Transient low-dose methotrexate induces tolerance to murine anti-thymocyte globulin and together they promote long-term allograft survival.

Rabbit anti-thymocyte globulin (Thymoglobulin) effectively treats transplant rejection but induces anti-rabbit Ab responses, which limits routine readministration. Aiming to tolerize anti-rabbit responses, we coadministered a brief methotrexate regimen with a murine version of Thymoglobulin (mATG) for effects on anti-mATG Abs and cardiac allotransplantation in mice. Although both single and three courses of methotrexate could significantly inhibit anti-drug Ab titers to repeated mATG treatment, surprisingly, the single course given at the first mATG administration was most effective (>99% reduction). The transient methotrexate treatment also significantly improved pharmacokinetics and pharmacodynamics of repeated mATG administration. In the cardiac allograft model, the combination of transient mATG and methotrexate given only at the time of transplant dramatically improved allograft survival (>100 d) over either agent alone (<30 d). Anti-drug Ab titers were reduced and mATG exposure was increased which resulted in prolonged rather than enhanced mATG-mediated effects when combined with methotrexate. Moreover, methotrexate administration significantly reduced alloantibodies, suggesting that methotrexate not only decreases anti-drug Ab responses but also reduces Ab responses to multiple tissue-derived alloantigens simultaneously. These data suggest that mATG and methotrexate together can provide long-term allograft survival potentially through the induction of immune tolerance.

Homeostatic role of transforming growth factor-beta in the oral cavity and esophagus of mice and its expression by mast cells in these tissues.

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor; its overexpression has been implicated in many diseases, making it a desirable target for therapeutic neutralization. In initial safety studies, mice were chronically treated (three times per week) with high doses (50 mg/kg) of a murine, pan-neutralizing, anti-TGF-beta antibody. Nine weeks after the initiation of treatment, a subset of mice exhibited weight loss that was concurrent with decreased food intake. Histopathology revealed a unique, nonneoplastic cystic epithelial hyperplasia and tongue inflammation, as well as dental dysplasia and epithelial hyperplasia and inflammation of both the gingiva and esophagus. In an effort to determine the cause of this site-specific pathology, we examined TGF-beta expression in these tissues and saliva under normal conditions. By immunostaining, we found higher expression levels of active TGF-beta1 and TGF-beta3 in normal tongue and esophageal submucosa compared with gut mucosal tissues, as well as detectable TGF-beta1 in normal saliva by Western blot analysis. Interestingly, mast cells within the tongue, esophagus, and skin co-localized predominantly with the TGF-beta1 expressed in these tissues. Our findings demonstrate a novel and restricted pathology in oral and esophageal tissues of mice chronically treated with anti-TGF-beta that is associated with basal TGF-beta expression in saliva and by mast cells within these tissues. These studies illustrate a previously unappreciated biological role of TGF-beta in maintaining homeostasis within both oral and esophageal tissues.

Ultrasound imaging accurately detects skin thickening in a mouse scleroderma model.

Systemic sclerosis (scleroderma) is characterized by initial thickening of the skin because of the accumulation of collagen within the dermis followed by progression of fibrosis to internal organs. Although ultrasound assessment of dermal thickening in scleroderma patients is well documented, whether this technique can accurately detect skin thickening in mice under similar disease conditions is not known. Unlike traditional histologic assessments performed for disease models, ultrasound does not require sacrifice of the animal, and assessments of the same individual mice can be made over time. For these reasons, we examined the feasibility of ultrasound imaging to detect changes in skin thickness in a mouse model of graft-vs.-host-induced scleroderma (GVH-scleroderma). These studies determined ultrasound measurements to be highly consistent, both between multiple measurements of the same mouse as well as within a group of normal mice (coefficient of variation <8%). Ultrasound analysis of skin thickening in a GVH-scleroderma model showed similar sensitivity to histologic measurements because changes in skin thickness were detected by both methods at similar time points and to similar degrees. Direct comparisons between histologic and ultrasound measurements in the same animals over the course of disease also demonstrated significant correlations. Thus, these studies demonstrate that ultrasound can accurately detect skin thickening in a mouse model of scleroderma.

Teaching tolerance: New approaches to enzyme replacement therapy for Pompe disease.

Babies born with Pompe disease require life-long treatment with enzyme-replacement therapy (ERT). Despite the human origin of the therapy, recombinant human lysosomal acid α glucosidase (GAA, rhGAA), ERT unfortunately leads to the development of high titers of anti-rhGAA antibody, decreased effectiveness of ERT, and a fatal outcome for a significant number of children who have Pompe disease. The severity of disease, anti-drug antibody (ADA) development, and the consequences thereof are directly related to the degree of the enzyme deficiency. Babies born with a complete deficiency GAA are said to have cross-reactive immunologic material (CRIM)-negative Pompe disease and are highly likely to develop GAA ADA. Less frequently, GAA ADA develop in CRIM-positive individuals. Currently, GAA-ADA sero-positive babies are treated with a combination of immunosuppressive drugs to induce immunological tolerance to ERT, but the long-term effect of these regimens is unknown. Alternative approaches that might redirect the immune response toward antigen-specific tolerance without immunosuppressive agents are needed. Methods leading to the induction of antigen-specific regulatory T cells (Tregs), using peptides such as Tregitopes (T regulatory cell epitopes) are under consideration for the future treatment of CRIM-negative Pompe disease. Tregitopes are natural T cell epitopes derived from immunoglobulin G (IgG) that cause the expansion and activation of regulatory T cells (Treg). Teaching the immune system to tolerate GAA by co-delivering GAA with Tregitope peptides might dramatically improve the lives of CRIM-negative babies and could be applied to other enzyme replacement therapies to which ADA have been induced.

