Adoptive cell transfer (ACT) is considered a promising modality for cancer treatment, but despite ongoing improvements many patients do not experience clinical benefits. The tumor microenvironment is an important limiting factor in immunotherapy that has not been addressed fully in ACT treatments. In this study, we report that upregualtion of the immunosuppressive receptor PD-1 expressed on transferred T cells at the tumor site, in a murine model of ACT, compared with its expression on transferred T cells present in the peripheral blood and spleen. Since PD-1 can attenuate T cell-mediated antitumor responses, we tested whether its blockade with an anti-PD-1 antibody could enhance the antitumor activity of ACT in this model. Co-treatment with both agents increased the number of transferred T cells at the tumor site and also enhanced tumor regressions, compared to treatments with either agent alone. While anti-PD-1 did not reduce the number of immunosuppressive Treg cells and MDSCs present in tumor-bearing mice, we found that it increased expression of IFN-γ and CXCL10 at the tumor site. Bone marrow transplant experiments using IFN-γR-/- mice implicated IFN-γ as a crucial nexus for controlling PD-1-mediated tumor infiltration by T cells. Taken together, our results imply that blocking the PD-1 pathway can increase IFN-γ at the tumor site, thereby increasing chemokine-dependent trafficking of immune cells into malignant disease sites.

Purpose Treatment of melanoma patients with selective BRAF inhibitors results in objective clinical responses in the majority of patients with BRAF mutant tumors. However, resistance to these inhibitors develops within a few months. In this study, we test the hypothesis that BRAF inhibition in combination with adoptive T-cell transfer (ACT) will be more effective at inducing long-term clinical regressions of BRAF-mutant tumors. Experimental Design BRAF-mutated human melanoma tumor cell lines transduced to express gp100 and H-2Db to allow recognition by gp100-specific pmel-1 T-cells were used as xenograft models to assess melanocyte differentiation antigen-independent enhancement of immune responses by BRAF inhibitor PLX4720. Luciferase expressing pmel-1 T cells were generated to monitor T-cell migration in vivo. The expression of vascular endothelial growth factor (VEGF) was determined by enzyme-linked immunosorbent assay, protein array and immunohistochemistry. Importantly, VEGF expression after BRAF inhibition was tested in a set of patient samples. Results We found that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced the antitumor activity of ACT. This increased T-cell infiltration was primarily mediated by the ability of PLX4720 to inhibit melanoma tumor cell production of VEGF by reducing the binding of c-myc to the VEGF promoter. Furthermore, analysis of human melanoma patient tumor biopsies before and during BRAF inhibitor treatment showed downregulation of VEGF consistent with the pre-clinical murine model. Conclusion These findings provide a strong rationale to evaluate the potential clinical application of combining BRAF inhibition with T-cell based immunotherapy for the treatment of melanoma patients.

Ack͞Ack1 is a nonreceptor protein tyrosine kinase that comprises a tyrosine kinase core, an SH3 domain, a Cdc42-binding region, a Ralt homology region, and a proline-rich region. Here we describe a detailed characterization of the Ack protein as well as the chromosomal localization of human Ack (chromosome 3q29) and the primary structure of murine Ack. We demonstrate that Ack is ubiquitously expressed, with highest expression seen in thymus, spleen, and brain. Activation of integrins by cell adhesion on fibronectin leads to strong tyrosine phosphorylation and activation of Ack. Upon cell stimulation with EGF or PDGF, Ack is tyrosine-phosphorylated and recruited to activated EGF or PDGF receptors, respectively. A pool of endogenous Ack molecules is constitutively tyrosine-phosphorylated, even in starved cells. Moreover, tyrosine-phosphorylated Ack forms a stable complex with the adapter protein Nck via its SH2 domain. Finally, we have characterized a membrane-targeting sterile ␣ motif-like domain in the amino terminus of Ack. Using several Ack mutants, we show that the amino-terminal and CRIB domains are necessary for Ack autophosphorylation, whereas the SH3 domain appears to have an autoinhibitory role. These experiments suggest a functional role for Ack as an early transducer of multiple extracellular stimuli.cell signaling ͉ tyrosine phosphorylation ͉ surface receptors ͉ growth factors ͉ cell adhesion

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca2+ to directly measure the Ca2+ sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca2+ sensitivity of release in that a small proportion of granules makes up a highly Ca2+-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca2+. A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca2+ are not located close to Ca2+ channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca2+-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca2+, suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca2+ sensor that is poised to respond to modest, global elevations of [Ca2+]i.

