A major question for gene therapy in brain concerns methods to administer therapeutic genes in a uniform manner over major portions of the brain. A second question in neuroimmunology concerns the extent to which monocytes migrate to the CNS in degenerative disorders. Here we show that CD11bϩ cells (largely monocytes) isolated from the bone marrow of GFP (green fluorescent protein)-expressing donors spontaneously home to compacted amyloid plaques in the brain. Injections of these cells as a single pulse show a rapid clearance from circulation (90 min half-life) and tissue residence half-lives of ϳ3 d. The uptake into brain was minimal in nontransgenic mice. In transgenic mice containing amyloid deposits, uptake was dramatically increased and associated with a corresponding decrease in monocyte uptake into peripheral organs comparedtonontransgeniclittermates.TwiceweeklyinfusionsoftheCD11bϩbonemarrowcellstransfectedwithageneticallyengineeredform of the protease neprilysin completely arrest amyloid deposition in an aggressively depositing transgenic model. Exploiting the natural homing properties of peripherally derived blood cells to deliver therapeutic genes has the advantages of access to the entire CNS, expression largely restricted to sites of injury, low risk of immune reactivity, and fading of expression if adverse reactions are encountered. These observations support the feasibility of testing autologous monocytes for application of therapeutic genes in human CNS disease. Moreover, these data support the results from bone marrow grafts that circulating CD11bϩ cells can enter the CNS without requiring the use of lethal irradiation.

No abstract

BackgroundPancreatic cancer is one of the most aggressive cancers, with tumor-induced myeloid-derived suppressor cells (MDSC) contributing to its pathogenesis and ineffective therapies. In response to cytokine/chemokine receptor activation, src homology 2 domain-containing inositol 5′-phosphatase-1 (SHIP-1) influences phosphatidylinositol-3-kinase (PI3K) signaling events, which regulate immunohomeostasis. We hypothesize that factors from murine pancreatic cancer cells cause the down-regulation of SHIP-1 expression, which may potentially contribute to MDSC expansion, and the suppression of CD8+ T cell immune responses. Therefore, we sought to determine the role of SHIP-1 in solid tumor progression, such as murine pancreatic cancer.Methodology and Principal FindingsImmunocompetent C57BL/6 mice were inoculated with either murine Panc02 cells (tumor-bearing [TB] mice) or Phosphate Buffer Saline (PBS) (control mice). Cytometric Bead Array (CBA) analysis of supernatants of cultured Panc02 detected pro-inflammatory cytokines such as IL-6, IL-10 and MCP-1. TB mice showed a significant increase in serum levels of pro-inflammatory factors IL-6 and MCP-1 measured by CBA. qRT-PCR and Western blot analyses revealed the in vivo down-regulation of SHIP-1 expression in splenocytes from TB mice. Western blot analyses also detected reduced SHIP-1 activity, increased AKT-1 and BAD hyper-phosphorylation and up-regulation of BCL-2 expression in splenocytes from TB mice. In vitro, qRT-PCR and Western blot analyses detected reduced SHIP-1 mRNA and protein expression in control splenocytes co-cultured with Panc02 cells. Flow cytometry results showed significant expansion of MDSC in peripheral blood and splenocytes from TB mice. AutoMACS sorted TB MDSC exhibited hyper-phosphorylation of AKT-1 and over-expression of BCL-2 detected by western blot analysis. TB MDSC significantly suppressed antigen-specific CD8+ T cell immune responses in vitro.Conclusion/SignificanceSHIP-1 may regulate immune development that impacts MDSC expansion and function, contributing to pancreatic tumor progression. Thus, SHIP-1 can be a potential therapeutic target to help restore immunohomeostasis and improve therapeutic responses in patients with pancreatic cancer.

Pancreatic cancer (PC) evades immune destruction by favoring the development of regulatory T cells (Tregs) that inhibit effector T cells. The transcription factor Ikaros is critical for lymphocyte development, especially T cells. We have previously shown that downregulation of Ikaros occurs as a result of its protein degradation by the ubiquitin-proteasome system in our Panc02 tumor-bearing (TB) mouse model. Mechanistically, we observed a deregulation in the balance between Casein Kinase II (CK2) and protein phosphatase 1 (PP1), which suggested that increased CK2 activity is responsible for regulating Ikaros’ stability in our model. We also showed that this loss of Ikaros expression is associated with a significant decrease in CD4+ and CD8+ T cell percentages but increased CD4+CD25+ Tregs in TB mice. In this study, we evaluated the effects of the dietary flavonoid apigenin (API), on Ikaros expression and T cell immune responses. Treatment of splenocytes from naïve mice with (API) stabilized Ikaros expression and prevented Ikaros downregulation in the presence of murine Panc02 cells in vitro, similar to the proteasome inhibitor MG132. In vivo treatment of TB mice with apigenin (TB-API) improved survival, reduced tumor weights and prevented splenomegaly. API treatment also restored protein expression of some Ikaros isoforms, which may be attributed to its moderate inhibition of CK2 activity from splenocytes of TB-API mice. This partial restoration of Ikaros expression was accompanied by a significant increase in CD4+ and CD8+ T cell percentages and a reduction in Treg percentages in TB-API mice. In addition, CD8+ T cells from TB-API mice produced more IFN-γ and their splenocytes were better able to prime allogeneic CD8+ T cell responses compared to TB mice. These results provide further evidence that Ikaros is regulated by CK2 in our pancreatic cancer model. More importantly, our findings suggest that API may be a possible therapeutic agent for stabilizing Ikaros expression and function to maintain T cell homeostasis in murine PC.

No abstract