PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation.

Dendritic cells (DC) are key players in the initiation and modulation of adaptive immune responses due to their ability to acquire and present antigen and stimulate T cells. For the induction of effector T cell functions, antigen must be presented by activated DC. In this study, we have compared uptake of antigen by mouse DC in the presence of different Toll-like receptor (TLR) agonists, which are potent inducers of DC activation. Here we show that the reduction in uptake of soluble antigen in the presence of the viral double-stranded RNA (dsRNA) analogues polyinosinic-polycytidylic acid and Ampligen is independent of TLR-mediated DC activation. A reduction in antigen uptake by bone marrow-derived and splenic DC was also observed in response to other RNA homopolymers such as polyinosinic and polyguanylic acids, which are known inhibitors of scavenger receptor-mediated endocytosis. Pinocytosis and mannose receptor-mediated uptake of soluble antigen were not affected by any of the tested nucleic acids. The reduction in antigen uptake by dsRNA did not negatively influence the T cell stimulating properties of the DC. In summary, we conclude that the decrease in antigen endocytosis observed in the presence of a variety of TLR agonists is independent of TLR signalling and is caused by competition for specific surface receptors that are involved in the uptake of these TLR agonists and the antigen.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Alteration of antigen-independent immunologic synapse formation between dendritic cells from HLA-B27-transgenic rats and CD4+ T cells: selective impairment of costimulatory molecule engagement by mature HLA-B27.

OBJECTIVE: To investigate the molecular mechanism responsible for the reduced capacity of dendritic cells (DCs) from HLA-B27-transgenic rats to form conjugates with naive T cells. METHODS: We monitored interactions between DCs derived from HLA-B27-transgenic, HLA-B7-transgenic control, and nontransgenic rats and naive CD4+ T cells. Chemoattraction was studied in Transwell assays, and the formation of an immunologic synapse was examined by videomicroscopy and electron microscopy. Involvement of specific molecules in the defective interaction was examined in antibody-blocking assays. RESULTS: T cells migrated normally toward B27 DCs, but upon contact, the frequency of T cells undergoing a Ca2+ response was decreased, indicating impaired immunologic synapse formation. The immunologic synapse formed between B27 DCs and T cells appeared to be normal, as assessed by electron microscopy and by the Ca2+ response. Blocking lymphocyte function-associated antigen 1 on T cells or blocking activated leukocyte cell adhesion molecules on DCs inhibited an equivalent proportion of conjugates from forming between B27 or control DCs and T cells, whereas blocking CD86 on DCs and blocking CD28, CD2, or CD4 on T cells inhibited a greater number of conjugates from forming with control DCs, indicating specific involvement of costimulatory molecules in the reduced formation of conjugates with B27 DCs. Mature B27 molecules on the DC surface were responsible for this decreased formation of conjugates. CONCLUSION: In the HLA-B27-transgenic rat model of spondylarthropathy, mature B27 molecules expressed by DCs impair the formation of an antigen-independent immunologic synapse with naive CD4+ T cells by interfering with the engagement of costimulatory molecules. This phenomenon could potentially affect the production and/or maintenance of regulatory T cells and contribute to the expansion of pathogenic CD4+ T cells.

Two HLA-B27 alleles differently associated with spondylarthritis, B*2709 and B*2705, display similar intracellular trafficking and oligomer formation.

OBJECTIVE: To examine whether and to what extent the intracellular trafficking features of HLA-B*2705, which is associated with the development of spondylarthritis (SpA), differ from those of HLA-B*2709 and HLA-B*0702, which are not associated with SpA. METHODS: HeLa cells were transfected with complementary DNA encoding for HLA-B proteins fused to Renilla luciferase or yellow fluorescent protein. The subcellular distribution of properly folded and unfolded/misfolded HLA-B proteins was examined by flow cytometry and confocal microscopy of cells labeled with ME1 and HC-10 antibodies, respectively. HLA-B/HLA-B interactions were monitored in endoplasmic reticulum (ER)- and plasma membrane-enriched subcellular fractions, by bioluminescence resonance energy transfer (BRET). RESULTS: All 3 HLA-B alleles displayed a similar distribution pattern (properly folded heavy chain at the cell surface, unfolded/misfolded proteins only in the cytoplasm). By means of BRET, we provided evidence that both HLA-B*2705 and HLA-B*2709 formed more oligomers in the ER and the plasma membrane than did HLA-B*0702. The propensity of HLA-B*2705 to form oligomers in the ER was partly attributable to residue Cys(67) of the molecule. For all 3 alleles, increased expression of HLA-B proteins was associated with intracytoplasmic accumulation of unfolded/misfolded proteins and intracellular vesicles, probably corresponding to expanded ER-Golgi intermediate compartments, in which these proteins accumulated together with the stress sensor BiP. CONCLUSION: Our results suggest that the difference in disease susceptibility conferred by HLA-B*2705 and HLA-B*2709 cannot be explained by their different propensity to form dimers or misfolded proteins, thus presumably implicating other, still unknown factors.