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The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

Current HLA-typing methods are typically designed to provide exquisitely-detailed identification of multiple HLA-alleles to satisfy the requirements for organ and bone marrow transplantation or genetic studies. Many human immunological studies, on the other hand, focus around only a small number of HLA alleles that are abundant or of relevance to specific diseases. Consequently, for such studies, many HLA typing approaches are not cost-effective and are potentially complicated, slow and not easily performed in-house. Work-flow would be streamlined by a simple, inexpensive and rapid typing method able to be performed in-house. We outline a straightforward approach that provides appropriate data for much immunological research. In a predominantly Caucasian population, flow cytometry using anti-HLA-A2, -B8 and -B7 antibodies consistently and accurately screened for samples carrying the highly-abundant HLA class I alleles HLA-A*02:01, -B*08:01 and -B*07:02 that form the focus of immunological studies. Next, we describe a straightforward and simple strategy for design and use of allele-specific PCR primers to identify, at high-resolution, alleles of interest. When combined with a simple gDNA extraction technique this provides reliable, simple and inexpensive in-house HLA typing demonstrated here for highly-abundant HLA class I alleles.

The dendritic cell receptor Clec9A facilitates processing of dead cell-derived antigens for cross-presentation and the induction of effective CD8+ T cell immune responses. Here, we show that this process is regulated by E3 ubiquitin ligase RNF41 and define a new ubiquitin-mediated mechanism for regulation of Clec9A, reflecting the unique properties of Clec9A as a receptor specialized for delivery of antigens for cross-presentation. We reveal RNF41 is a negative regulator of Clec9A and the cross-presentation of dead cell-derived antigens by mouse dendritic cells. Intriguingly, RNF41 regulates the downstream fate of Clec9A by directly binding and ubiquitinating the extracellular domains of Clec9A. At steady-state, RNF41 ubiquitination of Clec9A facilitates interactions with ER-associated proteins and degradation machinery to control Clec9A levels. However, Clec9A interactions are altered following dead cell uptake to favor antigen presentation. These findings provide important insights into antigen cross-presentation and have implications for development of approaches to modulate immune responses.

Dendritic cells are a specialized subset of hematopoietic cells essential for mounting immunity against tumors and infectious disease as well as inducing tolerance for maintenance of homeostasis. DCs are equipped with number of immunoregulatory or stimulatory molecules that interact with other leukocytes to modulate their functions. Recent advances in DC biology identified a specific role for the conventional dendritic cell type 1 (cDC1) in eliciting cytotoxic CD8+ T cells essential for clearance of tumors and infected cells. The critical role of this subset in eliciting immune responses or inducing tolerance has largely been defined in mice whereas the biology of human cDC1 is poorly characterized owing to their extremely low frequency in tissues. A detailed characterization of the functions of many immunoregulatory and stimulatory molecules expressed by human cDC1 is critical for understanding their biology to exploit this subset for designing novel therapeutic modalities against cancer, infectious disease and autoimmune disorders.

Objectives\ud Vaccines that prime Wilms' tumor 1 (WT1)‐specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C‐type lectin‐like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T‐cell responses. We developed a new vaccine comprising a human anti‐CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1‐specific CD8+ T cells.\ud \ud Methods\ud WT1 was genetically fused to antibodies specific for human CLEC9A, DEC‐205 or β‐galactosidase (untargeted control). Activation of WT1‐specific CD8+ T‐cell lines following cross‐presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1‐specific CD8+ T cells, were used to investigate naïve WT1‐specific CD8+ T‐cell priming.\ud \ud Results\ud The CLEC9A‐WT1 vaccine promoted cross‐presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC‐205‐WT1 and untargeted control‐WT1 vaccines.\ud \ud Conclusions\ud Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag‐presenting cells via DEC‐205, suggesting that cross‐presentation by CD141+ DCs is sufficient for effective CD8+ T‐cell priming in humans. The CLEC9A‐WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1.

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.

BackgroundDendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs.MethodsHuman anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, tumor necrosis factor and CD107a and by lysis of target tumor cells.ResultsCLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro.ConclusionsThese data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.

