The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin

Exogenous HIV-1 Nef Upsets the IFN-γ-Induced Impairment of Human Intestinal Epithelial Integrity

The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions

The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin

Effect of HIV-1 Nef on cytokine and chemokine secretion by untreated or IFN-γ treated intestinal epithelial cells.

<p>Caco-2 cells were grown and differentiated on inserts and then left untreated (medium) or treated with graded amounts of Nef (ranging from 0.01 to 1 µg/ml), boiled Nef (0.1 µg/ml, bNef) or Nef_F12 (0.1 µg/ml). After 48 h supernatants were harvested from the basolateral chambers and tested for cytokines (TNF-α, IL-6, IL-10) and chemokines (MIP-3α, IL-8) by ELISA. In some experiments Nef and IFN-γ were preincubated with an anti-Nef mAb (10 µg/ml) or anti-Tat mAb (10 µg/ml) for 1 h at 37°C and then added to Caco-2 cells for 48 h. Results are expressed as pg/ml and are means ± SEM from 4 independent experiments in duplicate. *p<0.05 vs medium; ^○p<0.05 vs IFN-γ; ^Δp<0.05 vs Nef.</p

Effect of HIV-1 Nef on paracellular flux of FITC-dextran and transepithelial electrical resistance (TEER) in untreated or IFN-γ treated intestinal epithelial cell monolayer.

<p>(A) Caco-2 cells were grown and differentiated on inserts and then left untreated (medium) or treated with graded amounts of Nef (ranging from 0.01 to 1 µg/ml), boiled Nef (0.1 µg/ml, bNef) or Nef_F12 (0.1 µg/ml) for 48 h. FITC-dextran (FITC-DX) was added in the apical compartment and after 3 h, the fluorescence intensity was measured in the basolateral compartment. Data represent means ± SEM of 3 independent experiments in duplicate. F.I. fold increase. (B) Caco-2 cells were treated for 48 h with supernatants from 293T cells transfected with either a pcDNA3.1 vector expressing wt Nef HIV-1 (SN wt Nef, containing 0.01 µg/ml Nef), or with the vector alone (SN mock). The amount of Nef in supernatants was estimated by semi-quantitative anti-Nef Western blot assay against quantified amounts of recombinant Nef. (C) Caco-2 cells were left untreated or treated with Nef (0.1 µg/ml), IFN-γ (600 U/ml) or their combination for 48 h. Nef and IFN-γ were preincubated with an anti-Nef mAb (10 µg/ml) or anti-Tat mAb (10 µg/ml) for 1 h at 37°C and then added to Caco-2 cells for 48 h. Means ± SEM from 4 independent experiments in duplicate, are shown. (D) Caco-2 cells were cultured on filter inserts for 21 days and then left untreated (medium ○) or treated with Nef (0.1 µg/ml ▪), IFN-γ (600 U/ml ▴) or IFN-γ/Nef (Δ). The TEER values were measured at the indicated time points and were normalized with values obtained at the start point of the analysis. Data represent means ± SEM of 8 independent experiments in duplicate. *p<0.05 vs medium; ^○p<0.05 vs IFN-γ; ^Δp<0.05 vs Nef.</p

Effect of HIV-1 Nef on tight junction protein expression and localization in untreated or IFN-γ treated intestinal epithelial cell monolayer.

<p>(A) Differentiated Caco-2 cells were left untreated (medium) or were treated for 48 h with Nef, IFN-γ or their combination. Cells were then lysed and proteins were probed on Western blots with anti-occludin or anti-ZO1 mAbs. (B) The expression of the proteins was estimated by densitometry after normalization with the β-actin signal. Data represent values from 1 experiment representative of 3 performed. (C) Immunofluorescence analysis of ZO1 distribution in untreated (medium) or 48 h Nef-, IFN-γ - or IFN-γ/Nef- treated Caco-2 monolayers. The experiment was repeated 3 times to ensure reproducibility.</p

HIV-1 Nef uptake in intestinal epithelial cells.

<p>Differentiated Caco-2 cells were left untreated (medium) or were treated for 1, 3, 6 and 24 h with biotynilated-Nef alone or in combination with IFN-γ. Nef uptake was analyzed by CLSM (central optical sections). Nuclei are reported in blue (DAPI). Nef was detected as a diffuse intracytoplasmatic positivity (green). Scale bar, 20 µm. Panels are representative of 3 independent experiments.</p