Nonclinical evaluation of GMA161--an antihuman CD16 (FcγRIII) monoclonal antibody for treatment of autoimmune disorders in CD16 transgenic mice.

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Murine antithymocyte globulin T-cell depletion is mediated predominantly by macrophages, but the Fas/FasL pathway selectively targets regulatory T cells.

BACKGROUND: Thymoglobulin is a T-cell-depleting polyclonal rabbit anti-human thymocyte antibody used clinically for immunosuppression in solid organ and hematopoietic stem-cell transplantation. By using a surrogate rabbit anti-mouse thymocyte globulin (mATG), we previously demonstrated that murine regulatory and memory T cells are preferentially spared from mATG depletion in vivo. The current studies were designed to determine whether different effector mechanisms are involved in differential depletion of T-cell subsets by mATG. METHODS: Complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and apoptotic mechanisms of depletion by mATG were evaluated in vitro and in vivo. RESULTS: In vitro, there was evidence of differential susceptibility of T-cell subsets by different effector mechanisms where naïve and CD4 effector memory T cells show reduced susceptibility to apoptosis, whereas regulatory T cells are less susceptible to mATG-mediated complement-dependent cytotoxicity and ADCC. However, mATG treatment of mice depleted of ADCC effector cell types (neutrophils, natural killer cells, or macrophages) or deficient in complement C5 or Fas demonstrated that mATG depletion of all T-cell subsets is mediated primarily by macrophages and that the role of neutrophils, natural killer cells, and complement is minimal in vivo. Interestingly, the Fas/FasL pathway does play a role in regulatory T-cell depletion, which is likely a result of increased basal expression of Fas on these cells. CONCLUSIONS: These data suggest that macrophages deplete most T cells by mATG in mice, but regulatory T cells are also uniquely susceptible to mATG-mediated Fas-dependent depletion.

In vivo characterization of rabbit anti-mouse thymocyte globulin: a surrogate for rabbit anti-human thymocyte globulin.

BACKGROUND: Polyclonal rabbit anti-human thymocyte globulin (Thymoglobulin) is used clinically for immunosuppression in solid organ transplantation; however, it is difficult to fully characterize the effects of this agent in humans. METHODS: A surrogate rabbit anti-murine thymocyte globulin (mATG) was generated analogously to the commercial product Thymoglobulin and in vivo activities were evaluated, including pharmacokinetics, T-cell depletion, dose response and kinetics, depletion/sparing of T-cell subsets or other leukocyte populations, and depletion in different lymphoid organs. RESULTS: Within 1 day, T cells are depleted by mATG in the blood, spleen, lymph node, and bone marrow down to doses of 1 mg/kg. Although mATG binds and depletes thymocytes in vitro, there is no thymocyte depletion in vivo at any dose level, suggesting decreased antibody accessibility to the thymus. After two doses of mATG given 3 days apart, T-cell reconstitution begins as early as day 9 and returns to basal levels by day 21 and 29 for CD4 and CD8 T cells, respectively. There is also preferential depletion of naïve T cells that results in increased ratios of regulatory and memory T cells within 1 day after mATG administration. Depletion of natural killer-T cells, natural killer cells, plasma cells, and plasmablasts occurs, but is modest and more transient compared with T cells. B cells, macrophages, dendritic cells, hematopoetic stem cells, and bone marrow stromal cells seem resistant to mATG depletion. CONCLUSIONS: These studies characterize the depletive effects of mATG in normal mice and provide insight into mechanisms of action of Thymoglobulin.

Characterization of in vitro antimurine thymocyte globulin-induced regulatory T cells that inhibit graft-versus-host disease in vivo.

Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin-induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4(+)CD25(+) T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

A modified model of graft-versus-host-induced systemic sclerosis (scleroderma) exhibits all major aspects of the human disease.

