Expression Of Immunomodulatory Molecules Research Articles

Recombinant Herpesvirus Glycoprotein G Improves the Protective Immune Response to Helicobacter pylori Vaccination in a Mouse Model of Disease

Alphaherpesviruses, which have co-evolved with their hosts for more than 200 million years, evade and subvert host immune responses, in part, by expression of immuno-modulatory molecules. Alphaherpesviruses express a single, broadly conserved chemokine decoy receptor, glycoprotein G (gG), which can bind multiple chemokine classes from multiple species, including human and mouse. Previously, we demonstrated that infection of chickens with an infectious laryngotracheitis virus (ILTV) mutant deficient in gG resulted in altered host immune responses compared to infection with wild-type virus. The ability of gG to disrupt the chemokine network has the potential to be used therapeutically. Here we investigated whether gG from ILTV or equine herpesvirus 1 (EHV-1) could modulate the protective immune response induced by the Helicobacter pylori vaccine antigen, catalase (KatA). Subcutaneous immunisation of mice with KatA together with EHV-1 gG, but not ILTV gG, induced significantly higher anti-KatA IgG than KatA alone. Importantly, subcutaneous or intranasal immunisation with KatA and EHV-1 gG both resulted in significantly lower colonization levels of H. pylori colonization following challenge, compared to mice vaccinated with KatA alone. Indeed, the lowest colonization levels were observed in mice vaccinated with KatA and EHV-1 gG, subcutaneously. In contrast, formulations containing ILTV gG did not affect H. pylori colonisation levels. The difference in efficacy between EHV-1 gG and ILTV gG may reflect the different spectrum of chemokines bound by the two proteins. Together, these data indicate that the immuno-modulatory properties of viral gGs could be harnessed for improving immune responses to vaccine antigens. Future studies should focus on the mechanism of action and whether gG may have other therapeutic applications.

Wharton’s jelly mesenchymal stem cells differentiation into hepatocyte-like cells: functional characterization and expression of immunomodulatory molecules

Mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) recently emerged as promising tools for cellular therapy due to their ability to differentiate into diverse cell types and their immunomodulatory features. Little is known on the expression of immunomodulatory molecules in mature cells differentiated from WJ-MSCs, therefore we aimed to characterize the extent of maintenance of the naive traits of these cells also in a highly specialized differentiated counterpart. WJ-MSCs were differentiated into hepatocyte-like cells (HLCs) with a four weeks protocol. RT-PCR, flow cytometry, IHC and ICC were performed to assess expression of key markers in both undifferentiated and differentiated cells. Hepatocyte specific assays such as PAS staining, CYP3A4 induction and activity, G6Pase activity, were performed to demonstrate the acquisition of traits of the mature hepatocyte phenotype. WJ-MSCs were successfully differentiated into HLCs using a multi-step protocol. These cells were able to store glycogen, incorporate specific live cells stains, perform enzymatic reactions and metabolic activities which are usually featured by mature hepatocytes. In addition, WJ-MSCs did express several immune-related molecules, for which an immunomodulatory role has been demonstrated both in vitro and in vivo. We demonstrated for the first time that key molecules as HLA-E and B7-H3 are expressed also by HLCs derived from WJ-MSCs. Our data showed for the first time that HLCs mostly maintain the expression of immune-modulating molecules, together with new markers and functional features of mature cells. In multiple pathologic conditions the intrinsic immunomodulatory ability of differentiated cells may help survive the interaction with the host immune system, even in the absence of a specific immunosuppressive therapy. This, together with the acquisition of key mature hepatocyte functions, may render WJ-MSCs promising in cell therapy applications for liver diseases.

Umbilical cord revisited: from Wharton's jelly myofibroblasts to mesenchymal stem cells.

The umbilical cord (UC) is an essential part of the placenta, contributing to foetal development by ensuring the blood flow between mother and foetus. The UC is formed within the first weeks of gestation by the enclosure of the vessels (one vein and two arteries) into a bulk of mucous connective tissue, named Wharton's jelly (WJ) and lined by the umbilical epithelium. Since their first identification, cells populating WJ were described as unusual fibroblasts (or myofibroblasts). Recent literature data further highlighted the functional interconnection between UC and the resident cells. The UC represents a reservoir of progenitor populations which are collectively grouped into MSCs (mesenchymal stem cells). Such cells have been sourced from each component of the cord, namely the sub-amnion layer, the WJ, the perivascular region, and the vessels. These cells mainly show adherence to the phenotype of adult MSCs (as bone marrow-derived ones) and can differentiate towards mature cell types belonging to all the three germ layers. In addition, cells from human UC are derived from an immunoprivileged organ, namely the placenta: in fact, its development and function depend on the elusion of the maternal immune response towards the semi-allogeneic embryo. This is reflected in the expression of immunomodulatory molecules by UC-derived MSCs. The present paper describes UC structural features and the cell types which can be derived, with a focus on their phenotype and the novel results which boosted the use of UC-derived cells for regenerative medicine applications.

