How B cells drive T-cell responses: A key role for cross-presentation of antibody-targeted antigens.

In this review we discuss an underexposed mechanism in the adaptive immune system where B cell and T cell immunity collaborate. The main function of B cell immunity is the generation of antibodies which are well known for their high affinity and antigen-specificity. Antibodies can bind antigens in soluble form making so-called immune complexes (ICs) or can opsonize antigen-exposing cells or particles for degradation. This leads to well-known effector mechanisms complement activation, antibody-dependent cytotoxicity and phagocytosis. What is less realized is that antibodies can play an important role in the targeting of antigen to dendritic cells (DCs) and thereby can drive T cell immunity. Here we summarize the studies that described this highly efficient process of antibody-mediated antigen uptake in DCs in vitro and in vivo. Only very low doses of antigen can be captured by circulating antibodies and subsequently trapped by DCs in vivo. We studied the handling of these ICs by DCs in subcellular detail. Upon immune complex engulfment DCs can sustain MHC class I and II antigen presentation for many days. Cell biological analysis showed that this function is causally related to intracellular antigen-storage compartments which are functional endolysosomal organelles present in DCs. We speculate that this function is immunologically very important as DCs require time to migrate from the site of infection to the draining lymph nodes to activate T cells. The implications of these findings and the consequences for the immune system, immunotherapy with tumor-specific antibodies and novel vaccination strategies are discussed.

Saponin-based adjuvants enhance antigen cross-presentation in human CD11c+ CD1c+ CD5- CD163+ conventional type 2 dendritic cells.

BACKGROUND: Adjuvants are key for effective vaccination against cancer and chronic infectious diseases. Saponin-based adjuvants (SBAs) are unique among adjuvants in their ability to induce robust cell-mediated immune responses in addition to antibody responses. Recent preclinical studies revealed that SBAs induced cross-presentation and lipid bodies in otherwise poorly cross-presenting CD11b+ murine dendritic cells (DCs). METHOD: Here, we investigated the response of human DC subsets to SBAs with RNA sequencing and pathway analyses, lipid body induction visualized by laser scanning microscopy, antigen translocation to the cytosol, and antigen cross-presentation to CD8+ T cells. RESULTS: RNA sequencing of SBA-treated conventional type 1 DC (cDC1) and type 2 DC (cDC2) subsets uncovered that SBAs upregulated lipid-related pathways in CD11c+ CD1c+ cDC2s, especially in the CD5- CD163+ CD14+ cDC2 subset. Moreover, SBAs induced lipid bodies and enhanced endosomal antigen translocation into the cytosol in this particular cDC2 subset. Finally, SBAs enhanced cross-presentation only in cDC2s, which requires the CD163+ CD14+ cDC2 subset. CONCLUSIONS: These data thus identify the CD163+ CD14+ cDC2 subset as the main SBA-responsive DC subset in humans and imply new strategies to optimize the application of saponin-based adjuvants in a potent cancer vaccine.

Simplified Monopalmitoyl Toll-like Receptor 2 Ligand Mini-UPam for Self-Adjuvanting Neoantigen-Based Synthetic Cancer Vaccines.

Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3 CysSK4 . A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells.

Sustained cross-presentation capacity of murine splenic dendritic cell subsets in vivo.

An exclusive feature of dendritic cells (DCs) is their ability to cross-present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen-antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α+ DCs showed sustained MHC class I-peptide specific CD8+ T-cell activation for more than 4 days. CD8α- DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α- DCs were able to present antigen in MHC class II to specific CD4+ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T-cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T-cell activation and have distinct roles in antigen presentation to specific T cells in vivo.

Adjuvants Enhancing Cross-Presentation by Dendritic Cells: The Key to More Effective Vaccines?

Over the last decades, vaccine development has advanced significantly in pursuing higher safety with less side effects. However, this is often accompanied by a reduction in vaccine immunogenicity and an increased dependency on adjuvants to enhance vaccine potency. Especially for diseases like cancer, it is important that therapeutic vaccines contain adjuvants that promote strong T cell responses. An important mode of action for such adjuvants is to prolong antigen exposure to dendritic cells (DCs) and to induce their maturation. These mature DCs are extremely effective in the activation of antigen-specific T cells, which is a pre-requisite for induction of potent and long-lasting cellular immunity. For the activation of CD8+ cytotoxic T cell responses, however, the exogenous vaccine antigens need to gain access to the endogenous MHCI presentation pathway of DCs, a process referred to as antigen cross-presentation. In this review, we will focus on recent insights in clinically relevant vaccine adjuvants that impact DC cross-presentation efficiency, including aluminum-based nanoparticles, saponin-based adjuvants, and Toll-like receptor ligands. Furthermore, we will discuss the importance of adjuvant combinations and highlight new developments in cancer vaccines. Understanding the mode of action of adjuvants in general and on antigen cross-presentation in DCs in particular will be important for the design of novel adjuvants as part of vaccines able to induce strong cellular immunity.

Synthesis and evaluation of fluorescent Pam3Cys peptide conjugates.

Chirally pure R- and S-epimers of TLR2 ligand Pam3CysSK4 were prepared and separately conjugated to an OVA model epitope, in which lysine was replaced by azidonorleucine. The azide function in the conjugate permitted labelling with different fluorophores by use of strain-promoted 3+2 cycloaddition. The R-epimer of the labelled conjugates induced TLR2-dependent DC maturation, while S-epimer proved to be inactive. Combining the lipophilicity of Pam3CysSK4 ligand with fluorophores influenced the solubility of the resulting conjugates in an unpredictable way and only the conjugates labelled with Cy-5 were suitable for confocal fluorescence microscopy experiments. It was shown that both epimers of the Cy-5 labelled lipopeptides were internalized equally well, indicating TLR2-independent cellular uptake. The presented results demonstrate the usefulness of strain-promoted azide-alkyne cycloaddition in the labelling of highly lipophilic lipopeptides without disturbing the in vitro activity of these conjugates with respect to activation of TLR-2.