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

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 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

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

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

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

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

Primer pairs used for quantitative gene expression analysis.

<p>Primer pairs used for quantitative gene expression analysis.</p

Increased expression of matrix degrading molecules by vSMCs and macrophages after type I NKT cell activation.

<p>Splenocytes of LDLr^-/- mice were incubated with or without type I NKT cell specific ligands α-GalCer and OCH for 48 hours. Subsequently, supernatant of the splenocytes was added to vSMCs (A-C) or macrophages (D-F) for 72 hours after which the mRNA expression of the matrix degrading molecules Cathepsin S (A and D), MMP-12 (B and E), Cathepsin K (C) and Cathepsin L (F) was determined. All values are mean±SEM and statistical analysis was performed using one-way ANOVA. *P<0.05, ***P<0.001, ****P<0.0001.</p