Horse fetal adnexa: a source of mesenchymal and epithelial stem cells-like

Horse fetal adnexa: a source of mesenchymal and epithelial stem cells-like

Lentiviral expression of CTLA4Ig inhibits primed xenogeneic lymphocyte proliferation and cytokine responses

Co-stimulatory blockade is known to inhibit lymphocyte responses and to prolong allograft and xenograft survival. The present study examines the effect of transgenic expression of cytotoxic T lymphocyte-associated molecule-4 immunoglobulin (CTLA4Ig) by a porcine endothelial cell line (PIEC) transduced by a lentiviral vector, on primed xenogeneic T-cell proliferative and cytokine responses. Splenocytes from mice primed with PIEC were used as responder cells in a secondary proliferative assay. CTLA4Ig transduced and wild-type PIEC were used as stimulator cells. Responder cells were assayed for proliferation and cytokine production. Proliferation was profoundly inhibited by CTLA4Ig transduced cells compared with control cells. Cytokine analysis by enzyme linked immunospot demonstrated that production of interferon-gamma, IL4 (interleukin 4) and IL10 was inhibited by CTLA4Ig transduced cells compared with control cells. CTLA4Ig inhibited primed indirect xenogeneic T-cell proliferative and cytokine responses in vitro. Expression of immunomodulatory molecules by xenogeneic tissues has potential therapeutic applications for future xenotransplantation.

Herpes simplex virus type-1 amplicon vectors for vaccine generation in acute lymphoblastic leukemia

For leukemia vaccine generation, high-efficiency gene transfer is required to express immunomodulatory molecules that stimulate potent antileukemic immune responses. In this context, herpes simplex virus type-1 (HSV-1)-derived vectors have proven to be a promising tool for genetic modification of lymphoblastic leukemia cells. Yet, vector-associated viral protein expression might inadvertently modulate vaccine efficacy facilitating both immune evasion and immune stimulation. To explore the issue of immune-stimulation versus immune-suppression in immature lymphoblastic leukemia cells, two types of HSV-1 amplicon vectors, helper virus-dependent and helper virus-free that express the immunomodulatory molecules CD70 and IL-2, were compared with regard to their vector-associated immunomodulatory potential. We first established that lymphoblastic cell lines and primary acute lymphoblastic leukemia (ALL) cells express HSV receptor genes. Lymphoblastic cell lines were transduced with high efficiency, and in primary ALL cells high gene transfer rates of 47+/-15 and 42+/-14% were obtained with helper virus-dependent and -free HSV-1 amplicon vectors, respectively. The efficacy of the two amplicon vectors to induce antineoplastic responses was assessed in a vaccine setting in mice with pre-existing highly malignant lymphoblastic disease. Treatment of mice with vaccine cells transgenically expressing CD70+IL2 significantly suppressed lymphoblastic cell proliferation and improved survival. Of note, when helper virus-dependent HSV-1 amplicon vectors were used for vaccine preparation, the high immunogenic potential of the vector itself, in the absence of transgenic CD70+IL2 expression, seemed to be sufficient to mediate protection comparable to the antineoplastic response achieved by expression of immunomodulatory molecules. Thus for vaccine generation in B lymphoblastic leukemia, the immunogenic potential of HSV-1 helper virus-dependent amplicon vectors does provide additional benefit to the high transduction efficiency of HSV-1-derived vectors.

