Use of a novel antigen expressing system to study the <i>Salmonella enterica</i> serovar Typhi protein recognition by T cells

<div><p><i>Salmonella enterica</i> serovar Typhi (<i>S</i>. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in <i>S</i>. Typhi protection, it is crucial to identify <i>S</i>. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>) expressing one of four <i>S</i>. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific <i>S</i>. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant <i>E</i>. <i>coli</i> also possess the ability to respond to purified proteins. We observed that CD4⁺ cell responses, but not CD8⁺ cell responses, to recombinant <i>E</i>. <i>coli</i> were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an <i>E</i>. <i>coli</i> expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4⁺ and CD8⁺ cell responses.</p></div

Ability of the <i>Hly</i> gene to improve antigen processing.

<p>B-LCL cells were infected with either <i>E</i>. <i>coli</i> strain BL21 (BL21) or its <i>Hly</i>-recombinant <i>E</i>. <i>coli</i> (Hly) counterpart at 1:30 or 1:100 multiplicity of infection (MOI). After 2 hours, the cells were washed and exposed to gentamicin for an additional 2 hours to kill and detach extracellular bacteria. After further washings, the ability of the <i>Hly</i> gene to improve antigen processing was assessed by detecting <i>E</i>. <i>coli</i> antigens at the B-cell surface over time by flow cytometry (up to 120 minutes). Cells exposed to media only were used as control (uninfected).</p

Expression of <i>S</i>. Typhi proteins and lysteriolysin on recombinant <i>E</i>. <i>coli</i>.

<p>(<b>A</b>) Anti-HisTag antibody revealing positive control protein Annexin3 (A3) as well as lysteriolysin (Hly) and <i>S</i>. Typhi gene encoded proteins (OmpC, FliC, GroEL and SifA). A negative control with pmark (P, stop codons) clones was used instead of the protein. (<b>B</b>) Anti-lyteriolysin antibody revealing Hly gene expression (red box) in all lanes except in the negative control. Expression of <i>S</i>. Typhi proteins and lysteriolysin on recombinant <i>E</i>. <i>coli</i> were detected by Western blot.</p

CD8+ T cell responses to <i>S</i>. Typhi proteins presented by targets exposed to one of the four recombinant <i>S</i>. Typhi proteins.

<p><i>Ex vivo</i> PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant <i>S</i>. Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD8⁺ T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.</p

CD4+ T cell responses to <i>S</i>. Typhi proteins presented by targets infected with recombinant <i>E</i>. <i>coli</i>.

<p><i>Ex vivo</i> PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi/Hly (<i>Hly</i>/<i>SifA</i> (SifA), <i>Hly</i>/<i>FliC</i> (FliC), <i>Hly</i>/<i>GroEL</i> (GroEL) and <i>Hly</i>/<i>OmpC</i> (OmpC)) or only <i>Hly</i> (control) proteins. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4+ T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.</p

Correlation between T cell subset responses to B-LCL targets exposed to recombinant <i>S</i>. Typhi proteins or infected with recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi proteins.

<p><i>Ex vivo</i> PBMC were analyzed as described in Figs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005912#pntd.0005912.g001" target="_blank">1</a> & <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005912#pntd.0005912.g002" target="_blank">2</a>. Shown are the correlation between T cell subset responses to either of the four proteins (SifA, FliC, GroEL and OmpC) after stimulation by B-LCL targets exposed to recombinant <i>S</i>. Typhi proteins or infected with recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi proteins. Samples are representative of two individuals collected 42 days after vaccination. Coefficients of determination “R2” and “p” values are shown. p values of <0.05 were considered statistically significant. Dashed lines represent 95% confidence intervals.</p

<i>S</i>. Typhi proteins evaluated in this manuscript.

<p><i>S</i>. Typhi proteins evaluated in this manuscript.</p

Percentage of T cell subsets specific to any <i>S</i>. Typhi protein.

<p><i>Ex vivo</i> PBMC from 7 volunteers collected before and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi and <i>Hly</i> genes: <i>Hly</i>/<i>SifA</i> (SifA), <i>Hly</i>/<i>FliC</i> (FliC), <i>Hly</i>/<i>GroEL</i> (GroEL) and <i>Hly</i>/<i>OmpC</i> (OmpC). After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was analyzed by flow cytometry. Two T cell subset responses (i.e., CD4⁺ and CD8⁺ T cells) were evaluated. Net responses were calculated by subtracting the T cell responses to B-LCLs infected with recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi/Hly antigens from the responses to the controls (B-LCL expressing <i>Hly</i> only). Increases over day 0 were calculated by subtracting the net responses of the PBMC collected 42 days after immunization from the net responses of PBMC collected before immunization. Bars represent mean ± SE.</p

Volunteer responses to <i>S</i>. Typhi proteins.

<p><i>Ex vivo</i> PBMC from 7 immunized volunteers collected before (day 0) and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at an 1:30 MOI with one of the four recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi and <i>Hly</i> antigens: <i>Hly</i>/<i>SifA</i> (SifA), <i>Hly</i>/<i>FliC</i> (FliC), <i>Hly</i>/<i>GroEL</i> (GroEL) and <i>Hly</i>/<i>OmpC</i> (OmpC). After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was analyzed by flow cytometry. Two T cell subset responses (i.e., CD4⁺ and CD8⁺ T cells) were evaluated. (<b>A</b>) Heat-map of the multifunctionality of CD4⁺ and CD8⁺ T cells based on expression of cytokines and CD107a/b antigens. Percentages correspond to the net responses calculated by subtracting the T cell responses to B-LCLs infected with recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi/Hly proteins from the responses to the controls (B-LCL expressing <i>Hly</i> only). Volunteers were considered responders if the net responses of the PBMC collected 42 days after immunization were greater than 0.1 from the net responses of PBMC collected before immunization. (<b>B</b>) Cumulative frequency of responders to any functional test.</p

Antigen presentation of <i>S</i>. Typhi proteins by targets infected with recombinant <i>E</i>. <i>coli</i>.

<p><i>Ex vivo</i> PBMC from a volunteer collected before (day 0) and 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi/Hly (<i>Hly</i>/<i>SifA</i> (SifA), <i>Hly</i>/<i>FliC</i> (FliC), <i>Hly</i>/<i>GroEL</i> (GroEL) and <i>Hly</i>/<i>OmpC</i> (OmpC)) or only <i>Hly</i> (control) proteins. After incubation, cells were stained and the ability of the PBMC to express IFN-γ was analyzed by flow cytometry. CD4⁺ and CD8⁺ T cells were evaluated. Numbers represent the percentage of positive cells. The data of a representative volunteer are shown.</p