Joanna Stanson

Antigen-Processing Machinery in Human Dendritic Cells: Up-Regulation by Maturation and Down-Regulation by Tumor Cells

It has been known for some time that functional properties of dendritic cells (DC), and in particular their ability to process and present Ags to T cells, can be modulated by cytokine-induced maturation and by interactions with tumor cells. However, the molecular basis for these functional changes is unknown. We have investigated whether changes in expression of Ag-processing machinery (APM) components in DC are associated with alterations in their ability to present tumor-derived Ags to T cells. Using a panel of mAbs specific for individual APM components and a quantitative flow cytometry-based method, the level of APM components was measured in DC generated from peripheral blood monocytes of 12 normal donors and of 8 patients with cancer. Immature DC had significantly lower (p < 0.01) expression of MB1, LMP-7, LMP-10, TAP-1, and tapasin than mature DC. However, maturation in the presence of a cytokine mixture up-regulated expression of these components in DC obtained from normal donors and patients with cancer. Immature DC incubated with tumor cells had significantly lower (p < 0.001) expression of MB1, LMP-2, LMP-7, LMP-10, and endoplasmic reticulum p75 than controls. These changes were associated with a decreased ability of DC to present tumor-derived Ags to T cells, as measured in ELISPOT assays and with apoptosis of T cells in DC-T cell cultures. Thus, tumor cells have a significant suppressive effect on DC; however, ex vivo maturation of DC derived from patients with cancer in a polarizing cytokine mix restores normal expression of APM components and Ag-processing capabilities.

Modulation of TcR/CD3-zeta chain expression by a circulating factor derived from ovarian cancer patients

In women with ovarian cancer, suppression of components of the immune system may promote tumour development. Previous studies in ovarian cancer have demonstrated that decreased expression and function of the T-cell receptor (TcR)-associated signal transducing zeta-chain correlates with deficient immune responsiveness of T cells. In this study, sera and ascitic fluids obtained from woman with advanced ovarian cancer were found to suppress the expression of TcR-associated zeta chain. This suppression of zeta chain expression was dose-dependent and was not observed with biologic fluids obtained from healthy women.The factor responsible for the loss of zeta chain was purified from ascites and characterized as a protein with an appropriate molecular weight of 14 kD. Suppression of T-cell TcR-zeta was specific, since neither lck nor ZAP-70 expression was affected, while zeta chain was almost completely suppressed. This selective suppression of TcR-zeta expression by the 14 kD ascites-derived factor was shown to operate at the mRNA level. By defining the mechanism through which this protein modulates TcR-zeta chain levels, it might be possible to ultimately prevent the suppressive influences of the tumour microenvironment and restor immune competence in patients with ovarian carcinoma.

Interleukin-2 expression in human carcinoma cell lines and its role in cell cycle progression

Human carcinomas were shown to express mRNA and protein for IL-2R α, β and γ chains. Recently, human carcinomas were also shown to constitutively express protein and mRNA for IL-2 in vivo and in vitro. Here we report that the expression levels of cytoplasmic IL-2 as well as IL-2Rβ- and γ-chain in human carcinoma cells change during the cell cycle progression. Carcinoma cells synchronized in the G2/M phase of the cell cycle expressed significantly more intracytoplasmic IL-2 as well as IL-2Rβ and γ proteins than tumor cells in the G0/G1 phase. The level of mRNA for IL-2 was 5–10-fold higher in the M phase than in the G0/G1-phase, as shown by quantitative competitive RT–PCR. Expression of the cyclin-dependent kinase (CDK) inhibitor p27kip1 in these carcinoma cells was found to be high in the G0/G1 phase, nearly absent in the S phase, and it increased again in the G2/M phase of the cell cycle. In synchronized cells, the decrease in p27 expression coincided with high levels of expression of IL-2. Using the IL-2 specific antisense oligonucleotide to block synthesis of endogenous IL-2 in tumor cells, we observed increased levels of p27 as well as p21. The antisense oligonucleotides specific for p27 or p21 blocked expression of these proteins but not of IL-2. Thus, endogenous IL-2 is important in regulating expression of p27 as well as p21 and, therefore, in controlling cell cycle progression of tumor cells, while its own expression remains independent of the CDK inhibitors.

