Fig. 5

Immunophenotyping of T cell subsets by flow cytometry. A Splenocytes collected at day 7 post last immunization were stained with CD3-FITC, CD4-APC, and CD8-PE monoclonal antibodies. Splenocytes were gated based on their FSC/SSC to remove the dead cells, while FSC-H vs. FSC-A density plots was used to exclude doublets (see Additional file 1: Fig. S2). Flow cytometric analysis was performed with a gated population of T cells (FITC), Th cells (APC), and Tc cells (PE) collected from various experimental groups. For immunophenotypic profiles of Th and Tc cells, triple staining was performed (CD3-FITC, CD4-APC, and CD8-PE). Channels FL1 and FL2 were used as filters or detectors for FITC and PE-labelled antibodies, respectively. B Live cell percentage of gated T cells and other subsets (Th and Tc) among different experimental groups. For each experiment, a total of three animals was randomly selected from two independent experiments performed under similar conditions (n = 6). The mean percentage (%) of live T cells population in splenocytes obtained from the mice of each experimental group showed a minor increment in T cell (CD3⁺) population in case CWA-Hcp group while a significant increase was observed in the group of mice that received rL. lactis expressing Sec-Hcp (**P ≤ 0.01) compared to the control group of mice those received PBS only (a). A significant increment of the mean live T (CD4⁺) cell percentage in the different experimental groups (CWA-Hcp, *P ≤ 0.05; Sec-Hcp, **P ≤ 0.01) was observed (b). The mean live T (CD8⁺) cell percentage in splenocytes obtained from the mice that received Sec-Hcp showed a significant increment (*P ≤ 0.05) in the T cell population compared to the control mice those received PBS only (c). Data represent live cell percentage ± SE of two independent experiments. Asterisks indicate a statistically significant difference (*P ≤ 0.05, **P ≤ 0.01) with respect to the control group (received PBS only)