Requirement of B7 costimulation for Th1-mediated inflammatory bone resorption in experimental periodontal disease

The CD28 costimulation at TCR signaling plays a pivotal role in the regulation of the T cell response. To elucidate the role of T cells in periodontal disease, a system of cell transfer with TCR/CD28-dependent Th1 or Th2 clones was developed in rats. Gingival injection of specific Ag, Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein, and LPS could induce local bone resorption 10 days after the transfer of Ag-specific Th1 clone cells, but not after transfer of Th2 clone cells. Interestingly, the presence of LPS was required not only for the induction of bone resorption but also for Ag-specific IgG2a production. LPS injection elicited the induction of expression of both B7-1 and B7-2 expression on gingival macrophages, which otherwise expressed only MHC class II when animals were injected with Ag alone. The expression of B7 molecules was observed for up to 3 days, which corresponded to the duration of retention of T clone cells in gingival tissues. Either local or systemic administration of CTLA4Ig, a functional antagonist of CD28 binding to B7, could abrogate the bone resorption induced by Th1 clone cells combined with gingival challenge with both Ag and LPS. These results suggest that local Ag-specific activation of Th1-type T cells by B7 costimulation appeared to trigger inflammatory bone resorption, whereas inhibition of B7 expression by CTLA4Ig might be a therapeutic approach for intervention with inflammatory bone resorption.     

Th1 and Th2 cytokine immunomodulation by gangliosides in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis, a classical model for multiple sclerosis, the cytokines provide the necessary signals to activate specific T cells for self-antigens. Gangliosides have multiple immunomodulatory activities, decreasing the lymphoproliferative responses and modulating cytokine production. Here, we tested the effects of gangliosides on the switching of Th1 to Th2 cytokine expression, in spleen cells obtained from Lewis rats during the acute phase of EAE, and after recovery from the disease. For this purpose, total RNA from spleen cells was isolated and submitted to RT-PCR to investigate Th1 (IL-2, TNF-alpha, and IFN-gamma) and Th2/Th3 (IL-10 and TGF-beta) cytokine gene expression. Results demonstrate that the group treated with gangliosides displays mild disease, with low expression of IFN-gamma mRNA and high TGF-beta mRNA expression. We conclude that the gangliosides may modulate Th1 cells by the synthesis of cytokines shifting the profile to the Th2/Th3 phenotype.     

Viral interference with B7-1 costimulation: a new role for murine cytomegalovirus fc receptor-1

Murine CMV (MCMV), a beta-herpesvirus, infects dendritic cells (DC) and impairs their function. The underlying events are poorly described. In this study, we identify MCMV m138 as the viral gene responsible for promoting the rapid disappearance of the costimulatory molecule B7-1 (CD80) from the cell surface of DC. This was unexpected, as m138 was previously identified as fcr-1, a putative virus-encoded FcR. m138 impaired the ability of DC to activate CD8+ T cells. Biochemical analysis and immunocytochemistry showed that m138 targets B7-1 in the secretory pathway and reroutes it to lysosomal associated membrane glycoprotein-1+ compartments. These results show a novel function for m138 in MCMV infection and identify the first viral protein to target B7-1.     

T cell dynamics during induction of tolerance and suppression of experimental allergic encephalomyelitis