Purpose One of the most important rate-limiting steps in adoptive cell transfer (ACT) is the inefficient migration of T cells to tumors. Since melanomas specifically express the chemokines CXCL1 and CXCL8 that are known to facilitate the CXCR2-dependent migration by monocytes, our aim is to evaluate whether introduction of the CXCR2 gene into tumor-specific T cells could further improve the effectiveness of ACT, by enhancing T-cell migration to tumor. Experimental Design In this study, we utilized transgenic pmel-1 T cells which recognize gp100 in the context of H-2Db, that were transduced with luciferase gene to monitor the migration of transferred T cells in vivo. In order to visualize luciferase-expressing T cells within a tumor, a non-pigmented tumor is required. Therefore, we utilized the MC38 tumor model which naturally expresses CXCL1. Results Mice bearing MC38/gp100 tumor cells treated with CXCR2/luciferase-transduced pmel-1 T cells showed enhanced tumor regression and survival compared to mice receiving control luciferase transduced pmel-1 T cells. We also observed preferential accumulation of CXCR2-expressing pmel-1 T cells in the tumor sites of these mice using bioluminescence imaging. A similar enhancement in tumor regression and survival was observed when CXCR2-transduced pmel-1 T cells were transferred into mice bearing CXCL1-transduced B16 tumors compared to mice treated with control pmel-1 T cells. Conclusions These results implicate that the introduction of the CXCR2 gene into tumor-specific T cells can enhance their localization to tumors and improve antitumor immune responses. This strategy may ultimately enable personalization of cancer therapies based on chemokine expression by tumors.

Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 Å. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.angiogenesis | cancer | phosphorylation | protein kinases | surface receptors V ascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis by binding to and activating the three members of the VEGF-receptor (VEGFR) family of receptor tyrosine kinases (RTKs) (1). VEGFR1 (Flt1), VEGFR2 (KDR/Flk1) and VEGFR3 (Flt4) are members of type-V RTK; a family containing a large extracellular region composed of seven Ig-like domains (D1-D7), a single transmembrane (TM) helix and cytoplasmic region with a tyrosine kinase activity, and additional regulatory sequences. The second and third Ig-like domains, D2 and D3, of VEGFR ectodomains function as binding sites for the VEGF family of cytokines (i.e., VEGF-A, -B, -C, -D, and placenta growth factor) (2, 3). These growth factors are covalently linked homodimers. Each protomer is composed of four-stranded β-sheets arranged in an antiparallel fashion in a structure designated cysteine-knot growth factors (4). It was shown that VEGF-A stimulation of VEGFR2 induces endothelial cell proliferation, survival, and migration resulting in blood vessel formation and sprouting (5). On the other hand, VEGF-C activation of VEGFR3 plays an important role in the formation of the lymphatic vessel system (6, 7). Aberrant activation or expression of VEGF receptors and their ligands has been implicated in tumor angiogenesis, coronary artery disease, diabetic blindness, and other diseases (5).Other members of the cysteine-knot family of cytokines include nerve growth factor and platelet-derived growth factors (PDGFs). However, the ectodomains of the PDGF-receptor (PDGFR) fami...

We have used flash photolysis of caged Ca 2؉ and membrane capacitance measurements to probe exocytosis in chromaffin cells at low concentrations of intracellular Ca 2؉ ([Ca 2؉ ]i) (<10 M). We observed a small pool of granules that is more sensitive to [Ca 2؉ ]i than the previously described ''readily releasable pool.'' Upon activation of PKC, this ''highly Ca 2؉ -sensitive pool'' is enhanced in size to a greater extent than the readily releasable pool but is eliminated upon expression of a C-terminal deletion mutant (⌬9) of synaptosome-associated protein of 25 kDa (SNAP-25). Thus, in chromaffin cells, PKC enhances exocytosis both by increasing the number of readily releasable vesicles and by shifting vesicles to a highly Ca 2؉ -sensitive state, enabling exocytosis at sites relatively distant from Ca 2؉ channels.

Pancreatic cancer is an aggressive malignancy with morbidity rates almost equal to mortality rates because of the current lack of effective treatment options. Here, we describe a targeted approach to treating pancreatic cancer with effective therapeutic efficacy and safety in noninvasive imaging models. We developed a versatile expression vector "VISA" (VP16-GAL4-WPRE integrated systemic amplifier) and a CCKAR (cholecystokinin type A receptor) gene-based, pancreatic-cancer-specific promoter VISA (CCKAR-VISA) composite to target transgene expression in pancreatic tumors in vivo. Targeted expression of BikDD, a potent proapoptotic gene driven by CCKAR-VISA, exhibited significant antitumor effects on pancreatic cancer and prolonged survival in multiple xenograft and syngeneic orthotopic mouse models of pancreatic tumors with virtually no toxicity.