CD8+ T-cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces IL-17. The role of this CD8+ T-cell subset in... Show moreCD8+ T-cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces IL-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis.\nFlow cytometry analysis of atherosclerotic lesions from apoE-/- mice revealed a pronounced increase in RORγt+CD8+ T-cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8-/-LDLr-/- mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of IL-17A production in vivo. Moreover, Tc17 cells produced lower levels of IFN-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice.\nThese findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart.\nCD8+ T-cells are present in high numbers in human atherosclerotic plaques, however their role in inflammation and the pathogenesis of atherosclerosis remains elusive. Our results indicate that the majority of CD8+ T-cells in atherosclerotic plaques of mice have lost their ability to produce the pro-inflammatory cytokine IFN-γ and gain traits of IL-17-producing CD8+ T-cells (Tc17 cells). We show that this subset of CD8+ T-cells is less atherogenic then IFN-γ producing Tc1 cells. Show less
This thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and... Show moreThis thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and atherosclerosis. For each of these treatment strategies, the liposomal formulation was tailored to obtain the desired therapeutic effect. Chapter 2 reviews some of the most important physicochemical properties (size, shape, and rigidity) that determine the immunological effects of liposomes and other nanoparticles. In chapter 3 we present a detailed study on the effect of the rigidity of anionic liposomes, as measured by atomic force microscopy, on antigen-specific regulatory T-cell (Treg) responses. In chapter 4, we show that our optimized anionic liposomes can induce potent antigen-specific Treg responses, and can be used to delay atherosclerosis progression in a mouse model. Chapter 5 also focuses on liposomal treatment of atherosclerosis, but here targeted liposomes were prepared to successfully deliver a small molecule to foam cells in atherosclerotic plaques. In Chapter 6, we used cationic liposomes in combination with an adjuvant for cancer immunotherapy in mice. Finally, we summarize the overall findings in chapter 7 and discuss perspectives of using liposomes for vaccination and targeted drug delivery. Show less
Atherosclerosis is characterized by the retention of lipids in foam cells in the arterial intima. The liver X receptor (LXR) agonist GW3965 is a promising therapeutic compound, since it induces... Show moreAtherosclerosis is characterized by the retention of lipids in foam cells in the arterial intima. The liver X receptor (LXR) agonist GW3965 is a promising therapeutic compound, since it induces reverse cholesterol transport in foam cells. However, hepatic LXR activation increases plasma and liver lipid levels, inhibiting its clinical development. Herein, a formulation that specifically enhances GW3965 deposition in the atherosclerotic lesion is aimed to be developed. GW3965 is encapsulated in liposomes functionalized with the cyclic peptide Lyp-1 (CGNKRTRGC), which binds the p32 receptor expressed on foam cells. These liposomes show preferential uptake by foam cells in vitro and higher accumulation in atherosclerotic plaques in mice compared to non-targeted liposomes as determined by in vivo imaging. Flow cytometry analysis of plaques reveals increased retention of Lyp-1 liposomes in atherosclerotic plaque macrophages compared to controls (p < 0.05). Long term treatment of established plaques in LDLR -/- mice with GW3965-containing Lyp-1 liposomes significantly reduces plaque macrophage content by 50% (p < 0.01). Importantly, GW3965-containing Lyp-1 liposomes do not increase plasma or hepatic lipid content. Thus, GW3965-containing Lyp-1 liposomes successfully target the atherosclerotic macrophages allowing plaque stabilization without commonly observed side effects of LXR agonists. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano Vigario, F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomescomposed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigenspecificTreg responses. We hypothesized that altering the rigidity of these liposomes while maintaining theirsize and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes isaffected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part bythe presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL(1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa),DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG(494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-deriveddendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation betweenliposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responsesin vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measureliposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Duijn, J. van; Lozano Vigario, F.; Leboux, R.; Veelen, P. van; Jiskoot, W.; ... ; Slütter, B. 2019
