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.

Dendritic cell (DC)-based immunotherapy has been hampered by the lack of suitable methods for antigen delivery. Here, we use biodegradable poly(D,L-lactide-co-glycolide) microspheres (PLGA-MS) as carriers of peptides and proteins for antigen delivery to human monocyte-derived DC (MoDC). Compared to soluble proteins, MHC classes I and II-restricted presentation of PLGA-MS-encapsulated proteins and peptides by MoDC was markedly prolonged and proteins were presented 50-fold more efficiently on class I molecules. The vaccination of mice with DC loaded with PLGA-MS-encapsulated proteins raised strong and persisting cytotoxic T cell responses. In conclusion, antigen delivery via PLGA-MS markedly enhanced the duration of antigen presentation by human MoDC and the potency of DC-based vaccination.

Lymphocyte exit from lymph nodes and their recirculation into blood is controlled by the sphingolipid sphingosine 1-phosphate (S1P). The cellular receptor mediating lymphocyte exit is S1P 1 , one of five S1P receptors. Nonselective agonists for S1P receptors lead to blood lymphocyte count reduction. The effects of selective S1P 1 agonists on blood lymphocyte count and their impact in models of lymphocyte-mediated tissue inflammation have been less investigated. We describe here the general pharmacology of ponesimod, (Z,Z)-5-[3-chloro-4-((2R)-2,3-dihydroxy-propoxy)-benzylidene]-2-propylimino-3-o-tolylthiazolidin-4-one, a new, potent, and orally active selective S1P 1 agonist. Ponesimod activated S1P 1 -mediated signal transduction with high potency (EC 50 of 5.7 nM) and selectivity. Oral administration of ponesimod to rats led to a dosedependent decrease of blood lymphocyte count. After discontinuation of dosing, blood lymphocyte count returned to baseline within 48 h. Ponesimod prevented edema formation, inflammatory cell accumulation, and cytokine release in the skin of mice with delayed-type hypersensitivity. Ponesimod also prevented the increase in paw volume and joint inflammation in rats with adjuvant-induced arthritis. These data show that selective activation of S1P 1 using ponesimod leads to blood lymphocyte count reduction and efficacy in models of lymphocyte-mediated tissue inflammation. Immunomodulation with a rapidly reversible S1P 1 -selective agonist may represent a new therapeutic approach in lymphocyte-mediated autoimmune diseases.

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.

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.