Kirsteen M. Tullett

Harnessing Human Cross-Presenting CLEC9A(+)XCR1(+) Dendritic Cells for Immunotherapy.

Dendritic cells (DC) are professional antigen presenting cells (APCs) that play a pivotal role in the induction and regulation of immune responses, including the induction of cytotoxic T lymphocyte (CTL) responses. They are an important focus for the development of vaccines against cancers and many pathogens, including HIV and malaria, where CTL responses are required for protection and disease eradication. DC loaded ex vivo with tumor antigen (Ag) have been administered as vaccines to cancer patients for over 15 years. They are well-tolerated and induce immune responses, including some clinical regressions, but there is clearly room for improvement (1). The DC network in both mice and humans is heterogeneous, with specialized DC subsets driving specific immune functions (2). New developments in our understanding of DC biology have identified a subset of DC characterized by the expression of novel markers CLEC9A (DNGR-1) (3, 4) and XCR1 (5, 6) as being important for the induction of CTL responses (7). Vaccine strategies that deliver Ag and activators directly to CLEC9A+XCR1+ DC in vivo promise to overcome many of the logistical issues associated with in vitro-derived vaccines, allowing precision and specificity of the desired immune response (8). Here, we discuss the biological properties of CLEC9A+XCR1+ DC that make them such attractive targets for CTL vaccines and new vaccine approaches to target them in vivo.

Dendritic cells and cancer immunotherapy

Dendritic cells (DC) play an essential role in the induction and regulation of immune responses, including the generation of cytotoxic T lymphocytes (CTL) for the eradication of cancers. DC-based cancer vaccines are well tolerated with few side effects and can generate anti-tumour immune responses, but overall they have been of limited benefit. Recent studies have demonstrated that CD141(+) DC play an important role in anti-tumour responses. These are now attractive targets for the development of vaccines that directly target DC in vivo. An understanding of the functional specialisations of DC subsets, strategies for the delivery of tumour Ag to DC and for enhancing immune responses, point to promising new avenues for the design of more effective DC-based cancer vaccines.

Targeting CLEC9A delivers antigen to human CD141+ DC for CD4+ and CD8+T cell recognition

DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines.

Differential uptake and cross‐presentation of soluble and necrotic cell antigen by human DC subsets

Cross‐presentation is the mechanism by which exogenous Ag is processed for recognition by CD8+ T cells. Murine CD8α+ DCs are specialized at cross‐presenting soluble and cellular Ag, but in humans this process is poorly characterized. In this study, we examined uptake and cross‐presentation of soluble and cellular Ag by human blood CD141+ DCs, the human equivalent of mouse CD8α+ DCs, and compared them with human monocyte‐derived DCs (MoDCs) and blood CD1c+ DC subsets. MoDCs were superior in their capacity to internalize and cross‐present soluble protein whereas CD141+ DCs were more efficient at ingesting and cross‐presenting cellular Ag. Whilst cross‐presentation by CD1c+ DCs and CD141+ DCs was dependent on the proteasome, and hence cytosolic translocation, cross‐presentation by MoDCs was not. Inhibition of endosomal acidification enhanced cross‐presentation by CD1c+ DCs and MoDCs but not by CD141+ DCs. These data demonstrate that CD1c+ DCs, CD141+ DCs, and MoDCs are capable of cross‐presentation; however, they do so via different mechanisms. Moreover, they demonstrate that human CD141+ DCs, like their murine CD8α+ DC counterparts, are specialized at cross‐presenting cellular Ag, most likely mediated by an enhanced capacity to ingest cellular Ag combined with subtle changes in lysosomal pH during Ag processing and use of the cytosolic pathway.

Self-adjuvanting nanoemulsion targeting dendritic cell receptor Clec9A enables antigen-specific immunotherapy

Non–antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalized neoepitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen–anti-Clec9A mAb conjugates target cross-presenting DCs, adjuvant must be codelivered for cytotoxic T lymphocyte (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating OVA antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation and CTL and antibody responses. OVA-Clec9A-TNE–induced DC activation required CD4 and CD8 epitopes, CD40, and IFN-&agr;. Clec9A-TNE encapsulating HPV E6/E7 significantly suppressed HPV-associated tumor growth, while E6/E7–CpG did not. Clec9A-TNE loaded with pooled B16-F10 melanoma neoepitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neoepitopes. Clec9A-TNE encapsulating 6 neoepitopes significantly suppressed B16-F10 melanoma growth in a CD4+ T cell–dependent manner. Thus, cross-presenting DCs targeted with antigen–Clec9A-TNE stimulate therapeutically effective tumor-specific immunity, dependent on T cell help.

Activation of human CD141+ and CD1c+ dendritic cells in vivo with combined TLR3 and TLR7/8 ligation

Mice reconstituted with human hematopoietic stem cells are valuable models to study aspects of the human immune system in vivo. We describe a humanized mouse model (hu mice) in which fully functional human CD141+ and CD1c+ myeloid and CD123+ plasmacytoid dendritic cells (DC) develop from human cord blood CD34+ cells in immunodeficient mice. CD141+ DC are the human equivalents of murine CD8+/CD103+ DC which are essential for the induction of tumor‐inhibitory cytotoxic T lymphocyte responses, making them attractive targets to exploit for the development of new cancer immunotherapies. We used CD34+‐engrafted NSG‐A2 mice to investigate activation of DC subsets by synthetic dsRNA or ssRNA analogs polyinosinic‐polycytidylic acid/poly I:C and Resiquimod/R848, agonists for TLR3 and TLR8, respectively, both of which are expressed by CD141+ DC. Injection of hu mice with these agonists resulted in upregulation of costimulatory molecules CD80, CD83 and CD86 by CD141+ and CD1c+ DC alike, and their combination further enhanced expression of these molecules by both subsets. When combined, poly I:C and R848 enhanced serum levels of key cytokines associated with cross‐presentation and the induction of cytotoxic T lymphocyte responses including IFN‐α, IFN‐β, IL‐12 and CXCL10. These data advocate a combination of poly I:C and R848 TLR agonists as means of activating human DC for immunotherapy.