Differential expression of LFA-3, Fas and MHC Class I on Ad5- and Ad12-transformed human cells and their susceptibility to lymphokine-activated killer (LAK) cells

Adenovirus (Ad) E1A is a potent oncogene and has been shown to deregulate the expression of a large number of cellular genes leading to cellular transformation. Here we have analysed the expression of several immunomodulatory molecules on the surface of a set of human cell lines transformed with either Ad12 or Ad5. Human cells transformed with Ad12 demonstrated reduced expression of cell surface LFA-3, Fas and MHC class I when compared to Ad5-transformed cells. Furthermore, Ad12-transformed human cell lines demonstrated greater susceptibility to lysis by lymphokine-activated killer (LAK) cells, compared to Ad5-transformed human cell lines. In contrast, previous studies with rodent cells showed that both Ad5- and Ad12-transformed rat cells were susceptible to LAK cells. Thus, transformation of human cells with Ad5 or Ad12 results in differences in the expression of immunomodulatory molecules on the cell surface and differential recognition of these virus-transformed cells by immune effector cells.

Interactions of fibroblasts, adipocytes and immunocompeent cells in the pathogenesis of endocrine ophthalmopathy

Graves' ophthalmopathy is thought to result from a complex interplay of genetic and environmental factors. Various genes including those coding for HLA may determine a patient's susceptibility to the disease and its severity, but in addition numerous and often unknown environmental factors may determine its course. The orbital immune process is thought to be initiated, on the background of a permissive immunogenetic milieu, by circulating T cells directed against certain antigens on thyroid follicular cells that also recognize antigenic epitopes which are shared by tissues contained in the orbital space. Analysis of variable region genes of T cell antigen receptors in orbital T cells of patients with active Graves' ophthalmopathy has revealed limited variability of TcR V gene usage, suggesting that antigen-driven selection and/or expansion of specific T cells may occur during the early stages of Graves' ophthalmopathy. T cell recruitment into the orbital tissues is facilitated by certain chemokines and cytokines, which attract T cells by stimulating the expression of several adhesion molecules (e.g. ICAM-1, VCAM-1, CD44) in vascular endothelium and connective tissue cells. Adhesion molecules are known to be important for a variety of interactions between immunocompetent cells, preadipocyte fibroblasts and adipocytes. In addition, these molecules play a central role in lymphocyte activation and localization, facilitating antigen recognition, T cell costimulation, and various effector-target cell functions at the inflammatory sites, which result in amplification of the cellular immune process in active Graves' ophthalmopathy. T cells and macrophages populate the orbital space and release a number of cytokines (most likely a Th-1-type spectrum) into the surrounding tissues. Cytokines, oxygen free radicals and fibrogenic growth factors, released both from infiltrating inflammatory and residential cells, act upon orbital preadipocytes in a paracrine and autocrine manner to stimulate adipogenesis, fibroblast proliferation, glycosaminoglycan synthesis, and the expression of immunomodulatory molecules. Smoking, a well-known aggravating factor in Graves' ophthalmopathy, may aggravate tissue hypoxia and exert important immunomodulatory and pro-oxidant effects. Differentiation of orbital preadipocyte fibroblasts into mature adipocytes expressing increased levels of TSHR may also be driven by stimulation with circulating or locally produced cytokines or effectors. TSHR-directed autoantibodies or T cells may thus play a direct role promoting adipogenesis, glycosaminoglycan synthesis and expression of immunomodulatory proteins within the orbits. Once the net effect of these changes has come to increase the volume of the fatty connective tissues within the orbit, then proptosis, extraocular muscle dysfunction, and periorbital congestion will ensue.

Clinical implications of somatostatin-receptor scintigraphy in ophthalmic Graves' disease.

