Matthew Wheeler

ER stress drives Lipocalin 2 upregulation in prostate cancer cells in an NF-κB-dependent manner.

BackgroundTumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production.MethodsWe interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways.ResultsInduction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation.ConclusionsWe conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.

KDEL-Retained Antigen in B Lymphocytes Induces a Proinflammatory Response: A Possible Role for Endoplasmic Reticulum Stress in Adaptive T Cell Immunity

Generally, APCs activate CD4 T cells against peptides derived from exogenous Ag in the context of MHC II molecules. In this study, using transgenic B lymphocytes as model APCs, we demonstrate CD4 T cell priming in vivo against peptides derived from endogenously synthesized Ag targeted either to the cytosol or to the endoplasmic reticulum (ER). Surprisingly, priming by Ag containing the KDEL-retention motif yielded higher levels of two important proinflammatory cytokines, IFN-γ and TNF-α, in responding CD4 T cells. Importantly, we found that KDEL-mediated retention of Ag up-regulates ER-stress responsive genes in primary B lymphocytes. We also found that thapsigargin treatment of A20 lymphoma cells up-regulates transcription of ER stress and proinflammatory genes along with IL-23p19. Induction of ER stress by thapsigargin also up-regulated IL-23p19 in primary B lymphocytes, macrophages, and bone marrow-derived dendritic cells. We conclude that perturbation of the secretory pathway and/or ER stress play an important role in modulating the gene program in professional APCs and in shaping CD4 T cell responses in vivo. These findings are relevant to a better understanding of the immune response after infection by viral and bacterial pathogens and the pathogenesis of certain autoimmune diseases.

B lymphocytes as antigen-presenting cell-based genetic vaccines.

Summary:  Inoculation of plasmid DNA is a simple way to immunize, but it is characterized by low immunogenicity, which has hampered the development of effective DNA vaccines for human use. Here, we discuss how poor immunogenicity can be solved and present our proposal: genetically programmed B lymphocytes as antigen‐presenting cell (APC) vaccines. First, we demonstrate that mature B lymphocytes take up plasmid DNA spontaneously, i.e., in the absence of any facilitating molecule or event, spontaneous lymphocyte transgenesis. Second, we demonstrate that transgenic B lymphocytes are easily and reproducibly turned into functional APCs with dual characteristics: upregulation of costimulatory molecules and endogenous synthesis of antigen. Used as immunogens in mice, transgenic B lymphocytes induce robust and long‐lasting T‐cell immunity after single intravenous injection. Surprisingly, immunity and protection against lethal virus challenge can be obtained with a single intravenous injection of 3 × 102 transgenic lymphocytes. The new approach is discussed relative to the advantage of targeting secondary lymphoid organs with genetically programmed B lymphocytes and the advantage offered with respect to low antigen dose. We suggest that these properties reflect on simple characteristics, such as time synchronization and initial localization to secondary lymphoid organs of APCs endowed with protracted synthesis and presentation of antigen to T cells.

Endoplasmic reticulum stress drives a regulatory phenotype in human T-cell clones.

T cells alter their functional phenotype during the evolution of an immune response (intra-lineage differentiation), but the driving forces to this plastic intra-lineage differentiation are poorly understood. The endoplasmic reticulum (ER) stress response is a possible critical event for the initial T cell differentiation upon antigen recognition. Here we studied the relationship between ER and Il-10 transcription in human Treg clones. The induction of ER stress with a canonical stressor, thapsigargin, enhances Il-10 transcription. Salubrinal, a small molecule inhibitor of the eukaryotic translation initiation factor 2α (eIF2α) dephosphporylation, dramatically inhibits it. Il-10 transcription is also enhanced by exogenous TNFα. These results disclose a role for ER stress in driving T cell plasticity.

Ex vivo programming of antigen-presenting B lymphocytes: considerations on DNA uptake and cell activation.

Plasmids used in DNA vaccination not only serve as a source of antigen, but also have an important adjuvant effect. This review focuses on recent advancements made in understanding how cells internalize DNA, and how internalized DNA activates immune response pathways. We also comment on the role of B cells in both of these processes.

179. Transgenesis of B Lymphocytes by Bacterial DNA Is TLR9 Independent

Among the 11 TLRs identified, TLR9 is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. TLR9 is an intracellular receptor so that its signaling requires that DNA is first internalized by the cell. Human and murine B lymphocytes are easily transfected by plasmid DNA. Here we show that transgenesis of B lymphocytes with plasmid DNA is CpG independent since it is not inhibited by cloroquine, it does not induce cell proliferation, and it occurs in TLR9-deficient murine B lymphocytes. Nevertheless, transfected B lymphocytes of both species up regulate B7.1 and CD40, events not inhibited by cloroquine treatment. Importantly, transfected B lymphocytes from TLR9-deficient mice can be used as antigen presenting cells to immunize mice and induce both CD4 and CD8 T cell responses. Collectively, these results suggest the existance of a yet unidentified pathway of activation of B lymphocytes and question the role of TLR9 in naked DNA transgenesis.