ALBERT

Notes: Abstract Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2a mice with thymocytes from either B6-Lyt-2a, Lyt-3a or B6-Lyt-2a, Lyt-3a, H-2k mice (Boos et al. 1978). Surprisingly, mice of the congenic strain B6 failed to produce either anti-Lyt-2.1 or anti-Lyt-3.1 cytotoxic antibodies after identical immunizations. To determine the genetic basis for the difference in response to Lyt-3.1, (B6 × B6-Lyt-2a)Fa mice and progeny of the backcross, (B6 × B6-Lyt-2a)F1 × B6-Lyt-2a, were immunized with B6-Lyt-2a, Lyt-3a, H-2k thymocytes. In addition, thymic biopsies of backcross progeny were performed and thymocytes tested for the Lyt-2.2 antigenic specificity. Results indicate that gene(s) governing the immune response to Lyt-3.1 is (are) linked to theLyt-2 locus, and that the responder allele (linked toLyt-2 a ) shows very poor penetrance in Lyt-2a/Lyt-2b mice.

Notes: Abstract Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10−6 or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1∶2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes. In addition, three new strains of mice differing from existing strains in the region of theLyt-2 and4Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearingLyt-2 a andLyt-3 a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b, congenic with AKR/J and bearing theLyt-2 b andLyt-3 b alleles of Balb/cJ.

Notes: Abstract A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Efl. Two mouse stocks with previously identified crossovers within the Ly2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3)-(Rn7s-6, Igk-Efl)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3 a-Igk-Efla haplotype.

Notes: Abstract Comparison of the nucleotide sequences of the C.C58 M75 myeloma ϰ chain gene and the BALB/c germ-line Jϰ segments suggested that the Jϰ regions of C.C58 and BALB/c might be distinguished by restriction enzyme polymorphisms. This was shown to be the case in Southern hybridizations of Hinf I and Ace I digests of liver DNA from these and other strains with a Jϰ-specific probe. Tests of a wide variety of inbred, congenic, recombinant, and recombinant-inbred strains provided evidence for three alleles, Igk-J a, Igk-J b, and Igk-J c, the type strains for which are C58/J, BALB/c, and SJL/J, respectively. Analysis of the B6.PL(85NS) congenic strain suggests that the Igk-J locus lies in the neighborhood of the Lyt-2/Lyt-3 loci, approximately 0.30 cM from the V gene segment determining the Igk-VSer and Igk-Efl polymorphisms. Finally, nucleotide substitutions lead to amino acid sequence differences between the C.C58 M 75 ϰ gene and the BALB/c germ line in Jϰ2 and Jϰ4. Two of these substitutions reflect true germ-line differences, raising the possibility that idiotype differences observed among strains could reflect Jϰ as well as Vϰ differences.

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 The expression of two idiotype (id) families (5AF6 and 3C6) associated with the BALB/c p-azophenylarsonate-specific antibody response was examined in 11 mouse strains. Eight strains produced some of one or the other of these two id families with the mean percent expression in the anti-Ar responses of id(+) strains ranging from 8 to 43% for the 5AF6 and from 2 to 10% for the 3C6 idiotype. Four strains of mice (C58, AKR, PL, and RF) thought to have Lyt-3.1-linked VL repertoire differences from other mouse strains (Lyt-3.2) were tested for their capacity to contribute to 5AF6 and 3C6 id expression. The RE strain was capable of producing 5AF6 id and small amounts of 3C6 id. Tests of Lyt-3.1 congenic strains C.AKR (AKR Lyt-3.1 on a BALB/c background) and C.C58 (C58 Lyt-3.1 on a BALB/c background) showed that C.AKR could produce 5AF6 id while C.C58 could not. 3C6 id expression was present but depressed in C.C58 mice compared with the high 3C6 id expression in C.AKR. Breeding studies mating C.C58 (bearing the required Igh-Ca-linked V H genes) to other 5AF6(−) strains showed that gene complementation could result in 5AF6 expression in F1 offspring. 5AF6(−) strains capable of complementation included CBA/J, C57BL/6J, AKR/J, and PL/J. C58/J (from which C.C58 were derived) was the only tested strain that failed to complement for 5AF6 id expression. Additional matings between C58/J[5AF6(−)] and CBA/J[5AF6 (−)] showed F1 offspring could produce 5AF6 id, indicating that C58/J can contribute functional V H genes necessary for 5AF6 id expression. Depressed expression of 5AF6 and 3C6 id was noted in mice where the C58/J-derived Lyt-3.1 genotype was present. The possibility that the depression of 5AF6 and 3C6 id expression derived from C58/J mice was due to regulatory influences rather than a lack of the V L structural genes is discussed.

Notes: Abstract Several strains of mice were tested for their capacity to provide immunoglobulin L chains required for the expression of the major cross-reactive idiotype (CRIA) associated with p-azophenylarsonate-specific antibodies of strain A mice. To facilitate testing, mice were bred that were homozygous for Igh-C e and Lyt-2 a , 3 a , i. e., they possessed genes controlling H chains but not L chains required for expression of the CRIA. Such male mice were mated to females of various strains and their offspring were tested; expression of CRIA indicated the presence in the female parent of genes controlling the appropriate L chains. All females bearing the Lyt-2 a , 3 b or Lyt-2 b , 3 b genotype yielded offspring, most of which were CRI A + , whereas all the offspring of females that were Lyt-2 a , 3 a were CRI A − . The female parents included mice of several strains that are congenic for Lyt-2 a , 3 b , Lyt-2 b , 3 b or Lyt-2 a , 3 b , thus demonstrating very close linkage between the Lyt loci and the expression of CRIA. In addition, doubly congenic strains of mice with the heavy chain allotype of the CRI A + AL/N strain and the Lyt-2 a , 3 a genotype on a BALB/c background failed to express CRIA. The data provide further evidence for the similarity of repertoires of L chains in Lyt-3 b mice of various strains. When genes were present controlling A/J H chains and L chains of C57BL/6 or BALB/c origin, the quantitative expression of CRIA was only slightly lower than that observed in A/J mice. Mice possessing genes controlling the H or L chains required for CRIA expression, but not both, did not express CRIA but synthesized Ar-specific antibodies which contained low but significant concentrations of the idiotype-associated chain.

Notes: Abstract Endogenous retroviruses are known to affect expression of cellular genes in the vicinity of their integration sites. The endogenous mouse mammary tumor provirus (Mtv-8) previously has been reported to reside on mouse chromosome 6 near the immunoglobulin kappa chain locus. Using pairs of mouse strains on the BALB/c (Mtv-8 positive) and C58 (Mtv-8 negative) backgrounds which are congenic for chromosome 6 genetic markers, we have confirmed the chromosome assignment of this provirus. Moreover, we have analyzed the N1 progeny of a (B6 × C58) × C58 backcross to determine the segregation of the Mtv-8 provirus with respect to polymorphisms in the Igk-VSer and Igk-J loci. The results with congenic and backcross mice together with results of others suggest that Mtv-8 is located approximately 0.52 cM from several closely linked kappa markers on chromosome 6.

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.