Inducible functions in hybrids of a Lyt-2+ BW5147 transfectant and the 2C CTL line

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-2L^d-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. Address correspondence and offprint requests to: Paul D. Gottlieb     

Structure and expression of the Lyt-3 agene of C.AKR mice

The mouse Lyt-3 ^agene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5 flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3 ^agene and the cDNA sequences reported for Lyt-3 ^b(Nakauchi et al. 1987, Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3 ^aencodes serine and Lyt-3 ^bencodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5 to the Lyt-3 ^agene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3 to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3 ^agene together with a cloned Lyt-2 ^agene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk^– and BW5147 cells. Transfection of the Lyt-3 ^agene without Lyt-2 ^aled to expression of Lyt-3-related cellular RNA but did not result in surface expression of Lyt-3.1, suggesting that the Lyt-3 glycoprotein is not expressed on the cell surface in the absence of Lyt-2.     

Evidence for a gene extinguishing cell-surface expression of the Thy-1 antigen

Hybrids between the Thy-1^– murine myeloma S194 and the Thy-l⁺ lymphoma BW5147 (Thy-l⁺) express neither the Thy-1.1 nor the Thy-1.2 antigen on their cell-surface. Subclones isolated from Thy-1^– clones express both Thy-1.1 and Thy-1.2 antigens in amounts similar to those present on wild-type Thy-1⁺ lymphomas, demonstrating that the Thy-1^– hybrids retain all the genes necessary for Thy-1 expression. The results are consistent with the idea that myeloma cells have a functional gene which acts to extinguish cell-surface Thy-1 expression in hybrids. The exact mechanism by which the gene acts to produce the Thy-1^– phenotype remains to be determined.     

Nucleotide sequence analysis of the C.AKR Lyt-2 agene: structural polymorphism in alleles encoding the Lyt-2.1 T-cell surface alloantigen

The Lyt-2 ^aallele of the C.AKR strain of mice (genotype Lyt-2 ^a, Lyt-3 ^a) was cloned, and its complete nucleotide sequence as well as that of 2 kb of 5 flanking DNA was determined. The sequence was comapred with the partial sequence of the Lyt-2 ^aallele of DBA/2 (genotype Lyt-2 ^a, Lyt-3 ^b) and the nearly complete sequence of the B10.CAS2 Lyt-2 ^ballele reported by Liaw and coworkers (1986). The coding regions of the two Lyt-2 ^aalleles differ from each other by two nucleotide substitutions in the three exons over which they could be compared, resulting in two amino acid substitutions in the leader and transmembrane segments. The coding region of the C.AKR Lyt-2 ^aallele differs from the Lyt-2 ^ballele by two nucleotide substitutions in the extracellular V-like domain, one of which is silent and the second of which leads to substitution of valine for methionine at amino acid position 78 giving rise to the Lyt-2.1 allotypic specificity. The coding region of the DBA/2 Lyt-2 ^aallele shares with C.AKR the allotypic substitution at position 78 and differs from Lyt-2 ^bby three additional nucleotide substitutions in the coding regions, two of which lead to amino acid substitutions in the leader and transmembrane segments. It would therefore appear that the Lyt-2 alleles of the three strains analyzed are distinct, and the nomenclature Lyt-2 ^a1 and Lyt-2 ^a2 is suggested to distinguish the alleles of C.AKR and DBA/2, respectively. These alleles share a common difference from the Lyt-2 ^bgene product at position 78, and since the amino acid substitutions which distinguish them from each other are in the leader and transmembrane segments, their mature Lyt-2 gene products appear antigenically identical.     

Activation of Lyt-2 associated with distant upstream insertion of an SL3-3 provirus

Two Lyt-2⁺ mutants of the T-cell lymphoma SL12.4.10 were selected by fluorescence activated cell sorting. Both mutants expressed Lyt-2 (CD8 -chain) but not Lyt-3 (CD8 -chain). Derivatives of one Lyt-2⁺ mutant that expressed Lyt-3 could be isolated by sorting for Lyt-3⁺ cells. Southern blotting analysis indicated that both mutants had structural rearrangements within or immediately 3 of the Lyt-3 gene, accompanied by demethylation of at least one Hpa II site within the Lyt-2 gene. Gene cloning analysis of one mutant demonstrated that the structural rearrangement was due to insertion of an SL3-3 provirus 35 kb 5 to the Lyt-2 gene. It is likely that Lyt-2 gene activation is a direct or indirect consequence of proviral insertion at this site. Address correspondence and offprint requests to: R. Hyman.     

