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.
BackgroundMaintenance of T cell immune homeostasis is critical for adequate anti-tumor immunity. The transcription factor Ikaros is essential for lymphocyte development including T cells. Alterations in Ikaros expression occur in blood malignancies in humans and mice. In this study, we investigated the role of Ikaros in regulating T cell immune balance in pancreatic cancer mouse models.Methodology and Principal FindingsUsing our Panc02 tumor-bearing (TB) mouse model, western blot analysis revealed a reduction in Ikaros proteins while qRT-PCR showed no differences in Ikaros mRNA levels in TB splenocytes compared to control. Treatment of naïve splenocytes with the proteasomal inhibitor, MG132, stabilized Ikaros expression and prevented Ikaros downregulation by Panc02 cells, in vitro. Western blot analyses showed a reduction in protein phosphatase 1 (PP1) and protein kinase CK2 expression in TB splenocytes while CK2 activity was increased. Immunofluorescence microscopy revealed altered punctate staining of Ikaros in TB splenocytes. Flow cytometry revealed a significant decrease in effector CD4+ and CD8+ T cell percentages but increased CD4+CD25+ regulatory T cells in TB splenocytes. Similar alterations in T cell percentages, as well as reduced Ikaros and CK2 but not PP1 expression, were observed in a transgenic, triple mutant (TrM) pancreatic cancer model. Ikaros expression was also reduced in enriched TB CD3+ T cells. MG132 treatment of naïve CD3+ T cells stabilized Ikaros expression in the presence of Panc02 cells. Western blots showed reduced PP1 and CK2 expression in TB CD3+ T cells.Conclusions/SignificanceThe results of this study suggest that the pancreatic tumor microenvironment may cause proteasomal degradation of Ikaros, possibly via dysregulation of PP1 and CK2 expression and activity, respectively. This loss of Ikaros expression may contribute to an imbalance in T cell percentages. Ikaros may potentially be a therapeutic target to restore T cell homeostasis in pancreatic cancer hosts, which may be critical for effective anti-tumor immunity.
A cell-engineered system to assess tumor cell sensitivity to CD8 + T cell-mediated cytotoxicity, OncoImmunology, 8:8, 1-10, ABSTRACT In vitro assays that evaluate CD8 + T cell-mediated cytotoxicity are important to aid in the development of novel therapeutic approaches to enhance anti-tumor immune responses. Here, we describe a novel cytotoxicity coculture assay that circumvents the problem of highly variable allogeneic responses and obviates the constraints of HLA-restriction between effector and target cells. We show that this assay can be easily applied to a panel of tumor cell lines to provide additional insights into intrinsic drivers of sensitivity/resistance to T cellmediated killing, and to evaluate the impact of targeted therapies on both tumor and T cell compartments.
MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer  . In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens 7 . It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts 7-10 . However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression 11,12 . Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC 13 . MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1 + leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1 + leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1 + leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1 + leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1 + enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor-bearing) in in vivo and in vitro assays.