The cell dynamics associated with induction of peripheral T cell tolerance remain largely undefined. In this study, an in vivo model was adapted to two-photon microscopy imaging, and T cell behavior was analyzed on tolerogen-induced modulation. FcγR-deficient (FcγR(-/-)) mice were unable to resist or alleviate experimental allergic encephalomyelitis when treated with Ig-myelin oligodendrocyte glycoprotein (MOG) tolerogen, an Ig carrying the MOG35-55 peptide. However, when FcγR(+/+) dendritic cells (DCs) are adoptively transferred into FcγR(-/-) mice, uptake and presentation of Ig-MOG occurs and the animals were able to overcome experimental allergic encephalomyelitis. We then fluorescently labeled FcγR(+/+) DCs and 2D2 MOG-specific TCR-transgenic T cells, transferred them into FcγR(-/-) mice, administered Ig-MOG, and analyzed both T cell-DC contact events and T cell motility. The results indicate that tolerance takes place in lymphoid organs, and surprisingly, the T cells do not become anergic but instead have a Th2 phenotype. The tolerant Th2 cells displayed reduced motility after tolerogen exposure similar to Th1 cells after immunization. However, the Th2 cells had higher migration speeds and took longer to exhibit changes in motility. Therefore, both Th1 immunity and Th2 tolerance alter T cell migration on Ag recognition, but the kinetics of this effect differ among the subsets.     

In trans T cell tolerance diminishes autoantibody responses and exacerbates experimental allergic encephalomyelitis

A number of Ag-specific approaches have been developed that ameliorate experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis. Translation to humans, however, remains a consideration, justifying the search for more insight into the mechanism underlying restoration of self-tolerance. Ig-proteolipid protein (PLP) 1 and Ig-myelin oligodendrocyte glycoprotein (MOG) are Ig chimeras carrying the encephalitogenic PLP 139-151 and MOG 35-55 amino acid sequence, respectively. Ig-PLP1 ameliorates EAE in SJL/J (H-2(s)) mice while Ig-MOG modulates the disease in C57BL/6 (H-2(b)) animals. In this study, we asked whether the chimeras would suppress EAE in F(1) mice expressing both parental MHC alleles and representing a polymorphism with more relevance to human circumstances. The results show that Ig-MOG modulates both PLP1 and MOG peptide-induced EAE in the F(1) mice, whereas Ig-PLP1 counters PLP1 EAE but exacerbates MOG-induced disease. This in trans aggravation of MOG EAE by Ig-PLP1 operates through induction of PLP1-specific T cells producing IL-5 that sustained inhibition of MOG-specific Abs leading to exacerbation of EAE. Thus, in trans T cell tolerance, which should be operative in polymorphic systems, can aggravate rather than ameliorate autoimmunity. This phenomenon possibly takes place through interference with protective humoral immunity.     

Correlation of cytotoxicity with experimental allergic encephalomyelitis.

Cytotoxicity of immune lymph node cells in experimental allergic encephalomyelitis (EAE) was maximal 9 days after injection of encephalitogenic emulsion. The ability of these cells to passively transfer EAE was also maximal at this time. Immune spleen cells were more cytotoxic than lymph node cells 9 days after injection; however, these cells did not passively transfer EAE. Twelve days after injection of encephalitogenic emulsion immune spleen cells passively transferred EAE with resulting mild histopathologic lesions. At this time the spleen cells were 50% more cytotoxic than comparable lymph node cells. Cyclophosphamide suppressed the development of clinical EAE and the development of cytotoxic lymphoid cells. It also reduced clinical signs and cytotoxic activity of lymph node cells. Spleen cell cytotoxic activity was enhanced by Cyclophosphamide. It was concluded that cytotoxic activity of lymph node and spleen cells was correlated with the ability of these cells to produce EAE. Lymph node cell populations differed qualitatively and/or quantitatively from immune spleen cell populations in EAE. Capacity to passively transfer EAE coincided with the maximal Cytotoxicity of the lymphoid cells from each tissue.     

Successful treatment of experimental allergic encephalomyelitis with transforming growth factor-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional cytokine with immunosuppressive effects on T cells in vitro. Experimental allergic encephalomyelitis is an archetypal T cell-mediated autoimmune demyelinating disease of the central nervous system that often serves as a model for multiple sclerosis. In vivo administration of TGF-beta 1 into SJL mice was successful in reducing the incidence of clinical disease and the histologic severity of inflammation and demyelination in the brain and spinal cord. Immunohistochemical studies performed on control animals showed that TGF-beta-1, -2, and -3 were present in inflammatory perivascular lesions in the brain. The use of a naturally occurring cytokine with immunoregulatory functions in the treatment of an autoimmune disease is novel. However, potential long term complications of such therapy must be addressed before its use in human autoimmune disease such as multiple sclerosis.     