A human homolog of the mouse CD8 molecule,Lyt-3: genomic sequence and expression

The CD8 antigen is a marker for those cytotoxic T cells that recognize antigen in the context of class I major histocompatibility antigens (MHC) and has now been identified in many species. In rodents the CD8 antigen is a heterodimer of two distinct chains, Lyt-2 and Lyt-3 in the mouse and OX-8 M _r 32 000 and 37 000 chains in the rat. Human CD8 has consistently been described as a homodimer/homomultimer on mature T cells made up of one chain homologous to the Lyt-2 and OX-8 M _r 32 000 chains. This paper identifies a human equivalent of the second rodent CD8 chain (Lyt-3 and OX-8 M _r 37 000 chains) at the genomic level and shows that this gene is transcribed in human thymocytes and in some acute leukemic T-cell lines. The existence of a human Lyt-3 homolog raises the possibility that human CD8, like mouse CD8, may exist as a heterodimer.     

Enlargement of Lyt-2-positive T cells is associated with functional impairment and autoimmune hemolytic anemia in New Zealand Black mice

We investigated the relationship between the increased cell diameter of Lyt-2+ T cells and the development of autoimmune disease in aging NZB and NZB X NZW F1 hybrid (BW) mice. Individual animals were analyzed for Lyt-2+ T cell size (by narrow-angle forward light scatter), anti-erythrocyte autoantibodies, anemia, proteinuria, and splenomegaly. The peak light scatter of the Lyt-2+ T cells correlated with the level of anti-erythrocyte autoantibodies and severity of hemolytic anemia, but not with proteinuria or splenomegaly. The cell size of this T cell subset did not increase in old BW or in NZB mice homozygous for the xid gene (NZB.xid). The in vivo administration of bacterial lipopolysaccharide to young NZB mice did not stimulate the enlargement of Lyt-2+ T cells. Ly-2+ T cells from old NZB mice could be stimulated by concanavalin A (Con A) to express interleukin 2 (IL 2) receptors and to synthesize DNA in vitro. However, in vivo administration of Con A to old NZB mice did not induce the expression of IL 2 receptors on Lyt-2+ T cells. Further, in vivo T suppressor function was impaired in old NZB mice with enlarged Lyt-2+ T cells. Thus, the enlargement of Lyt-2+ T cells in old NZB mice appears related to impaired T cell function in vivo and is associated with the development of anti-erythrocyte autoantibodies and autoimmune hemolytic anemia.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.     

Analysis of H-2-linked immune responses involved in resistance to AKR tumor growth

H-2-associated immune response gene(s) govern resistance to growth of a spontaneous AKR lymphoma, BW5147. The antigenic specificities recognized by the anti-BW5147 humoral response have been characterized and include: Thy-1, a T -cell differentiation antigen; gp70, a retroviral envelope protein; and several previously uncharacterized proteins, including a 78 000 molecular mass protein, p78, which is restricted to expression on BW5147 cells and five phosphoproteins with molecular masses of 33 000, 29 000, 23 000, 17 000, and 16 000. Only mice which are able to respond to Thy-1, p78, and the phosphoproteins can survive an inoculation of BW5147. Thus, resistance to BW5147 is complex and involves multiple antigens with possible roles in tumor rejection.Abbreviations used in this paper DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - Ir immune response - MuLV murine leukemia virus - NMS normal mouse serum - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

