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.

Immunogenic modulation of cholangiocarcinoma cells by chemoimmunotherapy.

BACKGROUND/AIM: Chemoimmunotherapy has been used to treat intrahepatic cholangiocarcinoma (ICC). However, little is known about the phenomena underlying the immunomodulation of ICC cells elicited by chemoimmunotherapy. MATERIALS AND METHODS: Primary ICC cells from a patient with ICC who received gemcitabine followed by 5-fluorouracil (5-FU), both combined with dendritic cells pulsed with Wilms' tumor 1 (WT1) peptides were cultured. ICC cells were treated with gemcitabine, 5-FU or interferon (IFN)-γ in vitro. The phenotype of the ICC cells was examined by flow cytometry and quantitative reverse transcription polymerase chain reaction. RESULTS: Stimulation of the ICC cells with gemcitabine resulted in up-regulation of WT1 mRNA, programmed death receptor ligand-1 (PDL1) and calreticulin. Gemcitabine, 5-FU and IFN-γ induced up-regulation of mucin-1. Moreover, human leukocyte antigen (HLA)-ABC, HLA-DR and PDL1 were extremely up-regulated by IFN-γ. CONCLUSION: Chemoimmunomodulating agents alter the immunogenicity of ICC cells, resulting in complex clinical efficacy results.

Treatment with chemotherapy and dendritic cells pulsed with multiple Wilms' tumor 1 (WT1)-specific MHC class I/II-restricted epitopes for pancreatic cancer.

PURPOSE: We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II-restricted epitopes, in combination with chemotherapy. EXPERIMENTAL DESIGN: Ten stage IV patients with pancreatic ductal adenocarcinoma (PDA) and 1 patient with intrahepatic cholangiocarcinoma (ICC) who were HLA-positive for A*02:01, A*02:06, A*24:02, DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01, or DPB1*09:01 were enrolled. The patients received one course of gemcitabine followed by biweekly intradermal vaccinations with mature DCs pulsed with MHC class I (DC/WT1-I; 2 PDA and 1 ICC), II (DC/WT1-II; 1 PDA), or I/II-restricted WT1 peptides (DC/WT1-I/II; 7 PDA), and gemcitabine. RESULTS: The combination therapy was well tolerated. WT1-specific IFNγ-producing CD4(+) T cells were significantly increased following treatment with DC/WT1-I/II. WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in 4 of the 7 patients with PDA vaccinated with DC/WT1-I/II and in 0 of the 3 patients with PDA vaccinated with DC/WT1-I or DC/WT1-II. The WT1-specific DTH-positive patients showed significantly improved overall survival (OS) and progression-free survival (PFS) compared with the negative control patients. In particular, all 3 patients with PDA with strong DTH reactions had a median OS of 717 days. CONCLUSIONS: The activation of WT1-specific immune responses by DC/WT1-I/II combined with chemotherapy may be associated with disease stability in advanced pancreatic cancer.

Prognostic factors related to add-on dendritic cell vaccines on patients with inoperable pancreatic cancer receiving chemotherapy: a multicenter analysis.

OBJECTIVE: Dendritic cell (DC)-based cancer vaccines may have a significant benefit to patients with advanced pancreatic cancer. However, variations among clinical studies make it difficult to compare clinical outcomes. Here, we identified factors that determined the clinical benefits by analyzing data obtained at seven Japanese institutions that employed the same DC preparation and treatment regimens. METHODS: Of 354 patients who met the inclusion criteria, 255 patients who received standard chemotherapy combined with peptide-pulsed DC vaccines were analyzed. RESULTS: The mean survival time from diagnosis was 16.5 months (95 % CI 14.4-18.5) and that from the first vaccination was 9.9 months (95 % CI 8.0-12.9). Known prognostic baseline factors related to advanced pancreatic cancer, namely ECOG-PS, peritoneal metastasis, liver metastasis, and the prognostic nutrition index, were also representative. Importantly, we found that erythema reaction after vaccination was an independent and treatment-related prognostic factor for better survival and that OK-432 might be a good adjuvant enhancing the antitumor immunity during DC vaccination. CONCLUSIONS: This is the first report of a multicenter clinical study suggesting the feasibility and possible clinical benefit of an add-on DC vaccine in patients with advanced pancreatic cancer who are undergoing chemotherapy. These findings need to be addressed in well-controlled prospective randomized trials.

Cellular prion protein: from physiology to pathology.