IL-10-dependent suppression of experimental allergic encephalomyelitis by Th2-differentiated, anti-TCR redirected T lymphocytes

We previously showed that transgenically expressed chimeric Ag-MHC-zeta receptors can Ag-specifically redirect T cells against other T cells. When the receptor's extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic zeta-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89-101-IAs-zeta receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8+ RMTC were therapeutically effective whereas CD4+ RMTC were not. We re-examine here the therapeutic potential of CD4+ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4+ MBP89-101-IAs-zeta RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.     

Functional and pathogenic differences of Th1 and Th17 cells in experimental autoimmune encephalomyelitis

Background

There is consensus that experimental autoimmune encephalomyelitis (EAE) can be mediated by myelin specific T cells of Th1 as well as of Th17 phenotype, but the contribution of either subset to the pathogenic process has remained controversial. In this report, we compare functional differences and pathogenic potential of “monoclonal” T cell lines that recognize myelin oligodendrocyte glycoprotein (MOG) with the same transgenic TCR but are distinguished by an IFN-γ producing Th1-like and IL-17 producing Th17-like cytokine signature.

Methods and Findings

CD4⁺ T cell lines were derived from the transgenic mouse strain 2D2, which expresses a TCR recognizing MOG peptide 35–55 in the context of I-A^b. Adoptive transfer of Th1 cells into lymphopenic (Rag2^−/−) recipients, predominantly induced “classic” paralytic EAE, whereas Th17 cells mediated “atypical” ataxic EAE in approximately 50% of the recipient animals. Combination of Th1 and Th17 cells potentiated the encephalitogenicity inducing classical EAE exclusively. Th1 and Th17 mediated EAE lesions differed in their composition but not in their localization within the CNS. While Th1 lesions contained IFN-γ, but no IL-17 producing T cells, the T cells in Th17 lesions showed plasticity, substantially converting to IFN-γ producing Th1-like cells. Th1 and Th17 cells differed drastically by their lytic potential. Th1 but not Th17 cells lysed autoantigen presenting astrocytes and fibroblasts in vitro in a contact-dependent manner. In contrast, Th17 cells acquired cytotoxic potential only after antigenic stimulation and conversion to IFN-γ producing Th1 phenotype.

Conclusions

Our data demonstrate that both Th1 and Th17 lineages possess the ability to induce CNS autoimmunity but can function with complementary as well as differential pathogenic mechanisms. We propose that Th17-like cells producing IL-17 are required for the generation of atypical EAE whereas IFN-γ producing Th1 cells induce classical EAE.     

Inhibition of Th2-mediated allergic airway inflammatory disease by CD137 costimulation

The engagement of CD137 (4-1BB), an inducible T cell costimulatory receptor and member of the TNF receptor superfamily, by agonistic Abs can promote strong tumor and viral immunity mediated by CD8(+) T cells and stimulate IFN-gamma production. However, its role in Th2-mediated immune responses has not been well defined. To address this issue, we studied the function of CD137 engagement using an allergic airway disease model in which the mice were sensitized with inactivated Schistosoma mansoni eggs followed by S. mansoni egg Ag challenge directly in the airways and Th1/2 cytokine production was monitored. Interestingly, treatment of C57BL/6 mice with agonistic anti-CD137 (2A) during sensitization completely prevents allergic airway inflammation, as shown by a clear inhibition of T cell and eosinophil infiltration into the lung tissue and airways, accompanied by diminished Th2 cytokine production and reduced serum IgE levels, as well as a reduction of airway hyperresponsiveness. At various time points after immunization, restimulated splenocytes from 2A-treated mice displayed reduced proliferation and Th2 cytokine production. In accordance with this, agonistic Ab to CD137 can directly coinhibit Th2 responses in vitro although it costimulates Th1 responses. CD137-mediated suppression of Th2 response is independent of IFN-gamma and T regulatory cells. Our study has identified a novel pathway to inhibit Th2 responses in a CD137-dependent fashion.     

