No abstract

Human sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29) are represented by at least one gene and several related sequences that may represent pseudogenes. By using a DNA probe that is specific for the complete gene (c-myc), different somatic cell hybrids possessing varying numbers of human chromosomes were analyzed by the Southern blotting technique. The results indicate that the hu-

Self-reactive B cells can be regulated by either deletion or inactivation. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRl/lpr mouse. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.

SlllllmsryTo explore mechanisms that prevent autoreactivity in nonautoimmune mice, endogenous immunoglobulin (Ig) light (L) chains that associate with a transgenic anti-DNA heavy chain were analyzed. The antibodies from splenic B cell hybridomas of such mice did not bind doublestranded DNA (dsDNA) and their L chain sequences showed a biased use of V~ and J~ gene segments. The 44 L chains in this survey were coded for by just 18 germline genes. Six of the genes, each belonging to a different V~ group, were used more than once and accounted for three fourths of all sequences. Based on the distribution of V~ genes, the L chain repertoire in this line of transgenic mice was estimated at 37 V~ genes. The most frequently observed gene, a member of the V~ 12/13 group, was identified in 16 hybrids. In addition, the majority of V~ genes used J~5. We interpret the skewed representation of V~ and J~ gene segments to result from negative selection. Based on the data, we suggest that V~ rearrangements giving rise to anti-dsDNA reactivity are removed from the repertoire by a corrective mechanism capable of editing self-reactive Ig.T olerance to self has been studied in Ig transgenic models of autoreactivity. By using Ig transgenes against facultative self-antigens, it has been shown that self-reactive B cells are selected against by anergy or deletion (1, 2). We have constructed mice with transgenes that code for anti-DNA antibodies and obtained similar results. The majority of splenic B lymphocytes from mice with the V,3H9 and V~8 genes express anti-single-stranded DNA (ssDNA) 1 antibodies on their surface, but we do not detect them in the serum (3). By analogy to Goodnow et al. (1), anti-ssDNA B cells in those mice appear to be anergic.Analysis of splenic B cells from mice containing just the V,3H9 transgene suggested a second feature of tolerance to DNA. No anti-double-stranded DNA (dsDNA) activity was detected among hybridomas from these mice. This was surprising, since previous studies have established that the 3H9 H chain can combine with a diverse range of L chains to yield antibodies capable of binding to both ssDNA and dsDNA (4, 5). The absence of hybrids producing anti-dsDNA suggested that dsDNA-spedfic B cells are functionally deleted.Nevertheless, V.3H9 mice have near normal numbers of splenic B cells, and hybridomas can be readily obtained (3). Some of the antibodies produced by these hybridomas bound ssDNA, whereas others did not bind DNA at all. We have now examined the V~ and J~ gene segment use of hybrids obtained from two of these mice. The mAbs were characterized by a sharply reduced repertoire of L chains. L chains that are found among spontaneous anti-DNA antibodies from MLR/lpr (4) or NZB x NZW (6) mice were absent from our sample. In addition, J~5 was overutilized. The data suggested that the major driving force shaping the L chain use in V.3H9 mice is selection against dsDNA-binding B cells. Furthermore, the data are consistent with the notion that B cells can escape deletion if their autoreactive surf...

Selection of recently formed B cells into the follicular or marginal zone (MZ) compartments is proposed to occur by way of proliferative intermediates expressing high levels of CD21/35 and CD23. However, we show that CD21/35high CD23+ splenocytes are not enriched for proliferative cells, and do not contribute substantially to the generation of follicular B cells. Instead, ontogenic relationships, steady-state labeling kinetics, and adoptive transfer experiments suggest that CD21/35high CD23+ splenocytes serve primarily as precursors for MZ B cells, although their developmental potential seems to be broader and is influenced by environmental cues that are associated with lymphopenia. Furthermore, CD21/35high CD23+ splenocytes share several key functional characteristics with MZ B cells, including their capacity to trap T-independent antigen and a heightened proliferative response to LPS. These observations challenge previous models of peripheral B cell maturation, and suggest that MZ B cells develop by way of CD21/35high CD23+ intermediates.