CD8+ T-cells can be atheroprotective in clinically relevant advanced stages of atherosclerosis, as their depletion results in less stable lesions with a more inflammatory phenotype. However, the... Show moreCD8+ T-cells can be atheroprotective in clinically relevant advanced stages of atherosclerosis, as their depletion results in less stable lesions with a more inflammatory phenotype. However, the phenotype and function of these cells in the lesional microenvironment remains to be determined. Here, we address how the atherosclerotic environment affects the functionality of CD8+ T-cells.We compared the cytokine production of CD8+ T-cells derived from spleens and aortas of apoE-/- mice with advanced atherosclerosis by flow cytometry.CD8+ T-cells isolated from atherosclerotic lesions produced lower amounts of IFN-γ and TNF-α than their splenic counterparts. The observed dysfunctional phenotype of the lesion-derived CD8+ T-cells was associated with an increased expression of the ectonucleotidase CD39, which converts inflammatory extracellular ATP into immunomodulatory adenosine. Indeed, pharmacological inhibition of CD39 in apoE-/- mice partly restored cytokine production by CD8+ T-cells. Using a bone-marrow transplantation approach, we showed that induction of CD39 was a consequence of antigen-specific CD8+ T-cell activation via T-cell receptor (TCR) signaling within the lesions. Importantly, analysis of human endarterectomy samples showed a clear microenvironment specific upregulation of CD39 on CD8+ T-cells in the plaques of human patients compared to matched CD8+ T-cells from the blood .Our results indicate that the continuous TCR signaling in the atherosclerotic plaque induces an immune regulatory CD8+ T-cell phenotype that is associated with decreased cytokine production through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosis patients. This provides a new understanding of atheroprotective immune regulation by CD8+ T-cells. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.A.C.; Wezel, A.; Smeets, H.; ... ; Slütter, B.A. 2019
CD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs.... Show moreCD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs. Theatherosclerotic lesion, however, is a complex microenvironment containing a plethora of inflammatory signals,which may affect CD8+T-cell activation. Here, we address how this environment affects the functionality ofCD8+T-cells. We compared the cytokine production of CD8+T-cells derived from spleens and en-zymatically digested aortas ofapoE−/−mice with advanced atherosclerosis byflow cytometry. Aortic CD8+T-cells produced decreased amounts of IFN-γand TNF-αcompared to their systemic counterparts. The observeddysfunctional phenotype of the lesion-derived CD8+T-cells was not associated with classical exhaustion mar-kers, but with increased expression of the ectonucleotidase CD39. Indeed, pharmacological inhibition of CD39 inapoE−/−mice partly restored cytokine production by CD8+T-cells. Using a bone-marrow transplantation ap-proach, we show that TCR signaling is required to induce CD39 expression on CD8+T-cells in atheroscleroticlesions. Importantly, analysis of human endarterectomy samples showed a strong microenvironment specificupregulation of CD39 on CD8+T-cells in the plaques of human patients compared to matched blood samples. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in thevessel wall induces an immune regulatory CD8+T-cell phenotype that is associated with decreased cytokineproduction through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosispatients. This provides a new understanding of immune regulation by CD8+T-cells in atherosclerosis. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.; Wezel, A.; Smeets, H.; ... ; Slütter, B. 2019
CD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs.... Show moreCD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs. Theatherosclerotic lesion, however, is a complex microenvironment containing a plethora of inflammatory signals,which may affect CD8+T-cell activation. Here, we address how this environment affects the functionality ofCD8+T-cells. We compared the cytokine production of CD8+T-cells derived from spleens and en-zymatically digested aortas ofapoE−/−mice with advanced atherosclerosis byflow cytometry. Aortic CD8+T-cells produced decreased amounts of IFN-γand TNF-αcompared to their systemic counterparts. The observeddysfunctional phenotype of the lesion-derived CD8+T-cells was not associated with classical exhaustion mar-kers, but with increased expression of the ectonucleotidase CD39. Indeed, pharmacological inhibition of CD39 inapoE−/−mice partly restored cytokine production by CD8+T-cells. Using a bone-marrow transplantation ap-proach, we show that TCR signaling is required to induce CD39 expression on CD8+T-cells in atheroscleroticlesions. Importantly, analysis of human endarterectomy samples showed a strong microenvironment specificupregulation of CD39 on CD8+T-cells in the plaques of human patients compared to matched blood samples. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in thevessel wall induces an immune regulatory CD8+T-cell phenotype that is associated with decreased cytokineproduction through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosispatients. This provides a new understanding of immune regulation by CD8+T-cells in atherosclerosis. