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Low-Dose Tolerance Is Mediated by the Microfold Cell Ligand, Reovirus Protein 1¹

Agnieszka Rynda^*, Massimo Maddaloni^*, Dagmara Mierzejewska, Javier Ochoa-Repáraz^*, Tomasz Malanka^*, Kathryn Crist^*, Carol Riccardi^*, Beata Barszczewska^*, Kohtaro Fujihashi, Jerry R. McGhee and David W. Pascual^2,*

^* Veterinary Molecular Biology, Montana State University, Bozeman, MT 59718; Department of Food Chemistry, Institute of Food Research, Polish Academy of Science, Olsztyn, Poland; and Departments of Pediatric Dentistry and Microbiology, Immunobiology Vaccine Center, University of Alabama at Birmingham, Birmingham AL 35294

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein 1 (p1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via p1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus p1, termed OVA-p1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-p1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-p1 could be adoptively transferred using cervical lymph node CD4⁺ T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-β1, with greatly reduced IL-17 and IFN-. The induced IL-10 was derived predominantly from FoxP3⁺CD25⁺CD4⁺ T cells. No FoxP3⁺CD25⁺CD4⁺ T cells were induced in OVA-p1-dosed IL-10-deficient (IL-10^–/–) mice, and despite showing increased TGF-β1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using p1 as a mucosal delivery platform specifically for low-dose tolerance induction.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Public Health Service Grants AI-56286, DE-13812, DE-12242, AI-18958, and AG-25873, and in part, by the Montana Agricultural Station and U.S. Department of Agriculture Formula Funds. The Veterinary Molecular Biology flow cytometry facility was, in part, supported by National Institutes of Health/National Center for Research Resources, Centers of Biomedical Excellence P20 RR-020185, and an equipment grant from the M. J. Murdock Charitable Trust.

² Address correspondence and reprint requests to Dr. David W. Pascual, Veterinary Molecular Biology, Montana State University, P.O. Box 173610, Bozeman, MT 59717-3610. E-mail address: dpascual{at}montana.edu

³ Abbreviations used in this paper: T_reg, T regulatory; FoxP3, forkhead box P3; PP, Peyer’s patch; LN, lymph node; CLN, cervical LN; p1, protein 1; CT, cholera toxin; ODN, oligodeoxynucleotide; DTH, delayed-type hypersensitivity; MLN, mesenteric LN; HNLN, head and neck LN; NALT, nasopharyngeal-associated lymphoid tissue; M cell, microfold cell; Tg, transgenic; sPBS, sterile PBS; p.i., postimmunization.