CD4+ T Cell Effects on CD8+ T Cell Location Defined Using Bioluminescence

T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are “helped” by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell “help” is to program the homing potential of CD8+ T cells

The role of FasL and IFNγ in a model of AAV-mediated chronic liver injury in the mouse (39.52)

Abstract As models of chronic liver injury in the mouse are limited, we have developed such a model in the C57B6 mouse that utilizes Adeno-associated viral vectors (AAV) engineered to express both enhanced Green Fluorescent Protein and the CD8+ epitope SIINFEKL. After injection of this vector directly into the liver, 1x104 SIINFEKL-specific OT-1 cells are administered. Injection of these cells results in an increase in OT-1 cell numbers in the liver until Day 20 correlating with liver damage from Days 10-25. Addition of these cells does not cause decreased vector DNA but significantly decreases vector mRNA expression. To define the mechanisms by which OT-1 cells mediate their effect, we focused on both IFNγ and Fas. In looking at the role of these two molecules, we have found that IFNγ is important in the activation and/or recruitment of the OT-1 cells as IFNγR KO hosts have a significant decrease in the number of OT-1 in the liver at Day 20 compared to their WT controls. The same has been found when comparing WT and Faslpr hosts indicating a role of FasL in the same activation or recruitment processes. FasL has been shown by Suzuki et al to play a role in costimulation in a peptide model, but this phenomenon has not been seen in other viral or vector models1. The role of these two molecules in our AAV model may give clues to the molecular events that occur in other types of hepatitis. Suzuki I JI 2000 Nov 15; 165(10):5537-43. Support: NIH RO1 AI064463</jats:p

The E2F-1 Transcription Factor Promotes Caspase-8 and Bid Expression, and Enhances Fas Signaling in T Cells

Abstract The immune system depends on the extensive proliferation of rare Ag-specific precursor T lymphocytes, followed by their differentiation, the delivery of effector function, and finally death by apoptosis. T cells that lack the E2F-1 transcription factor, which is activated as cells pass the restriction point and enter S phase, show defects in activation-induced cell death. We now report that E2F-1 increases the activity of an apoptotic pathway that is important in murine primary T cells. Thus, E2F-1 promotes the transcription of Bid, a molecule that links death receptor signaling to the activation of apoptotic mechanisms in mitochondria. It also promotes the transcription of caspase-8, the enzyme that cleaves and activates Bid. Enforced expression of Bid can partially restore apoptosis in E2F-1-deficient T cells. Thus, E2F-1 integrates cell cycle progression with apoptosis.</jats:p

Effects of Neddylation and mTOR Inhibition in Acute Myelogenous Leukemia

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Recently, the neddylation (NEDD8) and the mammalian target of rapamycin (mTOR) signaling pathways have emerged as promising pharmaceutical targets for AML therapy. However, the interaction of these two pathways remains unclear. Here we evaluated the effects of pevonedistat, an inhibitor of the NEDD8 activating enzyme (NAE), and sapanisertib (TAK-228), an inhibitor of mTORC1 and mTORC2 as single agents or in combination on AML cell lines. We found that inhibition of neddylation with pevonedistat partially inhibited mTOR signaling transduction and vice versa, inhibition of mTOR signaling with sapanisertib partially inhibited neddylation in AML cell lines. Pevonedistat alone was able to induce cytotoxicity in most AML cell lines as well as in primary AML, whereas sapanisertib alone decreased cell metabolic activity, reduced cell size and arrested cells in G0 phase with only minimal induction of cell death. In addition, pevonedistat was able to induce cell differentiation, arrest cells in G2/M cell cycle phases, and induce DNA re-replication and damage. However, co-treatment with sapanisertib suppressed pevonedistat induced apoptosis, differentiation, S/G2/M arrest, and DNA damage. Taken together, our data demonstrate that pevonedistat and sapanisertib exhibit distinct anti-tumor effects on AML cells, i.e. cytotoxic and cytostatic effects, respectively; however, sapanisertib can attenuate pevonedistat-induced cellular responses in AML cells. Understanding mTOR and neddylation pathway interaction could provide therapeutic strategies for treatment of AML and other malignancies

