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

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

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

IgG1/IgG2a (A) and IgG1/IgG2c (B) ratios calculated for TTd-specific antibodies in the sera of TTd-immunized BALB/c and C57BL/6 mice, respectively.

<p>Serum samples were collected two weeks after the completion of the immunizations and were assayed by ELISA (dilution 1∶100). Ratios were determined using the A₄₀₅ values obtained upon the measurement of TTd-specific IgG1 and IgG2a or IgG2c in sera. The results are presented as the mean A₄₀₅(IgG1)/A₄₀₅(IgG2a,c) ± SE (n = 10). The significance of the differences between syngeneic mice immunized subcutaneously with TTd (reference group) and those immunized via the conjunctiva was calculated by <i>t</i>-test (<i>P<</i>0.05*, <i>P<</i>0.005**, <i>P<</i>0.0005***).</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