DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.5-Methylcytosine, a minor base in the DNA of a variety of organisms, is formed by postreplicative methylation of DNA by S-adenosylmethionine (AdoMet) in reactions catalyzed by DNA-cytosine methyltransferases (DCMTases). In recent years, much evidence has been obtained which indicates that 5-methylcytosine residues in DNA play an important role in eukaryotic gene expression (for reviews see refs. 1 and 2). Consequently, there has been wide interest in the pyrimidine analog 5-azacytidine (azaCyd), which inhibits formation of 5-methylcytosine in DNA and results in dramatic effects on gene expression and cell differentiation (e.g., see refs. 3-9).Current evidence indicates that the mechanism by which azaCyd causes decreased DNA methylation involves incorporation of 5-azacytosine (azaC) into DNA and subsequent inhibition of DCMTase. Incorporation of small amounts of azaC into DNA of mammalian cells results in a loss of DCMTase activity in extracts obtained from such cells (5,10). Further, incubation of DNA containing azaC (azaC-DNA) with mammalian or bacterial DCMTases results in a very potent inhibition of enzyme activity (11, 12), but kinetic studies have not revealed the mechanism of inhibition. On the basis of the probable catalytic mechanism of DCMTases and known chemical properties of azaC, we recently speculated that the mechanism o...

Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.

CD4 and CD8 are cell-surface glycoproteins expressed on mutually exclusive subsets of peripheral T cells. T cells that express CD4 have T-cell antigen receptors that are specific for antigens presented by major histocompatibility complex class II molecules, whereas T cells that express CD8 have receptors specific for antigens presented by MHC class I molecules (reviewed in ref. 1). Based on this correlation and on the observation that anti-CD4 and anti-CD8 antibodies inhibit T-cell function, it has been suggested that CD4 and CD8 increase the avidity of T cells for their targets by binding to MHC class II or MHC class I molecules respectively. Also, CD4 and CD8 may become physically associated with the T-cell antigen receptor, forming a higher-affinity complex for antigen and MHC molecules, and could be involved in signal transduction. Cell-cell adhesion dependent CD4 and MHC II molecules has recently been demonstrated. To determine whether CD8 can interact with MHC class I molecules in the absence of the T-cell antigen receptor, we have developed a cell-cell binding assay that measures adhesion of human B-cell lines expressing MHC class I molecules to transfected cells expressing high levels of human CD8. In this system, CD8 and class I molecules mediate cell-cell adhesion, showing that CD8 directly binds to MHC class I molecules.

SUMMARYRepetitive stimulation of naõ Ève T cells by immature splenic dendritic cells (DC) can result in the differentiation of T-cell lines with regulatory properties. In the present study we identi®ed a population of DC in the mucosae that exhibit the plasmacytoid phenotype, secrete interferon-a (IFN-a) following stimulation with oligodeoxynucleotides containing certain cytosine-phosphateguanosine (CpG) motifs and can differentiate naõ Ève T cells into cells that exhibit regulatory properties. Although these DC appear to be present in both spleen and mesenteric lymph nodes (MLN), only CpG-matured DC from the MLN (but not the spleen) were able to differentiate naõ Ève T cells into T regulatory 1-like cells with regulatory properties. The activity of these DC failed to sustain robust T-cell proliferation and thereby enhanced the suppressive ef®cacy of CD4 CD25 T regulatory cells. These DC are the major CD8a DC population in the Peyer's patches (PP). Given their signi®cant presence in mucosal tissue, we propose that these DC may provide a mechanistic basis for the homeostatic regulation in the gut by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation at a site of high antigenic stimulation like the intestine.

Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.