Conformational regulation of Escherichia coli DNA polymerase V by RecA and ATP.

Mutagenic translesion DNA polymerase V (UmuD'2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD'2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD'2C-RecA-ATP.

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V.

T cell receptor (TCR)-transgenic CD8 lymphocytes rendered insensitive to transforming growth factor beta (TGFβ) signaling mediate superior tumor regression in an animal model of adoptive cell therapy.

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma.

Enhanced antitumor activity induced by adoptive T-cell transfer and adjunctive use of the histone deacetylase inhibitor LAQ824.

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells.

Quantitative PET reporter gene imaging of CD8+ T cells specific for a melanoma-expressed self-antigen.

Adoptive transfer (AT) T-cell therapy provides significant clinical benefits in patients with advanced melanoma. However, approaches to non-invasively visualize the persistence of transferred T cells are lacking. We examined whether positron emission tomography (PET) can monitor the distribution of self-antigen-specific T cells engineered to express an herpes simplex virus 1 thymidine kinase (sr39tk) PET reporter gene.

Immunosensitization with a Bcl-2 small molecule inhibitor.

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models.

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain.

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain.

NK cells represent a potent immune effector cell type that have the ability to recognize and lyse tumors. However, the existence and function of NK cells in the traditionally "immune-privileged" CNS is controversial. Furthermore, the cellular interactions involved in NK cell anti-CNS tumor immunity are even less well understood.

Immunosensitization of tumor cells to dendritic cell-activated immune responses with the proteasome inhibitor bortezomib (PS-341, Velcade).

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.

Broad antitumor protection by dendritic cells administered to CD8alpha knock out mice.

Dendritic cell (DC) administration to CD8alpha knock-out (CD8alphaKO) mice results in a strong antigen-non-specific protection to a B16 murine melanoma tumor challenge. This response is mediated by lytic NK cells and cytokine-producing CD4 cells. We aimed to determine the signals that guide tumor targeting of this response.