Functional redundancy of MyD88-dependent signaling pathways in a murine model of histidyl-transfer RNA synthetase-induced myositis.

We have previously shown that i.m. administration of bacterially expressed murine histidyl-tRNA synthetase (HRS) triggers florid muscle inflammation (relative to appropriate control proteins) in various congenic strains of mice. Because severe disease develops even in the absence of adaptive immune responses to HRS, we sought to identify innate immune signaling components contributing to our model of HRS-induced myositis. In vitro stimulation assays demonstrated HRS-mediated activation of HEK293 cells transfected with either TLR2 or TLR4, revealing an excitatory capacity exceeding that of other bacterially expressed fusion proteins. Corresponding to this apparent functional redundancy of TLR signaling pathways, HRS immunization of B6.TLR2(-/-) and B6.TLR4(-/-) single-knockout mice yielded significant lymphocytic infiltration of muscle tissue comparable to that produced in C57BL/6 wild-type mice. In contrast, concomitant elimination of TLR2 and TLR4 signaling in B6.TLR2(-/-).TLR4(-/-) double-knockout mice markedly reduced the severity of HRS-induced muscle inflammation. Complementary subfragment analysis demonstrated that aa 60-90 of HRS were absolutely required for in vitro as well as in vivo signaling via these MyD88-dependent TLR pathways--effects mediated, in part, through preferential binding of exogenous ligands capable of activating specific TLRs. Collectively, these experiments indicate that multiple MyD88-dependent signaling cascades contribute to this model of HRS-induced myositis, underscoring the antigenic versatility of HRS and confirming the importance of innate immunity in this system.

Suppression of Th1 and Th17, but not Th2, responses in a CD8(+) T cell-mediated model of oral tolerance.

The role of CD8(+) T cells in oral tolerance remains unclear. To address this, we developed a model to induce CD8(+) Tregs by feeding the major histocompatibility complex class I immunodominant epitope of OVA, OVA((257-264)). OVA((257-264)) feeding induced tolerance similar to that observed in OVA protein-fed mice, capable of suppressing the production of Th1 and Th17 cytokines and inhibiting a Th1-driven delayed-type hypersensitivity response following immunization with whole OVA (wOVA) protein. OVA((257-264)) peptide-induced suppression could be transferred to naive mice with CD8(+) cells, but not CD8-depleted cells, isolated from mesenteric lymph nodes of peptide-fed mice. Interestingly, while capable of inhibiting Th1 and Th17 responses, OVA((257-264)) feeding could not suppress any feature of a Th2 inflammatory response, though OVA protein feeding could, suggesting that these cells function through a different mechanism than their CD4(+) counterparts generated in response to feeding with wOVA. Thus, CD8(+) T cells are functionally capable of mediating tolerance to Th1 and Th17 responses.

Delineation of the minimal encephalitogenic epitope of proteolipid protein peptide(91-110) and critical residues required for induction of EAE in HLA-DR3 transgenic mice.

Previously, we have reported that proteolipid protein (PLP) peptide 91-110 can induce experimental autoimmune encephalomyelitis (EAE) in HLA-DR3 transgenic (tg) mice. Here we, report that residues spanning 97-108 are the minimal epitope required for induction of EAE in DR3 mice. Utilizing a series of alanine-substituted peptides, positions 99, 101, 102, 103, 104, and 106 are identified as residues necessary for an immune response. Further analysis indicated that amino acid isoleucine (99), aspartate (102) and lysine (104) are anchor residues facilitating binding to HLA-DR3 molecules. These results may have applications in the future design of peptide based immunotherapy.

Protection against experimental autoimmune encephalomyelitis generated by a recombinant adenovirus vector expressing the V beta 8.2 TCR is disrupted by coadministration with vectors expressing either IL-4 or -10.

Adenovirus vectors are increasingly being used for genetic vaccination and may prove highly suitable for intervention in different pathological conditions due to their capacity to generate high level, transient gene expression. In this study, we report the use of a recombinant adenovirus vector to induce regulatory responses for the prevention of autoimmune diseases through transient expression of a TCR beta-chain. Immunization of B10.PL mice with a recombinant adenovirus expressing the TCR Vbeta8.2 chain (Ad5E1 mVbeta8.2), resulted in induction of regulatory type 1 CD4 T cells, directed against the framework region 3 determinant within the B5 peptide (aa 76-101) of the Vbeta8.2 chain. This determinant is readily processed and displayed in an I-A(u) context, on ambient APC. Transient genetic delivery of the TCR Vbeta8.2 chain protected mice from Ag-induced experimental autoimmune encephalomyelitis. However, when the Ad5E1 mVbeta8.2 vector was coadministered with either an IL-4- or IL-10-expressing vector, regulation was disrupted and disease was exacerbated. These results highlight the importance of the Th1-like cytokine requirement necessary for the generation and activity of effective regulatory T cells in this model of experimental autoimmune encephalomyelitis.

Immunogenic and encephalitogenic epitope clusters of myelin proteolipid protein.

To understand and develop strategies to intervene in autoimmune responses to myelin proteolipid protein (PLP), encephalitogenic epitopes must be identified. To expedite the identification of potentially immunogenic and encephalitogenic epitopes of PLP, overlapping synthetic 20-mer PLP peptides covering the whole PLP molecule were screened for their ability to bind to purified mouse I-Ad, I-Ak, and I-As molecules. The peptides that bound to the I-A molecules were tested for their ability to induce immune responses in corresponding inbred mouse strains. Immunogenic peptides were then tested for their ability to induce experimental autoimmune encephalomyelitis. Moderate to strong I-A binding was essential for development of immune responses, but immunogenicity was not sufficient for encephalitogenicity. Rather, encephalitogenic epitopes clustered in three regions of the molecule, namely within residues 40-70, 100-119, and 178-209. These were also the regions of the PLP that showed the greatest promiscuity in binding to I-A molecules. Except for PLP 139-151, which is an encephalitogenic determinant in mice expressing I-As, all encephalitogenic epitopes of PLP previously identified, regardless of their MHC class II restriction, are located within or adjacent to these epitope clusters. None of the encephalitogenic epitopes occur in regions of the molecule that have a high degree of homology with the neuronal M6a protein, a member of the DM20/PLP superfamily. Atypical clinical and histologic patterns of experimental autoimmune encephalomyelitis were observed in some strains of mice sensitized with certain PLP peptides and may reflect induction of T cells with different disease-inducing potentials.