Many adenocarcinomas, including colorectal cancer (CRC), overexpress the MUC13 cell surface mucin, but the functional significance and mechanisms are unknown. Here, we report the roles of MUC13 in colonic tumorigenesis and tumor progression. High-MUC13 expression is associated with poor survival in two independent patient cohorts. In a comprehensive series of in vivo experiments, we identified a critical role for MUC13 in the development of this malignancy, by promoting survival and proliferation of tumor-initiating cells and driving an immunosuppressive environment that protects tumors from checkpoint inhibitor immunotherapy. In Muc13-deficient mice, fewer tumors are generated after exposure to carcinogens and inflammation, they have markedly reduced β-catenin signaling, have more tumor-infiltrating CD103+ dendritic cells and CD8+ T lymphocytes, fewer myeloid-derived suppressor cells, and are rendered sensitive to checkpoint inhibitor immunotherapy (anti-PD-L1). Mechanistically, we show that MUC13 protects β-catenin from degradation, by interacting with GSK-3β, which increases β-catenin nuclear translocation and promotes its signaling, thereby driving cancer initiation, progression, invasion, and immune suppression. Therefore, MUC13 is a potential marker of poor prognosis in colorectal cancer, and inhibiting MUC13 may be useful in the treatment of colitis-associated cancer and sensitizing tumors to immunotherapy.

The method of choice for the development of new vaccines is to target distinct dendritic cell subsets with antigen in vivo and to harness their function in situ to enhance cell-mediated immunity or induce tolerance to specific antigens. The innate functions of dendritic cells themselves may also be targeted by inhibitors or activators that would target a specific function such as interferon production, potentially important in autoimmune disease and chronic viral infections. Importantly targeting dendritic cells requires detailed knowledge of both the surface phenotype and function of each dendritic cell subset, including how they may respond to different types of vaccine adjuvants, their ability to produce soluble mediators and to process and present antigens and induce priming of naïve T cells. This review summarizes our knowledge of the functional attributes of the human dendritic cell subsets in the steady state and upon activation and their roles in human disease.

Preclinical studies have suggested that purified populations of CD1c (BDCA-1) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use of CD1c BDC in patients with advanced metastatic hormone refractory prostate cancer. HLA-A*0201-positive patients with advanced metastatic prostate cancer were recruited and consented. The vaccine was manufactured by pulsing autologous CD1c BDC, prepared by magnetic bead immunoselection from apheresed peripheral blood mononuclear cells, with a cocktail of HLA-A*0201-restricted peptides (prostate-specific antigen, prostate acid phosphatase, prostate specific membrane antigen, and control influenza peptide) and keyhole limpet hemocyanin. The vaccine was administered intradermally or intravenously and peripheral blood was taken at predetermined intervals for clinical and immunologic monitoring. The vaccine was manufactured with a median purity of 82% CD1c BDC and administered successfully to 12 patients. Each patient received between 1 and 5 × 10 fresh CD1c BDC on day 0, followed by cryopreserved product in the same dose on days 28 and 56. The vaccine was well tolerated in all patients, with the most frequent adverse events being grade 1-2 fever, pain, or injection-site reactions. Vaccination with CD1c BDC is therefore feasible, safe, and well tolerated in patients with advanced-stage metastatic prostate cancer.

Background and aims Interferon lambda (IFN-λ) is a novel type of interferon produced by dendritic cells (DC). Despite its binding to a different receptor, IFN-λ shares functional similarities with type I IFN (IFN-I) by upregulating the expression of IFN-stimulated genes. The role of IFN-λ in DC biology and in autoimmunity remains unknown. to identify the DC subsets producing IFN-λ. to investigate the role of IFN-λ in DC functions. to investigate the role of IFN-λ in SLE. Methods Mouse and human DC subsets were stimulated ex vivo and the IFN-λ expression was measured. The maturation and the capacity of DC to cross-prime T cells was compared in WT and IFN-λR-/- mice. T cell cross-priming by human DCs was measured ex vivo in the presence of exogenous IFN-λ. Serum levels of IFN-λ was measured in lupus-prone mice and in SLE patients. The phenotype of the blood DC subsets from SLE patients was also characterised. Results Mouse plasmacytoid DC (pDC) and CD8+ DC highly secrete IFN-λ. In humans, the CD141+ DC are the major IFN-λ producers. IFN-λ enhances the capacities of mouse and human DCs to maturate and to cross-prime T cells. High serum levels of IFN-λ were detected in lupus-prone mice and in some SLE patients. SLE patients display increased activation of the IFN-producing DC subsets: the pDCs (producing IFN-I) and the CD141+ DCs (producing IFN-λ). Conclusions IFN-λ is produced by some DC subsets and enhances their functions. Furthermore, IFN-λ is expressed during SLE, suggesting a potential role of the cytokine in the aetiology of SLE.

