in minor research focus. Indeed, cytotrophoblast cells, mesenchymal stem cells, and cancer cells were recently described to secrete HLA-G-bearing EVs, displaying immunosuppressive effects and modulating the tumor microenvironment. However, numerous functional and clinical open questions persist. Here, we (i) introduce basic aspects of EVs biology, (ii) summarize the functional knowledge, clinical implications and open questions of HLA-G-bearing EVs, and (iii) discuss HLA-G-bearing EVs as a future element in HLA-G biology.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states.

Ocular Immune Privilege and Transplantation.

Allografts are afforded a level of protection from rejection within immune-privileged tissues. Immune-privileged tissues involve mechanisms that suppress inflammation and promote immune tolerance. There are anatomical features, soluble factors, membrane-associated proteins, and alternative antigen-presenting cells (APC) that contribute to allograft survival in the immune-privileged tissue. This review presents the current understanding of how the mechanism of ocular immune privilege promotes tolerogenic activity by APC, and T cells in response to the placement of foreign antigen within the ocular microenvironment. Discussed will be the unique anatomical, cellular, and molecular mechanisms that lessen the chance for graft destroying immune responses within the eye. As more is understood about the molecular mechanisms of ocular immune privilege greater is the potential for using these molecular mechanisms in therapies to prevent allograft rejection.

Retinal innate immune activation in health and disease

SummaryRetina is considered as an immune privileged tissue, yet many inflammatory retinal diseases occur. Circulating immune cells are not able to freely migrate into the healthy retina due to the blood retina barrier. Retina is protected by its own innate immune system, including microglia, perivascular macrophages, a small number of dendritic cells and the complement system. When retina suffers from exogenous/endogenous insults, a well‐controlled innate immune response is initiated to maintain homeostasis and restore functionality, such response is therefore beneficial. Dysregulation or malfunction of the innate immune response may result in excessive production of inflammatory mediators that may contribute to retinal pathology. The presentation will discuss how innate immune response is controlled in the retina, and the potential contribution of uncontrolled or dysregulated innate immune activation to retinal lesion development in common sight‐threatening diseases such as age‐related retinal degeneration.

Parainflammation, chronic inflammation, and age-related macular degeneration.

Inflammation is an adaptive response of the immune system to noxious insults to maintain homeostasis and restore functionality. The retina is considered an immune-privileged tissue as a result of its unique anatomic and physiologic properties. During aging, the retina suffers from a low-grade chronic oxidative insult, which sustains for decades and increases in level with advancing age. As a result, the retinal innate-immune system, particularly microglia and the complement system, undergoes low levels of activation (parainflammation). In many cases, this parainflammatory response can maintain homeostasis in the healthy aging eye. However, in patients with age-related macular degeneration, this parainflammatory response becomes dysregulated and contributes to macular damage. Factors contributing to the dysregulation of age-related retinal parainflammation include genetic predisposition, environmental risk factors, and old age. Dysregulated parainflammation (chronic inflammation) in age-related macular degeneration damages the blood retina barrier, resulting in the breach of retinal-immune privilege, leading to the development of retinal lesions. This review discusses the basic principles of retinal innate-immune responses to endogenous chronic insults in normal aging and in age-related macular degeneration and explores the difference between beneficial parainflammation and the detrimental chronic inflammation in the context of age-related macular degeneration.

Jean Elizabeth Clark

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain–containing receptor (ILDR)2 as a...

Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye.

Immune privilege of the CNS is not the consequence of limited antigen sampling

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Immune Privilege and the Philosophy of Immunology

