Figure 1.
Immunization with YopE69–77 peptide protects mice against Y. pestis.
Wild-type C57BL/6 mice were immunized intranasally with CT adjuvant alone (CT) or CT mixed with YopE69–77 peptide (YopE) and then challenged intranasally with (A) 20 MLD (2×105 CFU) or (B) 200 MLD (2×106 CFU) Y. pestis strain D27 or (C) 10 MLD (1×104 CFU) Y. pestis strain CO92. In comparison with CT–immunized mice (n = 10–40), YopE69–77–immunized mice (n = 15–39) exhibited significantly increased survival. Data were pooled from 2–5 independent experiments.
Figure 2.
Mice immunized with YopE69–77 exhibit perforin-dependent cytotoxic activity.
Wild-type (WT) and perforin-deficient (PKO) C57BL/6 mice were immunized with CT adjuvant alone or CT mixed with 1 or 10 µg of YopE69–77 or OVA257–264 peptides. Splenocytes from naïve congenic WT mice (CD45.1+) that were either pulsed with YopE69–77 peptide and labeled with 10 µM of CFSE or pulsed with OVA257–264 peptide and labeled with 1 µM of CFSE were mixed together at a 1∶1 ratio and injected into the immunized recipient mice. Splenocytes of recipient mice were then collected 20–22 h later and stained for congenic marker. The target cells (CD45.1+CD45.2−) were gated and the proportion of each CFSE-labeled population was analyzed by flow cytometry. The percent specific lysis was then calculated as described in the Materials and Methods. (A) Representative plots of the flow cytometry analysis of naïve or immunized WT recipient mice. The numbers on the lower right corner of the plots depict the percentage of specific lysis of YopE69–77-pulsed target cells or OVA257–264-pulsed target cells. (B and C) The percentages of specific lysis of YopE69–77-pulsed target cells by YopE69–77-immunized mice (B) or OVA257–264-pulsed target cells by OVA257–264-immunized mice (C). In comparison with WT mice, PKO mice displayed significantly decreased cytotoxicity (one-way ANOVA). Data shown are pooled from 5 independent experiments. (D and E) The percentages of PBL staining positive for CD8 and KbYopE69–77 (D) or KbOVA257–264 (E) tetramers two days before the cytotoxicity assay. In comparison with WT mice, PKO mice immunized with the same amount of peptide have comparable Ag-specific CD8 T cell frequencies (one-way ANOVA). Data shown are pooled from 5 independent experiments.
Figure 3.
Perforin is dispensable for YopE69–77–specific CD8 T cell-mediated protection against Y. pestis and Y. pseudotuberculosis.
Wild-type (WT) and perforin-deficient (PKO) C57BL/6 mice were immunized intranasally with CT adjuvant alone, or CT mixed with YopE69–77 or OVA257–264 peptides and then challenged with (A and B) 20 MLD (2×105 CFU) Y. pestis strain D27 intranasally, (C) 10 MLD (5×109 CFU) Y. pseudotuberculosis strain 32777 intragastrically or (D) 10 MLD (1.2×102 CFU) Y. pseudotuberculosis strain 32777 intravenously. (A) Y. pestis survival data pooled from 3 independent experiments (n = 9–30 mice/group). (B) Day 4 bacterial burden in lung and liver tissues after Y. pestis challenge (Kruskal-Wallis test, compared with CT- or OVA257–264–immunized PKO or WT mice). Data are pooled from 2 independent experiments (n = 9–11 mice/group). Solid bar depicts median; broken line depicts the limit of detection. (C and D) Y. pseudotuberculosis survival data (n = 6–7 mice/group for CT, n = 10–11 mice/group for YopE). Data were pooled from 2 independent experiments.
Figure 4.
TNFα and IFNγ are critical for T cell-mediated protection against Y. pestis.
(A and B) Wild-type (WT), TNFα-deficient (TNFaKO), IFNγ-deficient (IFNgKO) C57BL/6 mice were immunized intranasally with CT adjuvant alone or CT mixed with YopE69–77 peptide and then challenged intranasally with 20 MLD Y. pestis strain D27. In comparison with YopE69–77–immunized WT mice (n = 15–25), YopE69–77–immunized TNFα-deficient (n = 22) and IFNγ-deficient (n = 18) mice exhibited significantly reduced survival. Data were pooled from 3–5 independent experiments. (C) Wild-type C57BL/6 mice were immunized intranasally with CT alone or CT mixed with YopE69–77 peptide and then challenged intranasally with 20 MLD Y. pestis strain D27. One day before the challenge, the YopE69–77-immunized mice received neutralizing mAb specific for TNFα (anti-TNF), IFNγ (anti-IFN), or an isotype-matched mAb (Ctrl Ig). In comparison with CT-immunized mice (n = 20), YopE69–77-immunized mice treated with isotype-matched mAb (n = 19) or IFNγ-neutralizing mAbs (n = 10) but not TNFα-neutralizing mAb (n = 20) were protected against Y. pestis challenge. Data were pooled from 4 independent experiments.
