This is the competing renewal of an RO1 grant examining the mucosal immune response that is responsible for the induction and regulation of IgA B-cell responses. The work focuses on the murine oral/nasal cavity (e.g., submandibular gland, SMG). Previous work has shown that Th2 cell derived IL-5 and IL-6 are of particular importance for inducing sIgA positive B cells to differentiate into IgA producing plasma cells, and high levels of mRNA for these cytokines as well as IFN-gamma were reported by the investigator from SMG alpha beta (ab) TCR CD4 positive T cells. The current application proposes to examine the role of gamma delta (gd) T cells in mucosal immune responses. This direction is based on the fact that gd T cells are known to be localized to mucosal sites where they constitute approximately 50 percent of IEL in the intestine, and the investigator has found that approximately 20 percent of the T cells in the SMG are gd T cells. The investigator has shown by ELISPOT assay that the dominant Ig producing cells in SMG were IgA indicating that SMG are an IgA effector tissue, like the intestinal LP. The SMG gd T cells included IL-4 and IL-5 producing cells but not IL-4 producers. Moreover, the investigator has found in very recently completed work that gd deficient (knock out) mice have impaired mucosal IgA responses. Besides the clear role for the mucosal immune system in the production of sIgA, mucosally induced tolerance is an important phenomenon in which orally administered antigens may produce mucosal IgA responses but systemic hyporesponsiveness. The investigator has shown that mucosal gd T cells from the intestinal epithelium of orally tolerized mice can abrogate antigen-specific unresponsiveness upon adoptive transfer of gd T cells. Studies from others have shown that gd T cells can down regulate IgE responses after orally administered antigens. The investigator proposes that gd T cells may play an important regulatory role in mucosal IgA and E responses at mucosal sites through cytokine production and through interactions with ab T cells. In the first aim, SMG mononuclear cells will be examined by ELISPOT assay for IgA producing cells in gd deficient and wild type mice in general and following specific mucosal immunization with TT. Similar analyses in ab deficient mice will provide evidence for whether gd T cells can support IgA production directly, or rather suggest that they act primarily through the activation of ab T cells. Adoptive transfer experiments will confirm the need for ab T cells and support the idea that the regulatory role of gd T cells may be mediated upon ab T cells.
In Aim 2, the effects of gd T cells as regulators of CD4 positive ab T cells in producing IgA will be analyzed directly in double chamber or in co-culture experiments in vitro and complemented by T cell transfer experiments using TCR knockout mice in vivo.
Aim 3 addresses the expression and function by SMG gd T cells of cell surface molecules B71/2 that may mediate direct interactions with ab T cells that determine their cytokine profiles.
Aim 4 examines a model of intranasal immunization to produce mucosal IgA responses along with systemic (i.e., serum and spleen) hyporesponsiveness, that is, nasal tolerance. Then gd deficient versus wild type mice will be compared to further demonstrate the role of gd T cells in maintaining mucosal responses in the setting of systemic hyporesponsiveness, and nasal gd T cells from tolerized mice will be used to determine if they can reverse the hyporesponsiveness of spleen (systemic) ab T cells from similarly tolerized animals.
In Aim 5, the role of IL-7 in the development and proliferation of SMG gd T cells will be determined in IL-7 deficient or IL-7R deficient mice, since IL-7 has recently been shown to be a growth factor for a subset of gd T cells that express its receptor.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
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Immunological Sciences Study Section (IMS)
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University of Alabama Birmingham
Schools of Dentistry
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Dohi, Taeko; Fujihashi, Kohtaro; Koga, Toshiya et al. (2003) T helper type-2 cells induce ileal villus atrophy, goblet cell metaplasia, and wasting disease in T cell-deficient mice. Gastroenterology 124:672-82
Hagiwara, Yukari; McGhee, Jerry R; Fujihashi, Keiko et al. (2003) Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue. J Immunol 170:1754-62
Kato, Hirotomo; Fujihashi, Kohtaro; Kato, Rie et al. (2003) Lack of oral tolerance in aging is due to sequential loss of Peyer's patch cell interactions. Int Immunol 15:145-58
Boyaka, Prosper N; Ohmura, Mari; Fujihashi, Kohtaro et al. (2003) Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit. J Immunol 170:454-62
Kweon, Mi-Na; Yamamoto, Masafumi; Watanabe, Fumiko et al. (2002) A nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced IgE antibodies. J Infect Dis 186:1261-9
Fujihashi, Kohtaro; Koga, Toshiya; van Ginkel, Frederik W et al. (2002) A dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants. Vaccine 20:2431-8
Kato, H; Fujihashi, K; Kato, R et al. (2001) Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses. J Immunol 166:3114-21
Fujihashi, K; Dohi, T; Rennert, P D et al. (2001) Peyer's patches are required for oral tolerance to proteins. Proc Natl Acad Sci U S A 98:3310-5
Fujihashi, K; Kato, H; van Ginkel, F W et al. (2001) A revisit of mucosal IgA immunity and oral tolerance. Acta Odontol Scand 59:301-8
Dohi, T; Rennert, P D; Fujihashi, K et al. (2001) Elimination of colonic patches with lymphotoxin beta receptor-Ig prevents Th2 cell-type colitis. J Immunol 167:2781-90

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