Anti-CtB antibody levels in serum and on mucosal surfaces.

<p>(A-C) Anti-CtB IgA levels and (C-E) anti-CtB IgG levels in the serum (A and D), tears (B and E) and vaginal washes (C and F) of BALB/c mice immunized via the conjunctiva and age-matched controls (nc). All samples were collected two weeks after the completion of the indicated immunization protocol and were assayed by ELISA. Results for each individual sample are presented. The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (<i>P</i> < 0.05*, <i>P</i><0.005**). Compared groups are indicated by two-head arrow.</p

Anti-PmpC antibody serum levels.

<p>Levels of (A) anti-N-PmpC IgA and (B) anti-N-PmpC IgG and (C) ratio of the levels of N-PmpC-specific IgG1 and IgG2a antibodies in the serum of BALB/c mice immunized via the conjunctiva and age-matched normal controls (nc). All serum samples were collected two weeks after completion of the indicated immunization protocol and were assayed by ELISA. Results for each individual serum sample are presented. The levels of N-PmpC-specific IgG1 and IgG2a were judged according to the A_492/620 values recorded for individual serum samples. The statistical significance of the observed differences was evaluated using Kruskal -Wallis test followed by Dunn's multiple comparisons test to compare between groups (<i>P</i> < 0.05*, <i>P</i><0.005**). Compared groups are indicated by two-head arrow.</p

Phenotyping of SMLN T lymphocytes by FACS analysis.

<p>Bar graph showing the contribution (expressed in %) of specific T cell populations to the overall T cell pool within SMLNs isolated from BALB/c mice two weeks after the completion of the indicated immunization protocol. The mean percentages of (A) CD4+ and CD8+, (B) CD25+, and (C) CD4-CD25+ and CD4+CD25+ cells within T lymphocytes pool are presented. Lymphocytes were gated according to their position within the forward scatter (FSC) vs side scatter (SSC) and T cells were marked according to the expression of CD3+ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157875#pone.0157875.s001" target="_blank">S1 Fig</a>). Assessment of Foxp3 and CD25 co-expression was performed for CD4+ lymphocytes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157875#pone.0157875.s002" target="_blank">S2 Fig</a>) and mean percentages of Foxp3+CD25+ within CD4+ lymphocytes (D) are presented. The mean percentage of corresponding T cell population recorded in SMLN isolated from age-matched control mice (nc) is presented on each graph as a solid line (mean value) and dotted lines (upper and lower standard error values). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc <i>P</i> < 0.05*, <i>P</i><0.005**; between immunized groups <i>P</i> < 0.05^#, <i>P</i><0.005^##).</p

Effect of N-PmpC and CtB stimulation on the <i>in vitro</i> cytokine pattern in SMLN cells isolated after ocular mucosal immunization with N-PmpC and N-PmpC/LB.

<p>Bar graphs representing the levels of IFNγ, IL-4, IL-10 and IL-17A in the supernatants of non-stimulated, N-PmpC- or CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva (bars). The corresponding measurements in SMLN cells isolated from age-matched controls (nc) are presented on the graphs as a solid line (mean value) and dotted lines (upper and lower standard error values). SMLN cells were cultured at 37°C under a 5% CO₂ atmosphere for 48 h in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10⁶ CFU/ml for CtB). The results are presented as the mean concentrations ± SE (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc stimulated in a same way <i>P</i> < 0.05*, <i>P</i><0.005**; vs non-stimulated samples of the same group <i>P</i> < 0.05^#, <i>P</i><0.005^##).</p

Anti-PmpC antibody levels from mucosal surfaces.

<p>(A-B) Anti-N-PmpC IgA levels and (C-D) anti-N-PmpC IgG levels in the tears (A and C) and vaginal washes (B and D) of BALB/c mice immunized via the conjunctiva and age-matched controls (nc). All samples were collected two weeks after the completion of the indicated immunization protocol and were assayed by ELISA. Results for each individual sample are presented. The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (<i>P</i> < 0.05*, <i>P</i><0.005**). Compared groups are indicated by two-head arrow.</p

The proliferation indices (PI) of TTd-stimulated SMLN cells from BALB/c and C57BL/6 mice immunized via the conjunctiva according to the assigned immunization protocol.

