Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-RasQ61L or K-RasG12D) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.

SUMMARYActivated human and rat T cells as well as mouse T-cell clones have been reported to synthesize and express major histocompatibility complex (MHC) class II molecules. However, the capacity of class II antigen (Ag) presenting T cells to induce proliferation of Ag-specific cloned T cells has been controversial. We analysed whether the failure of some T-cell clones to proliferate in response to Ag presented by class II T cells is because of a lack of costimulatory cytokine production by the antigenpresenting cells (APC). As a model system the mouse class II cloned BI/O4.1 T cells were used as APC in order to activate the T cell clone KIII5. This T-helper 1 (Th1) type, GAT (synthetic copolymer of Lglutamic acid, L-alanine and L-tyrosine)-specific clone is characterized by an efficient downregulation of interleukin-2 receptor (IL-2R) with time following antigenic stimulation. KIII5 cells respond to GATpresenting splenic antigen-presenting cells (APC) by IL-2 production, IL-2R upregulation and proliferation. When BI/O4.1 T cells were used as APC, KIII5 cells produced IL-2, but did not proliferate. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed a lack of IL-12 production by BI/O4.1 cells. Addition of IL-12 to a coculture of Ag-presenting BI/O4.1 cells and KIII5 cells fully reconstituted a proliferative response. IL-12 in synergy with IL-2 upregulated IL-2Ra chain expression and enhanced proliferation of KIII5 cells. Our data suggest, that class II T cells are not functional in inducing Ag-mediated expansion of resting Th1 cells owing to their failure to produce IL-12, but rather that they play a role in amplification loops during an ongoing immune response.

An important aspect of immunity is the recruitment and accumulation of lymphocytes into target tissues where Ags are localized. Because the TCR recognizes antigenic peptides presented by MHC molecules, the subset of T cells that exert effector function is determined by the class of MHC molecules expressed on a given tissue. We and others have demonstrated that CD8 T cells are preferentially recruited into B7-1-transfected class II-negative plasmacytoma J558, and the virus-infected central nervous system. However, the mechanism for such specificity has not been addressed. Here we analyzed the mechanism for selective recruitment of CD8 T cells into B7-1-transfected plasmacytoma J558. We show that sustained accumulation of CD8 T cells in vivo requires local expression of B7-1. In addition, we have observed a striking correlation between expression of macrophage-inflammatory protein 1alpha (MIP1alpha) and selective accumulation of CD8 T cells in the tumors. The selective recruitment of CD8 T cells is blocked by in vivo administration of neutralizing anti-MIP1alpha antisera. Moreover, gene-transfer studies reveal that locally produced MIP1alpha is sufficient to induce selective recruitment of CD8 T cells. Taken together, our study reveals that costimulation by B7 leads to sustained local production of MIP1alpha, which selectively recruits CD8 T cells into tumors. These results have important implications for T cell recruitment in vivo and for tumor immunotherapy.