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.

Dendritic cells (DC) are a heterogeneous cell population, with specialist subtypes driving specific immune responses. In mice, the cDC1 subset (also referred to as Batf3-dependent DC, XCR1+ DC, CD8+ DC in lymphoid tissues and CD103+ DC in peripheral tissues) is essential for the induction of tumor immune responses and for the efficacy of checkpoint inhibitor blockade and adoptive T-cell immunotherapies. Vaccines that can deliver antigens (Ag) directly to DCs in vivo are more effective than cell-based therapies in mouse models and are promising approaches to translate to humans. CD141+ DC are the human cDC1 equivalent and specifically express the C-type lectin-like receptor CLEC9A, that facilitates cross-presentation of dead cell Ag. Targeting tumor-associated Ag (TAA) to human CD141+ DC using CLEC9A antibody (Ab) is therefore an attractive strategy to induce or boost tumor immune responses. NYESO1 and WT1 are well characterised, highly immunogenic TAA expressed by a broad array of tumor types. We developed recombinant human chimeric IgG4 Ab specific for human CLEC9A genetically fused to NYESO1 or WT1. For comparison we developed TAA fusions with chimeric IgG4 Ab specific for human DEC-205, which is expressed by many human leukocytes, and β-galactosidase as an irrelevant isotype control. CLEC9A-NYESO1 and CLEC9A-WT1 Abs retained their binding specificity for CD141+ DC. Following uptake of CLEC9A-WT1, CD141+ DC cross-presented a WT-1 HLA-A24-restricted epitope for recognition by specific CD8+ cytotoxic T-cells. Likewise, a HLA-A2-restricted NYESO1 epitope was cross-presented Ag specific CD8+ T-cells by CD141+ DC following uptake of CLEC9A-NYESO1. For both TAA, the CLEC9A Abs were more efficient at delivery of Ag for cross-presentation than DEC-205 or isotype control Abs. Moreover, using a humanized mouse model in which functional human CD141+ DC and Ag-specific T-cells develop, CLEC9A-TAA Ab induced priming of Ag-specific T-cells. Our data advocate further development of human CLEC9A targeting Abs as cancer vaccines. Citation Format: Kristen Radford, Frances Pearson, Kelly-Anne Masterman, Kirsteen Tullett, Oscar Haigh, Carina Walpole, Ghazal Daraj, Ingrid Leal Rojas, Mireille Lahoud. Targeting human CD141+ DC using CLEC9A antibodies for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B125.

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.

The distribution and function of the C-type lectin Mincle has not previously been investigated in human cells, although mouse models have demonstrated a non-redundant role for Mincle in the host response to fungal infections. This study identified an unusual pattern of reciprocal expression of Mincle on peripheral blood monocytes or neutrophils isolated from the same donor. Expression on monocytes was inversely correlated with phagocytosis and yeast killing, but was necessary for the induction of inflammatory cytokines in response to ex vivo Candida challenge. In contrast, Mincle expression on neutrophils was associated with phagocytic and candidacidal potential of those cells. Candida challenge upregulated Mincle expression but only in Mincle+ cells. These data highlight species-specific differences between the regulation of Mincle expression in mouse and man. Reciprocal expression of Mincle modified the candidacidal potential of monocytes or neutrophils, suggesting it may also polarize the type of host response to fungal infection.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C–activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Dendritic cells (DC) are specialized white blood cells that initiate and direct immune responses. Targeting DC surface proteins to deliver liposomes carrying antigens has demonstrated potential for eliciting antigen-specific immune responses. To evaluate this strategy in preclinical studies, we prepared anti-human DEC-205 immunoliposomes (anti-hDEC-205 iLPSM) and compared their uptake by monocyte-derived DC (MoDC) and blood DC (BDC) with conventional liposomes (cLPSM). Antibody conjugation increased the number of immature MoDC taking up liposomes to 70-80%, regardless of the antibody coupled, whereas less than 20% endocytosed cLPSM. Anti-hDEC-205-IgG specifically increased cell uptake by 15% and the total iLPSM uptake six-fold. The non-specific iLPSM uptake was unlikely to be Fc receptor-mediated as excess immunoglobulins failed to block the uptake. Only a small population (7-24%) of mature MoDC took up cLPSM and control iLPSM. In contrast, approximately 70% of mature MoDC took up anti-hDEC-205 iLPSM, endocytosing 10-fold more iLPSM than the control iLPSM. Anti-hDEC-205 iLPSM uptake by CD1c+ BDC was similar to the immature MoDC, but was five-fold increased compared to the control iLPSM. Confocal microscopy confirmed that the anti-hDEC-205 iLPSM were phagocytosed by DC and available for antigen processing. Thus, DEC-205 is an effective target for delivering liposomes to human DC.