Optimizing Effects of mTOR Inhibition in Acute Myelogenous Leukemia

Abstract Introduction : Mammalian target of rapamycin (mTOR) signaling has previously been identified as a possible therapeutic target in acute myelogenous leukemia (AML), as the PI3K/AKT/mTOR pathway has been shown to be upregulated in blasts of up to 90% of AML cases through the constitutive loss of the tumor suppressor PTEN. The activation of this pathway is implicated in synthesis of prosurvival transcription and translational factors responsible for cellular resistance to apoptosis, resistance to chemotherapy, and enhanced survival in the marrow microenvironment. Unfortunately, exploitation of this pathway has been largely ineffective in clinical studies. A second mTOR complex (mTORC2) will reestablish the activation of the first when the first mTOR complex (mTORC1) is shut down under treatment with traditional rapalogs, such as rapamycin and everolimus. Additionally, the cellular process of autophagy has been postulated to be a protective mechanism for leukemic blasts during treatment with mTOR inhibitors, therefore decreasing their efficacy. For these reasons, this work explores means to optimize mTOR pathway inhibition by examining effects of dual mTORC inhibition (OSI027), dual PI3K/mTOR inhibition (BEZ235/Dactilosib), combination of mTOR inhibitors with a histone deacetylase inhibitor with ability to inhibit AKT (LBH589/Panobinostat), and the combination of mTOR inhibitors with hypomethylating agents able to overcome the hypermethylation of mTOR pathway components such as TSC1, TSC2 and PTEN (decitabine/5-azacytidine). Materials and Methods : Primary AML leukapheresis samples obtained with informed consent were cultured for 48 hours in the presence of OSI027, LBH589 (Novartis), BEZ235 (Novartis), decitabine, or OSI027 in combination with each of these individual inhibitors and compared with control culture conditions. Cells were stained with annexinV and DAPI as previously described, and the percent of living, pre-apoptotic, necrotic and dead cells were determined via flow cytometry. Synergy calculations were completed using Calcusyn™ software as described by Chao and Talalay (1977). Autophagic flux was determined in the OSI027 and LBH589 combination exposure via flow cytometry using anti-LC3. Lastly, protein expression under treatment with various combinations was determined via standard western blotting techniques. Results: The MV411 AML cell line was utilized to establish efficacy of combination exposures. These cells expressed phosphorylated (p)Akt (ser 473), p4EBP1, and pmTOR. OSI027 10µM, LBH589 1µM, and BEZ235 10nM were able to inhibit pmTOR, pAKT, and p4EBP1 expression as determined by western blotting. In the MV411 cell line, IC50s were determined, and combination indices were determined for OSI027+BEZ235, panobinostat+BEZ235, and panobinostat+OSI027; all of which demonstrated synergy. In primary AML samples, the combination of OSI027 10uM and LBH589 10nM significantly increased the percentage of dead cells in comparison to OSI027 treatment alone. Additionally in primary AML blasts, pmTOR and pAKT expression were decreased in the combination of OSI027 and LBH589 when compared to single agent treatment via western blot. The combination of OSI027 and LBH589 demonstrated a significant reduction in the autophagic flux in comparison to OSI027 treatment alone, suggesting an anti-autophagic effect, which correlates with the increased rates of cellular death in the combination. It was also found in AML cell lines that the combination of 10 µM azacytidine and 100 nM rapamycin resulted in synergistic suppression of U937, MV411, and KG1a survival as measured by MTT. No synergy was noted in the HL60 cell line. This combination effectively suppressed CFU-L in primary AML cases and suppressed p70S6K and p4EBP1 expression more than either agent alone by western blotting. The combination of decitabine 500nM with OSI027 10uM did not demonstrate an increase in apoptotic cells in primary blasts compared to OSI027 alone. Conclusions : Based on these findings, we conclude that mTOR inhibition in AML cell lines and primary samples can be enhanced through dual mTORC inhibition, PI3K modulation, and histone deacetylase inhibition. This may occur through suppression of activated pathway mediators or through suppression of autophagic flux. These results suggest that there is merit in exploring these combinations for therapeutic potential in AML. Disclosures Portwood: ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding. </jats:sec