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, 8 (1), e1002499

Temporal Expression of Bacterial Proteins Instructs Host CD4 T Cell Expansion and Th17 Development

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Temporal Expression of Bacterial Proteins Instructs Host CD4 T Cell Expansion and Th17 Development

Seung-Joo Lee et al. PLoS Pathog.

Abstract

Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS) effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Discovery of novel Salmonella I-Ab epitopes.
(A) C57BL/6 mice were immunized with a mixture of 4 groups of 11 peptides from Table 1 at 5 µg/peptide in CFA. Eight days later, draining lymph nodes were isolated and purified CD4 T cells restimulated with individual peptides (Table 1) in the presence of irradiated splenocytes. IFN-γ production was measured 24–36 hours later by ELISPOT. (B) 129Sv mice were orally infected with virulent 5x107 Salmonella (SL1344) and spleens harvested 11 weeks later. Purified CD4 T cells were restimulated with peptides from Table 2 for 24–36 h in the presence of irradiated splenocytes and IFN-γ production measured by ELISPOT. ConA and no peptide wells constitute the positive and negative controls respectively. Each plate is representative of 2–3 individual experiments.
Figure 2
Figure 2. Differential regulation of bacterial Flagellin (FliC) and SseJ in vitro and in vivo.
For in vitro detection of bacterial mRNA, Salmonella (BRD509) were cultured under SPI1-inducing conditions (LB broth, for 3hours) or SPI2-inducing conditions (modified N minimal medium, for 6hours) (Top). For in vivo detection, C57BL/6 mice were infected intravenously with 5X105 Salmonella (BRD509), and spleens harvested 30 min or 5 hours later (Bottom). Bacterial RNA was isolated as described in Materials and Methods. Expression of Flagellin (FliC) or SseJ mRNA was quantified by real-time qPCR. Bar graphs show the mean number ± SEM of Flagellin (FliC) or SseJ mRNA transcripts normalized to the respective amount of 16S rRNA in each sample. Data are pooled from two separate experiments (in vitro) or three separate experiments using a total of nine mice (in vivo). Numbers above indicate statistical significance and show the p value of a comparison between the groups.
Figure 3
Figure 3. Expansion of endogenous flagellin427–441- or SseJ329–341-specific CD4 T cells after peptide immunization or Salmonella infection of mice.
C57BL/6 mice were immunized sub-cutaneously with either 100 µg of flagellin427–441 or SseJ329–341 peptide in the presence of CFA or infected with 5x105 Salmonella (BRD509). On day 7, inguinal, axillary, and brachial lymph nodes were isolated from peptide-immunized mice (Immunized). On day 35, spleens were harvested from C57BL/6 mice infected intravenously with Salmonella BRD509 (Infected). Endogenous flagellin427–441- or SseJ329–341-specific CD4 T cells were detected using Flagellin:I-Ab or SseJ:I-Ab tetramers, enriched with anti-fluorochrome microbeads, stained with antibodies to several surface markers, and examined by flow cytometry. CD4 T cells from naive, immunized, and Salmonella-infected mice, were detected among CD11cCD11bF4/80B220CD3+ cells, and further analyzed for expression of CD44 and Flagellin:I-Ab or SseJ:I-Ab tetramer positive cells. CD4 T cells from the unbound column fraction and CD8 T cells within the bound fraction are also shown as controls. FACS plots are representative of three mice per group and three replicate experiments.
Figure 4
Figure 4. Kinetics of endogenous Salmonella flagellin427–441-, and SseJ329–341-specific CD4 T cells after Salmonella infection.