The number and function of human T cells in the periphery are regulated by homeostatic signals received from antigen-presenting cells (APCs) and the common gamma chain (gammac) cytokines interleukin (IL)-7 and IL-15. We found that, in the absence of introduced antigen, blood monocytes or myeloid dendritic cells (MDCs) in the presence of IL-7 and IL-15 (IL-7/IL-15) can regulate CD4(+) T memory (Tm) cell numbers by polyclonal cell proliferation. The dynamics of CD4(+) Tm cell proliferation, in the presence of IL-7/IL-15, was dependent on contact with MDCs and to a lesser extent on contact with monocytes. IL-7/IL-15 either alone or combined with monocytes or MDCs enhanced the proportion of CD4(+) Tm cells with activated and effector phenotype and diminished the helper function of CD4(+) Tm cells. These CD4(+) Tm cells, preconditioned with IL-7/IL-15 alone or with monocytes or MDCs and IL-7/IL-15, reduced T cell-dependent immunoglobulin M (IgM) and IgG responses. This appeared to be a contact-dependent effect involving a reduction in antibody-producing CD27(+) B memory cells, but contact-independent suppression by soluble factors also contributed to the antibody-producing capacity of CD27(+) B memory cells. These results indicate that blood monocytes, MDCs and the cytokines IL-7/IL-15 contribute to homeostasis of CD4(+) Tm cells by regulating their number, activation state and helper/suppressor (regulatory) function. In healthy individuals, this mode of regulating CD4(+) Tm cell homeostasis may provide a basis for the control of autoimmune responses.

Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed "cross-presentation." The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.

The generation of tumour-specific cytotoxic T-lymphocyte (CTL) responses is the primary focus in the design of immunotherapeutic cancer vaccines. We have recently demonstrated generation of ovalbumin (OVA)-specific CTLs and tumour-protection in a murine tumour model using vaccination with dendritic cells (DCs) pulsed with E. coli expressing listeriolysin O (LLO) and OVA as a model antigen. In this system paraformaldehyde fixation of E. coli/LLO provided an additional safety feature without compromising vaccine efficacy. We therefore reasoned that paraformaldehyde-fixed recombinant E. coli expressing LLO would be an efficient vehicle for the delivery of human tumour antigens to human DCs. In the present study, we demonstrate that fixed E. coli expressing LLO are taken up efficiently by human monocyte-derived DCs (MoDCs) with minimal toxicity. As a consequence of the interaction with bacteria, human DCs undergo marked phenotypic and functional maturation. Furthermore, we show that fixed E. coli/LLO expressing the well-characterised human melanoma antigen, MART1, efficiently deliver the HLA-A2-restricted MART1(27-35) epitope for processing and presentation on human MoDCs, suggesting the potential of this system as a novel strategy for human tumour immunotherapy.

We have examined the potential of recombinant Escherichia coli expressing listeriolysin O (LLO) to deliver tumour antigens to dendritic cells (DCs) for cancer immunotherapy. Using OVA as a model tumour antigen, we have shown in murine DCs that E. coli expressing cytoplasmic LLO and OVA proteins can deliver the OVA K(b)-restricted epitope SIINFEKL for MHC class I presentation. In contrast, when E. coli expressing OVA alone were used, MHC class II presentation of the OVA 323-339 I-A(b)-restricted peptide was predominant. When injected in vivo, DCs pulsed with E. coli expressing LLO and OVA induced production of cytotoxic T-lymphocytes capable of lysing an OVA-expressing melanoma cell line (B16-OVA) and resulted in suppression of tumour growth following challenge with B16-OVA. Immunisation of mice by direct injection of E. coli LLO/OVA provided a more potent anti-tumour response, resulting in complete protection in 75% of mice. Injection of live bacteria was not necessary as immunisation with paraformaldehyde-fixed E. coli LLO/OVA provided an even stronger anti-tumour response against B16-OVA. Altogether, our data highlight the potential of this system as a novel and efficient strategy for tumour immunotherapy.

