Murine Pancreatic Adenocarcinoma Reduces Ikaros Expression and Disrupts T Cell Homeostasis

Background Maintenance 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 Findings Using 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/Significance The 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

TFE3 contains two activation domains, one acidic and the other proline-rich, that synergistically activate transcription.

TFE3 is a basic-helix-loop-helix-zipper (bHLHZIP) domain-containing protein that binds mu E3 sites in regulatory elements in the immunoglobulin heavy chain gene. The protein is a transcriptional activator that is expressed in vivo as two alternately spliced isoforms with different activating properties: TFE3L contains an N-terminal acidic activation domain; TFE3S lacks this activation domain and is a dominant negative inhibitor of TFE3L. We show that TFE3L and TFE3S contain a second, C-terminal activation domain rich in proline residues. This pro-rich activation domain has activity in a Gal4 fusion assay comparable to the N-terminal acidic activation domain present in TFE3L. The TFE3 pro-rich activation domain contains regions of strong homology with the related proteins microphthalmia and TFEB, suggesting that these regions are important for function. Using two different assays, we show that the N- and C-terminal activation domains of TFE3 act synergistically. This synergism explains in part the ability of TFE3S to act as a dominant negative. Our domain analysis of TFE3 is incorporated into a general structural model for the TFE3 protein that predicts that the activation domains of TFE3 will be widely separated in space

Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation.

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant-negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Here we report the cloning of Aiolos, a gene which encodes an Ikaros homologue that heterodimerizes with Ikaros proteins. In contrast to Ikaros--which is expressed from the pluripotent stem cell to the mature lymphocyte--Aiolos is first detected in more committed progenitors with a lymphoid potential and is strongly up-regulated as these differentiate into pre-T and pre-B cell precursors. The expression patterns of Aiolos and Ikaros, the relative transcriptional activity of their homo- and heteromeric complexes, and the dominant interfering effect of mutant Ikaros isoforms on Aiolos activity all strongly suggest that Aiolos acts in concert with Ikaros during lymphocyte development. We therefore propose that increasing levels of Ikaros and Aiolos homo- and heteromeric complexes in differentiating lymphocytes are essential for normal progression to a mature and immunocompetent state

E2F1 and E2F2 Determine Thresholds for Antigen-Induced T-Cell Proliferation and Suppress Tumorigenesis

E2F activity is critical for the control of the G(1) to S phase transition. We show that the combined loss of E2F1 and E2F2 results in profound effects on hematopoietic cell proliferation and differentiation, as well as increased tumorigenesis and decreased lymphocyte tolerance. The loss of E2F1 and E2F2 impedes B-cell differentiation, and hematopoietic progenitor cells in the bone marrow of mice lacking E2F1 and E2F2 exhibit increased cell cycling. Importantly, we show that E2F1 and E2F2 double-knockout T cells exhibit more rapid entry into S phase following antigenic stimulation. Furthermore, T cells lacking E2F1 and E2F2 proliferate much more extensively in response to subthreshold antigenic stimulation. Consistent with these observations, E2F1/E2F2 mutant mice are highly predisposed to the development of tumors, and some mice exhibit signs of autoimmunity

Harnessing of the nucleosome-remodeling-deacetylase complex controls lymphocyte development and prevents leukemogenesis

Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage-determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell-specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks. © 2012 Nature America, Inc. All rights reserved.link_to_subscribed_fulltex