Dynamic MHCII presentation and delayed activation of MΦs during co-infection.

<p>Spleen mononuclear cells were extracted on consecutive days from single and co-infected mice. They were stained and counted in a LCRII flow cytometer (N = 6 per group) experiment performed in duplicate. (A) The percentage fraction of MHCII expressing cells in the F4/80+ population of SMCs. (B) The proportion of F4/80+MHCII+ MΦs expressing MHCII to a high extent (MHCII+^high). (C) The fraction of CD11c+ DCs expressing the activation marker CD86. (D) The fraction of F4/80+MHCII+^low cells expressing CD86. (E) The fraction of F4/80+MHCII+^high cells expressing CD86, (▪) indicates mice infected with both <i>P. berghei</i> and <i>B. duttonii</i>, (○) <i>B. duttonii</i> infected animals, and (Δ) mice infected with <i>P. berghei</i>.</p

Dendritic cell responses are delayed during co-infection.

<p>Spleen mononuclear cells (SMC) were extracted on consecutive days from single and co-infected mice. Splenocytes were stained and acquired by flow cytometry (N = 6 per group), experiment performed in duplicate. (A) The percentage fraction of MHCII expressing cells in the CD11c+ population of SMCs. (B) The proportion of CD11c+MHCII+ DCs expressing MHCII to a high extent (MHCII+^high). (C) The fraction of CD11c+ DCs expressing the activation marker CD83. (D) The fraction of CD11c+MHCII+^low cells expressing CD83. (E) The fraction of CD11c+MHCII+^high cells expressing CD83, (▪) indicates mice infected with both <i>P. berghei</i> and <i>B. duttonii</i>, (○) <i>B. duttonii</i> infected animals, and (Δ) mice infected with <i>P. berghei</i>.</p

Co-infection causes brain endothelial damage and reduced L-Arginine levels.

<p>(A) Mice were monitored and scored for clinical signs of ECM using the rapid murine coma and behavior scale (RMCBS) (N = 6 per group; values are means ±SD, experiment performed in duplicate) (B) Images of brain tissue from mice co- and single infected day 5 p.i. stained with Hematoxylin-Eosin. (C) Plasma was sampled from infected mice, and the concentration of L-Arginine in plasma is shown. (▪) indicates mice infected with both <i>P. berghei</i> and <i>B. duttonii</i>, (○) <i>B. duttonii</i> infected animals and (Δ) mice infected with <i>P. berghei</i> (N = 10 per group; values are means ±SD, experiment performed in duplicate). (D) Concentration of N^G, N^G-dimethylarginine (ADMA) in plasma on day 5 p.i. UI is an acronym for uninfected mice. <i>P. berghei</i>/<i>B. duttonii</i> indicates mixed infection, P.b mice infected with <i>P. berghei</i> and B.d animals infected with <i>B. duttonii. *</i> indicates a significant difference of P<0.05 tested with Students t-test (N = 10 per group; values are means ±SD, experiment performed in duplicate).</p

Co-infection causes conflicting cytokine signaling and persisting nTreg populations.

<p>Mice were injected with 1×10⁷<i>P. berghei</i> NK65-infected RBCs and/or 1×10⁵<i>B. duttonii</i> 1120K3 bacteria, and then sacrificed at indicated time points (N = 6 per group). Cytokine level experiments performed in triplicate, nTreg experiment performed in duplicate. Plasma levels of cytokines were measured with multiplex ELISA (A) Plasma IL-10 concentration (pg/mL) (B) Plasma IL-1β concentration (pg/mL) (C) Plasma TNF-α concentration (pg/mL) (D) The percentage fraction of CD4+CD25^highFoxP3+ cells, in the total CD4+ cell pool in the spleen determined with flow cytometry. (▪) Represents mice infected with both <i>P. berghei</i> and <i>B. duttonii</i>, (○) indicates animals infected with <i>B. duttonii</i> and (Δ) mice infected with <i>P. berghei</i>.</p

The brain in co-infected mice contain high amounts of CD8+ cells and activated MΦs.

<p>Brains from mice infected with <i>P. berghei</i>, <i>B. duttonii</i> or both as per M&M were collected day 5 p.i. (N = 6 per group), experiments performed in duplicate (A) Cryo-sectioned tissue double stained for CD3 and CD8 positive cells and DNA visualized with DAPI. Representative regions in the frontal cortex were imaged by confocal laser microscopy. Tissue stained in absence of primary CD3 and CD8 antibodies served as negative controls. (B) The relative fold-change differences in absolute numbers of CD8+ cells. (C) The percentage frequency of CD86+ MΦs in the total pool of F4/80+ cells extracted from the brains (N = 6 per group, experiment performed in duplicate). (D) Cryo-sectioned brain tissue, immuno-stained for ICAM and VCAM.</p

Histograms indicating the frequency of mono-, bi- and tri-functional cell subsets in CD45RO⁺ and CD45RA⁺ T-cell populations of vaccinees.

<p>Mean values are illustrated throughout. The black axis indicates percentages for mono-functional IFN-γ-positive and all bi- and tri-functional T-cell subsets. The red axis indicates percentages for MIP-1β- and CD107a-positive mono-functional T cell subsets.</p

Levels of cytokines secreted by human PBMC after recall stimulation with ffLVS or ffSchu S4 for five days.

<p>Cytokine concentrations were measured in cell culture supernatants using multiplex analysis. Median values ± SEM from PBMC samples of 14–16 individuals per donor group are shown (black bars indicate convalescent patients; grey bars indicate LVS vaccinees; white bars indicate naïve donors). Statistically significant differences between immune and naïve donors are marked by asterisks (<i>P</i><0.05). For IL-7, 36 out of 40 values were below the detection limit and therefore not included in the data analysis.</p

Stimulation indices for the proliferative responses of human PBMC to recall stimulation with ffLVS for five days.

<p>Median values ± SEM per donor group of 12–16 individuals are shown. “pat” indicates convalescent tularemia patients, “vc” LVS vaccinees, and “nv” naïve individuals.</p

Heat map of Spearman correlation coefficients for iMFI values in various cell populations after intra-individual comparison for all donors.

<p>Integrated MFI values were obtained for all three functional markers (IFN-γ, MIP-1β, CD107a) in all mono-, bi- and trifunctional T-cell subsets and for all donors using <i>Clust</i> semi-automated gating. The iMFI values (24 values per PBMC sample, identified by numbers 1–24) from recall stimulation with 0.1 cfu ffLVS/PBMC were compared and lined up in a two-dimensional matrix. Correlated data are marked by shades of grey depending on the strength of the correlation coefficients; a coefficient above 0.7 is considered to indicate very strong correlation.</p

Histograms indicating the frequency of mono-, bi- and tri-functional cell subsets in CD45RO⁺ and CD45RA⁺ T-cell populations of non-vaccinated individuals.

<p>Mean values are illustrated throughout. The black axis indicates percentages for mono-functional IFN-γ-positive and all bi- and tri-functional T-cell subsets. The red axis indicates percentages for MIP-1β- and CD107a-positive mono-functional T cell subsets.</p