DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells.

KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.

A stimulation-dependent alternate core promoter links lymphotoxin α expression with TGF-ß1 and fibroblast growth factor-7 signaling in primary human T cells.

Lymphotoxin (LT)-α regulates many biologic activities, yet little is known of the regulation of its gene. In this study, the contribution to LTA transcriptional regulation of the region between the transcription and translation start sites (downstream segment) was investigated. The LTA downstream segment was found to be required for, and alone to be sufficient for, maximal transcriptional activity in both T and B lymphocytes. The latter observation suggested that an alternate core promoter might be present in the downstream segment. Characterization of LTA mRNAs isolated from primary and from transformed human T cells under different stimulation conditions identified eight unique transcript variants (TVs), including one (LTA TV8) that initiated within a polypyrimidine tract near the 3' end of the downstream segment. Further investigation determined that the LTA downstream segment alternate core promoter that produces the LTA TV8 transcript most likely consists of a stimulating protein 1 binding site and an initiator element and that factors involved in transcription initiation (stimulating protein 1, TFII-I, and RNA polymerase II) bind to this LTA region in vivo. Interestingly, the LTA downstream segment alternate core promoter was active only after specific cellular stimulation and was the major promoter used when human T cells were stimulated with TGF-ß1 and fibroblast growth factor-7. Most importantly, this study provides evidence of a direct link for crosstalk between T cells and epithelial/stromal cells that has implications for LT signaling by T cells in the cooperative regulation of various processes typically associated with TGF-ßR and fibroblast growth factor-R2 signaling.

A single polymorphism disrupts the killer Ig-like receptor 2DL2/2DL3 D1 domain.

Genetic polymorphisms found in the killer Ig-like receptor (KIR), two domains, long cytoplasmic tail 2/3 (KIR2DL2/3) locus are responsible for the differential binding of KIR2DL2/3 allelic products with their HLA-C ligands and have been associated with the resolution of hepatitis C infection. In our study, a KIR CD3zeta fusion-binding assay did not detect any interaction between the KIR2DL2*004 extracellular domain and several putative KIR2DL2/3 ligands. To determine the amino acid polymorphism(s) responsible for the KIR2DL2*004 phenotype, we mutated the polymorphic residues of full-length KIR and expressed them in human Jurkat cells. Flow cytometry analysis failed to detect the surface expression of receptors containing a threonine at position 41 (T41), a polymorphism specific to KIR2DL2*004. Confocal microscopy showed that receptors containing T41 were retained inside the cell and had a perinuclear localization, possibly indicating that their extracellular domain was misfolded. Most KIR2DL2/3 alleles possess an arginine at position 41 (R41), and we predicted through molecular modeling and demonstrated by mutagenesis that R41 most likely interacts with the nearby residues Y77 and D47. Interaction between these residues would maintain C strand contact with the C' and F strands of the D1 domain beta-sheet. Furthermore, R41 and Y77 are conserved in the C and F strand amino acid alignments of Ig-like superfamily members, and may therefore be necessary for the structural integrity of other immune response proteins. Our data indicate that the extracellular T41 polymorphism encoded by the KIR2DL2*004 allele most likely results in misfolding of the D1 domain and complete intracellular retention of the receptor.

A molecular mechanism for the differential regulation of TGF-beta1 expression due to the common SNP -509C-T (c. -1347C > T).

Transforming growth factor beta1 (TGF-beta1) levels influence many cellular, immunologic and pathologic processes. Activator protein 1 (AP1) and hypoxia are key regulators of TGF-beta1 expression levels. The common TGFB1 promoter SNP c.-1347C > T (-509C-T, rs1800469) has been linked to a nearly twofold difference in plasma levels among individuals and with risk, progression, and outcome of numerous diseases. We demonstrate exclusive in vitro and in vivo recruitment of AP1 containing JunD to -1347C. This study also is the first to demonstrate hypoxia inducible factor 1 (HIF-1) binding to the TGFB1 promoter. HIF-1 was found to associate with both -1347C and -1347T and compete with AP1 for binding to -1347C. Reporter constructs demonstrate that expression differences between -1347C and -1347T are due to selective AP1 recruitment to the TGFB1 promoter. As AP1 is known to down-regulate transcription of other genes, we suggest that the molecular mechanism for the difference in TGF-beta1 plasma levels linked to -1347 is due to transcriptional suppression by AP1 binding to -1347C. These data should aid in our understanding of the association of the -1347 SNP with the pathogenesis of certain TGF-beta1-related diseases.

The effect of KIR ligand incompatibility on the outcome of unrelated donor transplantation: a report from the center for international blood and marrow transplant research, the European blood and marrow transplant registry, and the Dutch registry.

Matching for HLA class I alleles, including HLA-C, is an important criterion for outcome of unrelated donor transplantation. However, haplotype-mismatched transplantations for myeloid malignancies, mismatched for killer immunoglobulin-like receptor (KIR) ligands in the graft-versus-host (GVH) direction, is associated with lower rates of graft-versus-host disease (GVHD), relapse, and mortality. This study investigated the effect of KIR ligand mismatching on the outcome of unrelated donor transplantation. The outcomes after 1571 unrelated donor transplantations for myeloid malignancies where donor-recipient pairs were HLA-A, -B, -C, and -DRB1 matched (n = 1004), GVH KIR ligand-mismatched (n = 137), host-versus-graft (HVG) KIR ligand-mismatched (n = 170), and HLA-B and/or -C-mismatched but KIR ligand-matched (n = 260) were compared using Cox regression models. Treatment-related mortality (TRM), treatment failure, and overall mortality were lowest after matched transplantations. Patients who received grafts from donors mismatched at the KIR ligand in the GVH or HVG direction and mismatched at HLA-B and/or C but matched at the KIR ligand had similar rates of TRM, treatment failure, and overall mortality. There were no differences in leukemia recurrence between the 4 groups. These results do not support the choice of an unrelated donor on the basis of KIR ligand mismatch determined from HLA typing.

Allelic diversity in the TGFB1 regulatory region: characterization of novel functional single nucleotide polymorphisms.

Altered TGF-beta1 expression due to polymorphisms affects a wide variety of normal cellular and disease processes such as T cell activation and proliferation, tumor progression, and asthma. In this study, a comprehensive examination of function and diversity was undertaken for the TGFB1 promoter region and exon 1 (-2,665 to +423). The known TGF-beta1 promoter was extended to encompass 463 bases by the identification of a strong enhancer activity for a distal segment (-2,665 to -2,204). Ten novel polymorphisms and 14 novel alleles were identified. Most single nucleotide polymorphisms (SNPs) appear to be randomly associated except c.-768_-769insC and c.+74G > C and a set of five novel polymorphisms present in a single allele in persons of African descent. The TGFB1 alleles clustered into three phylogenetic groups based on the common functional SNPs c.-1347C > T (commonly known as -509C-T) and c.+29T > C (commonly known as +869T-C) suggesting three phenotypic groups. Two SNPs unique to African-Americans affect the TGFB1 regulatory region. The c.-1287G > A SNP in the promoter alters the binding affinity of two unidentified transcription factor complexes which translates into a significant difference in reporter gene expression and the c.-387C > T SNP in the 5' UTR alters the binding of Stimulating protein 1 and 3. Thus, TGFB1 possesses a highly polymorphic, extensive regulatory region that likely impacts the pathogenesis of numerous TGF-beta1 related diseases.