C-C chemokine-encoding DNA vaccines enhance breakdown of tolerance to their gene products and treat ongoing adjuvant arthritis.

Depending on the method of immunization, a single administration of CFA may result in the development of a local inflammatory process or chronic polyadjuvant-induced arthritis (AA). We administered naked DNA vaccines encoding MIP-1 alpha, MCP-1, MIP-1 beta, and RANTES to Lewis rats and confirmed that each of these vaccines induced immunological memory to the corresponding gene product. Upon induction of disease, this memory effectively inhibited the development of the autoimmune condition. Self-specific Ab's developed in DNA-vaccinated animals were neutralizing in vitro and could adoptively transfer the beneficial effect of each vaccine. Repeated administration of the constructs encoding MCP-1, MIP-1 alpha, or RANTES inhibited the development and progression of AA, even when each vaccine was administered only after the onset of disease. This suggests a highly effective way by which the immune system could be re-educated to generate protective immunity against its own harmful activities.

Expansion of neonatal tolerance to self in adult life: I. The role of a bacterial adjuvant in tolerance spread.

T cell neonatal tolerance to self evolves perturbation of the Th1/Th2 balance towards Th2-type self-specific T cells. In the current study we have demonstrated that a tolerant state could be extended to another encephalitogenic determinant only if the neonatally tolerizing determinant was co-administered in adult life with an emulsion of Mycobacterium tuberculosis (i.e. complete Freund's adjuvant). The mechanisms underlying tolerance elicitation and expansion were then explored by an in vitro system in which indirect suppression could be measured. Addition of a tolerizing epitope to splenic T cells from neonatally tolerized animals induced a marked suppression of the anti-MT response. This response could be restored by neutralizing antibodies to IL-4. In contrast, the neutralizing antibodies to IL-4 had no affect on the response of these cells to the tolerizing determinant. These findings are highly significant not only because they explore the important role of microbial antigens in neonatal tolerance, but also because they distinguish, for the first time, between tolerizing and tolerized T cells.

Expansion of neonatal tolerance to self in adult life: II. Tolerance preferentially spreads in an intramolecular manner.

Newborn rats exposed to a myelin basic protein determinant acquired long-lasting resistance to experimental autoimmune encephalomyelitis induced by another determinant only if both determinants are co-administered in adult life. We demonstrate here that during the course of disease both the anti-self response and the tolerant state spread in an intramolecular and not an intermolecular manner. Mechanisms involved in tolerance elicitation and expansion are then explored using an in vitro system in which indirect suppression could be measured.

Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis.

Specific oligonucleotide primers were used to identify and isolate IFN-gamma-inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as a model for multiple sclerosis. IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein that was used to induce anti-IGIF neutralizing Abs. These Abs significantly reduced the production of IFN-gamma by primed T cells proliferating in response to their target myelin basic protein epitope and by Con A-activated T cells from naive donors. When administered to rats during the development of either active or transferred EAE, these Abs significantly blocked the development of disease. Splenic T cells from protected rats were cultured with the encephalitogenic myelin basic protein epitope and evaluated for production of IL-4 and IFN-gamma. These cells, which proliferated, exhibited a profound increase in IL-4 production that was accompanied by a significant decrease in IFN-gamma and TNF-alpha production. Thus, we suggest that perturbation of the Th1/Th2 balance toward Th2 cells is the mechanism underlying EAE blockade by anti-IGIF immunotherapy.

Long-lasting protective immunity to experimental autoimmune encephalomyelitis following vaccination with naked DNA encoding C-C chemokines.

DNA vaccination represents a novel means of expressing Ag in vivo for the generation of both humoral and cellular immune responses. The current study uses this technology to elicit protective immunity against experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as an experimental model for multiple sclerosis. RT-PCR verified by Southern blotting and sequencing of PCR products of four different C-C chemokines, macrophage-inflammatory protein-1alpha (MIP-1alpha), monocyte-chemotactic protein-1 (MCP-1), MIP-1beta, and RANTES, were performed on brain samples from EAE rats to evaluate mRNA transcription at different stages of disease. Each PCR product was then used as a construct for naked DNA vaccination. The subsequent in vivo immune response to MIP-1alpha or MCP-1 DNA vaccines prevented EAE, even if disease was induced 2 mo after administration of naked DNA vaccines. In contrast, administration of the MIP-1beta naked DNA significantly aggravated the disease. Generation of in vivo immune response to RANTES naked DNA had no notable effect on EAE. MIP-1alpha, MCP-1, and MIP-1beta mRNA transcription in EAE brains peaked at the onset of disease and declined during its remission, whereas RANTES transcription increased in EAE brains only following recovery. Immunization of CFA without the encephalitogenic epitope did not elicit the anti-C-C chemokine regulatory response in DNA-vaccinated rats. Thus, modulation of EAE with C-C chemokine DNA vaccines is dependent on targeting chemokines that are highly transcribed at the site of inflammation at the onset of disease.

Short peptide-based tolerogens without self-antigenic or pathogenic activity reverse autoimmune disease.

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in brain lesions of multiple sclerosis (MS), and this peptide can trigger experimental autoimmune encephalomyelitis (EAE). We designed truncated peptides based on this pathogenic 13-mer that are not antigenic. These short peptides reduced production of IFN-gamma and TNF-alpha in vivo. Moreover, paraplegic rats given the 7-mer FKNIVTP in soluble form showed total reversal of paralysis in 24 h. Truncated peptides that are too small to stimulate antigenic responses to pathogenic regions of myelin basic protein are nevertheless effective tolerogens and are able to anergize autoreactive T cells. Short peptide-based tolerogens, devoid of immunogenic and pathogenic potential, may be attractive for therapy of autoimmune diseases.