A multicenter comparison study between the Endosafe® PTS™ rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products

BACKGROUND Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Healths Department of Transfusion Medicine, USA. METHODS Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS Valid paired results, based on each centers LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

FAP‐1‐mediated activation of NF‐κB induces resistance of head and neck cancer to fas‐induced apoptosis

Molecular mechanisms responsible for tumor resistance to apoptosis often involve the Fas/FasL pathway. While squamous cell carcinomas of the head and neck (SCCHN) express both Fas and FasL, their resistance to self‐induced apoptosis or apoptosis mediated by Fas agonistic antibody (CH‐11Ab) was independent of the level of Fas surface expression or the presence of soluble Fas in supernatants of primary or metastatic SCCHN cell lines. By in vitro immunoselection, using PCI‐15A cell line treated with successive cycles of CH‐11 Ab, Fas‐resistant sublines with the parental genotype were selected. Such sublines failed to cleave caspase‐8 upon Fas engagement and were resistant to CH‐11 Ab, although they remained sensitive to VP‐16 or staurosporin. In the presence of cycloheximide, the selected SCCHN sublines become susceptible to CH‐11 Ab, and showed cleavage of caspase‐8, suggesting that apoptosis resistance was mediated by an inhibitory protein(s) acting upstream of caspase‐8. Overexpression of Fas‐associated phosphatase 1 (FAP‐1), but not cellular FLICE‐inhibitory protein (cFLIP) in SCCHN sublines was documented by Western blots and RT‐PCR analyses. The FAP‐1+ selected sublines also downregulated cell surface Fas. A high phosphorylation level of IκBκ, NFκB activation and upregulation of Bcl‐2 expression were observed in the FAP‐1+ sublines. Treatment with the phosphatase inhibitor, orthovanadate, or silencing of FAP‐1 with siRNA abolished their resistance to apoptosis, suggesting that FAP‐1 phosphatase activity could be responsible for NF‐κB activation and resistance of SCCHN cells to Fas‐mediated apoptosis. J. Cell. Biochem. 100: 16–28, 2007.

Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo

This article describes a DNA-based vaccination strategy evaluated ex vivo with human cells. The vaccine was prepared by transferring tumor-derived genomic DNA to PCI-13 cells, a highly immunogenic tumor cell line (“recipient cell”), which had been genetically modified to secrete IL-2 (PCI-13/IL-2). PCI-13 cells expressed class I MHC determinants (HLA-A2) shared with the tumor from which the DNA was obtained as well as allogeneic determinants. DNA from a gp100+ melanoma cell line was transduced into gp100− PCI-13/IL-2 cells (PCI-13/IL-2/DNA). A T cell line specific for the gp100 epitope responded to PCI-13/IL-2/DNA cells by IFN-γ-secretion measured in enzyme-linked immunospot assays. The T cell line also recognized the gp100 epitope presented by dendritic cells that ingested PCI-13/IL-2/DNA cells, which had been induced by UVB irradiation to undergo apoptosis. After up-take and processing of apoptotic PCI-13/IL-2/DNA cells, the dendritic cells primed normal peripheral blood lymphocytes to generate effector T cells specific for the tumor donating the DNA. The results indicate that tumor epitopes encoded in such DNA are expressed in recipient cells and can induce tumor-specific T cells. The findings support translation of this vaccination strategy to a phase I trial in patients with cancer.

Production of a Dendritic Cell-Based Vaccine Containing Inactivated Autologous Virus for Therapy of Patients with Chronic Human Immunodeficiency Virus Type 1 Infection

ABSTRACT In preparation for a pilot clinical trial in patients with chronic human immunodeficiency virus type 1 (HIV-1) infection, a novel dendritic cell (DC)-based vaccine is being manufactured. The trial will test the hypothesis that isolated endogenous virus presented by DCs serves as a potent immunogen for activation of CD8+ and CD4+ T cells specific for a broad range of autologous HIV-1 antigens. Production of the vaccine under good manufacture practice conditions involves (i) autologous virus isolation; (ii) superinfection of CD4+ T cells with the virus; (iii) inactivation of the virus in CD4+ T cells, T-cell apoptosis, and coincubation of T cells with autologous DCs; and (iv) product testing and release. Endogenous virus was isolated from peripheral blood-derived CD4+ T cells of three HIV-1-positive subjects by coincubation with autologous OKT-3-stimulated CD4+ T cells. CD4+ T-cell supernatants were tested for p24 levels by enzyme-linked immunosorbent assay (>25 ng/ml) and for the 50% tissue culture infective doses (TCID50; which ranged from 4,642 to 46,416/ml on day 19 of culture). Autologous CD4+ T cells that were separated on immunobeads (>95% purity) and superinfected with virus-expressed p24 (28 to 54%) had TCID50 of >400/ml on days 5 to 10. Virus inactivation with psoralen (20 μg/ml) and UVB irradiation (312 nm) reduced the TCID50 of the supernatants from 199,986 to 11/ml (>99%). 7-Amino-actinomycin D-positive, annexin V-positive CD4+ T cells were fed to autologous DCs generated by using the Elutra cell separation system and the Aastrom system. Flow analysis showed that DC loading was complete in 24 h. On the basis of these translational results and experience with the generation of DCs from HIV-1-infected patients in a previous clinical trial, the Investigational New Drug application for clinical vaccination was submitted and approved by the FDA (application no. BB-IND-13137).

