Effective recovery of highly purified CD326(+) tumor cells from lavage fluid of patients treated with a novel cell-free and concentrated ascites reinfusion therapy (KM-CART).

For the production of tumor-specific vaccines, including dendritic cell (DC) vaccines, the tumor cells themselves are an ideal source. Floating tumor cells in the ascites fluid from patients with malignant ascites are a good candidate source, but it is not easy to obtain pure tumor cells from ascites because of various types of cell contamination as well as protein aggregates. We here report an effective method to recover pure tumor cells from malignant ascites. We used lavage fluid from 13 patients with malignant ascites who were treated with modified cell-free and concentrated ascites reinfusion therapy (KM-CART). Cellular components were separated from the lavage fluid by centrifugation, enzymatic digestion and hemolysis. Tumor cells were purified by depleting CD45(+) leukocytes with antibody-conjugated magnetic beads. The tumor cell lysate was extracted by freeze-and-thaw cycles. The mean obtained total cell number was 7.50 × 10(7) cells (range 4.40 × 10(6)-2.48 × 10(8) cells). From this fraction, 6.39 × 10(6) (range 3.23 × 10(5)-2.53 × 10(7)) CD45(-) cells were collected, and the tumor cell purity was over 80 % defined as CD45(-)CD326(+). A sufficient amount of tumor lysate, average  = 2416 µg (range 25-8743 µg), was extracted from CD45(-)CD326(+) tumor cells. We here established an effective method to produce highly purified tumor cells from KM-CART lavage fluid. The clinical feasibility of this simple preparation method for generating tumor lysate should be examined in clinical studies of DC vaccines.

Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma.

OBJECTIVES: In the current study, we have evaluated the clinical and immunological responses in patients with advanced pancreatic carcinoma who received dendritic cell (DC)-based immunotherapy in combination with gemcitabine and/or S-1. METHODS: Dendritic cell-based immunotherapy (DC vaccine alone or DC vaccine plus lymphokine-activated killer [LAK] cell therapy) in combination with gemcitabine and/or S-1 has been carried out in 49 patients with inoperable pancreatic carcinoma refractory to standard treatment. RESULTS: Of 49 patients, 2 patients had complete remission, 5 had partial remission, and 10 had stable disease. Prolongation of survival in this cohort was highly likely (median survival, 360 days). Survival of patients receiving DC vaccine and chemotherapy plus LAK cell therapy was longer than those receiving DC vaccine in combination with chemotherapy but no LAK cells. Increased numbers of cancer antigen-specific cytotoxic T cells and decreased regulatory T cells were observed in several patients on immunotherapy, but increased overall survival time tended to be associated only with the latter. None of the patients experienced grade 3 or worse adverse events during the treatment period. CONCLUSIONS: Dendritic cell vaccine-based immunotherapy combined with chemotherapy was shown to be safe and possibly effective in patients with advanced pancreatic cancer refractory to standard treatment.

C-type lectin SIGNR1 enhances cellular oxidative burst response against C. albicans in cooperation with Dectin-1.

We investigated the role of SIGNR1 in the recognition of Candida albicans and the subsequent cellular oxidative burst response. Soluble SIGNR1 (sSIGNR1) tetramer bound equally to zymosan and both heat-killed (HK) and live C. albicans in an EDTA-sensitive manner, whereas sDectin-1 tetramer predominantly bound to zymosan and HK-microbes in an EDTA-independent manner. In cellular response, enhanced oxidative burst was observed in RAW264.7 cells expressing SIGNR1 (RAW-SIGNR1) compared with RAW-control cells upon stimulation with HK-C. albicans and zymosan. This response was independent of TLR2 and the cytosolic portion of SIGNR1 but dependent on the recognition by SIGNR1 via carbohydrate recognition domain. Antagonistic laminarin and anti-Dectin-1 mAb cooperatively reduced the response with mannan and anti-SIGNR1 mAb, respectively, although they had no effect by themselves. Moreover, oxidative response and bactericidal activity largely relied on Syk-mediated signaling. RAW-SIGNR1 cells not only captured microbes more efficiently but also showed higher responses than RAW-control cells. Similar enhanced responses were observed in SIGNR-1-expressing resident peritoneal Mϕ. Interestingly, Dectin-1 was recruited to the phagosomal membrane upon the stimulation and physically associated with SIGNR1. These results suggest that SIGNR1 plays a significant role in inducing oxidative response to C. albicans by Syk-dependent signaling, possibly through Dectin-1.

Development of murine plasmacytoid dendritic cells defined by increased expression of an inhibitory NK receptor, Ly49Q.

Plasmacytoid dendritic cells (PDCs) are defined in mice by a unique combination of markers: CD11c, B220, and Ly6C/G. We have reported previously that PDCs express Ly49Q, a lectin-type killer cell inhibitory receptor. We now find that different expression levels of Ly49Q define sequential developmental stages of PDCs in bone marrow. Although PDCs in spleen and lymph nodes express high levels of Ly49Q, a significant portion of CD11c(+)B220(+) PDCs in bone marrow lack Ly49Q, as well as the CD4 and MHC II. Purified Ly49Q(-) marrow PDCs spontaneously up-regulate Ly49Q after overnight culture without cell proliferation and acquire most features of typical PDCs in spleen. When exposed to TLR ligands, such as CpG-oligodeoxynucleotide and hemagglutinating virus of Japan (Sendai virus), Ly49Q(-) PDCs increase CD86 and MHC class II expression but produce less IFN-alphabeta, IL-6, and IL-12p70 than Ly49Q(+) PDCs, although they are able to produce comparable amounts of TNF-alpha. However, interestingly, Ly49Q(-) PDCs do not produce TNF-alpha in response to the TLR2 ligand, Pam3SCK(4), whereas Ly49Q(+) PDCs did. Therefore, Ly49Q is a new marker to identify a precursor form of PDCs that participates in innate immunity.

Immune tolerance after delivery of dying cells to dendritic cells in situ.

Peripheral immune tolerance is believed to be induced by the processing and presentation of self-tissues that die during physiologic tissue turnover. To examine the mechanism that mediates tolerance, we injected mice with dying syngeneic TAP(-/-) splenocytes loaded with small amounts of the protein antigen, ovalbumin (OVA). After ingestion and presentation of cell-associated OVA by the CD8(+) subset of dendritic cells in situ, large numbers of antigen-reactive, CD8(+) T cell receptor (TCR) transgenic T lymphocytes were driven into cell cycle, but then the T cells were deleted. The animals were also tolerant to challenge with OVA in complete Freund's adjuvant. An agonistic anti-CD40 monoclonal antibody was then administered together with the OVA-loaded splenocytes, so that the dendritic cells in the recipient mice would mature. In contrast to observations made in the steady state, the antigen-reactive T cells expanded in numbers for 1-2 wk and produced large amounts of interleukin 2 and interferon gamma, while the animals retained responsiveness to antigen rechallenge. The specific tolerance that develops when dendritic cells process self tissues in the steady state should prevent or reduce the development of autoimmunity when dying cells are subsequently processed during infection.