Prevention of experimental allergic encephalomyelitis by nonencephalitogenic basic peptides

The biological properties of the chymotrypsin-treated encephalitogenic basic protein are described. The basic protein, isolated from bovine spinal cord, was digested with chymotrypsin and filtered through Sephadex gel resulting in three distinct well-separated peaks starting at the void volume of the column. Tubes common to each peak were combined into Fractions I, II, and III, respectively. More than 90% of the original protein was recovered in the three fractions. Fraction II, representing 76% of the original protein, was nonencephalitogenic when tested in guinea pigs at 0.010-, 0.025-, and 0.500-mg doses emulsified in complete Freund's adjuvant. Guinea pigs immunized with Fraction II were protected from EAE when challenged with encephalitogenic emulsions. A critical dose of 1.0 mg completely protected the animals from disease, while partial protection was obtained with lower doses. In addition to producing circulating antibodies, animals sensitized with Fraction II, the encephalitogenic tryptophan peptide or the basic protein displayed a delayed-type hypersensitivity response when skin tested with either of the three antigens. The positive skin reactivity in animals sensitized with Fraction II was not followed by EAE during 5 months of observation. In contrast, animals sensitized with extracts from bovine tissues other than the central nervous system were not protected from disease when challenged with encephalitogenic emulsions.The main finding here reported is the prevention of EAE with nonencephalitogenic peptides derived from the parent EAE-producing protein. The peptides retain the ability to induce delayed-type hypersensitivity and provide antigens to study the role of delayed hypersensitivity in experimental allergic encephalomyelitis.     

Apoptosis of neurons in rats with experimental allergic encephalomyelitis

NPP2, also known as phosphodiesterase-I alpha/autotaxin, is a type-II membrane protein that belongs to the nucleotide pyrophosphatase/phosphodiesterase family (NPP). We have recently demonstrated that NPP2 is expressed and released by differentiating oligodendrocytes during the critical stages of CNS myelination. The structural domains of this secreted macromolecule suggest a functional role in the regulation of oligodendrocyte adhesion. Here, we present data that demonstrates that NPP2 interferes with the ability of oligodendroglial cells to adhere to known CNS adhesion molecules present during the onset of myelination, such as fibronection, vitronectin, and merosin (laminin2). Responses to NPP2 appear to be regulated by a different mechanism depending on the developmental stage of the oligodendrocyte. Although the exact mechanisms for NPP2 mediated counter-adhesion are unknown, our studies have implicated that an active signalling mechanism involving heterotrimeric G proteins is responsible for adhesion modulation. These studies clearly define a role of NPP2 as a matricellular protein modulating oligodendrocyte adhesion and suggest that NPP2 function may represent the first step of oligodendrocyte remodelling when differentiating oligodendrocytes are actively involved in the formation of the myelin sheath.     

Haplotype-specific suppression of experimental allergic encephalomyelitis with anti-IA antibodies

Treatment with monoclonal antibodies directed against the IA antigens of the MHC is known to alter the course and prevent a number of experimental autoimmune diseases. To determine whether the treatment in vivo with anti-IA antibodies is haplotype-specific, we studied the development of EAE in F1 (SJL/J X BALB/c) mice following anti-IA antibody therapy. We report that treatment of animals with monoclonal antibody directed against the high responder allele product, I-As, was successful in preventing disease when therapy was begun either at the time of immunization with antigen, or following passive transfer of MBP-sensitized T cells. Therapy with antibody directed to the low responder allele product (I-Ad), while effective when used at the time of immunization with antigen, was ineffective following passive transfer of MBP-sensitized lymphocytes.     

