The murine buccal mucosa is an inductive site for priming class I-restricted CD8+ effector T cells in vivo

Abstract

The present study shows that Langerhans cells of the buccal mucosa and the skin share a similar phenotype, including in situ expression of MHC class II, the mannose receptor DEC-205 and CD11c, and absence of the costimulatory molecules B7.1, B7.2 and CD40 as well as Fas. Application of 2,4-dinitrofluorobenzene (DNFB) onto the buccal mucosa is associated with a rapid migration of dendritic cells (DC) to the epithelium and induction of B7.2 expression on some DC. Buccal sensitization with DNFB elicited a specific contact sensitivity (CS) in response to skin challenge, mediated by class I-restricted CD8+ effector T cells and down-regulated by class II-restricted CD4+ T cells, demonstrated by the lack of priming of class I-deficient mice and the enhanced response of class II-deficient mice, respectively. CS induced by buccal immunization is associated with priming of class I-restricted CD8+ effector T cells endowed with hapten-specific cytotoxic activity. Thus, the buccal epithelium is an inductive site, equivalent to the epidermis, for the generation of CS independent of CD4 help, and of cytotoxic T lymphocyte (CTL) responses mediated by class I-restricted CD8+ T cells. We propose that immunization through the buccal mucosa, which allows antigen presentation by epithelial DC efficient for priming systemic class I-restricted CD8+ CTL, may be a valuable approach for single-dose mucosal vaccination with subunit vaccines.

Fig. 1

Immunohistochemical analysis of dendritic cells (DC) of the buccal and skin epithelium. Epithelial sheets from the buccal mucosa (a,b,c) and the skin (d,e,f) from BALB/c mice were stained with MoAbs specific for MHC class II (CD311) (a and d), CD11c (N418) (b and e) and the mannose receptor DEC-205 (NLDC-145) (c and f). Staining is exclusively observed on cells with dendritic morphology. (Final mag. × 400.)

Fig. 2

Up-regulation of B7.2 expression on dendritic cells (DC) after 2,4-dinitrofluorobenzene (DNFB) application onto the buccal mucosa. Cryostat sections (5 μm thick) from the buccal mucosa of C57Bl/6 mice either untreated (a,b), or 2 h (c,d) or 24 h (e,f) after buccal sensitization with DNFB were stained for MHC class II (CD311) (a,c,e) or B7.2 (CD86, clone GL1) (b,d,f) molecule expression. Sections were counterstained with Harris haematoxylin. (Final mag. × 400.)

Fig. 3

Dose–effect and kinetics of the contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) generated by buccal or epicutaneous sensitization. BALB/c mice were sensitized by topical application on the buccal mucosa (a) or on abdominal skin (b) of 0.5% (▵), 1% (○) or 2% (□) of DNFB diluted in acetone:olive oil. Five days later all mice were challenged on the right ear with 0.2% DNFB. The skin CS response was evaluated by the increment in ear thickness at various days after challenge. The results (mean ± s.d.) are representative of one experiment out of three using six mice per group. Ear swelling was < 10 μm in unsensitized mice that were ear-challenged with DNFB and in DNFB-sensitized mice that were ear-challenged with the irrelevant hapten oxazolone (not shown).

Fig. 4

Contact sensitivity (CS) induced by buccal immunization with 2,4-dinitrofluorobenzene (DNFB) is mediated by class I-restricted CD8⁺ effector T cells, independently of CD4⁺ T cells. (a) Heterozygote β2m⁺/° (•) or homozygote β2m°/° (○) mice and (b) heterozygote Aβ⁺/° (▪) or homozygote Aβ°/° (□) mice were buccally sensitized with 0.5% of DNFB and ear-challenged 5 days later with 0.2% DNFB. Ear swelling, expressed as mean ± s.d. of six mice per group, was determined at various time points after challenge. The data shown are representative of two to three experiments.

Fig. 5

Buccal immunization can prime hapten-specific CD8⁺ T cells in vivo. Unfractionated T cells, CD4⁺ or CD8⁺ T cells were purified from C57Bl/6 mice spleens on day 5 after buccal sensitization with 2,4-dinitrofluorobenzene (DNFB) and restimulated in vitro for 3 days in the presence of either unmodified, 2,4-dinitrobenzene sulfonate (DNBS)-modified or 2,4,6-trinitrobenzene sulfonate (TNBS)-modified syngeneic spleen cells. T cell proliferation was determined by ³H-thymidine uptake over the last 6 h of culture. The result represents mean ± s.d. of quadruplicate wells and is representative of two experiments.

Fig. 6

Buccal immunization induces specific class I-restricted CD8⁺ cytotoxic T lymphocytes (CTL). (a) Pooled spleen cells from either C57Bl/6 mice day 5 after buccal sensitization with 2,4-dinitrofluorobenzene (DNFB) were restimulated for 6 days with syngeneic 2,4-dinitrobenzene sulfonate (DNBS)-modified spleen cells. DNBS-specific CTL activity was determined by testing specific lysis of ⁵¹Cr EL-4 target cells that were either unmodified (•), DNBS-modified (▪) or 2,4,6-trinitrobenzene sulfonate (TNBS)-modified (▴). (b) Pooled spleen cells from MHC class I-deficient mice β2m°/° (□,○), or β2m⁺/° heterozygote control (▪,•) mice were restimulated for 6 days with syngeneic DNBS-modified spleen. DNBS-specific CTL activity was determined by testing lysis of unmodified (○,•) or DNBS-modified (□,▪) ⁵¹Cr EL-4 target cells.