The genes that encode the immunoglobulin proteins made by B lymphocytes are made up of segments that are separately encoded in the germ-line genome and brought together by recombination during B-cell ontogeny. There are two types of immunoglobulin light chain, kappa and lambda, but only a single type is expressed in individual B cells. It is thought that kappa gene recombination precedes lambda gene recombination during B-cell ontogeny. We describe here unusual recombinations that have occurred in two lambda-producing B-cell lines and suggest that they are involved in the developmental switch from kappa to lambda gene expression in maturing B cells. These recombinations involve the J kappa-C kappa introns of V-J joined but nonfunctional kappa genes and a sequence that in the germ line occurs downstream of the C kappa exon (called RS, for recombining sequence).

We isolated and characterized the germ-line counterpart of a DNA segment designated RS (for recombining sequence), that is frequently recombined in mouse X light chain-producing B lymphocytes. Using Southern blot analyses of myelomas and mouse-Chinese hamster fusion cell lines, we found that RS DNA sequences are located on mouse chromosome 6, evidently more than 15 kilobases downstream of the K light-chain locus. We find that a typical recognition site for Ig gene recombination is situated within germ-line RS sequences near the recombination points observed in at least two X chain-producing cell lines. This represents a complete and functional Ig recognition site that is not directly associated with Ig genes. We also characterized a recombined RS segment isolated from the cell line BM18-4.13.9. This recombined segment has a variable region K light chain gene (V,) joined directly to RS sequences. Our results suggest that the deletion of the K light chain constant region (C,) exon in many X chain-producing B cells is the result of RS recombination and that C, deletion may be mediated by the same processes as antibody gene V-J joining (J = joining segment gene). We discuss the potential biological significance of RS DNA recombination in B-cell maturation.We have reported that two X chain-producing cell lines, MOPC315 and CH2, display novel DNA recombinations within the K chain locus different from all previously observed K chain gene recombination events (7). In these two cell lines, a segment of DNA [designated RS for recombining sequence] has recombined into the Je-CK intron region and replaced the C,, exon (QK = K chain joining region gene; CK = K constant region gene). In addition to being recombined in MOPC315 and CH2, RS DNA also was found to be recombined frequently in X chain-producing, but not in K chain-producing, hybridomas. Although no functional role for RS DNA has yet been demonstrated, these results raised the possibility that RS recombinations might be involved in the switch from K to X chain gene recombination in maturing B cells.In this article we report that germ line RS sequences are located on mouse chromosome 6 and that, within germ-line RS DNA, an Ig gene recognition site is located contiguous to the sites of RS recombination observed in MOPC315 and CH2. We also have detected and characterized a recombined RS segment that contains a VK gene joined directly to RS.

The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS +/+ APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS -/-T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination.

Immunization of transgenic mice carrying an immunoglobulin ,s heavy chain resulted in a response dominated by expression of the transgene variable region. Unexpectedly, in a large proportion of the antibody produced by immunized mice, the transgene MATERIALS AND METHODSAnimals. Transgenic C57BL/6 mice were prepared as described (13).DNA Hybridizations. DNAs digested with restriction enzymes were separated by electrophoresis through a 0.8% agarose gel, transferred to nitrocellulose, and hybridized with 32P-labeled fragments (14).Quantitation of Antibodies. Anti-Ar antibodies were quantitated as described (15); wells were developed with 125I-labeled affinity-purified rabbit anti-IgM or anti-IgG. Reactivity with the anti-idiotypic rat mAb AD8 (16) was quantitated by a solid-phase radioimmunoassay similar to that used for anti-Ar, except that wells were coated with mAb AD8 and the labeled developing reagent was preadsorbed with rat immunoglobulin. CRIA was measured as described (17). The R16.7 private idiotype was measured by the method used for CRIA; rabbit anti-R16.7 antiserum was rendered specific for R16.7 by adsorption with normal A/J immunoglobulin and with another CRIA-positive mAb, CB9. mAb CB9 appears to express the germ-line VH36-65 sequence that controls CRIA (P.F.R. and A.N., unpublished results) whereas the VH of R16.7 expresses several somatic mutations (see below). This adsorbed anti-idiotype antiserum reacts with R16.7 but not with nonspecific IgG or any other CRIA+ mAb tested.Sequence Analysis of mRNA.t The primer-extension dideoxynucleotide sequencing procedure was used (18,19 Fig. 1A) by joining a Abbreviations: Ar, p-azophenylarsonate; CRIA, cross-reactive idiotype of anti-Ar antibodies from A/J

The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS +/+ APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS -/-T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination.