MABF2734-100UG Sigma-AldrichAnti-PVR (CD155) Antibody, clone L95

Histone deacetylase inhibitors (HDIs) are emerging as potent anti-tumour agents which induce cell cycle arrest, differentiation and/or apoptosis in many tumour cells. Furthermore, they render tumour cells more sensitive to other therapeutic regimens like ionizing radiation, chemotherapy and recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we show that the HDIs suberoylanilide hydroxamic acid (SAHA; vorinostat), sodium butyrate (NaB) and MS-275 sensitized DAOY and PC3 tumour cells for the cytotoxic effects of IL-2-activated PBMCs. In (51)Cr-release assays, blockade of the activating NK receptors DNAM-1, NKG2D and the NCRs completely abrogated tumour cell lysis, revealing that NK cells were the main effector cells involved. HDIs increased the tumour surface expression of ligands for the activating NK receptors NKG2D and DNAM-1 thereby facilitating tumour cell recognition by NK cells. These results suggest that the combination of HDIs and immunotherapy may be an effective strategy for anti-cancer therapy.

Purpose: The aim of this study was to characterize the immune response of patients affected by renal cell carcinoma (RCC). <br />Methods: Long-term RCC lines were established by retroviral-mediated transfer of the large T-antigen of SV40 into fresh carcinoma cells. Reactive T cell effectors were generated by iterative stimulations of patients' PBMC with autologous tumour cells. <br />Results: This protocol led to the induction of CD8(+) T cell clones reactive against the autologous tumour, but not against NK-sensitive cell lines. However, some of these effectors recognize normal renal cells, allogeneic renal carcinoma cell lines and colon and non-small cell lung carcinomas but not melanomas and lymphoblastoid lines, without evidence of shared classical HLA class I (HLA-I) molecules. Further characterization performed on the CD8(+) TCR alpha/beta(+) clone, CTL30, demonstrated that neither expression of CD1, HLA-Ia nor HLA-Ib, correlated with the T cells' recognition. Moreover, beta2m expression by target cells was not required to achieve interaction of tumour-effector cells. In agreement with this observation, the lytic activity of CTL30 was not inhibited by anti-HLA-I Ab, and antigen expression was not affected by inhibitors of antigen processing. Lytic activity of CTL30, while partially inhibited by anti-NKG2D, could not be abolished by anti-CD3 Abs. Moreover, growth and expansion of CTL30 was sustained only by T cell interaction with antigen-expressing tumour cells; unspecific mitogenic stimuli, such as anti-CD3 and PHA, did not allow T cell expansion. These results demonstrated the existence of an alpha/beta T cell population, recognizing epithelial tumour cells through an HLA-unrestricted, CD3-independent mechanism.

Mast cells have critical effector functions in various immune reactions. In allergic inflammation, mast cells interact with tissue-infiltrating eosinophils, forming a regulatory unit in the late and chronic phases of the allergic process. However, the pathways and molecules within this unit are still largely undefined. Here, we show that human mast cells and eosinophils express DNAX accessory molecule 1 (DNAM-1, CD226) and its ligand Nectin-2 (CD112). CD226 synergizes with FcepsilonRI on mast cells, and its engagement augments degranulation through a pathway involving Fyn, linker of activation of T-cells, phospholipase C gamma2, and CD18. This pathway is subject to negative interference by inhibitory receptors and is completely inhibited by linking IgE with IRp60 (CD300a) using a bispecific antibody. Moreover, blocking CD112 expressed on eosinophils using neutralizing antibodies normalized the hyperactivity resulting from IgE-dependent activation of mast cells co-cultured with eosinophils. Our findings demonstrate a novel interface between these two effector cells, implicating relevance for in vivo allergic states. Moreover, costimulatory responses might be a critical component in allergic reactions and may therefore become novel targets for anti-allergic therapy.

The capability of NK lymphocytes to kill tumor cells depends on different receptors/ligands interactions. In order to identify the cellular ligands recognized by "orphan" triggering receptors, mice were immunized with NK susceptible target cells. mAbs were selected that inhibited NK cytotoxicity and recognized two different molecules of 70 and 60-65 kDa. Tryptic digestion and mass spectra analysis of purified proteins identified these molecules as PVR and Nectin-2, respectively. PVR-Fc and Nectin-2-Fc chimeric molecules stained COS-7 cells expressing the DNAM-1 activating receptor and conversely, PVR and Nectin-2 CHO-K cell transfectants were stained by DNAM-1-Fc. Thus, both PVR and Nectin-2 represent specific ligands for DNAM-1. Importantly, the specific interaction between DNAM-1 (in NK cells) and PVR or Nectin-2 (in target cells) enhanced the NK-mediated lysis of tumor cells that was downregulated by mAb-mediated masking of the receptor or its ligands.

Human natural killer (NK) cells express a series of activating receptors and coreceptors that are involved in recognition and killing of target cells. In this study, in an attempt to identify the cellular ligands for such triggering surface molecules, mice were immunized with NK-susceptible target cells. On the basis of a functional screening, four mAbs were selected that induced a partial down-regulation of the NK-mediated cytotoxicity against the immunizing target cells. As revealed by biochemical analysis, three of such mAbs recognized molecules of approximately 70 kD. The other mAb reacted with two distinct molecules of approximately 65 and 60 kD, respectively. Protein purification followed by tryptic digestion and mass spectra analysis, allowed the identification of the 70 kD and the 65/60 kD molecules as PVR (CD155) and Nectin-2 delta/alpha (CD112), respectively. PVR-Fc and Nectin-2-Fc soluble hybrid molecules brightly stained COS-7 cells transfected with the DNAM-1 (CD226) construct, thus providing direct evidence that both PVR and Nectin-2 represent specific ligands for the DNAM-1 triggering receptor. Finally, the surface expression of PVR or Nectin-2 in cell transfectants resulted in DNAM-1-dependent enhancement of NK-mediated lysis of these target cells. This lysis was inhibited or even virtually abrogated upon mAb-mediated masking of DNAM-1 (on NK cells) or PVR or Nectin-2 ligands (on cell transfectants).