Figure 5.
Selective depletion of TNFα from either macrophages/neutrophils or T cells does not impact the protection conferred by YopE69–77 immunization.
Mice with (A) monocyte/neutrophil-specific (MN-TNF KO) or (B) T cell-specific (T-TNF KO) deletion of TNFα were immunized with CT adjuvant alone or CT mixed with YopE69–77 peptide and challenged intranasally with 20 MLD Y. pestis strain D27. Littermate TNF-floxed (TNF flox/flox) mice were used as controls. In comparison with CT-immunized mice (n = 10–14), YopE69–77-immunized MN-TNF KO mice (n = 20) and T-TNF KO mice (n = 19) were protected against Y. pestis challenge (p<0.0001) with no significant difference from YopE69–77-immunized TNF flox/flox mice (n = 12–13 mice/group). Data were pooled from 3 (A) and 5 (B) independent experiments.
Figure 6.
TNFα and IFNγ produced by YopE69–77-specific CD8 T cells have complementary roles during protection against Y. pestis.
(A) Wild-type (WT), TNFα-deficient (TNFaKO) or IFNγ-deficient (IFNgKO) C57BL/6 mice were immunized with CT mixed with YopE69–77 peptide. WT mice immunized with CT adjuvant only or CT mixed with OVA257–264 peptide were used as controls. CD8+ splenocytes were then purified and transferred intravenously to naïve WT C57BL/6 mice, which were challenged intranasally with 20 MLD Y. pestis strain D27 the next day. In comparison with mice that received CD8+ T cells from CT-immunized WT mice (n = 50), mice that received CD8+ T cells from YopE69–77-immunized WT (n = 56), TNFα-deficient (n = 17) and IFNγ-deficient (n = 15) mice were protected against Y. pestis challenge. Notably, mice that received CD8+ T cells from OVA257–264-immunized WT mice (n = 25) were also protected (p<0.05). Data were pooled from 7 independent experiments. (B) Splenocytes isolated from naïve WT, TNFαKO, IFNγKO, perforin-deficient (PKO) or TNFα/IFNγ-deficient (TNFαIFNγ DKO) mice were transferred to TCRβδ-deficient mice. The mice were then immunized with CT mixed with YopE69–77. Control mice received naïve WT splenocytes and were immunized with CT mixed with OVA257–264 peptide. Mice were then challenged intranasally with 20 MLD Y. pestis strain D27. In comparison with control mice (n = 15), YopE69–77 immunized mice that received WT (n = 22), TNFαKO (n = 25), IFNγKO (n = 20), or PKO (n = 11) splenocytes were protected against Y. pestis challenge. YopE69–77-immunized mice that received TNFαIFNγ DKO splenocytes were also protected (n = 11) but the survival was significantly lower in comparison with the mice received WT, TNFαKO or IFNγKO splenocytes. Data were pooled from 3 independent experiments.
Figure 7.
YopE69–77-specific CD8 T cells lacking the capacity to produce TNFα and IFNγ fail to protect mice and control bacterial burden.
TCRβδ-deficient (TCRbdKO) mice were lethally irradiated and reconstituted with 75% TCRβδKO bone marrow cells and 25% of either WT, TNFαKO, IFNγKO, PKO, or TNFαIFNγ DKO bone marrow cells. Six weeks later they were immunized with CT mixed with YopE69–77 or OVA257–264 and then challenged intranasally with 20 MLD Y. pestis strain D27. (A) Survival. In comparison with OVA257–264-immunized mice reconstituted with WT T cells (n = 20), the YopE69–77-immunized chimeric mice reconstituted with WT (n = 25), TNFαKO (n = 17), IFNγKO (n = 19), PKO (n = 8) or TNFαIFNγ DKO (n = 33) T cells all showed significant protection. (B) The percentage of CD8+ T cells that stained positive for MHC class I tetramer KbYopE69–77 in PBL on the day before challenge. Solid bar depicts the mean. All groups of chimeric mice that were immunized with YopE69–77 had significantly increased frequency of KbYopE69–77+CD8+ T cells in compared with the chimeric mice immunized with OVA257–264 (p<0.001). YopE69–77-immunized chimeric mice reconstituted with TNFαKO T cells had significantly higher frequency of KbYopE69–77+CD8+ T cells in compared with YopE69–77-immunized chimeric mice reconstituted with WT T cells (p<0.01). Data for (A) and (B) are pooled from 6 independent experiments. (C and D) Bacterial burden in lung (C) and liver (D) tissues was measured at day 4 after challenge (Kruskal-Wallis test). Data are pooled from 3 independent experiments. Solid bar depicts median; broken line depicts the limit of detection.