Hicok, K. C.

Abstract The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual’s T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.

Abstract Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID−1, ID−2, ID−3, and ID−4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor β (CBFβ), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.

Giant cell arteritis (GCA) is a spontaneous vasculitic syndrome that specifically targets the walls of medium and large arteries. Vascular lesions are characterized by patchy granulomatous infiltrates composed of T cells, macrophages, histiocytes, and giant cells. To test the hypothesis that a locally residing antigen recruits T cells into the vessel walls, we have analyzed T cell receptor (TCR) molecules of tissue infiltrating T cells. A total of 638 CD4+ T cell clones were isolated from temporal artery specimens of three patients with GCA. Analysis of TCR molecules for the usage of V beta 1-V beta 20 revealed that all TCR V beta elements were represented, demonstrating that interleukin 2 (IL-2)-responsive T cells infiltrating the tissue are highly diverse. To detect expanded T cell specificities, we made use of the patchy character of the inflammatory disease and compared the TCR repertoire of T cells established from independent vasculitic foci of the same artery. Sequence analysis of TCR V beta chains documented that individual TCR specificities were present in multiple copies, indicating clonal expansion. T cells with identical beta chains were isolated from distinct inflammatory foci of the same patient. These specificities represented only a small fraction of tissue-infiltrating T cells and involved the V beta 5.3 gene segment in the two patients sharing the HLA-DRB1*0401 allele. The third complementarity determining region of clonally expanded TCR beta chains was characterized by a cluster of negatively and positively charged residues, suggesting that the juxtaposed antigenic peptide is charged. The sharing of identical T cell specificities by distinct and independent regions of the granulomatous inflammation suggests that these T cells are disease relevant and that their repertoire is strongly restricted. These data suggest that an antigen residing in the arterial wall is recognized by a small fraction of CD4+ T cells in the inflammatory process characteristic for GCA.