A pilot study of peripheral blood BDCA-1 (CD1c) positive dendritic cells pulsed with NY-ESO-1 ISCOMATRIX™ adjuvant.

AIM: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. PATIENTS & METHODS: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+ B cells followed by positive selection of CD1c+ PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. RESULTS: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. CONCLUSION: This method was feasible and safe but was minimally immunogenic.

Effective Clinical Responses in Metastatic Melanoma Patients after Vaccination with Primary Myeloid Dendritic Cells.

PURPOSE: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro-generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c(+) myeloid DCs, naturally circulating in the blood. EXPERIMENTAL DESIGN: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c(+) myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100. RESULTS: Our results show that therapeutic vaccination against melanoma with small amounts (3-10 × 10(6)) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12-35 months), which directly correlated with the development of multifunctional CD8(+) T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth. CONCLUSIONS: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155-66. ©2015 AACR.

Natural human plasmacytoid dendritic cells induce antigen-specific T-cell responses in melanoma patients.

Vaccination against cancer by using dendritic cells has for more than a decade been based on dendritic cells generated ex vivo from monocytes or CD34(+) progenitors. Here, we report on the first clinical study of therapeutic vaccination against cancer using naturally occurring plasmacytoid dendritic cells (pDC). Fifteen patients with metastatic melanoma received intranodal injections of pDCs activated and loaded with tumor antigen-associated peptides ex vivo. In vivo imaging showed that administered pDCs migrated and distributed over multiple lymph nodes. Several patients mounted antivaccine CD4(+) and CD8(+) T-cell responses. Despite the limited number of administered pDCs, an IFN signature was observed after each vaccination. These results indicate that vaccination with naturally occurring pDC is feasible with minimal toxicity and that in patients with metastatic melanoma, it induces favorable immune responses.

Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF.

CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8+ T-cells. Two major MDSC subsets were recently shown to play an equal role in MDSC-induced immune dysfunctions: monocytic- and granulocytic-like. We isolated three fractions of MDSC, i.e. CD11b+/Gr-1high, CD11b+/Gr-1int, and CD11b+/Gr-1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b+/Gr-1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8+ T-cell activation, while CD11b+/Gr-1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b+/Gr-1int but not CD11b+/Gr-1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b+/Gr-1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM-CSF was necessary to induce preferential expansion of both CD11b+/Gr-1int and CD11b+/Gr-1low subsets in the spleen of tumor-bearing mice and mediate tumor-induced tolerance whereas G-CSF, which preferentially expanded CD11b+/Gr-1high cells, did not create such immunosuppressive environment. GM-CSF also acted on granulocyte-macrophage progenitors in the bone marrow inducing local expansion of CD11b+/Gr-1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor-released GM-CSF.

Enhanced dendritic cell-induced immune responses mediated by the novel C-type lectin receptor mDCAR1.

The dendritic cell (DC) immunoreceptors (DCIR) and DC-immunoactivating receptors (DCAR) represent a subfamily of cell surface C-type lectin receptors (CLR), whose multifunctional capacities range from classical Ag uptake and immunoregulatory mechanisms to the involvement in DC ontogeny. On the basis of the generation of specific mAbs, we functionally characterized mouse DCAR1 (mDCAR1) as a member of the DCIR/DCAR family. Expression of mDCAR1 was strongly tissue dependent. mDCAR1 expression on DCs was restricted to the CD8(+) DC subset in spleen and thymus and on subpopulations of CD11b(+) myeloid cells in bone marrow and spleen, whereas the molecule was not detectable on both cell types in lymph nodes and peripheral blood. With respect to the function of CLRs as pattern recognition receptors, Ag delivered via mDCAR1 was internalized, was trafficked to early and late endosomes/lysosomes and, as a consequence, induced cellular and humoral responses in vivo even in the absence of CD40 stimulation. Intriguingly, upon triggering mDCAR1, CD8(+) DCs increased the secretion of bioactive IL-12, whereas IL-10 release is markedly reduced, thereby indicating that Ag recognized by mDCAR1 induces enhanced proinflammatory responses. These data indicate that mDCAR1 is a functional receptor on cells of the immune system and provides further insights into the regulation of immune responses by CLRs.

CD303 (BDCA-2) signals in plasmacytoid dendritic cells via a BCR-like signalosome involving Syk, Slp65 and PLCgamma2.

