Fibrin Facilitates Both Innate and T Cell-Mediated Defense against Yersinia pestis

The gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a non-diffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for antibody-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin in order to withstand Y. pestis encounters and effectively clear bacteria

TNFα and IFNγ but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias

YopE_69–77-specific CD8 T cells lacking the capacity to produce TNFα and IFNγ fail to protect mice and control bacterial burden.

<p>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 YopE_69–77 or OVA_257–264 and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. (A) Survival. In comparison with OVA_257–264-immunized mice reconstituted with WT T cells (n = 20), the YopE_69–77-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 K^bYopE_69–77 in PBL on the day before challenge. Solid bar depicts the mean. All groups of chimeric mice that were immunized with YopE_69–77 had significantly increased frequency of K^bYopE_69–77+CD8+ T cells in compared with the chimeric mice immunized with OVA_257–264 (p<0.001). YopE_69–77-immunized chimeric mice reconstituted with TNFαKO T cells had significantly higher frequency of K^bYopE_69–77+CD8+ T cells in compared with YopE_69–77-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.</p

Immunization with YopE_69–77 peptide protects mice against <i>Y. pestis</i>.

<p>Wild-type C57BL/6 mice were immunized intranasally with CT adjuvant alone (CT) or CT mixed with YopE_69–77 peptide (YopE) and then challenged intranasally with (A) 20 MLD (2×10⁵ CFU) or (B) 200 MLD (2×10⁶ CFU) <i>Y. pestis</i> strain D27 or (C) 10 MLD (1×10⁴ CFU) <i>Y. pestis</i> strain CO92. In comparison with CT–immunized mice (n = 10–40), YopE_69–77–immunized mice (n = 15–39) exhibited significantly increased survival. Data were pooled from 2–5 independent experiments.</p

Perforin is dispensable for YopE_69–77–specific CD8 T cell-mediated protection against <i>Y. pestis</i> and <i>Y. pseudotuberculosis</i>.

<p>Wild-type (WT) and perforin-deficient (PKO) C57BL/6 mice were immunized intranasally with CT adjuvant alone, or CT mixed with YopE_69–77 or OVA_257–264 peptides and then challenged with (A and B) 20 MLD (2×10⁵ CFU) <i>Y. pestis</i> strain D27 intranasally, (C) 10 MLD (5×10⁹ CFU) <i>Y. pseudotuberculosis</i> strain 32777 intragastrically or (D) 10 MLD (1.2×10² CFU) <i>Y. pseudotuberculosis</i> strain 32777 intravenously. (A) <i>Y. pestis</i> survival data pooled from 3 independent experiments (n = 9–30 mice/group). (B) Day 4 bacterial burden in lung and liver tissues after <i>Y. pestis</i> challenge (Kruskal-Wallis test, compared with CT- or OVA_257–264–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) <i>Y. pseudotuberculosis</i> survival data (n = 6–7 mice/group for CT, n = 10–11 mice/group for YopE). Data were pooled from 2 independent experiments.</p

Mice immunized with YopE_69–77 exhibit perforin-dependent cytotoxic activity.

<p>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 YopE_69–77 or OVA_257–264 peptides. Splenocytes from naïve congenic WT mice (CD45.1+) that were either pulsed with YopE_69–77 peptide and labeled with 10 µM of CFSE or pulsed with OVA_257–264 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004142#s4" target="_blank">Materials and Methods</a>. (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 YopE_69–77-pulsed target cells or OVA_257–264-pulsed target cells. (B and C) The percentages of specific lysis of YopE_69–77-pulsed target cells by YopE_69–77-immunized mice (B) or OVA_257–264-pulsed target cells by OVA_257–264-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 K^bYopE_69–77 (D) or K^bOVA_257–264 (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.</p

TNFα and IFNγ are critical for T cell-mediated protection against <i>Y. pestis</i>.

<p>(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 YopE_69–77 peptide and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. In comparison with YopE_69–77–immunized WT mice (n = 15–25), YopE_69–77–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 YopE_69–77 peptide and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. One day before the challenge, the YopE_69–77-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), YopE_69–77-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 <i>Y. pestis</i> challenge. Data were pooled from 4 independent experiments.</p

Selective depletion of TNFα from either macrophages/neutrophils or T cells does not impact the protection conferred by YopE_69–77 immunization.

<p>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 YopE_69–77 peptide and challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. Littermate TNF-floxed (TNF flox/flox) mice were used as controls. In comparison with CT-immunized mice (n = 10–14), YopE_69–77-immunized MN-TNF KO mice (n = 20) and T-TNF KO mice (n = 19) were protected against <i>Y. pestis</i> challenge (p<0.0001) with no significant difference from YopE_69–77-immunized TNF flox/flox mice (n = 12–13 mice/group). Data were pooled from 3 (A) and 5 (B) independent experiments.</p