Multiple sclerosis is an autoimmune disease in which T lymphocytes reactive to myelin basic protein (BP) could play a central role. T cells specific for BP were cloned from the blood of multiple sclerosis patients and normal individuals, and expression of T-cel receptor variable region genes was analyzed. A remarkable bias for use of f-chain variable region (VP) 5.2 and, to a lesser extent, Vj36.1 was seen among BP-specific clones from patients but not from controls. Multiple sclerosis (MS) is an immune-mediated disease characterized by central nervous system mononuclear cell infiltration and demyelination. Although the pathogenesis of MS is unknown, both genetic and environmental factors have been implicated in the disease process. Major elements ofthe genetic predisposition include an association of disease with particular class II major histocompatibility complex (MHC) haplotypes, in particular HLA-DR2 and -DQwl (1-5), as well as with certain polymorphisms within the T-cell receptor (TCR) a-chain and (3-chain gene complexes (6-9). These studies suggest that the disease involves CD4+ T cells bearing af3 TCR. In support of this idea, CD4+ T cells represent a major component of mononuclear cells in the brains of active patients (10), and limited sets of a-chain TCRs are present within central nervous system tissue of MS patients but not controls (11).T lymphocytes that recognize myelin basic protein (BP) have been shown to have potent demyelinating and encephalitogenic activity in animals (12-18). Accumulating evidence also suggests that BP-specific T cells may contribute to the pathogenesis ofMS. Thus, cells selected from MS patients on the basis of in vivo activation have specificity for BP (19). In addition, the frequencies of BP-reactive T cells are increased Monoclonal antibodies directed to these regions or synthetic peptides with sequences common to these TCR V regions can both protect and treat animals with clinical signs of experimental autoimmune encephalomyelitis (21,22,(25)(26)(27). For a similar approach to be applied to MS patients, it is critical to know if potentially pathogenic T cells also preferentially use a limited set of V region genes. In this manuscript, we analyze the TCR Va and Vf3 genes expressed in BP-specific T cells selected from the blood of MS patients and normal individuals. MATERIALS AND METHODSPatients. Blood samples were obtained from seven patients being followed at the Oregon Health Sciences University MS clinic. All patients had clinically and laboratory-supported definite MS with an average ambulation index of 3.2 + 1.7 (range 2-6) and an average Kurtzke disability status score of 3.8 + 2.0 (range 2-6). Four patients had relapsing/remitting disease, and three patients had chronic progressive disease. The normal individuals were from the Veterans Affairs Medical Center, selected on the basis of a positive proliferative response of peripheral blood mononuclear cells (PBMC) to human BP in culture (20). All subjects were HLA-typed by standard serological methods at the...
Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor a/6 locus were isolated from a genomic library. The structure of the germ-line Vis1 variable gene segment was determined. V1al is located 8.5 kb downstream of the Val3.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The Vd7.1 segment is located between V81 and the Li, J;, Cc, region (containing the diversity, joining, and constant gene segments). Thus, Vs and V% segments -are interspersed along the chromosome. The germ-line organization of the L 2, Jr,1, and Jr,2 segments was determined. Linkage of Cc, to the J. region was established by identification of J,, segments within 20 kb downstream of C8.The organization of the locus was also analyzed by fieldinversion gel electrophoresis. The unrearranged Vsl and D8, Jl, Cat regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.In addition to T-cell antigen receptor (TCR) a43 (a heterodimer ofpolypeptides termed a and f3), a second TCR, 'y8, has recently been identified (1). Whereas TCR a,3 is expressed on the majority of peripheral blood T lymphocytes, TCR y5 is expressed on a small fraction of peripheral blood TYlymphocytes, as well as on some thymic T cells and dendritic epidermal cells. The-function of the lymphocytes that bear TCR y5 is unknown.The genes encoding the TCR a, /3, -y, and 8 polypeptides are all assembled from multiple gene segments that rearrange to form a functional gene during T-cell differentiation (1-3). The diversity of TCR a/3 is immense due to the use of large numbers of variable (V) and joining (J) segments, and in the case of TCR 3, diversity (D) segments as well. TCR y8 apparently displays a more limited repertoire of germ-line V and J elements but nevertheless displays extraordinary diversity at the V-J junction. This is due in part to the novel use of two Do elements that can be incorporated together into the junctional region (4-6).Whereas the TCR a, ,/, and ygenes are all unlinked, studies in mice (7), as well as preliminary studies in humans (8-10), indicate that the TCR 8 gene lies within the TCR a locus, upstream ofthe estimated 50-100J, segments and between V,, and J,,,. However, rearrangement at this locus appears to be highly regulated. Whereas TCR 8 genes rearrange early in thymic ontogeny, TCR a genes rearrange much later. Further, the utilization of V segments appears to be selective.For example, to date the human V.] segment has only been observed to be utilized in TCR y8 lymphocytes, whereas V. segments have not been found to be similarly utilized. The details of the organization of the TCR a/8 locus may shed light on the manner in which rearrangements at this locus are controlled. To better understand the structure of the germline elements that contribute to the diversity ofTCR 8, as well as the organization of these elements with respect to those of TCR a, we have undertaken an analysis of the germ-line organization of the T...
The human T cell receptor delta (TCR delta) gene encodes one component of the TCR gamma delta-CD3 complex found on subsets of peripheral blood and thymic T cells. Human TCR delta diversity was estimated by characterizing rearrangements in TCR gamma delta cell lines and determining the structures of complementary DNA clones representing functional and nonfunctional transcripts in these cell lines. One V delta segment and one J delta segment were identified in all functional transcripts, although a distinct J delta segment was identified in a truncated transcript. Further, one D delta element was identified, and evidence for the use of an additional D delta element was obtained. Thus human TCR delta genes appear to use a limited number of germline elements. However, the apparent use of two D delta elements in tandem coupled with imprecise joining and extensive incorporation of N nucleotides generates unprecedented variability in the junctional region.
The molecular organization of HLA-DQ regions derived from DR7, DQw2, and DR4, DQw3 parental haplotypes and DR7, DQw3, a presumed recombinant haplotype, have been studied to define the sequences between DQA1 and DQB1 which may have been involved in this recombinational event. The breakpoint was localized in the intergenic region near the 3' end of the DQB1 gene by restriction mapping. DNA sequences in the immediate vicinity of the breakpoint in DR7, DQw2 (parental), and DR7, DQw3 (recombinant) haplotypes revealed the presence of (CA)22 repeats, minisatellite-related sequences and GC-rich sequences. The intergenic regions varied considerably depending on the haplotype and contained several additional types of repetitive sequences including Alu and LINE repeats. Some of these sequences are related to sequences previously suggested to be involved in meiotic or somatic recombination. In particular, (CA)n repeats, which can adopt the Z-DNA conformation, have previously been shown to promote recombination in several systems.
Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.
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