Inna Lindner

Regulation of RelB Expression during the Initiation of Dendritic Cell Differentiation

ABSTRACT The transcription factor RelB is required for proper development and function of dendritic cells (DCs), and its expression is upregulated early during differentiation from a variety of progenitors. We explored this mechanism of upregulation in the KG1 cell line model of a DC progenitor and in the differentiation-resistant KG1a subline. RelB expression is relatively higher in untreated KG1a cells but is upregulated only during differentiation of KG1 by an early enhancement of transcriptional elongation, followed by an increase in transcription initiation. Restoration of protein kinase CβII (PKCβII) expression in KG1a cells allows them to differentiate into DCs. We show that PKCβII also downregulated constitutive expression of NF-κB in KG1a-transfected cells and restores the upregulation of RelB during differentiation by increased transcriptional initiation and elongation. The two mechanisms are independent and sensitive to PKC signaling levels. Conversely, RelB upregulation was inhibited in primary human monocytes where PKCβII expression was knocked down by small interfering RNA targeting. Altogether, the data show that RelB expression during DC differentiation is controlled by PKCβII-mediated regulation of transcriptional initiation and elongation.

Induced dendritic cell differentiation of chronic myeloid leukemia blasts is associated with down-regulation of BCR-ABL.

Although differentiation of leukemic blasts to dendritic cells (DC) has promise in vaccine strategies, the mechanisms underlying this differentiation and the differences between leukemia and normal progenitor-derived DC are largely undescribed. In the case of chronic myeloid leukemia (CML), understanding the relationship between the induction of DC differentiation and the expression of the BCR-ABL oncogene has direct relevance to CML biology as well as the development of new therapeutic approaches. We now report that direct activation of protein kinase C (PKC) by the phorbol ester PMA in the BCR-ABL+ CML cell line K562 and primary CML blasts induced nonterminal differentiation into cells with typical DC morphology (cytoplasmic dendrites), characteristic surface markers (MHC class I, MHC class II, CD86, CD40), chemokine and transcription factor expression, and ability to stimulate T cell proliferation (equivalent to normal monocyte-derived DC). PKC-induced differentiation was associated with down-regulation of BCR-ABL mRNA expression, protein levels, and kinase activity. This down-regulation appeared to be signaled through the mitogen-activated protein kinase pathway. Therefore, PKC-driven differentiation of CML blasts into DC-like cells suggests a potentially novel strategy to down-regulate BCR-ABL activity, yet raises the possibility that CML-derived DC vaccines will be less effective in presenting leukemia-specific Ags.

Tumor-Induced STAT3 Signaling in Myeloid Cells Impairs Dendritic Cell Generation by Decreasing PKCβII Abundance

Tumor-derived factors repress immune responses by reducing protein kinase C signaling in dendritic cell progenitors. Preventing the Antitumor Immune Response Within the tumor microenvironment, tumor cells use many mechanisms to subvert the immune response, such as secreting factors that suppress the differentiation of myeloid progenitor cells into dendritic cells (DCs), which are critical for the activation of antitumor T cells. Farren et al. found that activation of signal transducer and activator of transcription 3 (STAT3) signaling in myeloid progenitor cells exposed to conditioned medium from breast cancer cells resulted in a decrease in the abundance of protein kinase C βII (PKCβII), which is required for their differentiation into DCs. PKCβII abundance in myeloid cells was decreased in patients with advanced breast cancer and in mouse models of breast cancer. STAT3 bound to the promoter of PRKCB (which encodes PKCβII), inhibiting its expression and the generation of DCs. Conversely, PKCβII signaling stimulated PRKCB expression, as well as reduced the cell surface abundance of STAT3-activating receptors. Identification of these dueling pathways suggests that therapies that enhance PKCβII signaling in myeloid progenitors may increase the effectiveness of STAT3-targeted therapies. A major mechanism by which cancers escape control by the immune system is by blocking the differentiation of myeloid cells into dendritic cells (DCs), immunostimulatory cells that activate antitumor T cells. Tumor-dependent activation of signal transducer and activator of transcription 3 (STAT3) signaling in myeloid progenitor cells is thought to cause this block in their differentiation. In addition, a signaling pathway through protein kinase C βII (PKCβII) is essential for the differentiation of myeloid cells into DCs. We found in humans and mice that breast cancer cells substantially decreased the abundance of PKCβII in myeloid progenitor cells through a mechanism involving the enhanced activation of STAT3 signaling by soluble, tumor-derived factors (TDFs). STAT3 bound to previously undescribed negative regulatory elements within the promoter of PRKCB, which encodes PKCβII. We also found a previously undescribed counter-regulatory mechanism through which the activity of PKCβII inhibited tumor-dependent STAT3 signaling by decreasing the abundance of cell surface receptors, such as cytokine and growth factor receptors, that are activated by TDFs. Together, these data suggest that a previously unrecognized cross-talk mechanism between the STAT3 and PKCβII signaling pathways provides the molecular basis for the tumor-induced blockade in the differentiation of myeloid cells, and suggest that enhancing PKCβII activity may be a therapeutic strategy to alleviate cancer-mediated suppression of the immune system.