Like other scientists, immunologists use two types of approaches to research: one reduces the problem to its parts; the other studies the emergent phenomenon produced by the parts. Scientists that reduce the problem to its parts are sometimes called reductionists. The conclusions of reductionist experiments are often applied to the greater whole, when in actuality they may only apply to that particular experimental set. We, reductionists, are the ones who think our immune behavior exists solely because of genes, the presence of TGFb, the presence of inflammatory cytokines, and appearance of a receptor. We tend to interpret the outcome in the colors of our interests (ergo “to a hammer, everything is a nail”). We measure parts and from the parts, we draw parallels to farremoved outcomes in terms of health and disease. More often than not, however, the results from the study of the parts do not predict the whole, and often the whole becomes greater than the sum of its parts. For example, a toll-like receptor does one thing when there is a bacterial invasion but the same toll-like receptor may lead to a different outcome when activated by danger signals during injury. The approach that is perhaps more applicable to biology, and more specifically, to immunology, is the chaos theory. The chaos theory deals with the multiple layers of conditions and unexpected turns and restarts that can effect the outcome. It is applicable to studies of dynamic systems like the weather, and in our case, dynamic biological/immunological systems. The chaos theory points out that small differences in initial conditions make it impossible to predict the outcome. Thus, the behavior of the parts does not make the outcome predictable. Our point? Immune privilege is broadly understood as the ability of the tissue to actively regulate and direct immune responses that take place in its “territory.” The articles in this Research Topic in Frontiers in Immunology, “Good news‐bad news”, support the idea that to understand how immune privilege works, one has to understand the dynamics of the different tissues in terms of initiation, expression, regulation, and behavior, before one can begin to predict an outcome. This Research Topic contains a number of papers that deal with immune privilege in the eye: a review that explores the relationship of immune privilege to ocular disease (1); a contribution on local regulation of immune CD8 C T cells in the eye (2); a review discussing the intriguing parallels between the mechanisms of ocular immune privilege and uveal melanoma (3); a thoughtful contribution on how CD8 C Treg cells might enable ocular tumor growth (4); a review proposing that (paradoxically) immune privilege emerges as an enabler of immune cells that heal, as well as of regulator immune cells that promote tissue damage (5). While the eye is considered the prototypic immune privileged tissue, it is not the only one. We therefore shift to reviews of immune privilege and its mechanisms in other tissues and organs, underscoring the universality of the phenomenon: the reproductive tract (6), the testis (7), tumor environment (8), and finally chronic inflammatory diseases (9). While the eye, testes, reproductive tract, tumors, and chronic immune diseases all seem to share some “immune privileged” mechanisms, each has developed unique features of its own. Recent reports have repeatedly shown that immune regulation is “tailored” to the individual tissue in

Chronic Inflammation: Synergistic Interactions of Recruiting Macrophages (TAMs) and Eosinophils (Eos) with Host Mast Cells (MCs) and Tumorigenesis in CALTs. M-CSF, Suitable Biomarker for Cancer Diagnosis!

Ongoing debates, misunderstandings and controversies on the role of inflammation in cancer have been extremely costly for taxpayers and cancer patients for over four decades. A reason for repeated failed clinical trials (90% ± 5 failure rates) is heavy investment on numerous genetic mutations (molecular false-flags) in the chaotic molecular landscape of site-specific cancers which are used for “targeted” therapies or “personalized” medicine. Recently, unresolved/chronic inflammation was defined as loss of balance between two tightly regulated and biologically opposing arms of acute inflammation (“Yin”–“Yang” or immune surveillance). Chronic inflammation could differentially erode architectural integrities in host immune-privileged or immune-responsive tissues as a common denominator in initiation and progression of nearly all age-associated neurodegenerative and autoimmune diseases and/or cancer. Analyses of data on our “accidental” discoveries in 1980s on models of acute and chronic inflammatory diseases in conjunctival-associated lymphoid tissues (CALTs) demonstrated at least three stages of interactions between resident (host) and recruited immune cells: (a), acute phase; activation of mast cells (MCs), IgE Abs, histamine and prostaglandin synthesis; (b), intermediate phase; down-regulation phenomenon, exhausted/degranulated MCs, heavy eosinophils (Eos) infiltrations into epithelia and goblet cells (GCs), tissue hypertrophy and neovascularization; and (c), chronic phase; induction of lymphoid hyperplasia, activated macrophages (Mϕs), increased (irregular size) B and plasma cells, loss of integrity of lymphoid tissue capsular membrane, presence of histiocytes, follicular and germinal center formation, increased ratios of local IgG1/IgG2, epithelial thickening (growth) and/or thinning (necrosis) and angiogenesis. Results are suggestive of first evidence for direct association between inflammation and identifiable phases of immune dysfunction in the direction of tumorigenesis. Activated MFs (TAMs or M2) and Eos that are recruited by tissues (e.g., conjunctiva or perhaps lung airways) whose principal resident immune cells are MCs and lymphocytes are suggested to play crucial synergistic roles in enhancing growth promoting capacities of host toward tumorigenesis. Under oxidative stress, M-CSF may produce signals that are cumulative/synergistic with host mediators (e.g., low levels of histamine), facilitating tumor-directed expression of decoy receptors and immune suppressive factors (e.g., dTNFR, IL-5, IL-10, TGF-β, PGE2). M-CSF, possessing superior sensitivity and specificity, compared with conventional markers (e.g., CA-125, CA-19-9) is potentially a suitable biomarker for cancer diagnosis and technology development. Systematic monitoring of interactions between resident and recruited cells should provide key information not only about early events in loss of immune surveillance, but it would help making informed decisions for balancing the inherent tumoricidal (Yin) and tumorigenic (Yang) properties of immune system and effective preventive and therapeutic approaches and accurate risk assessment toward improvement of public health.

Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates

Complex barriers separate immune-privileged tissues from the circulation. Here, we propose that cell entry to immune-privileged sites through barriers composed of tight junction-interconnected endothelium is associated with destructive inflammation, whereas border structures comprised of fenestrated vasculature enveloped by tightly regulated epithelium serve as active and selective immune-skewing gates in the steady state. Based on emerging knowledge of the central nervous system and information from other immune-privileged sites, we propose that these sites are endowed either with absolute endothelial-based barriers and epithelial gates that enable selective and educative transfer of trafficking leukocytes or with selective epithelial gates only.

Extracellular Matrix Protein Lumican Promotes Clearance and Resolution of Pseudomonas aeruginosa Keratitis in a Mouse Model

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum −/−) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that in vitro Lum −/− macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum −/− mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum −/− mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum −/− mice compared to wild types. Infected Lum −/− corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24–48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum+/− mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the Lum −/− compared to Lum +/− infected corneas by 2–5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.

The gateway theory: bridging neural and immune interactions in the CNS

The central nervous system (CNS) is considered an immune-privileged tissue protected by a specific vessel structure, the blood-brain barrier (BBB). Upon infection or traumatic injury in the CNS, the BBB is breached, and various immune cells are recruited to the affected area. In the case of autoimmune diseases in the CNS like multiple sclerosis (MS), autoreactive T cells against some CNS-specific antigens can theoretically attack neurons throughout the CNS. The affected CNS regions in MS patients can be detected as multiple focal plaques in the cerebrum, thoracic cord, and other regions. Vision problems are often associated with the initial phase of MS, suggesting a disturbance in the optic nerves. These observations raise the possibility that there exist specific signals that direct autoreactive T cells past the BBB and into particular sites of the CNS. Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we recently defined the mechanism of the pathogenesis in which regional neural stimulations modulate the status of the blood vessel endothelium to allow the invasion of autoreactive T cells into specific sites of the CNS via the fifth lumbar cord. This gate for autoreactive T cells can be artificially manipulated by removing gravity forces on the hind legs or by electric pulses to the soleus muscles, quadriceps, and triceps of mice, resulting in an accumulation of autoreactive T cells in the intended regions via the activation of regional neurons. Gating blood vessels by regional neural stimulations, a phenomenon we call the gateway theory, has potential therapeutic value not only in preventing autoimmunity, but also in augmenting the effects of cancer immunotherapies.

