Vaccine-induced inflammation and inflammatory monocytes promote CD4+ T cell-dependent immunity against murine salmonellosis.

Prior infection can generate protective immunity against subsequent infection, although the efficacy of such immunity can vary considerably. Live-attenuated vaccines (LAVs) are one of the most effective methods for mimicking this natural process, and analysis of their efficacy has proven instrumental in the identification of protective immune mechanisms. Here, we address the question of what makes a LAV efficacious by characterising immune responses to a LAV, termed TAS2010, which is highly protective (80-90%) against lethal murine salmonellosis, in comparison with a moderately protective (40-50%) LAV, BRD509. Mice vaccinated with TAS2010 developed immunity systemically and were protected against gut-associated virulent infection in a CD4+ T cell-dependent manner. TAS2010-vaccinated mice showed increased activation of Th1 responses compared with their BRD509-vaccinated counterparts, leading to increased Th1 memory populations in both lymphoid and non-lymphoid organs. The optimal development of Th1-driven immunity was closely correlated with the activation of CD11b+Ly6GnegLy6Chi inflammatory monocytes (IMs), the activation of which can be modulated proportionally by bacterial load in vivo. Upon vaccination with the LAV, IMs expressed T cell chemoattractant CXCL9 that attracted CD4+ T cells to the foci of infection, where IMs also served as a potent source of antigen presentation and Th1-promoting cytokine IL-12. The expression of MHC-II in IMs was rapidly upregulated following vaccination and then maintained at an elevated level in immune mice, suggesting IMs may have a role in sustained antigen stimulation. Our findings present a longitudinal analysis of CD4+ T cell development post-vaccination with an intracellular bacterial LAV, and highlight the benefit of inflammation in the development of Th1 immunity. Future studies focusing on the induction of IMs may reveal key strategies for improving vaccine-induced T cell immunity

Cryo-EM structure of the extracellular domain of murine Thrombopoietin Receptor in complex with Thrombopoietin

Abstract Thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for haematopoetic stem cell maintenance. Tpo functions by binding its receptor (TpoR, a homodimeric Class I cytokine receptor) and initiating cell proliferation or differentiation. Here we characterise the murine Tpo:TpoR signalling complex biochemically and structurally, using cryo-electron microscopy. Tpo uses opposing surfaces to recruit two copies of receptor, forming a 1:2 complex. Although it binds to the same, membrane-distal site on both receptor chains, it does so with significantly different affinities and its highly glycosylated C-terminal domain is not required. In one receptor chain, a large insertion, unique to TpoR, forms a partially structured loop that contacts cytokine. Tpo binding induces the juxtaposition of the two receptor chains adjacent to the cell membrane. The therapeutic agent romiplostim also targets the cytokine-binding site and the characterisation presented here supports the future development of improved TpoR agonists

The <i>S</i>. Typhimurium live-attenuated vaccine (LAV) strain TAS2010 shows enhanced growth in tissues and confers potent protection against challenge.

A-C) Wild-type C57BL/6 mice were given an oral gavage of 5×109cfu S. Typhimurium LAV strain BRD509 (ΔaroA) or TAS2010 (ΔpfkAΔpfkBΔedd) as the vaccine (vac), or an oral gavage of PBS only (PBS). At week 10–12 post-vaccination, mice were challenged with an oral gavage of 107cfu wild-type S. Typhimurium SL1344. A) The bacterial load of S. Typhimurium LAV strains was determined in the mesenteric lymph nodes (mLNs) (n = 9–11), faeces (n = 10–35), spleen (n = 9–17) and liver (n = 9–17) at the indicated time points post-vaccination. B) Shown is the percentage of mice remaining protected at the indicated time points post-challenge. C) The growth of wild-type S. Typhimurium SL1344 in challenged mice was determined in the mLNs (n = 8–10), faeces (n = 10), spleen (n = 6–12) and liver (n = 6–12) at the indicated time points post-challenge. Symbols indicate geometric mean of bacterial load ± SEM, with data pooled from 2–4 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used for comparing the three groups, and asterisks indicate significant differences between the indicated group and the TAS2010-vaccinated group. D, E) Wild-type C57BL/6 mice were i.v. injected with 200cfu TAS2010 or BRD509. D) The bacterial load was determined in the spleen and liver at the indicated time points post-vaccination. Symbols indicate geometric mean of bacterial load ± SEM (n = 5–23), data at each time point are pooled from 2–4 independent experiments. E) At week 10–12 post-vaccination, naïve or vaccinated mice were challenged with an oral gavage of 107cfu wild-type S. Typhimurium SL1344. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. F) Wild-type C57BL/6 mice were i.v. vaccinated with either a single dose of 200cfu TAS2010 and challenged 30 weeks later, or four doses of 5×107cfu heat-killed S. Typhimurium SL1344 (HKSTm) or PBS at day 0, 3, 7 and 14, then challenged at day 28. Challenge was given as 107cfu wild-type S. Typhimurium SL1344 by oral gavage. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used to compare the PBS group with HKSTm-vaccinated and 30-week TAS2010-vaccinated groups, respectively.</p

