The ALCAM shedding by the metalloprotease ADAM17/TACE is involved in motility of ovarian carcinoma cells.

Previous findings indicated that the activated leukocyte cell adhesion molecule (ALCAM) is expressed by tumors and plays a role in tumor biology. In this study, we show that ALCAM is shed from epithelial ovarian cancer (EOC) cells in vitro, leading to the generation of a soluble ALCAM (sALCAM), consisting of most of the extracellular domain. A similar sALCAM molecule was also found in the ascitic fluids and sera from EOC patients, suggesting that this process also occurs in vivo. sALCAM is constitutively produced by EOC cells, and this process can be enhanced by cell treatment with pervanadate, phorbol 12-myristate 13-acetate (PMA), or epidermal growth factor (EGF), a known growth factor for EOC. Pharmacologic inhibitors of matrix metalloproteinases (MMP) and of a disintegrin and metalloproteases (ADAM), and the tissue inhibitor of metalloproteinase-3, significantly inhibited sALCAM release by EOC cells. The ADAM17/TACE molecule was expressed in EOC cell lines and ADAM17/TACE silencing by specific small interfering RNA-reduced ALCAM shedding. In addition, inhibitors of ADAM function blocked EOC cell motility in a wound-healing assay. Conversely, a recombinant antibody blocking ALCAM adhesive functions and inducing ALCAM internalization enhanced EOC cell motility. Altogether, our data suggest that the disruption of ALCAM-mediated adhesion is a relevant step in EOC motility, and ADAM17/TACE takes part in this process, which may be relevant to EOC invasive potential.

Resistance to CD95-mediated apoptosis of CD40-activated chronic lymphocytic leukemia B cells is not related to lack of DISC molecules expression.

In B-cell chronic lymphocytic leukemia (CLL), accumulation of neoplastic B cells may be the result of dysregulated apoptosis. One of the major molecules triggering apoptosis, CD95 (FAS), is not expressed on CLL B cells at resting conditions. However, CD40 triggering of CLL B cells upregulates receptors belonging to the tumor necrosis factor (TNF) superfamily, like CD95. In the present study, we analyzed in B cells from 20 CLL patients the effect of CD40/CD40L interaction on: (i) CD95 modulation; (ii) CD95-mediated apoptosis and (iii) mRNA and protein expression of the death-inducing signaling complex (DISC) molecules.CD40 activation of CLL B cells was carried out by coculture with CD40L-transfected cells and cytofluorimetric analyses were performed to study CD95 modulation and apoptosis induction by an anti-CD95 moAb. Despite strong CD95 upregulation on the membrane of all the cases studied, only a minority of cases analyzed (3/20) proved weakly responsive to CD95-mediated apoptosis. Multiplex RT-PCR was used to analyze FLICE, FAS, FADD and TRADD mRNAs before and after CD40 triggering. In agreement with the cytofluorimetric data, FAS mRNA appeared significantly increased after CD40 triggering; the other molecules involved in DISC formation and in CD95-mediated apoptosis were also expressed without relevant differences between resting and activated conditions. Western blot analyses further confirmed FLICE and FADD protein expression by resting and activated CLL cells. Our findings demonstrate that, following CD40 triggering, CLL B cells are resistant to CD95-mediated apoptosis despite a strong CD95 upregulation on the membrane and a normal mRNA or protein expression of the DISC components.

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine.

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.

IFN-gamma-independent synergistic effects of IL-12 and IL-15 induce anti-tumor immune responses in syngeneic mice.

IL-15 and IL-12 display anti-tumor activity in different models and IFN-gamma has been reported as a secondary mediator of both IL-12 and IL-15 effects. TS/A murine adenocarcinoma cells were engineered to secrete IL-12, IL-15 or both cytokines. TS/A cells secreting IL-15 (TS/A-IL-15) displayed a reduced tumorigenicity (50%) when implanted subcutaneously in syngeneic mice, while both TS/A-IL-12 and TS/A-IL-12/IL-15 were rejected by 100% of animals. In contrast, TS/A-IL-15 and TS/A-IL-12 were tumorigenic in syngeneic IFN-gamma knockout mice (100% and >90% of take rate, respectively), but TS/A-IL-12/IL-15 were completely rejected by 90% of these mice. All IFN-gamma-deficient mice rejecting TS/A-IL-12/IL-15 developed protective immunity against wild-type TS/A, as indicated by re-challenge experiments. Immunohistochemical analysis of the TS/A-IL-12/IL-15 tumor rejection area in IFN-gamma-deficient mice showed a marked reactive cell infiltration constituted of CD8+ cells, granulocytes, NK cells, macrophages and dendritic cells associated with the expression of IL-1beta, TNF-alpha, GM-CSF, MCP-1 and MIP-2. In vivo depletion experiments showed that rejection of TS/A-IL-12/IL-15 cells required CD8+ T lymphocytes and also involved granulocytes, while CD4+ and NK cells played a minor role. These data show IFN-gamma-independent synergistic anti-tumor effects of IL-12 and IL-15, involving CD8+ cells and secondary chemokines and cytokines, such as TNF-alpha.