The human cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI) anchored membrane glycoprotein with two N-glycosylation sites at residues 181 and 197. This protein migrates in several bands by Western blot analysis (WB). Interestingly, PNGase F treatment of human brain homogenates prior to the WB, which is known to remove the N-glycosylations, unexpectedly gives rise to two dominant bands, which are now known as C-terminal (C1) and N-terminal (N1) fragments. This resembles the ß-amyloid precursor protein (APP) in Alzheimer disease (AD), which can be physiologically processed by α-, ß-, and γ-secretases. The processing of APP has been extensively studied, while the identity of the cellular proteases involved in the proteolysis of PrP(C) and their possible role in prion biology has remained limited and controversial. Nevertheless, there is a strong correlation between the neurotoxicity caused by prion proteins and the blockade of their normal proteolysis. For example, expression of non-cleavable PrP(C) mutants in transgenic mice generates neurotoxicity, even in the absence of infectious prions, suggesting that PrP(C) proteolysis is physiologically and pathologically important. As many mouse models of prion diseases have recently been developed and the knowledge about the proteases responsible for the PrP(C) proteolysis is accumulating, we examine the historical experimental evidence and highlight recent studies that shed new light on this issue.

Cross priming amplification: mechanism and optimization for isothermal DNA amplification.

CPA is a class of isothermal amplification reactions that is carried out by a strand displacement DNA polymerase and does not require an initial denaturation step or the addition of a nicking enzyme. At the assay temperature of 63°C, the formation of a primer-template hybrid at transient, spontaneous denaturation bubbles in the DNA template is favored over re-annealing of the template strands by the high concentration of primer relative to template DNA. Strand displacement is encouraged by the annealing of cross primers with 5' ends that are not complementary to the template strand and the binding of a displacement primer upstream of the crossing primer. The resulting exponential amplification of target DNA is highly specific and highly sensitive, producing amplicons from as few as four bacterial cells. Here we report on the basic CPA mechanism - single crossing CPA - and provide details on alternative mechanisms.

Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

The cellular form of the prion protein, PrP(C), undergoes extensive proteolysis at the alpha site (109K [see text]H110). Expression of non-cleavable PrP(C) mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C) physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C) mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C).

Evaluation of immunological status in tumor-bearing dogs.

The purpose of this study was to evaluate immunological status in dogs with cancers at different stages, in comparison with normal dogs. The population of canine peripheral blood lymphocytes (cPBL), lymphocyte phenotypes, interleukin (IL)-6 activity and alpha 1-acid glycoprotein (alpha(1)-AG) level were analyzed. The tumor-bearing dogs had higher numbers of leukocytes than normal dogs, the count being higher in dogs with more advanced tumors. In the tumor-bearing dogs, differential leukocyte counts revealed higher percentages of inflammatory cells such as neutrophils, acidophils and monocytes, and lower numbers of CD4(+)T cells, than in normal dogs, the lymphocyte counts becoming much lower with tumor progression. In the tumor-bearing dogs, the CD8(+)T cell count at the early tumor stage was similar to that in normal dogs, but decreased with tumor progression, possibly reflecting the development of humoral immunity (Th2). Plasma IL-6 and TGF-beta activities were high in the tumor-bearing dogs. The plasma alpha(1)-AG concentration was also significantly high in the tumor-bearing dogs. Our findings suggest that assay of IL-6, TGF-beta and alpha(1)-AG may be very useful for prognostication in dogs with cancer, and that anti-tumor immunity is potently suppressed in such dogs.

KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2).

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.

NKp44 triggers NK cell activation through DAP12 association that is not influenced by a putative cytoplasmic inhibitory sequence.

NKp44 (NCR2) is a member of the natural cytotoxicity receptor (NCR) family that is expressed on activated human NK cells. We dissected structural attributes of NKp44 to determine their contributions to receptor function. Our results demonstrate that surface expression and NK cell activation by NKp44 is mediated through noncovalent association with the immunoreceptor tyrosine-based activation motif-containing protein, DAP12. Physical linkage to DAP12 requires lysine-183 in the NKp44 transmembrane domain. Intriguingly, the cytoplasmic domain of NKp44 also contains a sequence that matches the immunoreceptor tyrosine-based inhibitory motif (ITIM) consensus. By expressing a chimeric receptor in an NK-like cell line, we found that this ITIM-like motif from NKp44 lacks inhibitory capacity in a redirected cytotoxicity assay. The NKp44 cytoplasmic tyrosine was efficiently phosphorylated in the chimeric receptor upon treating the cells with pervanadate, but it was unable to recruit ITIM-binding negative effector phosphatases. We also generated NK-like cell lines expressing epitope-tagged wild-type or tyrosine to phenylalanine mutant (Y238F) versions of NKp44 and compared their capacities to induce activation marker expression, promote IFN-gamma production, or stimulate target cell cytotoxicity. We did not detect any tyrosine-dependent reduction or enhancement of NK cell activation through wild-type vs. Y238F mutant NKp44. Finally, the cytoplasmic tyrosine-based sequence did not provide a docking site for the AP-2 clathrin adaptor, nor did it potentiate receptor internalization. In summary, all activating properties and surface expression of NKp44 are mediated through its association with DAP12, and the putative ITIM in the NKp44 cytoplasmic domain does not appear to attenuate activating function.

