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TCF21 is a basic helix-loop-helix transcription factor that has recently been implicated as contributing to susceptibility to coronary heart disease based on genome wide association studies. In order to identify transcriptionally regulated target genes in a major disease relevant cell type, we performed siRNA knockdown of TCF21 in in vitro cultured human coronary artery smooth muscle cells and compared the transcriptome of siTCF21 versus siCONTROL treated cells. The raw (FASTQ) as well as processed (BED) data from 3 technical replicates per treatment has been deposited with Gene Expression Omnibus (GSE44461).

Osteoprotegerin (OPG) is a CD40-regulated gene in B cells and dendritic cells (DCs). We investigated the role of OPG in the immune system by generating opg(-/-) mice. Like its role as a regulator of bone metabolism, OPG also influences processes in the immune system, notably in B cell development. Ex vivo, opg(-/-) pro-B cells have enhanced proliferation to IL-7, and in opg(-/-) spleen, there is an accumulation of type 1 transitional B cells. Furthermore, opg(-/-) bone marrow-derived DCs are more effective in stimulating allogeneic T cells than control DCs. When challenged with a T-dependent Ag, opg(-/-) mice had a compromised ability to sustain an IgG3 Ag-specific response. Thus, in the immune system, OPG regulates B cell maturation and development of efficient Ab responses.

The interaction of the T cell receptor (TCR) with peptide in the binding site of the major histocompatibility complex molecule provides the basis for T cell recognition during immune surveillance, repertoire development, and tolerance. Little is known about the extent to which repertoire selection is influenced directly by variation of the structure of the class I heavy chain. We find that the 2C TCR, normally positively selected in the context of the K(b) molecule, is minimally selected into the CD8 lineage in the absence of antigen-processing genes. This finding underscores the importance of peptides in determining the positive-selecting class I ligands in the thymus. In contrast, K(bm3), a variant class I molecule that normally exerts a negative selection pressure on 2C-bearing T cells, positively selects 2C transgenic T cells into the CD8 lineage in an antigen-processing gene-deficient environment. These findings indicate that structural changes in the heavy chain can have direct influence in T cell recognition, from which we conclude that the nature of TCR interaction with class I heavy chain influences the array of TCRs selected during development of the functional adult repertoire.

Recent research on the formation and maintenance of the vasculature in the embryo and in the adult has provided a greater understanding of the cellular signals involved in these processes. With this understanding comes the potential means of controlling vascularization in pathological situations such as tumorigenesis and wounding. For the purpose of this review, we will discuss the key receptor tyrosine kinases involved in vascular function and the molecules which relay signals downstream of receptor activation. The receptor tyrosine kinases discussed include the vascular endothelial cell growth factor receptors, Eph receptors, Tie1, and Tie2, all of which are expressed on vascular endothelial cells. We also discuss the roles of the platelet derived growth factor receptors which are expressed on vascular smooth muscle cells. While all of these receptor tyrosine kinases activate many similar effector molecules, some of the signals initiated appear to be distinct. This may explain, at least in part, how different receptor tyrosine kinases expressed in overlapping patterns on the developing vasculature, direct unique biological functions.

The well-defined 2C T cell was used to investigate alloreactive degeneracy. A panel of class I molecules that are known ligands for the 2C TCR were sensitized with three known peptide ligands, p2Ca (LSPFPFDL), dEV-8 (EQYKFYSV), and SIYR-8 (SIYRYYGL). The peptide p2Ca was originally identified as the allopeptide seen in the Ld class I molecule by 2C T cells, 2C recognizes the dEV-8 peptide as the ligand in the Kbm3 class I molecule, and SIYR-8 was recently identified as a peptide ligand for 2C in the context of the Kb class I molecule. Strong recognition of all three Ag-presenting molecules occurred in the context of their respective allopeptides, but 2C recognized all three peptides to a measurable extent in the context of Kb. Molecular modeling of these Kb/peptide complexes revealed a high degree of similarity between dEV-8 and SIYR-8, but very little conformational similarity of either of these peptides with p2Ca. Furthermore, the structural changes in the mutant Kbm3 binding site resulted in generalized changes in the conformation of each of five bound peptides compared with those of the same peptides bound to Kb. The finding that degenerate recognition occurs on Kb, the restriction element responsible for selecting 2C T cells, suggests a unique relationship between a TCR and the Ag-presenting molecule that mediates its positive selection.

Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

Microsequence analysis of peptides eluted from the murine class I H-2Kb molecule together with the three-dimensional structure of the molecule co-crystallized with a homogeneous population of peptides suggests that pocket B is a minor pocket that does not play a major role in peptide presentation. This is in contrast to most other class I molecules in which pocket B plays a central role in selecting and presenting antigenic peptides. To investigate the role of pocket B in antigen presentation by the Kb molecule, we analyzed site-directed mutants of position 45 in pocket B for their effect on both allo- and peptide-specific recognition. We made an identical set of mutations in Kbm8 at residue 45 in order to evaluate their influence in the context of a more open pocket B which results from the bm8 substitution at amino acid 24 (E-->S). We demonstrated that this minor pocket did play a significant role in the antigenicity of both molecules and that this role was more readily apparent in the context of the more open pocket B of Kbm8. In addition, we found that some substitutions of residue 45 in the Kbm8 molecule restored recognition by some alloreactive and peptide specific anti-Kb T cell clones which are normally restricted to Kb, indicating that multiple configurations of amino acids in a pocket could result in similar binding and presentation capabilities.