Original Article
Virosome-bound antigen enhances DC-dependent specific CD4+ T cell stimulation, inducing a Th1 and Treg profile in vitro

https://doi.org/10.1016/j.nano.2017.02.004Get rights and content

Abstract

There is considerable interest to develop antigen-carriers for immune-modulatory clinical applications, but insufficient information is available on their effects on antigen-presenting cells. We employed virosomes coupled to ovalbumin (OVA) to study their interaction with murine bone marrow-derived dendritic cells (BMDCs) and modulation of downstream T cell responses. BMDCs were treated in vitro with virosomes or liposomes prior to determining BMDC phenotype, viability, and intracellular trafficking. Antigen-specific CD4+ T cell activation was measured by co-culture of BMDCs with DO11.10 CD4+ T cells. Compared to liposomes, virosomes were rapidly taken up. Neither nanocarrier type affected BMDC viability, nor did a moderate degree of activation differ for markers such as CD40, CD80, CD86. Virosome uptake occurred via clathrin-mediated endocytosis and phagocytosis, with co-localization in late endosomes. Only BMDCs treated with OVA-coupled virosomes induced enhanced OVA-specific CD4+ T cell proliferation. Antigen-coupled virosomes are endowed with an intrinsic ability to modulate DC-dependent adaptive immune responses.

Graphical Abstract

Virosomes and liposomes are spherical unilamellar structures comprised of phosphilipids. Virosomes additionally contain fractions from the influenza virus envelope, hemagglutinin (HA) and neuraminidase (NA). To study the interaction of such nanocarriers with dendritic cells (DCs), we fluorescently labeled and coupled them with ovalbumin (OVA). Murine bone marrow-derived dendritic cells (BMDCs) were treated with virosomes or liposomes prior to determining OVA-specific CD4+ T cell proliferation in an in vitro co-culture of BMDCs and T cells. BMDCs treated with OVA-coupled virosomes induced enhanced antigen-specific CD4+ T cell proliferation compared to treatment with empty virosomes and soluble OVA protein or OVA-coupled liposomes.

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Section snippets

Mice

8-12 week old female BALB/c and BALB/c DO11.10 T cell receptor transgenic mice were bred specific pathogen free at the Department of Clinical Research, University of Bern (Bern, Switzerland) in compliance with the Swiss Federal Veterinary Office guidelines under animal experimentation permission (BE71/15).

Cell cultures

BMDCs were derived from bone marrow of BALB/c mice as previously described.22 Briefly, BMDCs were allowed to differentiate for 8 days at 37 °C in a 5% CO2 humidified incubator in Iscove's Modified

Nanocarrier characterization

Both nanocarriers, virosomes and liposomes (Figure 1), were extensively characterized for size, homogeneity, zeta potential, HA, OVA, as well as endotoxin content. Size was measured by dynamic light scattering (DLS) and routinely resulted in a hydrodynamic diameter of approximately 100 nm (Table 1). Nanocarrier quality was uniform and reproducible throughout all experiments. Protein concentration of both OVA and HA was determined by performing SDS-PAGE, Spotblot and Western blot, and HA

Discussion

Influenza virosomes and liposomes are already being utilized in a broad range of clinical and therapeutic applications such as vaccinations10, 15, 29 or cancer therapy.30, 31, 32, 33, 34 To date insufficient information is available on the interaction and the fate of nanocarriers when taken up by APCs. With the current study we investigated how interaction of such bio-mimetic antigen carriers affect DC activation, function and T cell stimulatory capacity. We showed that both virosomes and

Acknowledgments

We gratefully acknowledge the expert technical assistance provided by Sandra Barnowski, Andrea Stokes and Dr. Amiq Gazdhar.

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    Financial and competing interest disclosure: This study was funded by the Swiss National Science Foundation SNF 146249. The authors have no other affiliations or financial involvement with any organization or entity with a financial interest.

    Funding: Flow Cytometry experiments were performed with the support of the FACSLab at the University of Bern, Switzerland. Microscopy acquisition and analysis were performed with the support of the Microscopy Imaging Center at the University of Bern, Switzerland. This study was supported by the Swiss National Science Foundation SNF grants 146249 and 316030_145003.

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    Contributed equally to this work as senior authors.

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