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141+ DC, CD1c+ DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c+ DC and CD141+ DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c+ DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141+ DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c+ DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8+ T cells, CD141+ DC are superior in their capacity to cross-present protein Ag to CD8+ T cells following activation with polyinosinic-polycytidylic acid. CD141+ DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141+ and CD1c+ DC and evaluate adjuvants and DC-targeting strategies in vivo.

Dendritic cells (DC) are a group of sparsely distributed “professional” antigen presenting cells. Most DC are relatively short-lived cells that must be continually renewed by precursors from bone marrow. Their characteristic form led to their recognition as a distinct cell type by Steinman and Cohn in 1973. Subsequent work revealed their key function in the adaptive immune system: the collection, processing, and presentation of peptide fragments of antigens to T cells on major histocompatibility complex (MHC) molecules. The exceptional ability of DC to process and present antigens and so to activate even naive T cells has endowed them with the title of “professional” antigen presenting cells. In steady state DC play a role in lymphocyte specificity selection and the maintenance of peripheral tolerance. However, DC serve as the sentinels of the immune system and are rapidly activated, via innate response mechanisms, by pathogens or host factors that signal danger or stress. Activated DC produce cytokines and other factors that contribute to inflammatory responses. Upon activation those sentinel DC in peripheral tissues migrate to lymph nodes to allow contact with T cells. The activated DC presenting antigen–MHC complexes then activate antigen-specific T cells to initiate immune responses. The nature of the subsequent response is in part determined by factors produced by the DC. DC thus link the innate and the adaptive immune systems. DC constitute 1% or less of the cells in various tissues of the body. Different types of DC occur in different anatomical locations and even within one location. These DC subsets differ in surface phenotype and in specialized aspects of DC function. This diversity of DC subsets is key to obtaining the appropriate immunological response to different pathogen challenges. However, together with the low number of DC it presents a daunting obstacle to determining the role of DC in human disease. This chapter aims to address the functional aspects of different types of DC relevant to autoimmunity, with specific examples of functions contributing to autoimmune disease etiology.

Human blood dendritic cells (DCs) are a rare, heterogeneous cell population that comprise approximately 1% of circulating peripheral blood mononuclear cells (PBMCs). Their isolation has been confounded by their scarcity and lack of distinguishing markers and their characterisation perplexed by the recent discovery of phenotypic and functionally distinct subsets. Human blood DCs are broadly defined as leukocytes that are HLA-DR positive and lack expression of markers specific for T cell, B cell, NK cell, monocyte and granulocyte lineages. They can be subdivided into the CD11c(-) (CD123(+)CD303(+)CD304(+)) plasmacytoid DC and CD11c(+) myeloid DC, which can be further subdivided into three subsets based on differential expression of CD1c, CD141 and CD16. DC can be isolated from peripheral blood by using an initial density gradient centrifugation step to enrich for mononuclear cells followed by immunomagnetic depletion of cells expressing markers specific for leukocyte lineages and undesired DC subsets. Subsequent flow cytometry-based cell sorting allows the isolation of highly pure individual DC subsets that can then be used for functional studies.

An invasive Escherichia coli expressing the inv gene from Yersinia pseudotuberculosis was used as a vector for protein delivery to mammalian epithelial cells. Upon incubation with beta1-integrin-expressing mammalian cells, the bacteria are internalized, allowing bacteria-encoded proteins to function from within the mammalian cell. These bacteria are eventually processed in the host phagosome where they are destroyed. Expression of listeriolysin O from Listeria monocytogenes in the bacterium and its subsequent release into the phagosome triggers the breakdown of the membrane, allowing the release of the bacterial content into the cytosol of host cells. Using this vector, we demonstrate delivery of a gene and intact, functional proteins into mammalian cells in which beta1-integrin is expressed and accessible. At a ratio of bacteria/mammalian cells compatible with the survival of the mammalian cells, protein delivery can be observed in the entire cell population in vitro, while gene transfer is far less efficient. Protein delivery can also be achieved in vivo in mouse tumour models and can be detected at least 96 h after inoculation. Functional, natural E. coli proteins are delivered in the process and can provide therapeutic benefit in vivo, when associated with prodrugs. This therapeutic effect is associated with infiltration of neutrophils, eosinophils, macrophages and to a lesser extent dendritic cells in the tumour mass.