CD4⁺ T cell deficiency leads to impaired control of primary and secondary infection.

Wild-type C57BL/6 or I-A-/-I-Enull mice were i.v. injected with 200cfu TAS2010. A) Infection-induced weight variation overtime is calculated as the percentage of initial body weight. Shown are mean ± SEM, n = 9–14, data pooled from 3 independent experiments. B) The bacterial load in the spleen and liver from individual mice is shown with geometric mean for each group at the indicated time points post-infection. C) C57BL/6 mice were intraperitoneally (i.p.) injected with αCD4 GK1.5 mAb or PBS at Wk 12 post-vaccination, a day prior to challenge with 107cfu wild-type S. Typhimurium SL1344 by oral gavage. The depletion was maintained by i.p. injection with GK1.5 mAb twice weekly thereafter. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used for statistical analysis between PBS-treated and CD4-depleted groups.</p

Mice with reduced immune control over <i>S</i>. Typhimurium growth are highly susceptible to infection with <i>S</i>. Typhimurium.

Wild-type C57BL/6 (black) or Ifng-/- (pink) mice were i.v. injected with 200cfu TAS2010 (square) or BRD509 (circle). A) The bacterial load in the spleen and liver from individual mice is shown with geometric mean for each group at day 7 post-infection. Data are pooled from 2 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used twice: first for comparing the two mouse genotypes, then for comparing the same genotype of mice infected with different S. Typhimurium strains. B) Shown is the percentage of mice remaining that were not considered moribund at the indicated time points post-infection. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used for statistical analysis. (TIF)</p

<i>S</i>. Typhimurium strain TAS2010 (Δ<i>pfkA</i>Δ<i>pfkB</i>Δ<i>edd</i>) is attenuated for <i>in vivo</i> growth due to genetic disruptions in carbon metabolism pathways.

A) Schematic diagram showing key metabolic steps in the Embden-Meyerhof-Parnas (EMP) and Entner-Doudoroff (ED) pathways, which are blocked in S. Typhimurium strain TAS2010 (ΔpfkAΔpfkBΔedd). Shaded boxes represent metabolites and arrows show the physiological direction of enzymatic reactions. Reactions blocked by the mutations are shown with a cross. DHAP, Dihydroxyacetone phosphate; Fru-6-P, D-Fructose 6-phosphate; Fru-1,6-P2, D-Fructose 2,6-bisphosphate; GADP, Glyceraldehyde 3-phosphate; Glu-6-P, D-Glucose 6- phosphate; Glul-6-P, D-Glucono-1,5-lactone 6-phosphate; Gln-6-P, D-Gluconate 6-phosphate; KDPG, 2-keto-3deoxy-6-phosphogluconate;PEP, Phosphoenolpyruvate; Ribu-5-P, D-Ribulose 5-phosphate; Rib-5-P, D-Ribose 5-phosphate. B) Wild-type C57BL/6 mice were given an oral gavage of 8×106cfu of indicated strain of S. Typhimurium, and the bacterial load in the spleen and liver was analysed at day 6 post-infection (dotted line represents detection limit). Note here the bacterial load in the spleen and liver was lower than shown in Fig 1A because a lower infection dose was used. The geometric mean of each group is shown, data are pooled from 3 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analysis in each organ. C-E) S. Typhimurium BRD509 (○) or TAS2010 (◼) were grown to stationary phase in Luria broth with streptomycin (50μg/ml) overnight and then normalised to OD600 of 0.8. The normalised culture was sub-cultured 1:100 into fresh Luria broth and grown for 24hr at 37°C in 96-well plates, with absorbance at 600nm measured every hour by the CARIOStar plate reader. The cultures were grown C) aerobically, with shaking at 300rpm in 200μl Luria broth per well, D) anaerobically, by static growth in 200μl Luria broth plus 80μl mineral oil overlay per well, or E) anaerobically, by static growth in 300μl Luria broth which filled the well fully. The mean of six technical replicates is shown, data representative of 2 independent experiments. (TIF)</p

IMs from <i>Ccr2</i>^<i>-/-</i> mice show potent activation in response to <i>S</i>. Typhimurium TAS2010.