C57BL/6 mice were infected intravenously with 5×105 Salmonella (BRD509). At various time points, endogenous CD4 T cells were stained using Flagellin:I-Ab or SseJ:I-Ab tetramers, enriched using magnetic selection, and examined by flow cytometry. CD4 T cells were gated from CD11cCD11bF4/80B220CD3+ cells, and further analyzed for Flagellin:I-Ab or SseJ:I-Ab tetramer positive cells. (A) The number of endogenous flagellin427–441- or SseJ329–341-specific CD4 T cells was calculated based on FACS analysis. Graph shows the mean number ± SEM of flagellin427–441- or SseJ329–341-specific CD4 T cells. (B and C) Line graphs show the mean percentage ± SEM of tetramer specific CD4 T cells positive for CCR7 (B) or CD27 (C) expression. (D) The number of viable Salmonella determined in the spleen at various time points. These data were pooled from several experiments and represent at least 3–5 mice at each time point. (E) Endogenous flagellin427–441- or SseJ329–341-specific CD4 were enriched from day 6 or day 48 Salmonella-infected mice, and stained intracellularly with antibodies specific for T-bet.
Figure 5
Figure 5. Salmonella-specific Th1 cells target flagellin and T3SS effectors at different time points after infection.
C57BL/6 mice were infected intravenously with 5×105 Salmonella (BRD509). On day 7 or day 40 post-infection, CD4 T cells from the spleens were purified and restimulated with 10 µM of various peptides (Table 3) for 16 h in the presence of irradiated splenocytes. IFN-γ production was measured by ELISPOT assay. (Top) Representative images from IFN-γ ELISPOT plates using CD4 T cells from Salmonella-infected mice at day 7 or day 40 after infection. (Bottom) Scatter graphs show the number of IFN-γ-producing CD4 T cells per infected mouse. Numbers above indicate statistical significance and show p value of a comparison between media alone (no stimulation) and peptide stimulation group.
Figure 6
Figure 6. Bacterial persistence is required for optimal expansion of SseJ-specific CD4 T cells.
C57BL/6 mice were infected intravenously with 5×105 Salmonella (BRD509). Some mice were treated with Enrofloxacin (Baytril) (2mg/ml in drinking water), beginning 5 days post-infection. At day 30 post-infection, endogenous SseJ-specific CD4 T cells were stained with SseJ:I-Ab tetramers, enriched, and examined by flow cytometry. (A) Representative FACS plots from control (without, left) or antibiotic-treated mice (with, right). Tetramer-specific CD4 T cells were gated from CD11cCD11bF4/80B220CD3+. (B) Scatter plots show the number of SseJ329–341-specific CD4 T cells in control (without) or treatment group (with). Data are pooled from three separate experiments. Numbers above indicate statistical significance and show p value of a comparison between control and treatment group.
Figure 7
Figure 7. Detection of flagellin-specific CD4 Th17 cells in intestinal tissues.
C57BL/6 mice were infected three times orally with 5×109 Salmonella (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from the spleens, MLN, including Peyer's patches, livers (LV), and laminar propria (LP) were purified. Isolated CD4 T cells (1×105) were restimulated with 10 µM of various peptides (Table 3) for 16 h in the presence of irradiated splenocytes. (A) IFN-γ or, (B) IL-17A production was measured by ELISPOT. Scatter graphs show the number of (A) IFN-γ or, (B) IL-17A-producing CD4 T cells in infected tissues from individual mice. (C) Bar graphs show the mean ratio of IFN-γ to IL-17 production after in vitro stimulation. Numbers above indicate statistical significance and show p value of a comparison between media alone (no stimulation) and peptide stimulation group.
Figure 8
Figure 8. Mucosal flagellin-specific CD4 T cells produce IL-22 upon restimulation.
C57BL/6 mice were infected three times orally with 5×109 Salmonella (BRD509) at one-month intervals. On day 7 after the third infection, CD4 T cells from spleen and MLN, including Peyer's patches, were isolated. Purified CD4 T cells were restimulated with 10 µM peptide (Table 3) for 16 h in the presence of irradiated splenocytes. Culture supernatants were collected and IL-22 production measured by ELISA. Data show IL-22 production in culture supernatants as scatter plots representing individual mice. Data are pooled from three separate experiments. Numbers above each group indicate statistical significance and show the p value of a comparison between media alone (no stimulation) and peptide stimulation group.

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