KIR2DL4 is an IL-2-regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production.

Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4(*)). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4(*) were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56(high) NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-gamma production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4(*) exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.

Expression of normal cellular prion protein (PrP(c)) on T lymphocytes and the effect of copper ion: Analysis by wild-type and prion protein gene-deficient mice.

The purpose of this report was to determine the effect of prion protein (PrP) gene disruption on T lymphocyte function. Previous studies have suggested that normal cellular prion protein (PrP(c)) binds to copper and Cu(2+) is essential for interleukin-2 (IL-2) mRNA synthesis. In this study, IL-2 mRNA levels in a copper-deficient condition were investigated using T lymphocytes from prion protein gene-deficient (PrP(0/0)) and wild-type mice. Results showed that Cu(2+) deficiency had no effect on PrP(c) expression in Con A-activated splenocytes. However, a delay in IL-2 gene expression was observed in PrP(0/0) mouse T lymphocyte cultures using Con A and Cu(2+)-chelator. These results suggest that PrP(c) expression may play an important role in rapid Cu(2+) transfer in T lymphocytes. The rapid transfer of Cu(2+) in murine T lymphocytes could be one of the normal functions of PrP(c).

The lck promoter-driven expression of the Wilms tumor gene WT1 blocks intrathymic differentiation of T-lineage cells.

In the thymi of WT1-transgenic (Tg) mice with the 17AA+/KTS- spliced form of the Wilms tumor gene WT1 driven by the lck promoter, the frequencies of CD4-CD8- double-negative (DN) thymocytes were significantly increased relative to those in normal littermates. Of the 4 subsets of CD4-CD8- DN thymocytes, the DN1 (CD44+CD25-) subset increased in both frequency and absolute cell number, whereas the DN2 (CD44+CD25+) and DN3 (CD44-CD25+) subsets decreased, indicating the blocking of thymocyte differentiation from the DN1 to the DN2 subsets. Furthermore, CD4-CD8+ T-cell receptor (TCR) -gammadelta T-cells increased in both frequency and absolute cell number in the spleen and peripheral blood of the WT1-Tg mice relative to those of normal littermates. The CD8 molecules of these CD4-CD8+ TCRgammadelta T-cells were CD8alphabeta, suggesting that they originated from the thymus. These results are the first direct evidence demonstrating that the WT1 gene is involved in the development and differentiation of T-lineage cells.

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) can play a direct role in the inhibitory function of killer cell Ig-like receptors in human NK cells.

The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.

SHP-1- and phosphotyrosine-independent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic domain in human NK cells.

Killer cell Ig-like receptors (KIR) are MHC class I-binding immunoreceptors that can suppress activation of human NK cells through recruitment of the Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) to two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. KIR2DL4 (2DL4; CD158d) is a structurally distinct member of the KIR family, which is expressed on most, if not all, human NK cells. 2DL4 contains only one ITIM in its cytoplasmic domain and an arginine in its transmembrane region, suggesting both inhibitory and activating functions. While 2DL4 can activate IFN-gamma production, dependent upon the transmembrane arginine, the function of the single ITIM of 2DL4 remains unknown. In this study, tandem ITIMs of KIR3DL1 (3DL1) and the single ITIM of 2DL4 were directly compared in functional and biochemical assays. Using a retroviral transduction method, we show in human NK cell lines that 1) the single ITIM of 2DL4 efficiently inhibits natural cytotoxicity responses; 2) the phosphorylated single ITIM recruits SHP-2 protein tyrosine phosphatase, but not SHP-1 in NK cells; 3) expression of dominant-negative SHP-1 does not block the ability of 2DL4 to inhibit natural cytotoxicity; 4) surprisingly, mutation of the tyrosine within the single ITIM does not completely abolish inhibitory function; and 5) this correlates with weak SHP-2 binding to the mutant ITIM of 2DL4 in NK cells and a corresponding nonphosphorylated ITIM peptide in vitro. These results reveal new aspects of the KIR-inhibitory pathway in human NK cells, which are SHP-1 and phosphotyrosine independent.