Blockade of costimulation through B7/CD28 inhibits experimental autoimmune uveoretinitis, but does not induce long-term tolerance

It has been reported that costimulation blockade can result in T cell anergy. We investigated the effects of blocking costimulatory molecules in vivo on the development of experimental autoimmune uveoretinitis (EAU), a model for autoimmune uveitis in humans that is induced in mice by immunization with the retinal Ag interphotoreceptor retinoid binding protein. B10.A mice immunized with a uveitogenic regimen of interphotoreceptor retinoid-binding protein were treated with Abs to B7.1 and B7.2 for 2 wk. Evaluation of EAU and immunological responses 1 wk later showed that disease had been abrogated, and cellular responses were suppressed. To determine whether the costimulation blockade resulted in tolerance, adult-thymectomized mice immunized for uveitis and treated with anti-B7 or anti-CD28 were rechallenged for uveitis induction 5 wk after the initial immunization. Although confirmed to be disease free after the initial immunization, both anti-B7- and anti-CD28-treated mice developed severe EAU and elevated cellular responses after the rechallenge, equivalent to those of control mice. We conclude that in this model costimulatory blockade in vivo prevents the development of autoimmune disease, but does not result in long-term tolerance. The data are compatible with the interpretation that B7/CD28 blockade prevents generation of effector, but not of memory, T cells.     

Kinetics and specificity of T and B cell responses in relapsing experimental allergic encephalomyelitis

T and B cell responses to myelin basic protein (MBP) and its relevant peptide fragments were examined throughout the course of MBP-induced relapsing experimental allergic encephalomyelitis (REAE) in (SJL X PL)F1 mice. T cell reactivity, measured by the antigen-driven proliferation of lymph node T cells in vitro, was directed predominantly against the encephalitogenic MBP-P2 peptide (amino acids 1 to 37) at all stages of disease. Levels of responsiveness did not correlate with disease expression, but declined over time to a relapse level that was four- to sixfold lower than that observed during peak acute stage reactivity. Relapse responses were further distinguished by the detection of host I-E restrictions on Lyt-1+ T cell recognition of P2, P2 recognition by acute-stage T cells occurring solely in the context of host I-A molecules. These data imply an increase in the heterogeneity of relapse T cell responses to MBP to include clones restricted by additional class II glycoproteins. A role for additional CNS autoantigens in the stimulation of relapse T cells is also considered. Serum antibody responses to MBP or the P2 fragment fluctuated randomly throughout R-EAE when total antibody activity (IgM plus IgG) was measured. However, analysis of individual isotypes of IgG immunoglobulins revealed an apparent correlation between peak antigen-binding activity and disease expression which may reflect either an effector or regulatory role for humoral immunity in recurrent EAE. Patterns of early antibody reactivity also distinguished F1 mice that developed or failed to develop disease signs after immunization, the latter exhibiting a consistent drop in antigen-binding activity 4 to 5 days before the usual onset of acute-stage paralysis. The results are considered with regard to possible mechanisms of chronic disease regulation in an environment of functional T cell suppression.     

Prevention and treatment of chronic relapsing experimental allergic encephalomyelitis by transforming growth factor-beta 1.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system. The effect of the immunosuppressive molecule transforming growth factor-beta, (TGF-beta 1) on chronic relapsing EAE produced by the transfer of myelin basic protein-specific T cell lines was studied. TGF-beta 1 markedly inhibited the activation and proliferation of myelin-basic protein-specific lymph node cells in vitro. This reduced the capacity of these cells to transfer EAE. In addition, administration of TGF-beta 1 in vivo consistently resulted in an improved clinical course, even when given during ongoing disease. Immunopathologic study demonstrated a marked reduction in central nervous system damage and expression of cell-surface lymphocyte function-associated Ag-1 and class II MHC molecules in TGF-beta 1-treated mice. These findings have identified TGF-beta 1 as a possible therapeutic agent for the human demyelinating disease multiple sclerosis.