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano, Vigario F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomescomposed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigenspecificTreg responses. We hypothesized that altering the rigidity of these liposomes while maintaining theirsize and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes isaffected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part bythe presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL(1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa),DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG(494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-deriveddendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation betweenliposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responsesin vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measureliposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano, Vigario F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is oftenassociated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomescomposed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigenspecificTreg responses. We hypothesized that altering the rigidity of these liposomes while maintaining theirsize and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes isaffected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part bythe presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL(1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa),DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG(494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-deriveddendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation betweenliposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responsesin vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measureliposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Leboux, R.J.T.; Glandrup, M.; Duijn, J. van; Lozano, Vigario F.; Neustrup, M.A.; ... ; Slütter, B. 2019
Regulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded... Show moreRegulatory T cells (Tregs) are vital for maintaining a balanced immune response and their dysfunction is often associated with auto-immune disorders. We have previously shown that antigen-loaded anionic liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) and cholesterol can induce strong antigen-specific Treg responses. We hypothesized that altering the rigidity of these liposomes while maintaining their size and surface charge would affect their capability of inducing Treg responses. The rigidity of liposomes is affected in part by the length and saturation of carbon chains of the phospholipids in the bilayer, and in part by the presence of cholesterol. We used atomic force microscopy (AFM) to measure the rigidity of anionic OVA323-containing liposomes composed of different types of PC and PG, with or without cholesterol, in a molar ratio of 4:1(:2) distearoyl (DS)PC:DSPG (Young's modulus (YM) 3611 ± 1271 kPa), DSPC:DSPG:CHOL (1498 ± 531 kPa), DSPC:dipalmitoyl (DP)PG:CHOL (1208 ± 538), DPPC:DPPG:CHOL (1195 ± 348 kPa), DSPC:dioleoyl (DO)PG:CHOL (825 ± 307 kPa), DOPC:DOPG:CHOL (911 ± 447 kPa), and DOPC:DOPG (494 ± 365 kPa). Next, we assessed if rigidity affects the association of liposomes to bone marrow-derived dendritic cells (BMDCs) in vitro. Aside from DOPC:DOPG liposomes, we observed a positive correlation between liposomal rigidity and cellular association. Finally, we show that rigidity positively correlates with Treg responses in vitro in murine DCs and in vivo in mice. Our findings underline the suitability of AFM to measure liposome rigidity and the importance of this parameter when designing liposomes as a vaccine delivery system. Show less
Benne, N.; Duijn, J. van; Lozano Vigario, F.L.; Leboux, R.J.T.; Veelen, P. van; Kuiper, J.; ... ; Slütter, B. 2018
Atherosclerosis is the predominant underlying pathology of many types of cardiovascular disease and is one of the leading causes of death worldwide. It is characterized by the retention of oxidized... Show moreAtherosclerosis is the predominant underlying pathology of many types of cardiovascular disease and is one of the leading causes of death worldwide. It is characterized by the retention of oxidized low-density lipoprotein (ox-LDL) in lipid-rich macrophages (foam cells) in the intima of arteries. Autoantigens derived from oxLDL can be used to vaccinate against atherosclerosis. However, a major challenge is the induction of antigen-specific Tregs in a safe and effective way. Here we report that liposomes containing the anionic phospholipid 1,2distearoyl-sn-glycero-3-phosphoglycerol (DSPG) induce Tregs that are specific for the liposomes' cargo. Mechanistically, we show a crucial role for the protein corona that forms on the liposomes in the circulation, as uptake of DSPG-liposomes by antigen-presenting cells is mediated via complement component lq (Clq) and scavenger receptors (SRs). Vaccination of atherosclerotic mice on a western-type diet with DSPG-liposomes encapsulating an LDL-derived peptide antigen significantly reduced plaque formation by 50% and stabilized the plaques, and reduced serum cholesterol concentrations. These results indicate that DSPG-liposomes have potential as a delivery system in vaccination against atherosclerosis. Show less
