Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo.

Dendritic cells (DC) were cultured from mouse bone marrow (BM) progenitors in low concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM(lo) DC) by two different protocols. The phenotype and functional properties of these GM(lo) DC were compared to those of standard BM-DC cultures generated in high concentrations of GM-CSF (GM(hi) DC) or in low GM-CSF plus IL-4 (GM(lo)/IL-4 DC). An effect of IL-4 on maturation was observed only at low but not high doses of GM-CSF. Compared to mature DC, GM(lo) DC were phenotypically immature, weak stimulators of allogeneic and peptide-specific T cell responses, but substantially more potent in presentation of native protein. Immature GM(lo) DC were resistant to maturation by lipopolysaccharide, TNF-alpha or anti-CD40 monoclonal antibodies, as the expression of co-stimulatory molecules was not increased, and stimulatory activity in oxidative mitogenesis was not enhanced. These maturation-resistant immature GM(lo) DC induced T cell unresponsiveness in vitro and in vivo. GM(lo) DC also prolonged haplotype-specific cardiac allograft survival (from 8 days to >100 days median survival time) when they were administered 7 days (but not 3, 14 or 28 days) before transplantation. Our findings may have important implications for future studies in T cell tolerance induction in vivo.

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival.

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.

Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation.

The capacity of dendritic cells (DC) to initiate immune responses is dependent on their specialized migratory and tissue homing properties. Chemotaxis and transendothelial migration (TEM) of DC were studied in vitro. Immature DC were generated by culture of human monocytes in granulocyte-macrophage colony-stimulating factor and IL-4. These cells exhibited potent chemotaxis and TEM responses to the CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and monocyte chemotactic protein-3, and weak responses to the CC chemokine MIP-3beta and the CXC chemokine stromal cell-derived factor (SDF)-1alpha. Maturation of DC induced by culture in lipopolysaccharide, TNF-alpha or IL-1beta reduced or abolished responses to the former CC chemokines but markedly enhanced responses to MIP-3beta and SDF-1alpha. This correlated with changes in chemokine receptor expression: CCR5 expression was reduced while CXCR4 expression was enhanced. These findings suggest two stages for regulation of DC migration in which one set of chemokines may regulate recruitment into or within tissues, and another egress from the tissues.

Bacterial lipopolysaccharide contamination of commercial collagen preparations may mediate dendritic cell maturation in culture.

Dendritic cells (DC) are potent antigen presenting cells, which are responsible for the initiation of naive T and T-dependent immune responses. The present studies were based upon recent reports that commercial collagen I preparations induce the maturation of human DC in vitro. We show that human blood monocyte-derived (GM-CSF and IL-4 cultured) DC pulsed on collagen I-coated plates undergo a dose-dependent increase in stimulatory capacity in oxidative mitogenesis assays. This is accompanied by the upregulation of costimulatory molecules (CD40, CD80, CD86), CD25, ICAM-1 and the DC-specific marker CD83. The maturation effect is more potent than TNF-alpha, which is a known mediator of DC function. However, bacterial lipopolysaccharide (LPS), a powerful inducer of DC maturation, was found to be present at very high levels in one commercial collagen solution that was tested. The effect of LPS upon DC maturation was similar to culture with collagen. Furthermore, a different collagen I preparation with low levels of LPS contamination was less effective at inducing DC maturation, while spiking the collagen solution with LPS prior to plastic coating equalised these effects. Finally, human monocyte-derived DC were found not to express typical collagen receptors VLA-1, 2 and 3. We therefore propose that LPS contamination may at least partially explain reported collagen I induced DC maturation.

Pulmonary arteriography for the assessment of technical feasibility of sleeve resection in lung cancer.

BACKGROUND: Pulmonary arteriography has been reported to be useful in the preoperative assessment of patients with lung cancer to determine the technical resectability and feasibility of pneumonectomy by imaging the main right and left pulmonary arteries. In this report, we describe the use of selective pulmonary arteriography in the assessment of lobar resectability. METHODS: Selective pulmonary arteriography provides a detailed anatomic view of the lobar branches and has been used at our institution for the past 30 years to preoperatively investigate patients who are candidates for a sleeve lobectomy. RESULTS: Three cases are described that demonstrate the usefulness of selective pulmonary arteriography in the assessment of the technical feasibility of sleeve resection in patients with lung cancer. CONCLUSIONS: Arteriographic findings may accurately show whether a sleeve lobectomy is technically possible, that only a pneumonectomy is possible, or that the only safe way to ensure clearance of the pulmonary artery is to perform arterioplasty. This information may obviate an unnecessary thoracotomy in patients who are judged on the basis of a physiologic assessment to be unable to tolerate a pneumonectomy.