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 (12). 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-2^a, Lyt-3^a and C.AKR-Lyt-2^a, Lyt-3^a, congenic with Balb/cAn and bearingLyt-2 ^a andLyt-3 ^a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2^b, Lyt-3^b, congenic with AKR/J and bearing theLyt-2 ^b andLyt-3 ^b alleles of Balb/cJ.Abbreviations used in this paper DMSO dimethylsulfoxide - NP40 Nonidet P-40 detergent - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoreses - NP-NET buffer 0.15 M NaCl, 0.005 M EDTA, 0.05 M Tris, 0.02% sodium azide, pH 7.4, containing 0.5% or 0.05% NP40 as stated in text     

Evidence for a trans-acting activator function regulating the expression of the human CD5 antigen

Interspecies somatic cell hybrids were generated by fusing the mouse T-lymphoma cell line, BW5147, with normal human T lymphocytes at different stages of differentiation. Thymocytes, activated peripheral T lymphocytes, or an activated T-cell clone were used as human partners, respectively, in three independent fusions. Irrespective of the human cell partner used for fusion, a certain number of hybrids lost CD5 surface expression over a period of time in culture. Analysis at the phenotype and genetic level showed that lack of CD5 expression was due neither to segregation of human autosome 11, on which the CD5 gene has been mapped, nor to deletion of the CD5 structural gene. Furthermore, loss of CD5 surface expression correlated with the absence of specific mRNA. Since these hybrids preferentially segregate human chromosomes, these results indicate the existence of a non-syntenic trans-active locus, or loci, positively controlling the expression of the human CD5 gene.     

Molecular analysis of an HLA-DP mutant cell line selected for its resistance to killing by HLA-DPw2-specific T-cell clones

A collection of HLA-DP mutants was generated, using ICR 191 as the mutagenic agent and resistance to lysis mediated by HLA-DPw2 allospecific cytotoxic T lymphocytes (CTLs) as the selection criterion. These mutants were derived from the HLA haploid lymphoblastoid cell line 45.1. Loss of HLA-DPw2 surface expression accounted for the lack of HLA-DPw2 CTL recognition in all the mutants. However, one of them, 45.EM19, binds to DPw2-specific monoclonal antibodies (mAb) after cell permeabilization. HLA-DPA1 and DPB1 mRNA expression studies permitted the classification of the mutants in four categories: 1) DPA1-negative, DPB1-positive; 2) DPA1-positive, DPB1-negative; 3) DPA1- and DPB1-negative, and 4) DPA1- and DPB1-positive mutants. Mutant 45.EM19 is included in the last group. The cloning and sequencing of the full-length DPA1 (DPA1*0103) and DPB1 (DPB1*02012) cDNAs from this mutant showed no changes in the DPA1 sequence compared to the wild-type sequence. However, a frame-shift mutation in the DPB1 gene exon coding for the transmembrane region was detected. The insertion of a guanine nucleotide provokes an extension of the open reading frame, increasing the length of the C-terminal domain and changing the hydropathicity pattern of the transmembrane domain. This change should be responsible for the phenotype of the 45.EM19 mutant. Correspondence to: Dr. M. Sánchez-Pérez.     

Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. I. Lyt-2.1 and Lyt-3.1 antigenic determinants reside on separable molecular species.

Anti-Lyt-2.1 and anti-Lyt-3.1 sera were employed for sequential precipitation of NP-40 extracts of 125I-labeled C57BL/6-Lyt-2a, Lyt-3a thymocytes (Lyt-2.1, Lyt-3.1) to determine whether these alloantigenic determinants are present on the same or different molecular species. Treatment of extracts with anti-Lyt-3.1 serum and SaCI completely precipitated both Lyt-3.1 and Lyt-2.1-specific components, whereas treatment with anti-Lyt-2.1 serum reduced by approximately 37% the quantity of labeled species subsequently precipitable by anti-Lyt-3.1 serum. When 125I-labeled thymocytes were subjected to mild trypsinization before NP-40 extraction, the quantity of radioactive components precipitated by anti-Lyt-2.1 serum was essentially unchanged, but that of anti-Lyt-3.1-precipitable components was greatly reduced. Moreover, sequential precipitation of extracts of trypsinized thymocytes with anti-Lyt-2.1 and anti-Lyt-3.1 sera demonstrated that these molecular species were precipitated independently. Thus 1) Lyt-2.1 and Lyt-3.1 antigenic determinants appear to reside on different molecular species; 2) some Lyt-2.1- and Lyt-3.1-positive molecules appear to be complexed with each other in the NP-40 extract; and 3) this association of Lyt-2.1- and Lyt-3.1-positive species was dependent upon components that were labile to trypsinization of intact thymocytes.     

