ALBERT

Notes: Abstract Cytolytic activity and release of interleukin 2 (IL-2) were induced in Lyt-2-positive T-T cell hybrids by incubation with either concanavalin A or irradiated stimulator cells. Since hybrids of Lyt-2-positive class I-specific cytotoxic T lymphocytes (CTLs) with the fusable mouse thymoma cell line, BW5147, are invariably Lyt-2-negative, a derivative of BW5147 was produced by transfection which constitutively expresses surface Lyt-2.1. This cell line, 3B2, was fused with the H-2Ld-specific long term CTL line, 2C. Such hybrids expressed the transfected Lyt-2 gene but not the endogenous gene of the 2C fusion partner. That Lyt-2 plays a functional role in hybrids of 3b2 with 2C is shown by the observations that: 1) cytolysis by Lyt-2-positive hybrids was inhibited by Lyt-2-specific monoclonal antibody (mAb); 2) Lyt-2-positive but not Lyt-2-negative subclones of one such line develop specific cytotoxicity when incubated with stimulator cells; 3) Less IL-2 was released from Lyt-2-negative subclones incubated with stimulator cells than from Lyt-2-positive subclones; 4) Lyt-2-specific mAb inhibits release of IL-2 from Lyt-2-positive hybrids incubated with stimulator cells. All Lyt-2-positive hybrids expressed functional surface Lyt-3 encoded by the CTL fusion partner, demonstrating that expression of the Lyt-3 gene is not sensitive to the negative regulation which shuts off the endogenous Lyt-2 gene in hybrids of classI-specific CTLs with the 3B2 or BW5147 cell lines. The existence of inducible T-T cell hybrids expressing functional Lyt-2 and Lyt-3 provides a system for evaluation of the role(s) of Lyt-2 and Lyt-3 in the induction of function independent of cell growth.

Notes: Abstract p-azophenylarsonate-specific antibodies of A/J mice which bear the Ars-A cross-reactive idiotype utilize the V K−Ars−A gene segment, a member of the V K 10 family. Southern hybridization of genomic DNA from several inbred strains using a probe from the 5′ flanking region of the V K−Ars−A gene demonstrated three patterns of restrictio fragment length polymorphisms (RFLP). Six genes corresponding to hybridizing bands were obtained from DNA libraries of C.AKR, PERU and A/J mice, and nucleotide sequence comparisons revealed two allelic groups: AKRI (Igk-V10.1 a ), AJ1 (Igk-V10.1 b ) and PERU1 (Igk-V10.1 c ); AKR2 (Igk-V10.2 a ), AJ2 (Igk-V10.2 b ), and PERU2 (Igk-V10.2 c ).The Igk-V10.1 b gene of the A/J strain is the V k−Ars−A gene used in Ars-A idiotype-positive antibodies. The product of the C.AKR allele (Igk-V10.1 a ) contained four amino acid substitutions in CDR3 as compared with Igk-V10.1 b . These substitutions probably explain the failure of AKR mice and other strains with the same VK10 RFLP pattern to provide in genetic crosses a L chain which, together with the A/J V H−ArsA gene product, form Ars-A idiotype-positive antibodies. Also, the nucleotide sequence identity between the Igk-V10.1 c and Igk-V10.1 b alleles and the Igk-V10.2 c and Igk-V10.2 b alleles is significantly greater than that seen in comparisons with the Igk-V10.1 a and Igk-V10.2 a alleles, respectively, suggesting an evolutionary pathway similar to that of the linked Igk-J locus. BALB/c antibodies bearing the A48 regulatory idiotype contain L chains encoded by the BALB/c Igk-V10.1 b and Igk-V10.2 b alleles. Strongly A48 idiotype-positive antibodies utilize the Igk-V10.1 b chain, and weakly A48-positive antibodies use the Igk-V10.2 b L chain. The possible effects of amino acid substitutions specified by the Igk-V10.1 a , Igk-V10.1 c , Igk-V10.2 a , and Igk-V10.2 c alleles on their ability to provide L chains used in A48 idiotype-positive are discussed. The locus name, Igk-V28 (D'Hoostelaere et al. 1988), will be used in this report in place of the name, Igk-VSer, used in the original publications (Goldrick et al. 1985; Boyd et al. 1986; Gottlieb et al. 1986; Ponath et al. 1988). The four alleles described at the Igk-VSer locus (Igk-VSer a , Igk-VSer b , Igk-VSer c , and Igk-VSer d ) are referred to as Igk-V28 a , Igk-V28 b , Igk-V28 c , and Igk-V28 d , respectively.

Notes: Abstract Hybrids of Lyt-2/Lyt-3-positive class I-specific cytotoxic T lymphocytes (CTLs) with the BW5147 thymoma cell line (Lyt-2/Lyt-3-negative) are known to be Lyt-2/Lyt-3-negative due to shutoff of transcription of the CTL's Lyt-2 gene. Hybrids of a constitutively Lyt-2-positive transfectant of BW5147 (3B2) with a long term CTL line, 2C, and with CTLs generated in a mixed leucocyte reaction (MLR) shut off the CTL's Lyt-2 gene as expected but express the CTL's Lyt-3 gene product as a heterodimer with the product of the transfected Lyt-2 gene. Thus the Lyt-3 gene is not subject to the same negative regulatory influences as the Lyt-2 gene. That expression of Lyt-2 is not necessary for Lyt-3 gene transcription to continue is demonstrated by the finding that hybrids of MLR-generated CTLs with either BW5147 (Lyt-2-negative) or 3B2 (Lyt-2-positive) cells express Lyt-3 RNA. Southern hybridization and structural analysis of DNA fragments generated using the polymerase chain reaction demonstrated that hybrids contain several species of Lyt-3 RNA, one of which lacks the exon encoding the extracellular V-like domain and appears to be the product of an alternatively-spliced RNA transcript.