OBJECTIVE: Diffuse systemic sclerosis (SSc; scleroderma) is a debilitating disease characterized by excessive dermal fibrosis with later progression to internal organs. In addition to the fibrotic component, major aspects of the disease include vascular or circulatory involvement and immune dysregulation evidenced by inflammatory cells in affected tissues and production of autoantibodies. Many animal models resembling this disease have been studied, including genetic models in mice and chickens, challenge with chemicals such as bleomycin or vinyl chloride to induce fibrosis, and models of graft-versus-host (GVH)-induced disease using certain strains of mice with differences in minor histocompatibility loci. The present studies were undertaken to determine if alteration of the induction of GVH-induced scleroderma could result in a model that more fully represented the human condition. METHODS: Disease was induced by injection of spleen cells from B10.D2 mice into BALB/c mice deficient in mature T and B cells (recombination-activating gene 2 targeted). Dermal thickening, collagen deposition, vasoconstriction, and parameters of immunity were analyzed. RESULTS: Similar to the human disease, this modified GVH model of SSc demonstrated evidence of dermal thickening, particularly in the extremities, progressive fibrosis of internal organs, vasoconstriction and altered expression of vascularity markers in skin and internal organs, early immune activation, inflammation in skin and internal organs, and autoantibody generation. CONCLUSION: This modified model of GVH-induced SSc exhibits all major components of human disease and is likely to contribute to better understanding of the disease mechanisms and, ultimately, improved treatments for patients.

Adenoviral vectors stimulate murine natural killer cell responses and demonstrate antitumor activities in the absence of transgene expression.

Adenoviral vector-mediated gene delivery is currently the focus of many efforts to administer therapeutic gene products for the treatment of cancer. Although these vectors are replication deficient, they can induce specific immune responses against both vector- and transgene-encoded proteins. We have extended these findings to determine the level of innate natural killer (NK) cell responses to adenoviral vector administration in vivo. Similar to many replicating viruses, the vectors induce prominent NK cell activation in mouse spleens within 2 days of injection. We also observed these NK cell responses regardless of the route of administration. Furthermore, stimulation of NK cells by adenoviral vectors is independent of viral gene transcription, as UV inactivation of the vectors does not reduce the NK cell response. In contrast, heat treatment of the vectors destroys their ability to activate NK cells, demonstrating the necessity for intact vector particles. In addition, we found that administration of "empty" (no transgene) adenoviral vectors delays tumor growth in mice bearing B16 melanomas, and this effect is abrogated by depletion of NK cells. Collectively, these results demonstrate in a murine system that the adenoviral vector gene delivery system itself stimulates NK cells, and this in turn can nonspecifically enhance antitumor immunity.

Minimal effects on immune parameters following chronic anti-TGF-beta monoclonal antibody administration to normal mice.

Mice genetically deficient in TGF-beta1 or TGF-beta signaling capacity in T or B cells demonstrate profound immune dysregulation, as evidenced by increased lymph node size, expression of markers of memory/activation on T cells, inflammation in a variety of tissues and development of autoantibodies. However, this constant and complete lack of TGF-beta1 or TGF-betaR signaling may not reflect effects of TGF-beta neutralization using antibodies in mature animals. Thus, the present studies were designed to determine if administration of an anti-TGF-beta monoclonal antibody (neutralizes TGF-beta1, 2 and 3) to mature, normal mice results in evidence of immune dysregulation or immune-mediated pathology. An initial study examined daily administration of 0.25, 0.75 and 2.5 mg/kg of anti-TGF-beta to mice for three weeks, achieving blood levels of as high as 9 mg/ml. Comprehensive hematological and histopathological evaluation showed no evidence of pathology. A second study was designed to extend the antibody treatment period and further examine the functional status of the immune system. Mice were injected with 1 mg/mouse (approximately 50 mg/kg) of anti-TGF-beta (1D11) three times per week achieving circulating blood levels of 1-2 mg/ml. Many parameters of immune status were assessed, including natural killer (NK) cell activity, lymphocyte proliferative responses, phagocytic activity, phenotypic assessment of leukocyte subsets, and serum measurements of proinflammatory cytokines, autoantibodies and immunoglobulin isotypes. In addition, histopathological assessment of heart, lungs, liver, kidney, salivary glands, skin, spleen and lymph nodes was also performed. Very few of the multiple immune parameters examined showed detectable changes in anti-TGF-beta-treated mice. Changes that were observed were primarily restricted to the spleen and included increased spleen cell recoveries, increased percentages of macrophages, decreased percentages of NK cells, decreased phagocytic activity, decreased proliferative responses to mitogens and slight increases in T and B cells displaying an activated phenotype. Many of these same parameters examined in the lymph nodes were not altered by the anti-TGF-beta treatment. The thymus was decreased in size, but altered only slightly in one population of developing T cells. Most of the changes observed were modest and returned to control levels after discontinuation of treatments. The only serological finding was an increase in IgA levels in anti-TGF-beta-treated mice, but not in any other isotype. Finally, there was no evidence of increased inflammation in any of the peripheral tissues examined in the anti-TGF-beta-treated mice. In conclusion, although there were changes in some of the immunological parameters examined in these studies, they were few and typically reversed following discontinuation of treatment. The modest nature of the changes observed in these studies is particularly evident when compared to published data of those same parameters examined in mice genetically deficient in TGF-beta1 or mice having TGF-beta unresponsive T or B cells. Thus, there does not appear to be any significant immune dysregulation detectable after long-term antibody-mediated neutralization of TGF-beta in normal mice.