An inducible enhancer required for Il12b promoter activity in an insulated chromatin environment.

Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that serve as critical regulators of T helper cell development. The Il12b gene, which encodes the p40 subunit of both IL-12 and IL-23, is expressed in macrophages and dendritic cells following induction by bacterial products. Although the Il12b promoter, like the promoters of most proinflammatory genes, can support transcriptional induction in typical transfection assays, we show that it is not sufficient for transcription in an insulated chromatin environment. Using a DNase I hypersensitivity assay, two potential distal control regions were identified. One region, DNase I-hypersensitive site 1 (HSS1), located 10 kb upstream of the transcription start site, exhibited hypersensitivity only in stimulated macrophages. In an insulated environment, a 105-bp fragment spanning HSS1 was sufficient for transcription when combined with the Il12b promoter. Although several elements are likely to contribute to activity of the endogenous HSS1 enhancer, including an evolutionarily conserved binding site for C/EBP proteins, the only element required for activity in transient- and stable-transfection assays bound Oct-1 and Oct-2, both of which are expressed constitutively in macrophages. Oct-1 and Oct-2 were recruited to the enhancer upon macrophage stimulation, and the Oct site appeared important for nucleosome remodeling at HSS1. These results suggest that the HSS1 enhancer and Oct proteins play central roles in Il12b induction upon macrophage activation.

Interleukin-10 inhibits interleukin-12 p40 gene transcription by targeting a late event in the activation pathway.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that suppresses the induction of proinflammatory cytokine genes, including the IL-12 p40 gene. Despite considerable effort examining the effect of IL-10 on specific transcription factors and signaling molecules, the mechanism by which IL-10 inhibits gene transcription has remained elusive. To provide a different perspective to this problem, we examined the effect of IL-10 on molecular events occurring at the endogenous IL-12 p40 locus in lipopolysaccharide-stimulated peritoneal macrophages. IL-10 abolished recruitment of RNA polymerase II to the p40 promoter. However, it only modestly reduced binding of C/EBPbeta, as monitored by genomic footprinting and chromatin immunoprecipitation. It also had little effect on NF-kappaB complexes that are critical for p40 induction. A substantial reduction in nucleosome remodeling at the p40 promoter was observed, but the magnitude of this reduction appeared insufficient to account for the strong inhibition of transcription. Finally, a lipopolysaccharide-inducible DNase I hypersensitive site identified 10 kb upstream of the start site was unaffected by IL-10. Thus, despite a dramatic reduction in p40 transcription, several events required for activation of the endogenous p40 gene occurred relatively normally. These findings suggest that IL-10 blocks one or more events that occur after p40 locus decondensation and nucleosome remodeling and after, or in parallel with, the binding of a subset of p40 transcriptional activators.

Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic.

Amino acid sequence differences in the variable region of immunoglobulin (Ig) cause wide variations in secretion outputs. To address how a primary sequence difference comes to modulate Ig secretion, we investigated the biosynthetic process of 2 human IgG2κ monoclonal antibodies (mAbs) that differ only by one amino acid in the light chain complementarity-determining region 1 while showing ∼20-fold variance in secretion titer. Although poorly secreted, the lower-secreting mAb of the 2 was by no means defective in terms of its folding stability, antigen binding, and in vitro biologic activity. However, upon overexpression in HEK293 cells, the low-secreting mAb revealed a high propensity to aggregate into enlarged globular structures called Russell bodies (RBs) in the endoplasmic reticulum. While Golgi morphology was affected by the formation of RBs, secretory pathway membrane traffic remained operational in those cells. Importantly, cellular protein synthesis was severely suppressed in RB-positive cells through the phosphorylation of eIF2α. PERK-dependent signaling was implicated in this event, given the upregulation and nuclear accumulation of downstream effectors such as ATF4 and CHOP. These findings illustrated that the underlining process of poor Ig secretion in RB-positive cells was due to downregulation of Ig synthesis instead of a disruption or blockade of secretory pathway trafficking. Therefore, RB formation signifies an end of active Ig production at the protein translation level. Consequently, depending on how soon and how severely an antibody-expressing cell develops the RB phenotype, the productive window of Ig secretion can vary widely among the cells expressing different mAbs.

Characterization of bispecific T-cell Engager (BiTE) antibodies with a high-capacity T-cell dependent cellular cytotoxicity (TDCC) assay.

The Bispecific T-cell Engager (BiTE) antibody modality is a clinically validated immunotherapeutic approach for targeting tumors. Using T-cell dependent cellular cytotoxicity (TDCC) assays, we measure the percentage of specific cytotoxicity induced when a BiTE molecule engages T-cells, redirects T-cell mediated cytolysis, and ultimately kills target cells. We establish a novel luminescence-based TDCC assay quantified by measuring cell viability via constitutive expression of luciferase. The luciferase-based TDCC assay performance is valid and comparable to an adenosine triphosphate (ATP)-based detection method. We demonstrate that the luciferase-based TDCC assay is an efficient homogeneous assay format that is amenable to both suspension and adherent target cells. The luciferase-based TDCC assay eliminates the need for plate-washing protocols, allowing for higher-throughput screening of BiTE antibodies and better data quality. Assay capacity is also improved by performing serial dilutions of BiTE antibodies in 384-well format with an automated liquid handler. We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors.

Selective and antagonistic functions of SWI/SNF and Mi-2beta nucleosome remodeling complexes during an inflammatory response.

Studies of mammalian genes activated in response to an acute stimulus have suggested diverse mechanisms through which chromatin structure and nucleosome remodeling events contribute to inducible gene transcription. However, because of this diversity, the logical organization of the genome with respect to nucleosome remodeling and gene induction has remained obscure. Numerous proinflammatory genes are rapidly induced in macrophages in response to microbial infection. Here, we show that in lipopolysaccharide-stimulated macrophages, the catalytic BRG1/BRM subunits of the SWI/SNF class of ATP-dependent nucleosome remodeling complexes are consistently required for the activation of secondary response genes and primary response genes induced with delayed kinetics, but not for rapidly induced primary response genes. Surprisingly, a Mi-2beta complex was selectively recruited along with the SWI/SNF complexes to the control regions of secondary response and delayed primary response genes, with the Mi-2beta complex acting antagonistically to limit the induction of these gene classes. SWI/SNF and Mi-2beta complexes influenced cell size in a similarly antagonistic manner. These results provide insight into the differential contributions of nucleosome remodeling complexes to the rapid induction of defined classes of mammalian genes and reveal a robust anti-inflammatory function of Mi-2beta.