The eye complications associated most typically withhyperthyroidism caused by Graves’ disease range fromdiscomfort and lid retraction to disfiguring proptosis,diplopia and sight loss (1, 2). Usually called Graves’ophthalmopathy, this condition is better termedthyroid-associated ophthalmopathy (TAO), emphasis-ing the wide spectrum of thyroid disorders in thesepatients. TAO is one of the most difficult autoimmunedisorders to investigate; it is characterised by enlarge-ment of the extraocular muscles and an increase inthe retrobulbar fat, which cause eye proptosis, themain clinical manifestation of the disease (Table 1).Involvement of the cornea, the optic nerve and orbitalsoft tissues may occur during the natural course ofthe disease (3).An exact in vivo characterisation of the underlyingpatho-physiological mechanism of TAO has remaineddifficult because the affected retrobulbar space is noteasily accessible. The identification of patients withGraves’ ophthalmopathy is based primarily on clinicalsigns and symptoms affecting one or both eyes (4).Additional information can be obtained from conven-tional imaging methods such as magnetic resonanceimaging (MRI) or computed tomography (CT), whichallow gross changes in the retrobulbar space (e.g.muscle swelling, oedema or fibrosis) to be recognised(1, 2). By using histology, orbital infiltration withactivated T lymphocytes and evidence of local release ofcytokines and interactions of lymphocytes with retro-bulbar fibroblasts have been demonstrated, suggestingthat TAO is an organ-specific autoimmune disease (1,2, 5, 6). Although it is accepted that the pathogenesisof TAO is almost certainly multifactorial, the triggerevent seems to be the activation of T cells directedagainst a thyroid follicular cell antigen(s) that thenrecognise and bind to a similar antigen(s) in orbitaltissue (1, 2, 6). Thereafter, these activated orbital T cellsrelease a variety of substances and stimulate excessiveproduction of glycosaminoglycans and collagen byorbital fibroblasts. This causes the inflammatory andoedematous changes of thyroid ophthalmopathy (7). Infact, during the early stages of the disease, macro-phages, highly specialised T cells, mast cells and,occasionally, plasma cells infiltrate the orbital connec-tive, adipose and muscle tissue. Several cytokines, suchas interferon-g, tumour necrosis factor-a, interleukin-1 and transforming growth factor-b, as well as growthfactors, such as insulin-like growth factor-I (IGF-I) andplatelet-derived growth factor, have been detectedwithin the orbital tissues (5–8). All these substancesare probably produced both by infiltrating immuno-competent cells and by residential fibroblasts, adipo-cytes, myocytes and microvascular endothelial cells(9). Cytokines and growth factors stimulate cellproliferation, glycosaminoglycan synthesis and expres-sion of immunomodulatory molecules in orbitalfibroblasts and microvascular endothelial cells (Table2).No consensus exists regarding the common anti-gen in the orbit and thyroid, the thyrotrophin(thyroid-stimulating hormone, TSH) receptor or avariant, or on the role of circulating antibodiesagainst extraocular muscle and fibroblast compo-nents. The finding of expression of the TSH receptorin retro-orbital tissue may provide the link, pre-viously lacking, between orbital involvement andthyroid disease in Graves’ disease. Thyroid-stimulat-ing antibodies directed against the TSH receptorcould interact with the orbital components thatexpress the receptor, thus stimulating glycosamino-glycans, cellular matrix proteins and connectivetissue production and bringing about antibody-dependent cell-mediated cytotoxicity mediated bynatural killer cells. On the other hand, T cells thatreact with the TSH receptor will be targeted to theorbit where, in the presence of the antigen, they willsecrete various cytokines that stimulate fibroblasts toproliferate, to express the major complex of histo-compatibility class II antigens and to synthesizecollagen and connective tissue (10).

Thyroid-associated eye disease

Graves' ophthalmopathy (GO) is thought to result from a complex interplay of genetic and environmental factors. Various genes, including those coding for HLA, may determine a patient's susceptibility to the disease and its severity, but in addition, numerous and often unknown environmental factors may determine its course. Once established, the chronic inflammatory process within the orbital tissues appears to take on a momentum of its own. Based upon our current state of knowledge, we propose the working scheme shown in Fig. 1 for the pathogenesis of GO: Against the background of a permissive immunogenetic milieu, circulating T cells in patients with Graves' Disease (GD), directed against certain antigens on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the orbital space. Here, preadipocytes and fibroblasts most likely act as target and effector cells of the orbital immune process. This includes preadipocyte fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically or metabolically different from those located in the orbital connective tissue. Differentiation of orbital preadipocyte fibroblasts into mature adipocytes expressing increased levels of TSHR may be driven by stimulation with circulating or locally produced cytokines or effectors. To date, it is still unknown how autoreactive T cells escape deletion by the immune system and become directed against a self-antigen that is presented by cells residing in the thyroid gland and in certain extrathyroidal locations. Mimicry of a host antigen by a microorganism or presentation of an altered self-antigen may promote proliferation and expansion of autoreactive T cell clones. T cell recruitment into the orbital tissues is facilitated by certain chemokines and cytokines, which help to attract T cells by stimulating the expression of several adhesion molecules (e.g. ICAM-1, VCAM-1, CD44) in vascular endothelium and connective tissue cells. Adhesion molecules are known to be important for a variety of interactions between immunocompetent cells, connective tissue cells and extracellular matrix components. In addition, these molecules play a central role in lymphocyte activation and localization, facilitating antigen recognition, T cell costimulation, and various effector-target cell functions at the inflammatory sites, many of which result in amplification of the cellular immune process in active GO. Analysis of variable region genes of T cell antigen receptors in orbital T cells of patients with active GO has revealed limited variability of TcR V gene usage, suggesting that antigen-driven selection and/or expansion of specific T cells may occur during the early stages of GO. T cells and macrophages populate the orbital space and release a number of cytokines (most likely a Th-1-type spectrum) into the surrounding tissues. Cytokines, oxygen free radicals and fibrogenic growth factors, released both from infiltrating inflammatory and residential cells, act upon orbital preadipocytes in a paracrine and autocrine manner to stimulate adipogenesis, fibroblast proliferation, glycosaminoglycan synthesis, and the expression of immunomodulatory molecules. Smoking, a well-known aggravating factor in GO with an uncertain mode of action, may aggravate tissue hypoxia and exert important immunomodulatory effects. Finally, the long-held hypothesis of a thyroid cross-reactive antigen within the orbital tissues has recently gained significant support from an animal model of ophthalmopathy, and from in vitro and ex vivo studies. If confirmed by immunological studies, these data may well explain the localized infiltration of the orbital tissues by autoreactive lymphocytes that share intriguing molecular features with intrathyroidal lymphocytes. Local release of certain cytokines, TSHR-directed autoantibodies, or other factors might further enhance adipogenesis, glycosaminoglycan synthesis and expression of