Wild-type C57BL/6 (black) or Ccr2-/- (orange) mice were either naïve or i.v. injected with 200cfu TAS2010 or BRD509. Mice were analysed at day 7 post-infection. A, C-D) The number of A) CD11b+Ly6GnegLy6Chi inflammatory monocytes (IMs), C) CD11b+Ly6G+ neutrophils and D) CD11c+MHC-IIhi conventional DCs were quantified. B, F) IMs were stained for MHC-II (surface) and CXCL9 (intracellular) and analysed by flow cytometry. B) Representative staining profiles are shown. F) the number of MHC-II- or CXCL9-expressing IMs was quantified. E) Serum concentration of IFN-γ was determined using the cytometric bead array (CBA). Data from individual mice are shown as symbols with group mean, pooled from 3–4 independent experiments. Brown-Forsythe and Welch ANOVA tests with Dunnett T3 corrections (a variation of One-way ANOVA that does not assuming equal variance) were used for statistical analyses. (TIF)</p

Inflammatory monocytes (IMs) show IFN-γ-dependent activation and become a potent source of CXCL9 and IL-12 following vaccination with <i>S</i>. Typhimurium TAS2010.

Wild-type C57BL/6 mice were either naïve or i.v. injected with 200cfu TAS2010 or BRD509, and the splenic CD4+ T cells were analysed at the indicated time points post-vaccination, where Wk 0 denotes data from naïve mice. A-C) A) The frequency of MHC-II expression, B) geometric mean fluorescence intensity (gMFI) of CD64 expression, and C) the frequency of CXCL9 production was determined for IMs in the spleen. Data are shown as group mean ± SEM for each time point, with data pooled from 3–6 independent experiments per time point (n = 9–29). Two-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between the two vaccination groups. D) Representative FACS plots showing staining of relevant markers in CXCL9-producing cells, using CXCL9-negative (CXCL9-) cells as the gating control. Plots show the vast majority of CXCL9-producing (CXCL9+) cells are MHC-II+ IMs. E) Representative FACS plots show IMs from mice that were either naïve or at day 7 post-vaccination with TAS2010. The expression of MHC-II and CXCL9 depends on IFN-γ. F) The percentage of IL-12p35-producing IMs was determined using intracellular staining for IL-12p35 after 4hr incubation with brefeldin A at 37°C ex vivo. Data are pooled from 2 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analyses. G) Representative spleen sections from C57BL/6 wild-type mice that were either naïve or vaccinated with TAS2010 for 2 or 3 weeks. Sections were stained with the indicated antibodies. White bars represent 50μm.</p

Escalated activation of IMs increased CD4⁺ T cell activation and led to improved BRD509-induced immune protection.

Wild-type C57BL/6 mice were i.v. injected with 200cfu TAS2010 or escalating doses of BRD509, as indicated. A) The bacterial load in the spleen increased with the dose of BRD509 given. Data points are shown for individual mice with geometric mean for each group. Data are pooled from 3 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analyses between 200cfu BRD509 and other vaccination groups. B-E) B) Representative FACS histogram overlay show upregulation of CD64 and MHC-II expression in IMs at the indicated time points post-vaccination, in contrast to naïve mice. The numbers of C) total IMs, D) MHC-II+ IMs, and E) CXCL9+ IMs are calculated per spleen, and shown as mean ± SEM (n = 9–20). Data are pooled from 2–4 independent experiments. Two-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between 200cfu BRD509 and other vaccination groups. F) Representative FACS plots show total viable CD4+ T cells in the spleen at week 2 post-vaccination. G,H) At week 10 post-vaccination, the frequency of CD4+ T cells G) expressing CXCL6, and H) producing IFN-γ in response to ex vivo re-stimulation was determined. Data from individual mice are shown as symbols with group mean, pooled from 3 independent experiments. One-way ANOVA with Bonferroni’s post-tests were used for statistical analysis between 200cfu BRD509 and other vaccination groups. I) At week 12 post-vaccination, mice were challenged with 107cfu S. Typhimurium wild-type SL1344 by oral gavage. Shown is the percentage of mice remaining protected at the indicated time points post-challenge. Data are pooled from 2 independent experiments. Log-rank Mantel-Cox test was used for statistical analysis between 200cfu BRD09 and the other vaccination groups.</p