Expression of interleukin-18 in human ovarian carcinoma and normal ovarian epithelium: evidence for defective processing in tumor cells.

Interleukin-18 (IL-18) is a proinflammatory monokine structurally related to IL-1beta that stimulates interferon-gamma (IFN-gamma) production. IL-18 is synthesized as an inactive precursor, pro-IL-18, which is cleaved by IL-1beta-converting enzyme (ICE)/caspase-1 in a mature protein. In view of the proposed use of IL-18 in cancer immuno/gene therapy, we have studied the expression of IL-18 in tumor cells. IL-18 mRNA was detected by reverse transcriptase polymerase chain reaction in all human ovarian carcinoma cell lines tested (9/9) and in one-half of tumor cell populations obtained from ovarian carcinoma patients (4/8). ICE mRNA was expressed in a smaller fraction of samples (3/9 cell lines and 3/8 samples from patients). IL-18 protein was also found in 7/13 ovarian carcinoma solid tumors by immunohistochemic analysis. In tumor cell lines we were able to detect abundant intracellular pro-IL-18 (24 kDa) by Western blotting, whereas the mature form of IL-18 was undetectable, irrespective of the presence of ICE mRNA and protein. Only pro-IL-18 was also found in the ovarian carcinoma cell supernatants, which did not display any IL-18 biologic activity in functional assays. Normal cultured ovarian epithelial cells revealed the presence of both IL-18 and ICE mRNA in all samples (5/5) and IL-18 protein was expressed by the thin epithelial cell layer surrounding normal ovary. More importantly, normal ovarian epithelial cells released low but detectable amounts of mature IL-18 in the culture supernatant, which displayed IL-18-like biologic activity in functional assays. These data suggest that mature biologically active IL-18 production is a feature of the normal ovarian surface epithelium lost during neoplastic transformation.

Identification of immunodominant epitopes in inactivated Tat-vaccinated healthy and HIV-1-infected volunteers.

We analyzed the epitopes and the molecular forms of Tat recognized by the antibodies raised by Tat-toxoid vaccination in both healthy and HIV-infected volunteers. Tat-toxoid-vaccinated healthy volunteer sera reacted predominantly with peptides covering amino acids 1 through 24 and 46 through 60, corresponding to the N-terminus and basic domains of Tat. In contrast, whereas all sera from vaccinated HIV-1-positive patients reacted with the N-terminus and (with a single exception) with the basic domain, most of these sera also recognized peptides encompassing distinct domains of Tat, particularly the C-terminus (79-86). The sera of vaccinated individuals recognized both monomeric and oligomeric forms of Tat 1 through 86 or of Tat 1 through 101 and also blocked the ability of cell-released extracellular Tat to transactivate the HIV-1 LTR promoter. Synthetic Tat preincubated with sera from vaccinated individuals lost its functional activity as well. This is probably because of its inability to enter the cells as a result of immune complex formation with anti-Tat IgG. These data demonstrate that Tat-toxoid vaccination induces an efficient antibody response blocking the functional activity of Tat.

IL-21 induces tumor rejection by specific CTL and IFN-gamma-dependent CXC chemokines in syngeneic mice.

IL-21 is an immune-stimulatory four alpha helix cytokine produced by activated T cells. To study the in vivo antitumor activities of IL-21, TS/A murine mammary adenocarcinoma cells were genetically modified to secrete IL-21 (TS/A-IL-21). These cells developed small tumors that were subsequently rejected by 90% of s.c. injected syngeneic mice. Five days after injection, TS/A-IL-21 tumors showed numerous infiltrating granulocytes, NK cells, and to a lesser extent CD8(+) T cells, along with the expression of TNF-alpha, IFN-gamma, and endothelial adhesion molecules ICAM-1 and VCAM-1. At day 7, CD8(+) and CD4(+) T cells increased together with IFN-gamma, and the CXC chemokines IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-inducible T cell alpha-chemoattractant. The TS/A-IL-21 tumor displayed a disrupted vascular network with abortive sprouting and signs of endothelial cell damage. In vivo depletion experiments by specific Abs showed that rejection of TS/A-IL-21 cells required CD8(+) T lymphocytes and granulocytes. When injected in IFN-gamma-deficient mice, TS/A-IL-21 cells formed tumors that regressed in only 29% of animals, indicating a role for IFN-gamma in IL-21-mediated antitumor response, but also the existence of IFN-gamma-independent effects. Most immunocompetent mice rejecting TS/A-IL-21 cells developed protective immunity against TS/A-pc (75%) and against the antigenically related C26 colon carcinoma cells (61%), as indicated by rechallenge experiments. A specific CTL response against the gp70-env protein of an endogenous murine retrovirus coexpressed by TS/A and C26 cells was detected in mice rejecting TS/A-IL-21 cells. These data suggest that IL-21 represents a suitable adjuvant in inducing specific CTL responses.