Molecular analysis of the IL-2 receptor β chain gene expressed in human tumor cells

Interleukin-2 (IL-2) is recognized as a T cell growth factor. We have previously reported that human carcinoma cell lines are inhibited in growth by exogenous IL-2, which binds to the IL-2 receptor β (IL-2Rβ) chain ubiquitously expressed on the surface of tumor cells. A possibility was considered that IL-2Rβ on carcinomas responsible for negative signaling was different from that expressed on hematopoietic cells. To investigate this possibility, mRNA for the IL-2Rβ chain was amplified and compared in carcinoma and lymphoid cells. Using RT–PCR with pairs of sense-antisense oligonucleotide primers specific for the various regions of extracellular, transmembrane and intracellular domains of the IL-2Rβ chain, we amplified mRNA obtained from three human carcinoma cell lines and human lymphoid cells as controls. The identity of the amplicons was confirmed by Southern analysis with the 32P-labeled cDNA probe coding for the entire span of the IL-2Rβ chain. In addition, genomic DNA obtained from the tumor cell lines was sequenced to examine the possibility that a mutation is present in the gene coding for the intracellular IL-2Rβ chain domain. No mutations or deletions were detected. The message for all three domains of the β chain was identical in tumor cells and in normal lymphoid cells used as controls. Also, by Western blot and northern analyses no differences between IL-2Rβ chain in tumors vs that expressed in lymphoid cells were demonstrable. The IL-2Rγ chain, which participates in IL-2/IL-2R signaling pathway, was expressed in tumor cells. Expression of JAK1 transcripts in these cells was comparable to that in lymphocytes. However, RT–PCR analysis identified differences in expression of JAK3 splice variants (B and M) in tumor cells. These differences may be responsible for altered downstream signaling by IL-2. Overall, our data indicate that the same IL-2/IL-2R pathway is operative in human carcinomas and in normal epithelial or lymphoid cells.

Shipping of therapeutic somatic cell products

BACKGROUND AIMS Shipment of therapeutic somatic cells between a current good manufacturing practice (cGMP) facility and a clinic or between different cGMP facilities requires validated standard operating procedures (SOP). Under National Heart Lung & Blood Institute (NHLBI) sponsorship, the Production Assistance for Cellular Therapies (PACT) group conducted a validation study for the shipping SOP it has created, including shipments of cryopreserved somatic cells, fresh peripheral blood specimens and apheresis products. METHODS Comparisons of pre- and post-shipped cells and cell products at the three participating facilities included measurements of viability, phenotypic profiles and cellular functions. The data were analyzed at the University of Pittsburgh Biostatistics Facility. RESULTS No consistent shipping effects on cell viability, phenotype or functions were detected for cryopreserved and shipped peripheral blood mononuclear cells (PBMC), monocytes, immature dendritic cells (iDC), NK-92 or cytotoxic T cells (CTL). Cryopreserved mesenchymal stromal cells (MSC) had a significantly decreased viability after shipment, but this effect was in part because of inter-laboratory variability in the viable cell counts. Shipments of fresh peripheral blood and apheresis products for the generation of CTL and dendritic cells (DC), respectively, had no significant effects on cell product quality. MSC were successfully generated from fresh bone marrow samples shipped overnight. CONCLUSIONS This validation study provides a useful set of data for guiding shipments of therapeutic somatic cells in multi-institutional clinical trials.

Transfection of human monocyte-derived dendritic cells with native tumor DNA induces antigen-specific T-cell responses in vitro.

Objective: Nucleofection of genomic tumor (Tu) DNA into human monocyte-derived dendritic cells (hMoDC) was evaluated for use in producing anti-tumor vaccines able to induce effective T-cell specific immune responses. Methods: Cultured hMoDC obtained from HLA-A2+ normal donors were nucleofected with genomic DNA extracted from an HLA-A2+gp100+ Mel 526 cell line and 3’end-labeled with biotinylated TdT nucleotides or from a genetically-modified Mel 526 expressing enhanced green fluorescent protein (EGFP). An Amaxa Nucleofector™ system was used for electroporation. Nucleofected hMoDC were matured in the presence of cytokines and examined in ELISPOT assays for the ability to present the gp100209-217 epitope to epitope-specific T cells or to prime autologous naïve T cells in culture. Results: The nucleofected hMoDC presented gp100 protein to HLA-A2+gp100-specific T cells as observed in IFN-γ ELISPOT assays. Spot formation was inhibited by anti-HLA class I and HLA-A2 but not anti-HLA class II antibodies (Abs). Tu DNA-nucleofected hMoDC also primed naïve autologous peripheral blood T cells in culture to develop into Tu-reactive effector cells (CTL). These CTL recognized Tu cells which had donated genomic DNA, and these responses were MHC class I- and class II-restricted. The CTL recognized shared Tu antigens encoded in Tu-derived DNA. Conclusion: Nucleofection of hMoDC with genomic Tu-derived DNA is a useful strategy for Tu vaccine production: it is feasible, does not require Tu epitope isolation, can be used when few Tu cells are available, and avoids Tu-induced DC suppression.