Dendritic cells phagocytose pathogens leading to maturation and cross-presentation on MHC class I. We found that the efficiency of cross-priming in mice after vaccination with biodegradable poly(D,L-lactide-co-glycolide) microspheres (MSs) was enhanced when ovalbumin was coencapsulated together with either a CpG oligonucleotide or polyI:C as compared to co-inoculation of ovalbumin-bearing MS with soluble or separately encapsulated adjuvants. A single immunization with MS containing coencaspsulated CpG and ovalbumin yielded 9% SIINFEKL/H-2K(b) tetramer positive CTLs, production of IFN-gamma, efficient cytolysis, and protection from vaccinia virus infection. Taken together, coencapsulation of adjuvant and antigen is an important paradigm for the generation of potent CTL responses.

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Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell–cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell–cell interactions by pulsed light activation results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic, compact and round multicellular clusters under thermodynamic control form, while otherwise branched and loose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction- and diffusion-limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell–cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.

Poly(lactide-co-glycolide) (PLGA) microparticles (MP) possess immunological adjuvant properties. Yet, exploitation of their full potential has just begun. The purpose of this study was to explore opportunities arising from surface modifications, and attachment and entrapment of combinations of antigen and a Toll-like receptor (TLR) ligand. The cytotoxic T lymphocyte (CTL)-restricted OVA ovalbumin peptide SIINFEKL was microencapsulated into bare, chitosan-coated, and protamine-coated PLGA MP using a microextrusion-assisted solvent extraction process. A TLR-ligand (CpG ODN) was either covalently coupled or physically adsorbed onto the MP surface. The peptide encapsulation efficiency decreased from 71% for uncoated particles to 62% and 45% upon coating with chitosan and protamine, respectively. CpG adsorption efficiency decreased from 93% for protamine-coated particles to 19% and 8% for chitosan and bare particles. Release of the adsorbed CpG was slow and incomplete (23% within 7 days) with the protamine coating, intermediate (>90% within 3 days) with the chitosan coating, and immediate (100% within 3 h) without coating. Interestingly, only the uncoated PLGA MP with adsorbed CpG mediated a prominent CTL response in mice at 6 days after immunization, as determined from IFN-gamma release from antigen-specific CD8+ cells; failure of the other MP formulations was ascribed to the low release of antigen and CpG within the first week after immunization. The study illustrates novel opportunities for PLGA MP vaccines by combining antigens and immunostimulatory ligands.

The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8+ and CD8− DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8− DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8+ DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8− DCs and MΦs.

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres (MS) deliver antigens and toll like receptor (TLR) ligands to antigen presenting cells (APC) in vitro and in vivo. PLGA-MS-microencapsulated model antigens are efficiently presented on MHC class I and II molecules of dendritic cells and stimulate strong cytotoxic and T helper cell responses enabling the eradication of pre-existing model tumors. The application of tumor lysates as a source of antigen for immunotherapy has so far not been very successful also due to a lack of suitable delivery systems. In this study we used PLGA-MS with co-encapsulated tumor lysates and CpG oligodeoxynucleotides (CpG-ODN) as well as microencapsulated polyI:C in order to elicit anti-tumor responses. Immunization of mice with such mixtures of MS yielded substantial cytotoxic T cell (CTL) responses and interfered with tumor growth in TRAMP mice, a pre-clinical transgenic mouse model of prostate carcinoma, which has previously resisted dendritic cell-based therapy. As an important step towards clinical application of PLGA-MS, we could show that γ-irradiation of PLGA-MS sterilized the MS, without reducing their efficacy in eliciting CTL and anti-tumor responses in subcutaneous tumor grafts. Since PLGA is approved for clinical application, sterilized PLGA-MS containing tumor lysates and TLR ligands hold promise as anti-tumor vaccines against prostate carcinoma in humans.

In experimental tumor immunotherapy, incomplete Freund's adjuvant (IFA) has been considered as the “gold standard” for T‐cell vaccination in mice and humans in spite of its considerable adverse effects. Recently, we succeeded in eliciting strong CTL responses in mice after vaccination with biodegradable poly(D,L‐lactide‐co‐glycolide) (PLGA) microspheres (MS). In our study, we compared the immune response to IFA and PLGA‐MS containing ovalbumin (OVA) and CpG‐oligodeoxynucleotide (MS‐OVA/CpG) or we used a mixture of MS‐OVA/CpG and MS‐polyI:C. A single vaccination with MS‐OVA/CpG elicited long‐lasting titers of IgG1 and IgG2a, but only low IgE titers, and also the T‐cell response was biased toward Th1 differentiation. Antigen presentation to CD4+ and CD8+ cells and activation of a cytotoxic T‐cell response in mice vaccinated with PLGA‐MS and IFA lasted for over 3 weeks. Preconditioning of the injection site with TNF‐α and heterologous prime‐boost regimen further enhanced the cytotoxic response. PLGA‐MS were as efficient or superior to IFA in eradication of preexisting tumors and suppression of lung metastases. Taken together, PLGA‐MS are well‐defined, biodegradable and clinically compatible antigen carrier systems that compare favorably with IFA in their efficacy of tumor immunotherapy in mouse models and hence deserve to be tested for their effectiveness against human malignant diseases.