Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO

International audienceBACKGROUND: Thyroid cancers are difficult to treat due to their limited responsiveness to chemo- and radiotherapy. There is thus a great interest in and a need for alternative therapeutic approaches. RESULTS: We studied the cytotoxic activity of anti-thyroperoxidase autoantibodies (anti-TPO aAbs, expressed in baculovirus/insect cell (B4) and CHO cells (B4') or purified from patients' sera) against a papillary thyroid cancer (NPA) cell line. Anti-TPO aAbs from patients' sera led to a partial destruction of NPA cell line by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) and exhibited an anti-proliferative activity. Comparison of the cytotoxic activity of anti-TPO aAbs shows that B4' induced an anti-proliferative effect and a better ADCC than B4, but a lower one than anti-TPO aAbs from patients' sera. Antibody-dependent cell-mediated cytotoxicity was increased when human peripheral blood mononuclear cells were used as effector cells, suggesting that FcgammaRs, CD64, CD32 and CD16 are involved. Indeed, anti-TPO aAbs from patients' sera, but not B4 and B4', exhibited CDC activity. CONCLUSIONS: These data indicate that anti-TPO aAbs display moderate ADCC and anti-proliferative activities on NPA cells; IgG glycosylation appears to be important for cytotoxic activity and ADCC efficiency depends on FcgammaR-bearing cells. Finally, recombinant human anti-TPO aAbs cannot yet be considered as an optimal tool for the development of a novel therapeutic approach for thyroid cancer

Perforin Rapidly Induces Plasma Membrane Phospholipid Flip-Flop

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells

TL1A Selectively Enhances IL-12/IL-18-Induced NK Cell Cytotoxicity against NK-Resistant Tumor Targets

# The Author(s) 2010. This article is published with open access at Springerlink.com Introduction TL1A (TNFSF15) augments IFN-γ production by IL-12/IL-18 responsive human T cells. Its ligand, death domain receptor 3 (DR3), is induced by activation on T and NK cells. Although IL-12/IL-18 induces DR3 expression on most NK cells, addition of TL1A minimally increases IFN-

Inhibition of Melanoma Growth by Subcutaneous Administration of hTERTC27 Viral Cocktail in C57BL/6 Mice

hTERTC27 is a 27 kDa C-terminal polypeptide of human telomerase reverse transcriptase that has previously been shown to reduce tumorigenicity of HeLa cells and suppress growth of xenografted glioblastoma in nude mice. Although ectopic expression of hTERTC27 upregulated genes that are involved in apoptosis, cell cycle, and immune response, the mechanism for hTERTC27-induced tumor suppression has not been completely elucidated. Since hTERT was identified as a universal tumor-associated antigen, we hypothesize that hTERTC27 inhibits tumor growth in vivo through activation of anti-tumor immune response. Immunocompetent C57BL/6 mice were used for mouse B16 melanoma model. Mice bearing B16 melanoma were administered rAAV-/rAdv viral cocktail expressing hTERTC27, and tumor growth was monitored after viral cocktail treatment. Blood and splenocytes were used to determine the level of cytokines and the activity of immune cells, respectively. B16 tumor growth was significantly inhibited by subcutaneous administration of a single dose of 1.5×10(11) vg rAAV-hTERTC27 and 2.5×10(9) pfu rAdv-hTERTC27 viral cocktail (rAAV-/rAdv-hTERTC27). The population and cytotoxicity of NK cells in the mice were significantly augmented by rAAV-/rAdv-hTERTC27 treatment, and selective depletion of the NK cell population in mice by intraperitoneal injection of anti-GM1 antibody abrogated the growth suppression of melanoma induced by rAAV-/rAdv-hTERTC27 administration. Activation of NK cells by administration of rAAV-/rAdv-hTERTC27 is critical for growth suppression of melanoma in mouse model.published_or_final_versio

Systemic IL-12 Administration Alters Hepatic Dendritic Cell Stimulation Capabilities

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2–3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections

Treatment of hepatocellular carcinoma with major portal vein thrombosis by combined therapy with subcutaneous interferon-α and intra-arterial 5-fluorouracil; role of type 1 interferon receptor expression

We previously reported the beneficial effects of combination therapy of interferon (IFN)-α/5-fluorouracil (FU) for advanced hepatocellular carcinoma (HCC) with tumour thrombi in the major portal branches. This report describes the results of longer follow-up and includes more than double the number of patients relative to the original report, and evaluates the role of IFN-α/type 2 interferon receptor (IFNAR2) expression on the response to the combination therapy. The study subjects were 55 patients with advanced HCC and tumour thrombi in the major branches of the portal vein (Vp3 or 4). They were treated with at least two courses of IFN-α/5-FU without major complication. In the 55 patients, 24 (43.6%) showed objective response (eight (14.5%) showed complete response, 16 (29.1%) partial response), four (7.3%) showed no response, and 27 (49.1%) showed progressive disease. Immunohistochemically, IFNAR2 expression was detected in nine out of 13 (69.2%) patients. There was significant difference in the time-to-progression survival (P=0.0002) and the overall survival (P<0.0001) between IFNAR2-positive and -negative cases. There was a significant correlation between IFNAR2 expression and response to IFN-α/5-FU combination therapy in univariate analysis (P=0.0070). IFN-α/5-FU combination therapy is a promising modality for advanced HCC with tumour thrombi in the major portal branches and could significantly depend on IFNAR2 expression

A Novel Role for IκBζ in the Regulation of IFNγ Production

IκBζ is a novel member of the IκB family of NFκB regulators, which modulates NFκB activity in the nucleus, rather than controlling its nuclear translocation. IκBζ is specifically induced by IL-1β and several TLR ligands and positively regulates NFκB-mediated transcription of genes such as IL-6 and NGAL as an NFκB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1β and IL-18, strongly synergize with TNFα for IFNγ production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNγ production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IκBζ protein expression in KG-1 cells upon stimulation with IL-1β, IL-18 and TNFα. Our results demonstrated that IL-18, as well as IL-1β, induced moderate IκBζ expression in KG-1 cells. However, TNFα synergized with IL-1β and IL-18, whereas by itself it had a minimal effect on IκBζ expression. NFκB inhibition resulted in decreased IL-1β/IL-18/TNFα-stimulated IFNγ release. Moreover, silencing of IκBζ expression led to a specific decrease in IFNγ production. Overall, our data suggests that IκBζ positively regulates NFκB-mediated IFNγ production in KG-1 cells