Thyroid-associated eye disease

Graves' ophthalmopathy (GO) is thought to result from a complex interplay of genetic and environmental factors. Various genes, including those coding for HLA, may determine a patient's susceptibility to the disease and its severity, but in addition, numerous and often unknown environmental factors may determine its course. Once established, the chronic inflammatory process within the orbital tissues appears to take on a momentum of its own. Based upon our current state of knowledge, we propose the working scheme shown in Fig. 1 for the pathogenesis of GO: Against the background of a permissive immunogenetic milieu, circulating T cells in patients with Graves' Disease (GD), directed against certain antigens on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the orbital space. Here, preadipocytes and fibroblasts most likely act as target and effector cells of the orbital immune process. This includes preadipocyte fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically or metabolically different from those located in the orbital connective tissue. Differentiation of orbital preadipocyte fibroblasts into mature adipocytes expressing increased levels of TSHR may be driven by stimulation with circulating or locally produced cytokines or effectors. To date, it is still unknown how autoreactive T cells escape deletion by the immune system and become directed against a self-antigen that is presented by cells residing in the thyroid gland and in certain extrathyroidal locations. Mimicry of a host antigen by a microorganism or presentation of an altered self-antigen may promote proliferation and expansion of autoreactive T cell clones. T cell recruitment into the orbital tissues is facilitated by certain chemokines and cytokines, which help to attract T cells by stimulating the expression of several adhesion molecules (e.g. ICAM-1, VCAM-1, CD44) in vascular endothelium and connective tissue cells. Adhesion molecules are known to be important for a variety of interactions between immunocompetent cells, connective tissue cells and extracellular matrix components. In addition, these molecules play a central role in lymphocyte activation and localization, facilitating antigen recognition, T cell costimulation, and various effector-target cell functions at the inflammatory sites, many of which result in amplification of the cellular immune process in active GO. Analysis of variable region genes of T cell antigen receptors in orbital T cells of patients with active GO has revealed limited variability of TcR V gene usage, suggesting that antigen-driven selection and/or expansion of specific T cells may occur during the early stages of GO. T cells and macrophages populate the orbital space and release a number of cytokines (most likely a Th-1-type spectrum) into the surrounding tissues. Cytokines, oxygen free radicals and fibrogenic growth factors, released both from infiltrating inflammatory and residential cells, act upon orbital preadipocytes in a paracrine and autocrine manner to stimulate adipogenesis, fibroblast proliferation, glycosaminoglycan synthesis, and the expression of immunomodulatory molecules. Smoking, a well-known aggravating factor in GO with an uncertain mode of action, may aggravate tissue hypoxia and exert important immunomodulatory effects. Finally, the long-held hypothesis of a thyroid cross-reactive antigen within the orbital tissues has recently gained significant support from an animal model of ophthalmopathy, and from in vitro and ex vivo studies. If confirmed by immunological studies, these data may well explain the localized infiltration of the orbital tissues by autoreactive lymphocytes that share intriguing molecular features with intrathyroidal lymphocytes. Local release of certain cytokines, TSHR-directed autoantibodies, or other factors might further enhance adipogenesis, glycosaminoglycan synthesis and expression of immunomodulatory proteins within the orbits. Once the net effect of these changes is to increase the volume of the fatty connective tissues within the orbit, then proptosis, extraocular muscle dysfunction, and periorbital congestion will ensue. Future studies will be aimed at identifying factors that might modulate adipogenesis in orbital cells and clarifying the link between adipogenesis and TSHR expression in the orbit. Taken together, a number of important details in the complex pathogenesis of GO have been resolved in recent years, but many challenges are still ahead. Elucidation of the primary antigen and how it is recognized by the immune system will be key issues.

