Biomedical subjects

Belma Ljutic

Publications and source records attributed to Belma Ljutic.

5 recordsLinked to original sources

PubMed2007

Chromosome transfer activates and delineates a locus control region for perforin.

Perforin gene (PRF1) transcription regulates perforin expression in NK cells and CTL. Here we identified the locus-wide ensemble of cis-acting sequences that drives PRF1 transcription physiologically. By using chromosome transfer, we revealed that de novo activation of a silent PRF1 locus was controlled by a 150 kb domain comprised of 16 DNase I hypersensitive sites (DHSs). These cis-acting sequences included a locus control region (LCR) and conferred developmentally appropriate and lineage-specific expression of human perforin from BAC transgenes. The LCR included four distal DHSs that were required for perforin expression from its natural locus, and their engineered deletion from the PRF1 BAC transgene abolished LCR function and led to rapid gene silencing. Thus, LCR function is central for regulating the developmental and activation-specific PRF1 promoter activity characteristic of NK cells and CTL.

Animals↗

PubMed2006

Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells.

NK1.1 alloantigen expression can be used to define NK cells in certain mouse strains, such as B6 (NKR-P1C) and SJL (NKR-P1B). However, BALB/c NK cells do not react with the anti-NK1.1 mAb, PK136. To investigate the NK1.1(-) phenotype of BALB/c NK cells, we have undertaken NK1.1 epitope mapping and genomic analysis of the BALB/c Nkrp1 region. Bacterial artificial chromosome library analysis reveals that, unlike the Ly49 region, the Nkrp1-Ocil/Clr region displays limited genetic divergence between B6 and BALB/c mice. In fact, significant divergence is confined to the Nkrp1b and Nkrp1c genes. Strikingly, the B6 Nkrp1d gene appears to represent a divergent allele of the Nkrp1b gene in BALB/c mice and other strains. Importantly, BALB/c NK cells express abundant and functional Nkrp1 transcripts, and the BALB/c NKR-P1B receptor functionally binds Ocil/Clr-b ligand. However, the BALB/c NKR-P1B/C sequences differ from those of the known NK1.1 alloantigens, and epitope mapping demonstrates that directed mutation of a single amino acid in the NKR-P1B(BALB) protein confers NK1.1 reactivity. Thus, PK136 mAb recognizes, in part, a distal C-terminal epitope present in NKR-P1B(Sw/SJL) and NKR-P1C(B6), but absent in NKR-P1A/D/F(B6) and NKR-P1B/C(BALB). Allelic divergence of the Nkrp1b/c gene products and limited divergence of the BALB/c Nkrp1-Ocil/Clr region explain a longstanding confusion regarding the strain-specific NK1.1 alloantigen reactivity of mouse NK cells.

Alleles↗

PubMed2006

NKR-P1 biology: from prototype to missing self.

Natural killer (NK) cells represent lymphocytes of the innate immune system capable of recognizing and destroying a broad array of target cells, including tumors, virus-infected cells, antibodycoated cells, foreign transplants, and "stressed" cells. NK cells eliminate their targets through two main effector mechanisms, cytokine secretion and cell-mediated cytotoxicity, which in turn depend on detection of target cells through a complex integration of stimulatory and inhibitory receptor-ligand interactions. The NKR-P1 molecules were the first family of NK cell receptors identified, yet they have remained enigmatic in their contribution to self-nonself discrimination until recently. Here, we outline a brief history of the NKR-P1 receptor family, then examine recent data providing insight into their genetic regulation, signaling function, cognate ligands, and gene organization and diversity.

Animals↗

PubMed2005

Functional requirements for signaling through the stimulatory and inhibitory mouse NKR-P1 (CD161) NK cell receptors.

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56(lck). The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.

Amino Acid Motifs↗

PubMed2003

Identification of upstream cis-acting regulatory elements controlling lineage-specific expression of the mouse NK cell activation receptor, NKR-P1C.

Mouse NKR-P1C (NK1.1) is a homodimeric type II transmembrane protein expressed on natural killer (NK) cells, NKT cells, and on CD117+ progenitor thymocytes capable of giving rise to cells of the T and NK lineages. Although its physiological ligands remain unknown, NKR-P1C engagement with a monoclonal antibody (mAb) leads to interferon-gamma (IFN-gamma) production and the directed release of cytotoxic granules from NK cells. We have cloned and sequenced a approximately 10-kb genomic fragment corresponding to the 5'-flanking region of the C57Bl/6 mouse NKR-P1C gene. A transcriptional initiation site has been mapped in NK cells and an NK1.1+ T cell line by primer extension and rapid amplification of 5'-cDNA ends (5'-RACE) techniques. Although the 5'-flanking region of NKR-P1C is TATA-less, we have identified an initiator region and a downstream promoter element, which together constitute the principal minimal functional promoter. Computational analysis of the 10-kb 5'-flanking region revealed potential regulatory factor binding sites. DNaseI hypersensitivity assays identified a single hypersensitive site (HS) about a 9-kb upstream of the transcriptional initiation site. This site, termed HS1, was able to act as a transcriptional enhancer element in an NK cell line, while minimally affecting transcription in non-NK cell lines. Moreover, the HS1 element was shown to function as a promoter, with a transcript detected only in fetal NK1.1+ cells. An additional promoter and two non-coding exons were also characterized. These results identify the minimal upstream cis-acting elements, and point to a complex regulatory mechanism involved in the lineage-specific control of NKR-P1C expression in NK lymphocytes.

Animals↗