The alternatively spliced transcription factor Ikaros is crucial for normal lymphocyte development. Ikaros plays a role as a tumor suppressor in murine T cells. In pancreatic cancer hosts, the balance between effector and regulatory T cells is lost, leading to immunosuppression and reduced anti-tumor immunity. In this study, we aim to identify the role of Ikaros in regulating T cell homeostasis and function in a pancreatic tumor microenvironment. Using our murine heterotopic model of pancreatic cancer, we isolated splenocytes from tumor-bearing (TB) and control mice and performed western blot and qRT-PCR analyses to evaluate Ikaros mRNA and protein expression, respectively. Flow cytometry analyses were also used to immunophenotype T cell populations in splenocytes from control and TB mice. Next, magnetic activated cell sorting (MACS) was used to sort CD3+ T cells from TB and control splenocytes for western blot analyses of Ikaros expression. We also carried out in vitro and in vivo experiments using apigenin, a plant flavonoid and protein kinase inhibitor with anti-inflammatory, anti-tumor and anti-proliferative properties, to shed insight into how Ikaros may be regulated and its involvement in T cell development. Our results showed no difference in Ikaros mRNA expression but reduced expression of all Ikaros isoforms in splenocytes from TB mice compared with controls. Ikaros protein expression was also downregulated in sorted TB T cells, compared with control Analyses of T cells by flow cytometry revealed a significant decrease in effector CD4+ and CD8+ T cell percentages but an increase in CD4+CD25+ regulatory T cells (Treg) in TB splenocytes compared with controls.. In vitro, treatment of splenocytes with apigenin stabilized Ikaros’ protein expression. In the presence of Panc02 cells, Ikaros protein expression was downregulated. However, the addition of apigenin to the co-culture blocked this downregulation. These correlated with in vivo findings that showed partial reversal of some Ikaros isoforms in TB mice treated with apigenin. Apigenin treatment also increased CD4+ and CD8+ T cell percentages but had no effect on Treg percentages. We are currently performing experiments to determine how Ikaros may be regulated in T cells and its effect on their functions. Thus far, our findings suggest that PC progression is associated with reduced Ikaros expression, which may lead to loss of T cell homeostasis and function and thus, dampened anti-tumor immune responses. Citation Format: Nadine Nelson, Shengyan Xiang, Xiaohong Zhang, Shari Pilon-Thomas, Nasreen Vohra, Tomar Ghansah. The role of Ikaros in T cell homeostasis and function in a murine pancreatic cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-270. doi:10.1158/1538-7445.AM2014-LB-270
Pancreatic cancer (PC) is currently the only major form of cancer that still has survival rates in the single digits. The limited success of cancer therapies in treating pancreatic cancer has been primarily due to the immunosuppressive tumor microenvironment. In particular, there is an increased prevalence of regulatory T cells (Tregs) (CD4+CD25+CD127−), which suppress anti-tumor immune responses in PC tumor-bearing (TB) hosts. Tregs express the Forkhead BoxP3 (FoxP3) gene, which is critical for their suppressive function. Studies have shown that Ikaros, a zinc finger transcription factor, is an important regulator of T lymphocyte development and function. The regulation of Ikaros’ expression and function is controlled by post-translational modification events. Deficiencies in Ikaros are observed in various T cell leukemias and lymphomas. However, little is known about the possible role of Ikaros in regulating immune cell development in response to solid cancers. In this study, we aim to identify the role of Ikaros in regulating Treg homeostasis and function in a pancreatic tumor microenvironment. Therefore, using our murine model of pancreatic cancer, we isolated splenocytes from TB and control mice and performed flow cytometry and magnetic activated cell sorting (MACS) to immunophenotype and enrich T cell populations for in vivo and in vitro analyses. Also, we performed quantitative real-time PCR (qRT-PCR) and western blot analyses to evaluate Ikaros and FoxP3 mRNA and protein expression in whole and enriched CD3+ T cells from TB and control splenocytes. Our results showed that effector T cell percentages (CD4+ and CD8+) were significantly lower in splenocytes from TB mice compared to control. However, our results also showed a significant expansion of Tregs in splenocytes from TB mice compared to control. In addition, enriched TB Tregs (CD4+CD25+) suppressed antigen-specific CD8+T cell immune responses in a dose-dependent manner, in vitro. Preliminary qRT-PCR results revealed no significant difference in Ikaros mRNA expression; whereas, Ikaros protein expression was reduced in TB whole splenocytes compared to control. Also, Ikaros protein expression was reduced in TB enriched CD3+ T cells compared to control. Furthermore, our results showed an increase in FoxP3 protein expression in TB CD3+ T cells compared to control. These findings suggest that the pancreatic tumor microenvironment potentially down-regulates Ikaros’ protein expression, which may contribute to the expansion of Tregs and their suppression of CD8+T cell (anti-tumor) immune responses. Citation Format: Nadine Nelson, Maya Jerald, Laura Pendleton, Karoly Szekeres, Nasreen Vohra, Shari Pilon-Thomas, Tomar Ghansah. The role of the Ikaros transcription factor in regulatory T cell (Treg) development and function in a murine pancreatic adenocarcinoma model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2013-4975
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