Construction of human-mouse T cell hybrids that express human T cell-associated surface antigens and allow the chromosomal localization of these antigens

We have constructed somatic cell hybrids between the murine T cell line BW5147 and cells from patients suffering from T cell acute lymphoblastic leukemia. The obtained hybrid clones were analyzed for expression of human T cell antigens and presence of human chromosomes. T cell hybrids derived from fusion between the BW5147 cell line and bone marrow cells from a patient with pre-T acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1-/T6-/T4-/T8-/T3-) appeared to express the human T cell antigen Tp41, which can be recognized by the monoclonal antibodies 3A1 and WT1. Although this panel of hybrid cells contained all human chromosomes, no other T cell antigens were expressed. Fusion of the BW5147 cell line with peripheral blood cells from a patient with a more mature T cell acute lymphoblastic leukemia (TdT+/HLA-DR+/Tp41+/T11+/T1+/T6-/T4+/T8+/T3-) resulted in a panel of hybrid clones that expressed not only the Tp41 antigen, but also the human T cell antigens T1 and T4; two hybrids even expressed the T3 antigen. This panel of hybrids also contained the whole human genome. The two panels of human-mouse T cell hybrids allowed us to assign the genes coding for the human T cell antigens Tp41, T1, and T4 to human chromosomes 17, 11, and 12, respectively. Furthermore, these data support our previous suggestion that the expression of human lymphoid differentiation antigens in human-mouse lymphoid hybrids is influenced by the differentiation stage of the fusion partners.     

The human Lyt-3 molecule requires CD8 for cell surface expression.

We have previously identified a monoclonal antibody, T8/2T8-5H7, which clustered serologically with CD8 monoclonal antibodies, but lacked reactivity with L cell transfectants expressing the human CD8 molecule (Lyt-2 homologue). Based on these observations, we postulated that T8/2T8-5H7 might recognize the human Lyt-3 gene product. To test this hypothesis, we have isolated a full-length cDNA encoding the human Lyt-3 molecule and have characterized its product in additional transfection experiments. The results of these studies indicate that the human Lyt-3 cDNA encodes a product recognized by the antibody T8/2T8-5H7. Interestingly, the human Lyt-3 molecule cannot be expressed alone, but requires the human Lyt-2 homologue for efficient cell surface expression. A heterodimer composed of the human Lyt-2 and Lyt-3 molecules may have importance in T cell-target cell interactions.     

Control of the antibody response of C57BL/6 Lyt-congenic strains to the Lyt-3.1 alloantigen by a gene linked to theLyt-2 locus

Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2^a mice with thymocytes from either B6-Lyt-2^a, Lyt-3^a or B6-Lyt-2^a, Lyt-3^a, H-2^k 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-2^a)F_a mice and progeny of the backcross, (B6 × B6-Lyt-2^a)F₁ × B6-Lyt-2^a, were immunized with B6-Lyt-2^a, Lyt-3^a, H-2^k 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-2^a/Lyt-2^b mice.     

Effect of gene dosage on cell-surface expression of Thy-1 antigen in somatic cell hybrids between Thy-1− abelson-leukemia-virus induced lymphomas and Thy-1+ mouse lymphomas

Hybrids between pseudodiploid Thy-1.1⁺ lymphomas and Thy 1.2^– pseudodiploid Abelson-leukemia-virus-induced (ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1⁺ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2⁺ or Thy 1.2^–. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1⁺, Thy 1.2^– hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1⁺ lymphomas and pseudotetraploid Thy 1.2^– Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1⁺ revertants were isolated from one of the Thy-1^– hybrids by cell sorting. — These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparentlycis-active, regulatory mechanisms which determine the ability of theThy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.