Article Figures & Data
Figures
- FIG. 1.
Surface expression of Salmonella serovar Typhi antigens on infected splenocytes and cell lines. Splenocytes from BALB/c (H-2d) and C57BL/6 (H-2b) mice and MHC-matched cell lines P815 (H-2d) and EL4 (H-2b) were infected in vitro with Salmonella serovar Typhi strain ISP1820 as described in Materials and Methods. Mock-infected cells were included as controls. Surface expression of bacterial antigens was measured in infected and mock-infected cells by multicolor flow cytometry by using FITC-conjugated anti-Salmonella CSA-1 antibodies. Cells were also incubated with FITC-labeled isotype control antibodies to determine nonspecific staining. Expression of bacterial antigens in BALB/c splenocytes was analyzed in the total cell population or gated on a particular cell phenotype (i.e., CD4+, CD8+, B220+, and CD11b+), as indicated. The percentage of specific staining is shown on each histogram. The data are representative of the data from four separate experiments in which similar results were obtained.
- FIG. 2.
Detection of Salmonella serovar Typhi-specific CTL responses in mice immunized i.n. with attenuated Salmonella serovar Typhi vaccine strains. BALB/c mice were immunized i.n. with two doses (28 days apart) of Salmonella serovar Typhi vaccine strains Ty21a, CVD 915, CVD 908, and CVD 908-htrA or PBS (control) as described in Materials and Methods. Spleens and CLN were collected 56 to 60 days after the primary immunization. Effector cells were stimulated in vitro for 7 days with Salmonella serovar Typhi-infected syngeneic splenocytes in the presence of IL-7 and IL-2 and assayed for CTL activity against Salmonella serovar Typhi-infected and mock-infected P815 cells in 51Cr release assays. (A) CTL responses in splenocytes from mice inoculated with different vaccine strains or PBS (control). (B) CTL responses in CLN and spleens of mice that received CVD 908-htrA. All curves show the mean percentages of specific cytotoxicity at different E/T ratios for triplicate or quadruplicate wells; the error bars indicate standard deviations. The percentage of background lysis of mock-infected P815 target cells was subtracted from the percentage of Salmonella serovar Typhi-infected P815 lysis. The data are representative of the data from two complete, separate experiments in which similar results were obtained.
- FIG. 3.
In vitro expansion of CTL effectors with Salmonella serovar Typhi-infected cells is required to detect cytotoxic activity. Splenocytes from mice immunized with Salmonella serovar Typhi strains CVD 908-htrA and CVD 915 were examined for CTL activity without expansion, after in vitro expansion for 7 days in the presence of Salmonella serovar Typhi-infected syngeneic splenocytes, or after two consecutive rounds of expansion for a total of 14 days. The curves show the mean percentages of specific cytotoxicity for replicate wells; the error bars indicate standard deviations. The data are representative of the data from four different experiments in which similar results were obtained.
- FIG. 4.
Increased proportion of CD8+ T cells in effector cell populations after expansion with Salmonella serovar Typhi-infected cells. The distribution of CD4+ and CD8+ T cells was measured by flow cytometry for splenocytes from immunized mice before and after expansion in the presence of Salmonella serovar Typhi-infected cells. The data are representative of the data from two complete, separate experiments in which similar results were obtained.
- FIG. 5.
Cytotoxic activity is mediated by CD8+ T cells. CD4+ and CD8+ T cells sorted by flow cytometry from expanded splenocyte cultures from mice immunized with the different vaccine strains were assayed for cytotoxicity in the presence of Salmonella serovar Typhi-infected and mock-infected targets. The purity of sorted populations in different experiments was between 92 and 98%. No CTL activity was observed in PBS (control) mice. The curves show the mean percentages of specific cytotoxicity for replicate wells; the error bars indicate standard deviations. The data are representative of the data from two complete, separate experiments in which similar results were obtained.
- FIG. 6.
CTL activity is mediated by CD8+ αβ+. Splenocytes from immunized mice were phenotypically evaluated for expression of CD4, CD8, TCRαβ, TCRγδ, CD3, and NK markers before and after expansion with Salmonella serovar Typhi-infected cells by multicolor flow cytometry. The values represent the percentages of positive cells in each quadrant. The percentages of CD4+ and CD8+ cells within the NK-T cell population (CD3+ DX5+) are also shown. The data are representative of the data from four different experiments performed with effector cells from mice immunized with CVD 908-htrA. PE, phycoerythrin.
- FIG. 7.
CTL responses in spleens and CLN are MHC restricted. Effector cells from CLN and spleens of mice immunized with CVD 908-htrA were expanded for 7 days or 14 days (two rounds of 7 days each) and tested for cytotoxic activity in the presence of Salmonella serovar Typhi-infected and mock-infected MHC-matched P815 cells and mismatched EL4 cells. The curves show the mean percentages of specific cytotoxicity for replicate wells; the error bars indicate standard deviations. The data are representative of the data from three separate experiments in which similar results were obtained.
- FIG. 8.
Inhibition of CTL activity by MAbs to MHC class I molecules. Salmonella serovar Typhi-infected target cells were treated with medium (No Ab), an isotype control, or MAbs to the class I molecules H-2Dd, Qa-1b, H-2Kd, and H-2Ld, and CTL assays were performed. Effector cells had been expanded in vitro with Salmonella serovar Typhi-infected splenocytes for 7 or 14 days. The curves show the mean percentages of specific cytotoxicity for replicate wells; the error bars indicate standard deviations. The data are representative of the data for mice immunized with CVD 908-htrA in three separate experiments.