Pathogenesis of ophthalmopathy in autoimmune thyroid disease.

What causes GO is still a mystery, but the disease process results from a complex interplay of genetic and environmental factors. Genes such as those for HLA genes may determine a patient's susceptibility to the disease and its severity, but environmental factors, often unknown, may determine its course. Once established, the chronic inflammatory process within the orbital tissues appears to take on a momentum of its own. Given our current state of knowledge, the following working scheme for the pathogenesis of GO can be proposed (Fig. 1): On the background of a permissive immunogenetic milieu, circulating T cells in patients with GD, directed against certain antigens on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the orbital space. Of the cell types residing in these tissues, preadipocytes and fibroblasts, most likely act as target and effector cells of the orbital immune process, respectively. This includes preadipocyte fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically different from those located in the orbital connective tissue. Orbital preadipocyte fibroblasts may be stimulated by unknown circulating or locally produced factors to differentiate into mature adipocytes that express increased levels of TSHr. How autoreactive T cells escape deletion and control by the immune system and come to be directed against a self-antigen presented by cells residing in the thyroid gland and extrathyroidal locations, is still unknown. Proliferation and expansion of autoreactive T cell clones may be due to mimicry of a host antigen by a microorganism, but this remains speculative. T cell recruitment into the orbital tissues is facilitated by certain chemokines and cytokines, which help to attract T cells by stimulating the expression of certain adhesion molecules (e.g., ICAM-1, VCAM-1, CD44) in vascular endothelium and connective tissue cells. These adhesion receptors are known to also play an important costimulatory role by activating T cells and facilitating antigen recognition, which amplifies the cellular immune process. Analysis of variable region genes of T cell antigen receptors in orbital T cells of patients with active GO has revealed their restricted TcR V gene usage, suggesting that antigen-driven selection and/or expansion of specific T cells may occur early in the evolution of GO. T cells and macrophages populating the orbital space are known to synthesize and release a [figure: see text] number of cytokines (most likely a Th1-type spectrum) into the surrounding tissue. Cytokines, oxygen free radicals and fibrogenic growth factors, released both from infiltrating inflammatory and residential cells, act upon orbital preadipocytes in a paracrine and autocrine manner to stimulate adipogenesis, fibroblast proliferation, glycosaminoglycan synthesis, and the expression of immunomodulatory molecules. Smoking, a well-known aggravating factor in GO, may aggravate tissue hypoxia and exert important immunomodulatory effects. The long held hypothesis of a thyroid cross-reactive antigen within the orbital tissues has recently gained significant support by an animal model of GO, and by in vitro and ex vivo studies. If confirmed in immunological studies, these data may well explain the localized infiltration of the orbital tissues by autoreactive lymphocytes that share intriguing molecular features with intrathyroidal lymphocytes. Local release of particular cytokines, TSHr-directed antibodies, or other factors might further enhance adipogenesis, glycosaminoglycan synthesis and expression of immunomodulatory proteins within the orbit. Other factors, including inflammatory cytokines, might act as counterbalancing inhibitors of these effects. However, if the net effect of these changes is to increase the volume of the fatty connective tissues within the orbit, then proptosis, extraocular muscle dysfunction, and periorbital congestion will ensue. Whether this hypothetical sequence of events will finally explain the involvement of the orbit in GD is unknown. Future studies will be aimed at identifying factors that might modulate adipogenesis in orbital cells and clarifying the link between adipogenesis and TSHr expression in the orbit. Taken together, a number of important details in the complex pathogenesis of GO have been resolved in recent years, but many challenges are still ahead. Elucidation of the primary antigen and how it is recognized by the immune system will be key issues.