Antigen Uptake After Intradermal Microinjection Depends on Antigen Nature and Formulation, but Not on Injection Depth.

The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.

Tc17 CD8+ T cells accumulate in murine atherosclerotic lesions, but do not contribute to early atherosclerosis development.

AIMS: CD8+ T cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces interleukin-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. METHODS AND RESULTS: Flow cytometry analysis of atherosclerotic lesions from apolipoprotein E-deficient 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-/- low-density lipoprotein receptor-deficient mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of interleukin-17A production in vivo. Moreover, Tc17 cells produced lower levels of interferon-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. CONCLUSION: These 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.

Atomic force microscopy measurements of anionic liposomes reveal the effect of liposomal rigidity on antigen-specific regulatory T cell responses.

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 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.

Anionic 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) liposomes induce antigen-specific regulatory T cells and prevent atherosclerosis in mice.

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 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 1,2-distearoyl-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 1q (C1q) 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.