Enhanced expression of myogenic differentiation factors and skeletal muscle proteins in human amnion-derived cells via the forced expression of MYOD1

Objectives: Mesenchymal stem cells are expected to be an ideal cell source for cellular and gene therapy. We previously showed that cells derived from the human placenta can be induced to differentiate into myotubes in vitro and to express dystrophin in mdx/scid mice in vivo. In this study, we examined whether amnion-derived cells can be efficiently transduced and differentiated using lentiviral vectors carrying human MYOD1. Methods: The amnion-derived cells were isolated from human preterm placentas. They were transduced with the MYOD1 vector, and mRNA levels for MYOD1, MYF5, MYOG, MYH2 and DMD were determined by quantitative-reverse transcriptase-polymerase chain reaction, and also examined immunocytochemically. Results: Approximately 70% of amnion-derived cells were efficiently transduced by the lentiviral vectors. MYOD1 activates MYF5 and MYOG, MYH2 and DMD after a 7-day culture. The concerted upregulations of these myogenic regulatory factors enhanced MYH2 and DMD expressions. PAX7 was below the detectable level. Both myosin heavy chain and dystrophin were demonstrated by immunocytochemistry. Conclusions: MYOD1 activates MYF5 and MYOG, the transcription factor genes essential for myogenic differentiation, and the concerted upregulation of these myogenic regulatory factors enhanced MYH2 and DMD expressions. The amniotic membrane is an immune-privileged tissue, making MYOD1-transduced amnion-derived cells an ideal cell source for cellular and gene therapy for muscle disorders. This is the first report showing that amnion-derived cells can be modified by exogenous genes using lentiviral vectors. Furthermore, MYOD1-transduced amnion-derived cells are capable of the dystrophin expression necessary for myogenic differentiation.

Arginine depletion as a mechanism for the immune privilege of corneal allografts.

The cornea is an immune privileged tissue. Since arginase has been found to modulate T-cell function by depleting arginine, we investigated the expression of arginase in the cornea and its possible role in immune privilege using a murine transplant model. We found that both the endothelium and epithelium of murine corneas express functional arginase I, capable of down-regulating T-cell proliferation in an in vitro culture system. The administration of the specific arginase inhibitor N-hydroxy-nor-l-Arg to recipient mice resulted in an accelerated rejection of allogeneic C57BL/6 (B6) corneal grafts. In contrast, in vivo blockade of arginase activity had no effect in altering the course of rejection of primary skin grafts that express little, if any, arginase. In addition, the inhibition of arginase did not alter systemic T-cell proliferation. These data show that arginase is functional in the cornea and contributes to the immune privilege of the eye, and that modulation of arginase contributes to graft survival.

Characterization of Langerin-Expressing Dendritic Cell Subsets in the Normal Cornea

In addition to Langerhans cells (LCs), other dendritic cells (CD11c(+)) have recently been shown to express Langerin (c-type lectin). In skin, (non-LC) Langerin+ dendritic cells initiate adaptive immunity. However, whether such dendritic cells (DC) reside in the cornea, an immune-privileged tissue, is unknown. Normal C57BL/6 corneas were harvested for qRT-PCR analyses of Langerin expression in the epithelium versus stroma. Immunohistochemistry for Langerin was also performed. Single-cell preparations of epithelium versus stroma were FACS analyzed for CD11c, CD11b, and CD103 expression. Fluorescence microscopy of corneas from muLangerin-eGFP mice (in which all CD11c(+) Langerin+ cells express eGFP), huLangerin-DTA mice (only LCs are constitutively deleted), and huLangerin-Cre eYFP-flox (only LCs express eYFP) was performed. qRT-PCR, immunohistochemistry, and FACS analysis identified CD11c(+) Langerin+ cells in the epithelium and stroma. Similarly, corneas of muLangerin-eGFP mice contained eGFP+ cells in the epithelium and stroma. However, FACS analysis indicated phenotypically differing CD11c(+) Langerin+ populations in the epithelium (CD11b(low)CD103(low)) versus stroma (CD11b(+)CD103(low)). Additionally, corneas from huLangerin-DTA mice were devoid of Langerin+ cells in the epithelium but were detectable in the stroma. In corneas from huLangerin-Cre eYFP-flox, eYFP+ cells were detectable in the epithelium but not in the stroma. The normal corneal epithelium is endowed with CD11c(+) Langerin+ cells that are LCs, whereas the stroma is endowed with a separate population of (non-LC) Langerin+ DCs. These findings should henceforth facilitate the examination of Langerin-expressing DC subsets in the immunopathogeneses of conditions such as keratoconjunctivitis sicca, allergic keratoconjunctivitis, and corneal allograft rejection.