We have analyzed tolerance-related clonal deletion of Mls-and I-E-reactive thymocytes at the RNA level using a multi-V beta probe RNAse protection assay, and used this phenomenon to identify the maturation stage of the abnormally expanded CD4-8-, TCR-alpha/beta + subset in lpr and gld homozygous mice, and of the phenotypically similar minor thymocyte subset found in normal mice. Essentially complete V beta clonal deletions were detected in lpr and gld cells of all appropriate background strains. Substantial, but not complete, V beta clonal deletions were also detected in the CD4-8- TCR-alpha/beta + subset of normal mice. Since expression of CD4/CD8 is required for V beta clonal deletions to occur, we conclude that lpr and gld cells, and at least a portion of CD4-8- TCR-alpha/beta + thymocytes in normal mice, are derived by secondary loss of CD4/CD8 accessory molecules from more mature CD4+8+ precursors. One possible interpretation of these findings is that such CD4/CD8 loss may affect a class of self-reactive thymocytes that have escaped direct clonal deletion. Exportation and expansion of such cells in the periphery may be an important contributory factor in the induction of systemic autoimmunity.
The autosomal recessive Ipr gene accelerates a systemic lupus erythematosus-like disease in genetically predisposed mice and induces autoantibodies in mice of normal genetic background. The molecular mode(s) of action of the lpr gene and its chromosomal location remain unknown, but it is primarily expressed as a massive T-cell proliferation manifested only in the presence of a thymus. To define the clonal diversity and maturational stage of the abnormally proliferating T cells found in enlarged lymph nodes of MRL-lpr/lpr mice, and their possible role in autoreactive B-cell activation, we analyzed their T-cell receptor /-chain variable region (Vp) gene sequences. Twenty-five VDJ-containing /-chain cDNA sequences were examined, each of which was found to derive from a distinct rearrangement in the correct reading frame, yielding translatable /3-chain mRNAs. An additional 10 clones were derived from truncated nonfunctional mRNAs. DB1 and DP2 elements were used equally in the sequenced clones, and 10 of the possible 12 mouse Jp elements were represented. Since T cells appear to be essential for the development of murine lupus, studies of the genetic loci encoding the antigenspecific T-cell receptor (TCR) of autoimmune mice have been initiated. In analyzing the a-and p-chain constant region genes (Ca and C,3, respectively) of this receptor, we (9) and others (10) identified a deletion of the C,01-Dp2-J,2 elements (D, diversity region; J, joining region) in the genome of the NZW mouse, the significance of which is currently being investigated. Here, we report our molecular analysis of the Vp gene repertoire and clonal diversity of cells from the enlarged lymph nodes of an MRL-lpr/lpr mouse. Our findings document the polyclonal nature of the Ipr cell population and indicate the presence of a significantly skewed Via gene repertoire in these cells. MATERIALS AND METHODSConstruction and Screening of the MRL-lpr/lpr cDNA Library. Total cellular RNA was prepared by the guanidinium isothiocyanate method from 3 x 108 cells isolated from the enlarged lymph node of a 5-month-old MRL-lpr/lpr mouse. Poly(A)+ RNA was purified (11), and a cDNA library was constructed in the pcDV-1 vector according to the procedure of Okayama and Berg (12). A portion of the final vector/cDNA preparation was used to transform Escherichia coli strain MC1061, and the transformants were plated directly at ==10,000 colonies per plate. Colonies were screened with an oligonucleotide probe complementary to Cp, picked, and purified by replating and rescreening with the same probe.Sequence Analysis of cDNA Clones. BamHI-Pst I fragments containing the full-length TCR p-chain cDNA inserts were purified by the agarose gel/DEAE paper method (13) and subcloned into M13mpll (14). VDJ sequences were then determined directly by dideoxy sequencing (15) using a primer hybridizing to a region of Cp adjacent to the C-J junction. Clone pMlpr2 was subcloned into the Gemini 3 vector (Promega Biotec, Madison, WI), and the lower-strand V sequence was determined by th...
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