Differentiation of acute and chronic myeloid leukemic blasts into the dendritic cell lineage: analysis of various differentiation-inducing signals.

Purpose: Ex vivo differentiation of myeloid leukemic blasts into dendritic cells (DCs) holds significant promise for use as cellular vaccines, as they may present a constellation of endogenously expressed known and unknown leukemia antigens to the immune system. Although variety of stimuli can drive leukemia→DC differentiation in vitro, these blast-derived DCs typically have aberrant characteristics compared with DCs generated from normal progenitors by the same stimuli. It is not clear whether this is due to underlying leukemogenic mechanisms (e.g., specific oncogenes), genetic defects, stage of maturation arrest, defects in cytokine receptor expression or signal transduction pathways, or whether different stimuli themselves induce qualitatively dissimilar DC differentiation. Methods: To assess what factors may contribute to aberrant leukemic blast→DC differentiation, we have examined how the same leukemic blasts (AML and CML) respond to different DC differentiation signals—including extracellular (the cytokine combination GM-CSF+TNF-α+IL-4) and intracellular (the protein kinase C agonist PMA, the calcium ionophore A23187, and the combination of PMA plus A23187) stimuli. Results: We have found that the same leukemic blasts will develop qualitatively different sets of DC characteristics in response to differing stimuli, although no stimuli consistently induced all of the characteristic DC features. There were no clear differences in the responses relative to specific oncogene expression or stage of maturation arrest (AML vs CML). Signal transduction agonists that bypassed membrane receptors/proximal signaling (in particular, the combination of PMA and A23187) consistently induced the greatest capability to activate T cells. Interestingly, this ability did not clearly correlate with expression of MHC/costimulatory ligands. Conclusions: Our findings suggest that signal transduction may play an important role in the aberrant DC differentiation of leukemic blasts, and demonstrate that direct activation of PKC together with intracellular calcium signaling may be an effective method for generating immunostimulatory leukemia-derived DCs.

Modulation of dendritic cell differentiation and function by YopJ of Yersinia pestis

Yersinia pestis evades immune responses in part by injecting into host immune cells several effector proteins called Yersinia outer proteins (Yops) that impair cellular function. This has been best characterized in the innate effector cells, but much less so for cells involved in adaptive immune responses. Dendritic cells (DC) sit at the crossroads between innate and adaptive immunity, and can function to initiate or inhibit adaptive immune responses. Although Y. pestis can target and inactivate DC, the mechanism responsible for this remains unclear. We have found that injection of Y. pestis YopJ into DC progenitors disrupts key signal transduction pathways and interferes with DC differentiation and subsequent function. YopJ injection prevents up‐regulation of the NF‐κB transcription factor Rel B and inhibits MAPK/ERK activation – both having key roles in DC differentiation. Furthermore, YopJ injection prevents costimulatory ligand up‐regulation, LPS‐induced cytokine expression, and yields differentiated DC with diminished capability to induce T cell proliferation and IFN‐γ induction. By modulating DC function through YopJ‐mediated disruption of signaling pathways during progenitor to DC differentiation, Yersinia may interfere with the adaptive responses necessary to clear the infection as well as establish a tolerant immune environment that leads to chronic infection/carrier state in the surviving host.

