IL-2/Neuroantigen Fusion Proteins as Antigen-Specific Tolerogens in Experimental Autoimmune Encephalomyelitis (EAE): Correlation of T Cell-Mediated Antigen Presentation and Tolerance Induction

Abstract

The purpose of this study was to assess whether the Ag-targeting activity of cytokine/neuroantigen (NAg) fusion proteins may be associated with mechanisms of tolerance induction. To assess this question, we expressed fusion proteins comprised of a N-terminal cytokine domain and a C-terminal NAg domain. The cytokine domain comprised either rat IL-2 or IL-4, and the NAg domain comprised the dominant encephalitogenic determinant of the guinea pig myelin basic protein. Subcutaneous administration of IL2NAg (IL-2/NAg fusion protein) into Lewis rats either before or after an encephalitogenic challenge resulted in an attenuated course of experimental autoimmune encephalomyelitis. In contrast, parallel treatment of rats with IL4NAg (IL-4/NAg fusion protein) or NAg lacked tolerogenic activity. In the presence of IL-2R+ MHC class II+ T cells, IL2NAg fusion proteins were at least 1,000 times more potent as an Ag than NAg alone. The tolerogenic activity of IL2NAg in vivo and the enhanced potency in vitro were both dependent upon covalent linkage of IL-2 and NAg. IL4NAg also exhibited enhanced antigenic potency. IL4NAg was ∼100-fold more active than NAg alone in the presence of splenic APC. The enhanced potency of IL4NAg also required covalent linkage of cytokine and NAg and was blocked by soluble IL-4 or by a mAb specific for IL-4. Other control cytokine/NAg fusion proteins did not exhibit a similar enhancement of Ag potency compared with NAg alone. Thus, the IL2NAg and IL4NAg fusion proteins targeted NAg for enhanced presentation by particular subsets of APC. The activities of IL2NAg revealed a potential relationship between NAg targeting to activated T cells, T cell-mediated Ag presentation, and tolerance induction.

Experimental Autoimmune Encephalomyelitis in Lewis rats: IFN-β Acts As a Tolerogenic Adjuvant for Induction of Neuroantigen-Dependent Tolerance

“…2D) or the synthetic gp69 -88 peptide (not shown). These data provide evidence that the cytokine domain may facilitate presentation of the NAg by professional APC, as was described for the IL4-NAg, IL2-NAg, and NAg-IL16 fusion proteins (25)(26)(27). Although the antigenic potency of IFN␤-NAg appeared greater than GPMBP as measured by the concentration eliciting a half-maximal response, the peak proliferative response stimulated by IFN␤-NAg was a Rats were pretreated with saline (first row), with 1 nmol of NAg (synthetic peptide gp69 -88) in saline (second row), or with 1 nmol of IFN␤-NAg in saline (third row) on days Ϫ21, Ϫ14, and Ϫ7 and were challenged with 50 g of DHFR-NAg in CFA on day 0.…”

“…Previous studies of IL2-NAg and NAg-IL16 fusion proteins showed that covalent linkage of the cytokine and NAg domains was necessary for inhibition of EAE (25,26). That is, administration of a fused cytokine-NAg protein was tolerogenic, whereas administration of cytokine and NAg as separate molecules to the same inoculation site was without activity.…”

“…Both IFN␤-NAg fusion proteins were active at titrations of 10 Ϫ4 , whereas baculovirus supernatants containing other cytokine-NAg fusion proteins, including IL1RA-NAg, IL2-NAg, IL4-NAg, IL10-NAg, IL13-NAg, and NAg-IL16, as well as mock baculovirus supernatants, had no activity. These control cytokine-NAg fusion proteins were active in bioassays specific for the respective cytokine domains (26,27). IFN␤-NAg caused cell death independently of the NAg domain, because BN-GP T cells did not recognize the 73-87 peptide sequence of MBP (not shown).…”

“…Several Ag-specific therapies are being developed, including those based on altered peptide ligands, DNA vaccines, and mucosal Ag delivery. Cytokine-Ag fusion proteins were originally developed for vaccination against cancer and infectious agents but have also been explored as tolerogenic vaccines based on the use of inhibitory, antiinflammatory, or tolerogenic cytokines as the cytokine fusion partner (25,26). Two cytokine-Ag fusion proteins in which the IL-2 or IL-16 cytokines were fused to the encephalitogenic deterEAE.…”

“…EAE was induced in Lewis rats by injection of an emulsion containing 50 g of dihydrofolate reductase (DHFR)-NAg (25,26) in CFA (200 g of Mycobacterium tuberculosis) in a total volume of 0.1 ml. DHFR-NAg was comprised of the mouse DHFR as the N-terminal domain and the encephalitogenic gp69 -87 peptide of GPMBP as the C-terminal domain.…”