A, Vaginal cells were extracted from PBS or E2 treated mice and pulsed with 10μg/ml of pSIINFEKL (pOVA) peptide or medium alone at 37°C for 1.5 hours. Pulsed cells were analyzed in Reverse Elispot assays containing either sorted naïve (IL-2) or in vitro-activated effector (IFNγ) OT-I cells. Graph shows mean±SEM of the frequency of vaginal APC inducing IFNγ or IL-2 secretion. Graph representative of three experiments. B, Cells were extracted from vaginal tissues (VAG) or spleens (SP) of PBS or E2 treated mice, and analyzed in Reverse Elispot assays using naïve anti-H2^d 2C cells (IL-2) or in vitro activated effector anti-H2^d 2C cells (IFNγ). Graphs show mean±SEM of the frequency of APC. N=3 mice per group. Graph representative of three independent experiments. Non-significant groups had a p-value greater than 0.05.

A, PBS or E2 treated mice were immunized IVAG with pSIINFEKL (pOVA) peptide at 0μg, 2μg, or 20μg per mouse. After 12 hours vaginal cells were extracted and analyzed by flow cytometry using the anti-pSIINFEKL/H2K^b antibody 25-D1.16. Left panel: representative histograms showing 25-D1.16 positive cells gated on live CD11b⁺ H2D^b+ vaginal cells; mean fluorescence intensity (MFI) is shown in the upper right hand corner of each histogram. Right panel: Mean±SEM of MFI from 25-D1.16. * indicates p<0.05; N=3 mice per group. B, The cell populations described in A were analyzed in Reverse Elispot assays using in vitro activated effector OT-I cells. * indicates p<0.05; N=3 mice per group. Graph representative of two independent experiments.

A, B6 female splenocytes were pulsed with FITC-labeled or unlabeled pSIINFEKL peptide at 0μg, 2μg, 20μg or 200μg/ml in cell culture medium for 1.5hrs, stained with 25-D1.16 antibody, then analyzed by flow cytometry. B, PBS or E2 treated mice were immunized IVAG with pSIINFEKL-FITC peptide at 20μg per mouse or HBSS alone. Twelve hours after immunization vaginal tissues were fixed, embedded, and sectioned. Sequential sections were stained with the nuclear dye DAPI or with haematoxylin and eosin. Exposure times were 0.5 seconds for DAPI and 15 seconds for FITC. Representative image of an experiment repeated twice. C, The percent of the area staining positive for pSIINFEKL-FITC binding (mean±SEM) is shown in the graph, and an example of boundary assignments is shown on the left. N=3 mice per group; * and ** indicates p<0.05; Lumen/mucus (L/M); Epithelial layer (EpC); Lamina propria (LP).

A, Estradiol-treated mice were immunized intravaginally with 10μg of pOVA peptide and 0U, 1.6U, 40U, 200U, or 1000U of mucinase hyaluronidase. PBS-treated mice were immunized with pOVA but not mucinase. The mean for PBS-treated mice is shown as a solid horizontal line and the confidence interval, for this group, as flanking dashed lines. After 12 hours, APC in vaginal tissues were enumerated by Reverse Elispot using in vitro effector OT-I cells. Antigen loading onto vaginal APC were quantitated by the ability of the extracted APC to stimulate effector T cells to secrete IFNγ. The graph shows mean±SEM; N=3 mice per group. B, Estradiol-treated mice were immunized intravaginally with 0μg, 0.1μg, or 1μg of pOVA peptide with 200U of mucinase. PBS-treated mice were immunized in the absence of mucinase. Reverse Elispot enumerated APC in the vaginal tissue. The graph shows mean±SEM; N=3 per group except 0μg (N=2); * indicates p<0.05. C, CFSE-labeled OT-I cells were adoptively transferred into OVX B6 females, which were treated with PBS or E2 then immunized intravaginally with 10μg of pOVA with or without 200U of mucinase. Three days post-immunization, OT-I proliferation in para-aortic lymph nodes was analyzed by flow cytometry. The graph shows percent divided of gated CD8α⁺ CD45.1⁺ Vα2⁺ Vβ5⁺ cells as mean±SEM; N=3 mice per group; * indicates p<0.05. Figures A, B, and C were each representative of three independent experiments.

A, PBS or estradiol (E2) treated ovariectomized (OVX) mice were immunized intravaginally (IVAG) with B6 or B6.mOVA male reproductive organ homogenates. After 12 hours, vaginal cells were extracted and analyzed for APC activity in Reverse Elispot assays that measure APC stimulation via the activation of antigen specific effector (measured by IFNγ secretion) or naïve (measured by IL-2 secretion) T cells. The graph shows mean±SEM of the frequency of vaginal APC inducing IFNγ synthesis by effector OT-I T cells. * indicates p<0.05; N=3 mice per group, graph representative of experiment repeated in duplicate. B, PBS or E2 treated OVX mice were immunized IVAG with HBSS alone or pSIINFEKL peptide (pOVA). Twelve hours post-insemination vagina were removed and processed for Reverse Elispots. The graph shows mean±SEM of the frequency of vaginal APC inducing IFNγ secretion by OT-I cells. * indicates p<0.05; N=3 mice per group, graph representative of experiment repeated in triplicate. C and D, Normal B6 female mice were treated with PMSG to induce a high proportion of estrus, and mice in estrus were identified by labial swelling. The ‘mixed’ group contained normal mice from all estrous states. Mice were immunized IVAG with B6.mOVA male reproductive organ homogenates (C) or pSIYRYYGL peptide (D). After 12 hours, vaginal cells were extracted and APC enumerated as in parts A and B, using OT-I (C) or 2C (D) effector T cells. * indicates p<0.05, ** indicates p<0.01 by the unpaired student's t test; N≥13 mice per group.