CD63 belongs to the Transmembrane 4 superfamily (TM4SF) of membrane proteins whose functions are largely unknown. Previous results have suggested that CD63 may play an important role in the regulation of melanoma progression. To explore the role of CD63 in melanoma we have examined its association with other molecules by immunoprecipitation of CD63 from detergent induced lysates of melanoma cells. These results are the first to demonstrate an association between CD63 and two other TM4SF members, CD9 and CD81 in 2 human melanoma cell lines. We were also able to identify an association between CD9 and CD63 with β1 integrins in melanoma. The results suggest that CD63 is capable of forming multicomponent complexes with TM4SF members and β1 integrins on the surface of melanoma. These findings provide further insights into the function of CD63.

CD63 has been identified in human melanoma cells by a number of different monoclonal antibodies (MAbs). Studies with MAbs have shown that expression is most marked in naevi and early forms of cutaneous melanoma and reduced in vertical growth phase and metastatic lesions. To investigate further the role of CD63 in progression of melanoma, genomic CD63 was transfected into a CD63-negative human melanoma cell line using an episomal vector. The stable transfected melanoma cells had similar growth rates to control transfected melanoma cells in vitro but much lower growth rates when injected intradermally into athymic nude mice. The CD63-transfected cells also had a reduced number of metastases in the peritoneal cavity and subcutaneous sites when injected intravenously. MAb against CD63 did not influence the growth of CD63-transfected melanoma cells in vitro. Our results confirm previous studies using H-ras-transformed NIH3T3 fibroblasts and suggest that CD63 may have a role as a tumor suppressor gene in human melanoma that acts to limit invasion and progression of melanoma.

We investigated the capacity of IL-15 to differentiate human dendritic cells (DC) from monocytes in the presence of GM-CSF (IL-15 MoDC) and compared them with MoDC differentiated in IL-4 and GM-CSF (IL-4 MoDC) as used in many immunotherapy protocols. IL-15 MoDC expressed higher levels of CD40 and HLA-DR and importantly, induced MART-1 specific cytotoxic T lymphocyte (CTL) responses with superior lytic capacity, when compared to IL-4 MoDC. In response to activation, IL-15 MoDC secreted high levels of IFN-γbut low or no IL-12, whereas IL-4 MoDC secreted high IL-12 but low or no IFN-γ. Using an IFN-γ blocking antibody, we demonstrated that IFN-γ production by the IL-15 MoDC did not account for the superior CTL responses induced. Despite immunoselecting monocytes to greater than 97% purity prior to DC differentiation, we noticed a small population (1–2%) of CD56+ and CD3+ lymphocytes in the IL-15 MoDC preparations that were less prominent in IL-4 MoDC differentiated from the same monocytes. Removal of the residual lymphocytes from monocytes prior to differentiation into IL-15 MoDC diminished their capacity to induce CTL but did not affect the expression of HLA-DR or CD40. These data suggest that IL-15-dependent cross-talk between the small lymphoid populations present and DC, during DC differentiation from monocytes results in superior CTL priming that is independent of IL-12 and IFN-γ. Based on these results, appropriately manufactured IL-15 MoDC preparations containing defined numbers of lymphoid cells should be considered for immunotherapy.

CD63 is a member of the tetraspan family of molecules that is expressed in melanoma and which appears to be involved in regulation of motility and adhesion of melanoma cells. In the present study we have screened a cDNA library from a melanoma cell with antisera from a patient with melanoma and have shown that one of the antigens identified was CD63. The clone isolated contained a mutation in the transmembrane region, but this did not appear to be involved in immunogenicity of the molecule. Antisera from the patient showed weak reactivity with CD63 from melanoma and recombinant CD63 in Western blots. The latter studies showed marked variation in the fractions of CD63 identified with monoclonal antibodies to CD63 consistent with variable glycosylation of CD63. We suggest that glycosylation may account for its recognition by the human immune system. Further studies are required to assess the role of CD63 as an immunogen in melanoma.