Signal Transduction in DC Differentiation: Winged Messengers and Achilles’ Heel

Dendritic cells (DC) are centrally involved in the initiation and regulation of the adaptive immune response, and different DC can have markedly different (e.g., opposing) function. Acquisition of specific functions is likely to be a result of both nature and nurture, namely differentiation of progenitors into distinct DC subsets as well as the influence of environmental signals. This is not unlike what is seen for T and B cells. This review will focus on the signal transduction pathways that allow an unusually wide range of hematopoietic progenitors to differentiate into DC, the functional characteristics regulated by these pathways, and the ability of pathogens to alter DC function by subverting these pathways during progenitor→DC differentiation.

6,7-Dihydroxy-3,4-Dihydroisoquinoline: A Novel Inhibitor of Nuclear Factor-κB and in vitro Invasion in Murine Mammary Cancer Cells

Background: The inhibition of nuclear factor (NF)-κB with nontoxic agents is a promising possible treatment approach that may inhibit tumor cell proliferation, counteract the prosurvival pathways that mediate resistance to cytotoxic therapy, and prevent tumor cell metastasis. Methods: An initial structure-activity relationship study of the NF-κB inhibitory activity of acetophenone-type compounds using electrophoretic mobility shift assay and Western blot analysis is presented. An in vitro cell invasion assay using DA3 cells, a murine breast cancer cell line, was conducted to model antimetastatic activity. Results: The carbonyl moiety is found to be the functional group responsible for inhibition of NF-κB, and a novel, more effective agent, 6,7-dihydroxy-3,4-dihydroisoquinoline, is postulated and confirmed. The compounds are characterized as active in the inhibition of both the canonical and noncanonical NF-κB signaling pathways. Lastly, 6,7-dihydroxy-3,4-dihydroisoquinoline is discovered to inhibit in vitro invasion in DA3 cells. Conclusion: 6,7-Dihydroxy-3,4-dihydroisoquionoline and its derivatives are presented as potential prototypes for a novel series of nontoxic antimetastatic agents that can be used in conjunction with current cancer therapeutic techniques.

Protein kinase C blockade inhibits differentiation of myeloid blasts into dendritic cells by calcium ionophore A23187

Direct differentiation of myeloid leukemia blasts into antigen-presenting dendritic cells (DCs) for use as cellular vaccines is unique in that identification of tumor-specific antigens may not be necessary because the antigens should already be endogenously expressed.We hypothesized that signaling through protein kinase C (PKC) is required for differentiation of HL-60 promyeloblasts into DCs upon stimulation with calcium ionophore A23187. To demonstrate the inhibitory effect of PKC blockade, we pretreated HL-60 myeloid blasts with the protein kinase inhibitor bisindolylmaleimide I (Bis-1) for 24 hours and then treated the cells with calcium ionophore A23187 for an additional 24 hours. Controls consisted of HL-60 blasts treated with A23187, Bis-1 alone, or media.We noted that blasts cultured in media, Bis-1, or Bis-1 then A23187 did not develop the morphologic and phenotypic DC characteristics, up-regulate Rel B, or activate allogeneic T-cells. Our findings suggested that PKC blockade inhibits morphologic, phenotypic, and functional differentiation of HL-60 promyeloblasts into antigen-presenting DCs. Our findings supported the role of PKC as an obligatory pathway for calcium ionophore A23187-induced differentiation of HL-60 myeloblasts into antigen-presenting DCs.