Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine that is considered a proinflammatory cytokine. However, our studies show that MIF, when produced in super-physiological levels by a murine neuroblastoma cell line (Neuro-2a) exceeding those normally seen during an immune response, inhibits cytokine-, CD3-, and allo-induced T-cell activation. MIF is also able to inhibit T cells that have already received an activation signal. The T-cell inhibitory effects of culture supernatants from neuroblastoma cells were reversed when the cells were transfected with dicergenerated si-RNA to MIF. When T cells were activated in vitro by co-culture with interleukin (IL)-2 and IL-15 and analyzed for cytokine production in the presence or absence of MIF-containing culture supernatant, inhibition of T-cell proliferation and induced cell death were observed even as the treated T cells produced high levels of interferon-gamma (IFN-γ). The inhibitory effects of MIF were partially reversed when lymphocytes from IFN-γ knockout mice were tested. We propose that the high levels of MIF produced by neuroblastoma cause activation induced T-cell death through an IFN-γ pathway and may eliminate activated T cells from the tumor microenvironment and thus contribute to escape from immune surveillance.

Plaen IG. Lack of VEGFR2 signaling causes maldevelopment of the intestinal microvasculature and facilitates necrotizing enterocolitis in neonatal mice.

R-Ras is a member of the RAS superfamily of small GTP-binding proteins. The physiologic function of R-Ras has not been fully elucidated. We found that RRas is expressed by lymphoid and nonlymphoid tissues and drastically upregulated when bone marrow progenitors are induced to differentiate into dendritic cells (DCs). To address the role of R-Ras in DC functions, we generated a R-Rasdeficient mouse strain. We found that tumors induced in Rras ؊/؊ mice formed with shorter latency and attained greater tumor volumes. This finding has prompted the investigation of a role for R-Ras in the immune system. Indeed, Rras ؊/؊ mice were impaired in their ability to prime allogeneic and antigen-specific T-cell responses. Rras ؊/؊ DCs expressed lower levels of surface MHC class II and CD86 in response to lipopolysaccharide compared with wild-type DCs. This was correlated with a reduced phosphorylation of p38 and Akt. Consistently, R-Ras- IntroductionDendritic cells (DCs) are potent antigen-presenting cells with key roles in initiating antigen-specific immune responses. 1 Although numerous reports have implicated members of the RAS superfamily of GTP-binding proteins in DC biology, 2 a complete understanding of their individual roles is still lacking. Small GTPases traditionally operate as molecular switches, being inactive in the GDP-bound state and active in the GTP-bound state, therein conferring signaling function through interactions with their downstream effectors. 3 Indeed, several small GTPases have been shown to play critical roles in immune cell functions by regulating actin cytoskeleton-dependent processes. [4][5][6][7][8][9][10] Small GTPases Rap1, Rac1/2, and Cdc42, with the latter 2 being part of the RHO family of GTPases, have been demonstrated to be important in DC biology. Active Cdc42 can reactivate endocytosis in mature DCs, whereas microinjection of a dominant negative Cdc42N17 mutant abrogates the uptake of dextran. 9 Interestingly, it was demonstrated in DCs that levels of active Cdc42 and Rac were not increased in response to lipopolysaccharide (LPS). 11 However, using mature DCs from Rac1 and Rac2 double-knockout mice (Rac1/2 Ϫ/Ϫ ), Rac1 and Rac2 were shown to be critical for dendrite formation and T-cell activation. 12 In addition, RAPL, a downstream effector of Rap1, has been shown to be critical for both lymphocyte and DC migration and adhesion. 5,6 Although both Rap1 and Cdc42/Rac GTPases are clearly involved in DC biology, little is known concerning the role of other key members of the Ras subfamily.R-Ras is a small GTPase that belongs to the Ras subfamily and shares approximately 55% amino acid sequence similarity with the RAS proto-oncogenes, H-RAS, N-RAS, and K-RAS (collectively referred to as Ras herein). 13,14 Distinctively, R-Ras possesses a unique 26-amino acid sequence in the aminoterminus that is necessary for Rac activation and Rac-dependent cell spreading. 15 Furthermore, active R-Ras in macrophages has been shown to positively regulate phagocytosis of C3bi-opsonized particles via the in...

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The goal of this study was to show that nonviral gene transfection technology can be used to genetically modify neuroblastoma cells with immune stimulatory molecules, and that the modified cells can generate an antitumor immune response. The authors found that an electroporation-based gene transfection method, nucleofection, could be used to modify mouse AGN2a (an aggressive variant of Neuro-2a) neuroblastoma cells to simultaneously express as many as four different immune stimulatory molecules encoded by separate plasmid vectors. Within 18 hours after nucleofection, greater than 60% of the cells typically expressed the transfected gene products, and the percentages of cells expressing the products often exceeded 96%. High levels of plasmid in cell nuclei immediately after nucleofection documented instantaneous availability of gene vectors to the transcriptional machinery. AGN2a cells nucleofected to express the co-stimulatory molecules CD80 and CD86 expressed higher levels of these molecules than cells that had been permanently transfected with these same plasmid vectors, and the nucleofected cells were as effective as the permanently transfected cells at inducing an antitumor response in vivo in a tumor prevention model. AGN2a cells nucleofected with four separate plasmid vectors encoding CD54, CD80, CD86, and CD137L induced a T-cell immune response in vitro and served as a potent tumor vaccine in the tumor prevention model. These data show that transient transfection using a nonviral based method, nucleofection, can be used to rapidly generate novel cell-based tumor vaccines.