Enhanced dendritic cell percentages and activation and decreased IL-10 expressing dendritic cells after agonistic anti-TIGIT treatment.

<p>At sacrifice, blood (A) and spleen (B) cells were isolated and stained for dendritic cells and activation markers and analyzed by flow cytometry (n = 5 per group). The effect of agonistic anti-TIGIT on IL-10 expression by DCs was determined by culturing splenocytes with increasing concentrations of agonistic anti-TIGIT (C). *<i>P</i><0.05, **<i>P</i><0.01.</p

Agonistic anti-TIGIT treatment does not reduce more advanced atherosclerosis.

<p>Agonistic anti-TIGIT treatment (n = 12) and Armenian Hamster IgG treatment (n = 11) reduces atherosclerosis development in LDLr^−/− mice fed a Western-type diet for 8 weeks in comparison with PBS treatment (n = 12). Representative cross-sections of lesion formation in the three valves area of the aortic root stained with Oil-Red-O and hematoxylin are shown and lesion size was determined (A). Sections of the aortic root were stained for collagen using Masson’s trichrome staining. The percentage of collagen relative to the lesion size was determined (B). Furthermore, relative macrophage content was determined with a Moma-2 staining and quantified (C).</p

Agonistic anti-TIGIT treatment does not reduce initial atherosclerotic lesion development.

<p>No difference in atherosclerotic lesion size between agonistic anti-TIGIT, Armenian Hamster IgG and PBS treated LDLr^−/− mice fed a Western-type diet for 4 weeks. Representative cross-sections of lesion formation in the three valves area of the aortic root stained with Oil-Red-O and hematoxylin are shown and lesion size was determined (A). Sections of the aortic root were stained for collagen using Masson’s trichrome staining. The percentage of collagen relative to the lesion size was determined (B). Furthermore, relative macrophage content was determined with a Moma-2 staining and quantified (C).</p

Additional file 1: Figure S1. of Mast cell depletion in the preclinical phase of collagen-induced arthritis reduces clinical outcome by lowering the inflammatory cytokine profile

Study outline of performed arthritis experiments in RMB-DBA/1 mice. (A) Mast cell depletion in clinical phase of CIA. (B) Collagen antibody-induced arthritis in mast cell-depleted RMB-DBA/1 mice. (C) Mast cell depletion in preclinical phase. (DT diphtheria toxin, CFA complete Freund’s adjuvant, CII collagen type II, IFA incomplete Freund’s adjuvant). (TIF 6909 kb

Agonistic anti-TIGIT strongly inhibits T cell function.

<p>Splenocytes from Western-type diet fed mice (n = 3) were cultured for 72 hours with αCD3/αCD28 in the presence or absence of agonistic anti-TIGIT (0–30 µg/ml). Activated T cells (CD4⁺CD25⁺) were determined with flow cytometry (A). Proliferation was assessed by the amount of ³H-thymidine incorporation in dividing cells and is expressed as stimulation index (B) and by the amount of IL-2 produced by the splenocytes as determined with ELISA (C). Splenocytes of PBS, Armenian Hamster IgG and agonistic anti-TIGIT-treated mice (n = 5 per group) were cultured in the presence of αCD3/αCD28 stimulation and proliferation was assessed by the amount of 3H-thymidine incorporation expressed as stimulation index (D). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Upregulation of TIGIT expression on CD4⁺ T cells under hypercholesterolemic conditions.

<p>Representative FACS dot plots of TIGIT surface expression on CD4⁺ T cells isolated from LDLr^−/− mice fed a Chow diet or a Western-type (WT) diet (A). Splenocytes from LDLr^−/− mice fed a Chow diet (n = 3) and Western-type diet (n = 3) were cultured for 48 hours in the presence or absence of anti-CD3/anti-CD28. Representative dot plots (B) and the mean percentage of TIGIT expressing CD4⁺ T cells in 4 different conditions (C) were obtained by flow cytometry. *<i>P</i><0.05.</p

Additional file 2: Figure S2. of Mast cell depletion in the preclinical phase of collagen-induced arthritis reduces clinical outcome by lowering the inflammatory cytokine profile

DT treatment in wild-type control animals. (A) Progression of CIA was monitored by clinical scoring of C57Bl/6-DBA/1 mice injected with either PBS or DT. (B) Serum levels of IL-6, IL-17, IFN-γ and IL-10 were quantified in serum of PBS and DT-treated C57Bl/6-DBA/1 mice. (C) FACS analysis of the blood compartment for peripheral leukocytes in PBS- and DT-treated C57Bl/6-DBA/1 mice. (D) Cytokine release of splenocytes from PBS- or DT-injected C57Bl/6-DBA/1 mice after restimulation with either αCD3/28 or collagen type II, or unstimulated medium control (n = 15/group). (E) Splenocytes from PBS- and DT-injected C57Bl/6-DBA/1 mice were stained intracellularly for IL-17 after stimulation with anti-CD3/28 (n = 15/group). Splenocytes from PBS- and DT-injected C57Bl/6-DBA/1 mice were stained intracellularly for FoxP3. (** p < 0.01, **** p < 0.001) All graphs n = 15/group). (TIF 7185 kb

Survival curve of angiotensin II treated LDLr^-/- and LDLr^-/-CD1d^-/- mice.

<p>Osmotic pumps filled with Ang II were implanted in LDLr^-/- (■, n = 12) and LDLr^-/-CD1d^-/- (●, n = 11) which were fed a Western-type of diet for 1 week. After implantation, LDLr^-/- mice started to die due to rupture of the abdominal aorta (A and B). Percent survival per group is depicted (C). Statistical analysis was performed using the Mantel-Cox test. *<i>P</i><0.05.</p

Increased NKT cell activity upon angiotensin II treatment.

<p>Osmotic pumps filled with PBS (n = 5) or Ang II (n = 5) were implanted in LDLr^-/- mice fed a Western type diet for 1 week. Two weeks after pump placement, the mice were sacrificed and the percentage of NKT (Tetramer⁺) cells in spleen and liver (A, white bars represent PBS treated mice, black bars Ang II treated mice) and the activation status of splenic NKT cells (B and C) were determined by FACS analysis. To confirm these effects, antigen-presenting cells (APCs) isolated from bone marrow of LDLr^-/- (white bars) and LDLr^-/-CD1d^-/- mice (black bars) were incubated with α-GalCer, AngII or a combination of both. Four hours after incubation, the APCs were co-cultured with DN32.D3 hybridoma cells. After 24 hours, the IL-2 concentration in the supernatant was determined (D). All values are mean±SEM and statistical analysis was performed using the unpaired two-tailed student’s T-test (A-C) or one-way ANOVA (D) *P<0.05, **P<0.01, ****P<0.0001.</p

Cytokine dependent decrease in vSMC viability after type I NKT cell activation.

<p>Splenocytes were cultured in presence of type I NKT cell specific ligands α-GalCer or OCH (50, 100 and 200 ng/ml) for 48 hours. Subsequently, supernatant of the splenocytes was added to vSMCs for 72 hours and viability of the vSMCs was assessed by an MTT assay (A). In addition, supernatant of the α-GalCer (200 ng/ml) stimulated splenocytes was pre-incubated with α-IFN-γ antibodies (B) and supernatant of OCH (200 ng/ml) stimulated splenocytes with α-IL4 (C) and α-IL10 (D) antibodies prior to addition of the supernatant to vSMCs. All values are mean±SEM and statistical analysis was performed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.</p