Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function.

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4(+) Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25(+)CD4(+) Tr cells in humans has not been reported. Here we show that human CD25(+)CD4(+) Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor-mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte-associated antigen (CTLA)-4. Human CD25(+)CD4(+) Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-beta, low levels of interferon (IFN)-gamma, and no IL-4 or IL-2. Importantly, CD25(+)CD4(+) Tr cells strongly inhibited the proliferative responses of both naive and memory CD4(+) T cells to alloantigens, but neither IL-10, TGF-beta, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25(+)CD4(+) Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25(+)CD4(+) Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell-mediated diseases.

Key role of Shc signaling in the transforming pathway triggered by Ret/ptc2 oncoprotein.

The RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of protoRET, a gene encoding two protein isoforms of a transmembrane tyrosine kinase receptor. By using Ret/ptc2 short isoform (iso9), we have previously demonstrated that Tyr586 (Tyr1062 of protoRet) is the docking site for both the PTB and the SH2 domains of Shc. To determine the relevance of this interaction for the transforming activity of Ret/ptc oncogenes, we have generated and characterized novel Ret/ptc mutants unable to activate Shc: Ret/ptc2 long isoform (iso51)-Y586F and both isoforms of Ret/ptc2-N583A. These mutants neither activate Shc nor transform NIH3T3 cells. Since Tyr1062 shows features of a multifunctional docking site, we have used a Shc mutant (Shc Y317F) to directly assess Shc role. We have demonstrated that in our cell system Shc Y317F behaves like a dominant interfering mutant on the activation of the Grb2-Sos pathway by endogenous Shc triggered by Ret/ptc2. A strong reduction of the transforming activity of Ret/ptc2 in presence of this mutant was also demonstrated. Our data suggest that Shc activation play a key role in the transforming pathways triggered by Ret/ptc oncoproteins. Moreover, we have shown that coexpression of the Shc-Y317F mutant with Ret/ptc2 specifically causes apoptosis, and that the surviving cells lose the long-term expression of one of the two genes.

IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells.

CD4(+) T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-alpha, but not TGF-beta, induces naive CD4(+) T cells derived from cord blood to differentiate into Tr1 cells: IL-10(+)IFN-gamma(+)IL-2(-/low)IL-4(-). Naive CD4(+) T cells derived from peripheral blood require both exogenous IL-10 and IFN-alpha for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-beta. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-alpha strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.

Tumor targeting with biotinylated tumor necrosis factor alpha: structure-activity relationships and mechanism of action on avidin pretargeted tumor cells.

We have recently described a new strategy for targeting biotinylated tumor necrosis factor-alpha (TNF-alpha) to tumors, based on pretargeting with biotinylated antibodies and avidin. Here, we have analyzed the structure-activity relationships of several biotin-TNF-alpha conjugates and studied the mechanism of their interaction with avidin and TNF-alpha receptors on tumor cells. The study has been carried out using an in vitro model based on human melanoma Colo 38 cells and monoclonal antibody 225, an antibody against the high molecular weight melanoma-associated antigen. Immunochemical and cytotoxicity studies showed that biotin-TNF-alpha but not TNF-alpha persists for several hours on the surface of cells pretargeted with biotin-monoclonal antibody 225 and avidin and triggers cytolytic effects. Studies on the mechanism of action showed that biotin-TNF-alpha trimers can slowly dissociate from targeted cells in a bioactive form, through trimer-monomer-trimer transitions. Structure-activity relationship studies showed that nonbiotinylated subunits must be present in the biotin-TNF-alpha trimers for efficient release of bioactive TNF-alpha. Colo 38 cells targeted with biotin-TNF-alpha were able to kill mouse L-M cells in coculture experiments, indicating that the released TNF-alpha can interact also with TNF-alpha receptors expressed by bystander cells. In conclusion, the targeting complex works as a system that slowly releases bioactive TNF-alpha in the microenvironment of the targeted cell. This opens up the possibility that cells other than those reached by the targeting antibody (e.g., endothelial cells and local cells of the immune system) can be affected in vivo.

Transient overexpression of human H- and L-ferritin chains in COS cells.

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.