Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a \u3e80% reduction in COL1A1 mRNA, and a \u3e70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease

Inhibition of basal and transforming growth factor-beta-induced stimulation of COL1A1 transcription by the DNA intercalators, mitoxantrone and WP631, in cultured human dermal fibroblasts

The Sp1 transcription factor plays a crucial role in COL1A1 transcriptional regulation under normal and pathologic conditions and under the effects of transforming growth factor-beta (TGF-beta). Sp1 activity is elevated in numerous diseases characterized by tissue fibrosis. Therefore, inhibition of Sp1 binding to COL1A1 regulatory elements may represent an effective treatment for these diseases. Here we examined the effect of two DNA intercalators that prevent Sp1 binding on the expression of COL1A1 in human dermal fibroblasts. Cultured human adult dermal fibroblasts were treated with WP631 (50 pm/ml to 500 nm/ml) or mitoxantrone (5-500 nm/ml). Cytotoxicity, cellular apoptosis, and collagen deposition were examined by fluorescence microscopy. Collagen production was examined by enzyme-linked immunosorbent assay and metabolic labeling, COL1A1 steady-state mRNA levels, and stability were assessed by Northern hybridizations, and COL1A1 transcription by in vitro nuclear transcription assays and transient transfections. Competition of the drugs for Sp1 binding and their effect on TGF-beta-induced stimulation of COL1A1 transcription was also examined. Both drugs caused a dose-related inhibition of COL1A1 production and mRNA levels without cytotoxicity or apoptosis. COL1A1 transcriptional activity showed a profound reduction mediated by a short proximal promoter region containing an Sp1-binding element at -87 to -82 bp. Furthermore, both drugs inhibited Sp1 DNA complex formation and abrogated the stimulation of COL1A1 transcription induced by TGF-beta. WP631 showed 10-fold higher potency than mitoxantrone. These data indicate that mitoxantrone and WP631 are very potent inhibitors of basal and TGF-beta-stimulated COL1A1 expression and suggest that Sp1-DNA intercalators may be an effective and novel approach for the treatment of fibrotic diseases and modulation of profibrogenic effects of TGF-beta

CCAAT binding transcription factor binds and regulates human COL1A1 promoter activity in human dermal fibroblasts: demonstration of increased binding in systemic sclerosis fibroblasts

OBJECTIVE: To determine the binding factors that interact with the proximal promoter region of the human type I collagen gene, COL1A1, and to examine their involvement in its transcriptional regulation in normal and systemic sclerosis (SSc) dermal fibroblasts. METHODS: Nuclear extracts from dermal fibroblasts from 4 patients with SSc and 4 age- and sex-matched control individuals were examined by electrophoresis mobility shift assays with a COL1A1 promoter fragment encompassing nucleotides -174 to -50 bp. Supershift assays with antibodies specific to various transcription factors, and competition experiments using consensus, wild-type, or mutated oligonucleotides corresponding to their specific binding sites, were performed. The effects of specific oligonucleotides as intracellular competitors were examined by transient transfection experiments in SSc fibroblasts using a COL1A1 construct containing -174 bp of the promoter. RESULTS: The findings demonstrate that the CCAAT binding transcription factor (CBF) binds the proximal CCAAT box located at -100 to -96 bp, but not the distal CCAAT box at -125 to -121 bp, of the human COL1A1 promoter in both SSc and normal fibroblasts. CBF binding activity was 3-5-fold higher in the SSc fibroblasts. Moreover, the promoter activity of the -174-bp COL1A1 construct was decreased by up to 50% when specific oligonucleotides were used as intracellular competitors. In addition, Sp1 and Sp3 were other transcription factors found to be involved in the formation of the DNA-protein complexes within this region of the COL1A1 promoter. CONCLUSION: These results indicate that the transcription factor CBF binds the human COL1A1 proximal promoter region in human dermal fibroblasts, and its binding activity is higher in SSc fibroblasts

Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory

Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor activity.

Antibody-dependent cellular cytotoxicity (ADCC) is a key effector mechanism of natural killer (NK) cells that is mediated by therapeutic monoclonal antibodies (mAbs). This process is facilitated by the Fc receptor CD16a on human NK cells. CD16a appears to be the only activating receptor on NK cells that is cleaved by the metalloprotease a disintegrin and metalloproteinase-17 upon stimulation. We previously demonstrated that a point mutation of CD16a prevents this activation-induced surface cleavage. This noncleavable CD16a variant is now further modified to include the high-affinity noncleavable variant of CD16a (hnCD16) and was engineered into human induced pluripotent stem cells (iPSCs) to create a renewable source for human induced pluripotent stem cell-derived NK (hnCD16-iNK) cells. Compared with unmodified iNK cells and peripheral blood-derived NK (PB-NK) cells, hnCD16-iNK cells proved to be highly resistant to activation-induced cleavage of CD16a. We found that hnCD16-iNK cells were functionally mature and exhibited enhanced ADCC against multiple tumor targets. In vivo xenograft studies using a human B-cell lymphoma demonstrated that treatment with hnCD16-iNK cells and anti-CD20 mAb led to significantly improved regression of B-cell lymphoma compared with treatment utilizing anti-CD20 mAb with PB-NK cells or unmodified iNK cells. hnCD16-iNK cells, combined with anti-HER2 mAb, also mediated improved survival in an ovarian cancer xenograft model. Together, these findings show that hnCD16-iNK cells combined with mAbs are highly effective against hematologic malignancies and solid tumors that are typically resistant to NK cell-mediated killing, demonstrating the feasibility of producing a standardized off-the-shelf engineered NK cell therapy with improved ADCC properties to treat malignancies that are otherwise refractory

iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy.

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer