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.

Epithelial stem cells as mucosal antigen-delivering cells: A novel AIDS vaccine approach.

A key obstacle limiting development of an effective AIDS vaccine is the inability to deliver antigen for a sufficient period of time resulting in weak and transient protection. HIV transmission occurs predominantly across mucosal surfaces; therefore, an ideal vaccine strategy would be to target HIV at mucosal entry sites to prevent infection. Such a novel strategy relies on the activation of mucosal immune response via presentation of viral antigens by the mucosal epithelial cells. The use of a terminally differentiated epithelial cell promoter to drive expression of antigens leading to viral protein production in the upper layers of the epithelium is central to the success of this approach. Our results show that when administered intradermally to mice, a GFP-reporter gene under the transcriptional control of the involucrin promoter is expressed in the upper layers of the epidermis and, although transduced cells were very low in number, high and sustained anti-GFP antibody production is observed in vivo. A subsequent experiment investigates the effectiveness of GFP-tagged replication-competent SIVdeltaNef and GFP-tagged replication-deficient SIVdeltaVifdeltaNef constructs under the transcriptional control of the involucrin promoter. Optimal conditions for production of pseudotyped VSV-G viral particles destined to transduce basal epithelial stem cells at the mucosal sites of entry of SIV in our animal model were determined. Altogether, the data demonstrate the feasibility of an epithelium-based vaccine containing involucrin-driven viral antigen encoding sequences that integrate into epithelial stem cells and show long-term expression in the upper layer of the epithelium even after multiple cycle of epithelia renewal. Such epithelium-based vaccine should elicit a long-term immunity against HIV/SIV infection at the site of entry of the virus.

The rs1024611 regulatory region polymorphism is associated with CCL2 allelic expression imbalance.

CC chemokine ligand 2 (CCL2) is the most potent monocyte chemoattractant and inter-individual differences in its expression level have been associated with genetic variants mapping to the cis-regulatory regions of the gene. An A to G polymorphism in the CCL2 enhancer region at position -2578 (rs1024611; A>G), was found in most studies to be associated with higher serum CCL2 levels and increased susceptibility to a variety of diseases such as HIV-1 associated neurological disorders, tuberculosis, and atherosclerosis. However, the precise mechanism by which rs1024611influences CCL2 expression is not known. To address this knowledge gap, we tested the hypothesis that rs1024611G polymorphism is associated with allelic expression imbalance (AEI) of CCL2. We used haplotype analysis and identified a transcribed SNP in the 3'UTR (rs13900; C>T) can serve as a proxy for the rs1024611 and demonstrated that the rs1024611G allele displayed a perfect linkage disequilibrium with rs13900T allele. Allele-specific transcript quantification in lipopolysaccharide treated PBMCs obtained from heterozygous donors showed that rs13900T allele were expressed at higher levels when compared to rs13900C allele in all the donors examined suggesting that CCL2 is subjected to AEI and that that the allele containing rs1024611G is preferentially transcribed. We also found that AEI of CCL2 is a stable trait and could be detected in newly synthesized RNA. In contrast to these in vivo findings, in vitro assays with haplotype-specific reporter constructs indicated that the haplotype bearing rs1024611G had a lower or similar transcriptional activity when compared to the haplotype containing rs1024611A. This discordance between the in vivo and in vitro expression studies suggests that the CCL2 regulatory region polymorphisms may be functioning in a complex and context-dependent manner. In summary, our studies provide strong functional evidence and a rational explanation for the phenotypic effects of the CCL2 rs1024611G allele.

An evolutionarily conserved TNF-alpha-responsive enhancer in the far upstream region of human CCL2 locus influences its gene expression.

Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.

Production of specific mRNA transcripts, usage of an alternate promoter, and octamer-binding transcription factors influence the surface expression levels of the HIV coreceptor CCR5 on primary T cells.

Surface levels of CCR5 on memory CD4(+) T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.

Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment.

The integral membrane adaptor protein linker for activation of T cells (LAT) couples the T-cell receptor (TCR) with downstream signalling and is essential for T-cell development and activation. Here, we investigate the dynamic distribution of LAT-GFP fusion proteins by time-lapse video imaging of live T lymphocytes interacting with antigen-presenting cells. We show that LAT forms two distinct cellular pools, one at the plasma membrane and one that co-distributes with transferrin-labelled intracellular compartments also containing the TCR/CD3-associated zeta chain. The distribution of LAT between these two pools is dependent on LAT intracytoplasmic residues. Whereas plasma membrane-associated LAT is recruited to immune synapses after a few seconds of cell conjugate formation, the intracellular pool is first polarized and then recruited after a few minutes. We further show that LAT intracytoplasmic amino acid residues, particularly the Tyr136, 175, 195 and 235 residues, are required for its own recruitment to the immune synapse and that a herein-identified juxtamembrane LAT region (amino acids 32-104) is involved in the localization of LAT in intracellular pools and in T-cell signalling. Altogether, our results demonstrate that LAT controls its own recruitment at the immune synapse, where it is required as a scaffold protein for the signalling machinery. The results also suggest that the intracellular pool of LAT might be required for T-cell activation.

Role of ICAM-3 in the initial interaction of T lymphocytes and APCs.

Antigen-independent adhesive interactions between T lymphocytes and antigen-presenting cells (APCs) are essential for scanning for specific antigens on the APC surface and for initiating the immune response. Here we show, through time-lapse imaging of live cells, that the intercellular adhesion molecule 3 (ICAM-3, also known as CD50) is clustered specifically at the region of the T lymphocyte surface that initiates contact with APCs. We describe the role of ICAM-3 in T cell-APC conjugate formation before antigen recognition, in early intracellular signaling and in cytoskeletal rearrangement. Our data indicate that ICAM-3 is important in the initial scanning of the APC surface by T cells and, therefore, in generating the immune response.