Benne, N.; Duijn, J. van; Vigario, F.L.; Leboux, R.J.T.; Veelen, P. van; Kuiper, J.; ... ; Stutter, B. 2018
Human endarterectomy samples analyzed by flow cytometry showed a negative correlation between the percentage of CD8+ T-cells and macrophages, suggesting a possible protective role for these cells... Show moreHuman endarterectomy samples analyzed by flow cytometry showed a negative correlation between the percentage of CD8+ T-cells and macrophages, suggesting a possible protective role for these cells in lesion development. To further test this hypothesis, LDLr-/- mice were fed a Western-type diet (WTD) for 10 weeks to induce atherosclerosis, after which they received CD8α-depleting or isotype control antibody for six weeks. Depletion of CD8+ T-cells in advanced atherosclerosis resulted in less stable lesions, with significantly reduced collagen content in the trivalve area, increased macrophage content and increased necrotic core area compared to controls. Mechanistically, we observed that CD8 depletion specifically increased the fraction of Th1 CD4+ T-cells in the lesions. Treatment of WTD-fed LDLr-/- mice with a FasL-neutralizing antibody resulted in similar changes in macrophages and CD4+ T-cell skewing as CD8+ T-cell depletion. T lymphocytes play an important role in atherosclerosis development, but the role of the CD8+ T-cell remains debated, especially in the clinically relevant advanced stages of atherosclerosis development. Here, we set out to determine the role of CD8+ T-cells in advanced atherosclerosis. These findings demonstrate for the first time a local, protective role for CD8+ T-cells in advanced atherosclerosis, through limiting accumulation of Th1 cells and macrophages, identifying a novel regulatory mechanism for these cells in atherosclerosis. Methods and Results Aims Conclusion Show less
Varypataki, E.M.; Benne, N.; Bouwstra, J.; Jiskoot, W.; Ossendorp, F. 2017
Atherosclerosis is a chronic inflammatory disease of the large and medium sized arteries affecting millions of people world-wide. It is characterised by sub-endothelial retention of low density... Show moreAtherosclerosis is a chronic inflammatory disease of the large and medium sized arteries affecting millions of people world-wide. It is characterised by sub-endothelial retention of low density lipoprotein (LDL), which, over time, leads to the formation of an atherosclerotic plaque. Regulatory T cells (Treg) have been shown to reduce chronic inflammation in atherosclerosis and provide a protective effect. Whereas repeated immunisations with high doses of plaque-specific antigens can induce Treg and reduce the atherosclerotic plaques, particulate formulations may require fewer immunisations and less antigen by specific targeting of the immune system. Therefore, we optimised liposomal formulations to elicit potent Treg responses. Liposomes (average diameter 150–200 nm) consisting of the lipids DSPC, DSPG and cholesterol in different molar ratios were loaded with an ovalbumin-derived CD4+ T cell-specific peptide (OVA323). Murine BMDCs were pulsed with formulations and subsequently cultured with OT-II transgenic CD4+T cells. Compared to free OVA323 or OVA323 mixed with empty liposomes, encapsulation of OVA323 induced increased numbers of Treg as measured by FoxP3 expression. Interestingly, liposomes with the composition 4:1:2 DSPC:DSPG:cholesterol induced the highest Treg responses and also prevented Th1 differentiation as determined by IFN-γ production. Subsequent studies suggest that the 4:1:2 liposomes achieve the optimal balance of antigen loading, flexibility and surface charge to induce Treg. In conclusion, this indicates that liposome composition influences Treg differentiation and liposomes may be used to elicit antigen-specific Treg responses for atherosclerosis vaccination. Show less
Benne, N.; Duijn, J. van; Kuiper, J.; Jiskoot, W.; Slütter, B. 2016
Particulate carrier systems are promising drug delivery vehicles for subunit vaccination as they can enhance and direct the type of T cell response. In order to develop vaccines with optimal... Show moreParticulate carrier systems are promising drug delivery vehicles for subunit vaccination as they can enhance and direct the type of T cell response. In order to develop vaccines with optimal immunogenicity, a thorough understanding of parameters that could affect the strength and quality of immune responses is required. Pathogens have different dimensions and stimulate the immune system in a specific way. It is therefore not surprising that physicochemical characteristics of particulate vaccines, such as particle size, shape, and rigidity, affect multiple processes that impact their immunogenicity. Among these processes are the uptake of the particles from the site of administration, passage through lymphoid tissue and the uptake, antigen processing and activation of antigen-presenting cells. Herein, we systematically review the role of the size, shape and rigidity of particulate vaccines in enhancing and skewing T cell response and attempted to provide a "roadmap" for rational vaccine design. Show less