<p>The numbers of viable SMLN cells were assessed by MTT assay following a 48 h cultivation in 10% FCS/50 µM β-mercaptoethanol/RPMI 1640 medium supplemented or not with TTd (5 µg/ml). PIs were calculated for each mouse. The results are presented as the mean PI ± SE for each experimental group (n = 10). The statistical significance of the differences in PIs between groups treated according to the assigned protocols was determined by <i>t</i>-test (<i>P<</i>0.05*, <i>P<</i>0.005**). The reference group is indicated by a dotted line, and the comparison group is indicated by an arrow.</p

<i>In vitro</i> analysis of proliferation of primary SMLN cells subjected to N-PmpC and CtB stimulation.

<p>Bar graph showing the proliferation indices (PI) of N-PmpC- and CtB-stimulated SMLN cells isolated from BALB/c mice immunized via the conjunctiva. The proliferation indices of SMLN cells isolated from age-matched control mice (nc) are presented on the graph as a solid line (mean value) and dotted lines (upper and lower standard error values). The number of viable SMLN cells was assessed using the Cell Counting Kit<i>-</i>8 following a 48 h culture period in 10% FCS/50 μM β-mercaptoethanol/RPMI 1640 medium supplemented or not with the indicated stimulator (10 μg/ml for N-PmpC or 1x10⁶ CFU/ml for CtB). PIs were calculated for each mouse. The results are presented as the mean PIs ± SE for each experimental group (n = 6). The statistical significance of the observed differences was evaluated using Kruskal-Wallis test followed by Dunn's multiple comparisons test to compare between groups (immunized vs nc <i>P</i> < 0.05*, <i>P</i><0.005**; between immunized groups <i>P</i> < 0.05^#, <i>P</i><0.005^##).</p

Levels of TTd-specific SIgA in tear washes from BALB/c and C57BL/6 mice that were immunized according to the assigned protocols.

<p>Samples were collected two weeks after the completion of the immunizations and were assayed by ELISA (dilution 1∶2). The results are presented as the mean A_492/620± SE (n = 10). The significance of the observed differences was calculated by <i>t</i>-test (<i>P<0.05*</i>, <i>P<0.005**</i>). The reference group is indicated by a dotted line, and the comparison group is indicated by an arrow.</p

Levels of IFNγ (A), IL-4 (B), IL-17A (C) and IL-10 (D) in the supernatants from <i>in vitro</i> TTd-stimulated SMLN cells obtained from age-matched control mice (n.c.) and mice immunized via the conjunctiva according to the assigned protocol (bars).

<p>SMLN cells were cultivated at 37°C in 5% CO₂ for 48 h in 10% FCS/RPMI 1640/50 µM β-mercaptoethanol supplemented with 5 µg/ml TTd. The levels of cytokines in the supernatants of corresponding SMLN cells incubated in 10% FCS/RPMI 1640/50 µM β-mercaptoethanol under similar conditions (non-stimulated cells) are indicated by solid lines. The results are presented as the mean concentration ± SE (n = 10). Concentrations of cytokines in supernatants of TTd-stimulated cultures were compared by <i>t-</i>tests. The reference group is indicated by a dotted line, and the comparison group is indicated by an arrow. A <i>t</i>-test was also used for the comparison of cytokine concentration in supernatants of corresponding non-stimulated and TTd-stimulated cultures, and the statistical significance of the differences is marked next to the solid bar, indicating the level of the cytokine within the non-stimulated culture. The levels of statistical significance are assigned as follows: <i>P<0.05*</i>, <i>P<0.005**</i>, <i>P<0.0005***</i>_.</p

The relative abundance (RA) of TTd-specific mIgG⁺ B cells within the total population of mIgG⁺ B cells in SMLN upon completion of the assigned immunization protocols.

<p>The RA of the TTd-specific mIgG⁺ B cell population was calculated for each mouse. The results are presented as the mean RA ± SE for each experimental group of mice (n = 5). The statistical significance of the observed differences in TTd-specific mIgG⁺ B cell pool abundances was determined by <i>t</i>-test (<i>P<0.05*</i>, <i>P<0.005**</i>, <i>P<0.0005***</i>). The reference group is indicated by a dotted line, and an arrow indicates the comparison group.</p