Plasmacytoid dendritic cells (PDC) are the main type I interferon (IFN-I) producers and play a central role in innate and adaptive immunity. CD303 (BDCA-2) is a type II c-type lectin specifically expressed by human PDC. CD303 signaling induces tyrosine phosphorylation and Src kinase dependent calcium influx. Cross-linking CD303 results in the inhibition of IFN-I production in stimulated PDC. Here, we demonstrate that PDC express a signalosome similar to the BCR signalosome, consisting of Lyn, Syk, Btk, Slp65 (Blnk) and PLCgamma2. CD303 associates with the signaling adapter FcR gamma-chain. Triggering CD303 leads to tyrosine phosphorylation of Syk, Slp65, PLCgamma2 and cytoskeletal proteins. Analogous to BCR signaling, CD303 signaling is likely linked with its internalization by clathrin-mediated endocytosis. Furthermore, CD303 signaling leads to reduced levels of transcripts for IFN-I genes and IFN-I-responsive genes, indicating that the inhibition of IFN-I production by stimulated PDC is at least partially regulated at the transcriptional level. These results support a possible therapeutic value of an anti-CD303 mAb strategy, since the production of IFN-I by PDC is considered to be a major pathophysiological factor in systemic lupus erythematosus patients.

Plasmacytoid dendritic cells of melanoma patients present exogenous proteins to CD4+ T cells after Fc gamma RII-mediated uptake.

Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons. Although human pDCs can induce T cell responses upon viral infection, it remains unclear if pDCs can present exogenous antigens. Here, we show that human pDCs exploit FcgammaRII (CD32) to internalize antigen-antibody complexes, resulting in the presentation of exogenous antigen to T cells. pDCs isolated from melanoma patients vaccinated with autologous monocyte-derived peptide- and keyhold limpet hemocyanin (KLH)-loaded dendritic cells, but not from nonvaccinated patients or patients that lack a humoral response against KLH, were able to stimulate KLH-specific T cell proliferation. Interestingly, we observed that internalization of KLH by pDCs depended on the presence of serum from vaccinated patients that developed an anti-KLH antibody response. Anti-CD32 antibodies inhibited antigen uptake and presentation, demonstrating that circulating anti-KLH antibodies binding to CD32 mediate KLH internalization. We conclude that CD32 is an antigen uptake receptor on pDCs and that antigen presentation by pDCs is of particular relevance when circulating antibodies are present. Antigen presentation by pDCs may thus modulate the strength and quality of the secondary phase of an immune response.

Isolation and generation of clinical-grade dendritic cells using the CliniMACS system.

Dendritic cells (DC) can either be generated from progenitors such as stem cells or CD14+ monocytes, or isolated directly from the blood. Blood-derived DC are present as at least two distinct populations-myeloid and plasmacytoid DC. Here we describe methods for the clinical-grade isolation of blood DC and DC precursors using the CliniMACS. We describe the isolation of ultra-pure monocytes in order to generate large numbers of monocyte-derived DC, and also new methods for the direct isolation of blood DC. Isolation of blood DC in large numbers means that natural DC with different properties can be investigated for their clinical function for the first time.

Plasmacytoid dendritic cells: from specific surface markers to specific cellular functions.

We have recently described a panel of monoclonal antibodies (mAb), that recognize two novel leukocyte surface antigens, BDCA-2 and BDCA-4. BDCA-2 is a novel type II C-type lectin specifically expressed by plasmacytoid dendritic cells (PDCs) that can internalize antigen for presentation to T cells. Furthermore, signaling via BDCA-2 may play a role in switching from interferon (IFN)-alpha/beta-controlled to interleukin (IL)-12-controlled immune response pathways, as triggering of BDCA-2 potently inhibits secretion of IFN-alpha/beta by PDCs and thereby promotes IL-12 p70 production in PDCs and other cells. Viruses may exploit this switch to escape innate antiviral immunity, but it may be beneficial for patients with systemic lupus erythematosus (SLE) if induced, for instance by anti BDCA-2 mAb treatment. BDCA-4 is shown here to be identical to neuropilin-1 (NP-1), a neuronal receptor for the axon guidance factors belonging to the class-3 semaphorin subfamily, and a receptor on endothelial and tumor cells for vascular endothelial growth factor (VEGF-A). In blood and bone marrow, BDCA-4/NP-1 is exclusively expressed on PDCs, but in tonsils also on a few other cells, primarily follicular B helper memory T cells (T(FH)).