Resistance to murine leukemia in mice rejecting syngeneic somatic hybrid cells.

(A/J X C3H/HeJ) F1 mice reject somatic cell hybrids of ASL-1 cells (A origin) and LM(TK)- cells (C3H origin), but die from leukemia within 10 days after the inoculation of approximately 10(6) viable ASL-1 cells. Mice rejecting hybrid cells survive for prolonged periods after challenge with otherwise lethal numbers of ASL-1 cells. The hybrid cells, rejected by syngeneic F1 recipients, retained their oncogenic potential as determined by the appearance and progressive growth of tumors in immunologically deficient nu/nu mice injected with the cells. Similar results were obtained using hybrids of a radiation-induced cell line (RADA-1), maintained by serial transfer in strain A mice and LM(TK)- cells. Syngeneic mice injected with RADA-1 X LM(TK)- cells failed to form tumors. Mice rejecting RADA-1 X LM(TK)- hybrid cells survived for prolonged periods after challenge with otherwise lethal numbers of RADA-1 cells.     

The effect of specific antiserum on the metabolism of three membrane-associated antigens of ASL-1 x LM(TK)- hybrid cells.

Fusion of ASL-1 cells, a murine leukemia forming thymus leukemia (TL) antigens, with LM(TK)- cells, a TL(--) murine cell line, resulted in a stable hybrid forming TL antigens. The hybrids failed to undergo modulation, the reversible dissappearance of TL antigens from the surfaces of the cells, stimulated by TL antiserum. Unlike ASL-1 cells, the rate of disappearance of the antigens from modulation negative hydrid cells was unaffected by TL antiserum. The t 1/2 of TL antigens of the hybrid was approximately 30 h. The t 1/2 of TL antigens of ASL-1 cells was 10 h in the presence of TL antiserum, 18 h in the absence of TL antiserum. The rate of metabolism of a putative tumor-associated antigen of ASL-1 cells formed by the hybrid was unaffected by exposure to specific antiserum, as was the metabolism of H-2 antigens formed by the cell types.     

Analysis of hybrids between an H-2+, TL− lymphoma and an H-2+, TL+ lymphoma and its H-2−, TL− variant subline

Hybrids between an H-2⁺, TL⁺ lymphoma and an H-2⁺, TL^– lymphoma were studied for their expression of H-2 and TL antigens. The H-2 antigens of both parents were expressed, the TL specificity of the TL⁺ parent was retained, and the TL specificity characteristic of the mouse strain from which the TL^– lymphoma was derived was not expressed. There was no evidence that the genome of either parent altered the expression of the TL antigens coded for by the genome of the opposite parent. Hybrids between the H-2⁺, TL^– lymphoma and an H-2^–, TL^– variant line derived from the H-2⁺, TL⁺ cell line expressed both theK- andD-regioncoded H-2 antigens and the TL specificity characteristic of the parental cell line from which the variant cell was derived. This result is consistent with the defect in the variant cell's being the result of a mutation affecting a gene coding for a positive element necessary for expression of both TL and the serologically detectable H-2 antigens on the cell surface.     

Biochemical genetics of TL antigens

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tla ^a, Tla ^d, Tla ^c), intermediate (Tla ^c, Tla ^f), and low (Tla ^b). (Relative amounts: 1000 : 100 : 1.) Some thymic leukemias arising in (Tla ^b, Tla ^c, Tla ^c) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.     

Inhibition of growth of ASL-1w murine leukemia cells by anti-thymus leukemia antigen (TL) serum in the absence of complement.

Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.     

Further polymorphism of the Tla locus defined by monoclonal TL antibodies

Six new monoclonal TL antibodies are described. At least one new TL antigen is defined (TL.7), and at least one more Tla allele, bringing the total number of known Tla alleles to six. Five of the monoclonal antibodies, and probably all six, identify distinct TL antigenic specificities. Four of these antigens conform in strain distribution and expression on leukemia cells to antigens defined by conventional antisera. The data contain a hint that monoclonal TL antibodies like TL.m6 may serve to identify a region of the Tla gene, which determines whether or not prothymocytes will respond to physiological induction by expressing TL, and thus may provide a means to study the regulatory mechanism that determines whether mouse strains are phenotypically TL⁺ or TL^– The nomenclature TL.m4–9 for the six monoclonal antibodies described follows McIntyre and coworkers (1980). The serial numbers 4–9 do not imply any correspondence with numbers assigned to TL antigens defined by conventional antisera. The corresponding hybridoma lines are available to interested investigators.     

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.     

Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

Characterization of a TL−variant of a homozygous TL+ mouse lymphoma

A homozygous antigen-loss variant for the TL antigen was isolated by immunoselectionin vitro. The variant expressed <0.01 the parental amount of TL antigen on its surface as measured by quantitative absorption. Neither theK nor theD end H-2^k antigens were detectable on the surface of the variant, although parental amounts of Thy 1.2 and GCSA were expressed. Karyotypic analysis showed that the variant had one less chromosome than the parental line.     

Selective cell surface expression of thymus leukemia antigen during S phase of the cell cycle.

The expression of the thymus leukemia antigen (TL) was studied on a murine leukemia cell line (ASL-1W) grown in vitro and separated into cell cycle phases by velocity sedimentation or growing synchronously in culture. The expression of TL was determined qualitatively by direct cytotoxicity and quantitatively by a modified inhibition of cytotoxicity assay. TL expression was found to vary with DNA synthesis. The hypothesis that expression is coordinately regulated with DNA synthesis, and the relationship of this to the restricted expression of TL on rapidly dividing cells is disucssed.     

Identification of chick thymidine kinase determinant in somatic cell hybrids of chick erythrocytes and thymidine kinase-deficient mouse cells

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses of chick-mouse somatic cell hybrids [LM(TK⁻)/CRB]isolated from fusion mixtures of chick erythrocytes and thymidine (TdR) kinase-deficient mouse [LM(TK⁻)]cells have demonstrated that the somatic cell hybrids contain only chick cytosol TdR kinase F and mouse mitochondrial TdR kinase A activities. Karyotypes were analysed by the method which sequentially reveals Q- and C-bands. Four hybrid clones contained the full complement of mouse chromosomes and 1 to 3 chick micro-chromosomes. Counterselection of the LM(TK⁻)/CRB hybrids in 5-bromodeoxyuridine (BUdR) medium resulted in the loss of chick cytosol TdR kinase F activity and at least one of the chick chromosomes, but mouse mitochondrial TdR kinase A activity was unaffected. Unlike the LM(TK⁻)/CRB somatic cell hybrids, the BUdR-resistant clones could not grow in HATG (hypoxanthine-aminopte-rin-thymidine-glycine) medium. The results demonstrate that: (1) the chick cytosol TdR kinase F gene is on a member of the micro-chromosomes; and (2) selection in HATG- and BUdR-containing medium involves only cytosol TdR kinase F.     

ACQUISITION OF CHICK CYTOSOL THYMIDINE KINASE ACTIVITY BY THYMIDINE KINASE-DEFICIENT MOUSE FIBROBLAST CELLS AFTER FUSION WITH CHICK ERYTHROCYTES

Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK^-)] cells. Autoradiographic studies demonstrated that 1 day after fusion, [³H]dT was incorporated into both red blood cell and LM(TK^-) nuclei of 23% of the heterokaryons. Self-fused LM(TK^-) cells failed to incorporate [³H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK^-)/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK^-) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK^-)/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK^-)/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK^-)/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK^-) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells.     

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

PA-I and PA-II lectin interactions with the ABO(H) and P blood group glycosphingolipid antigens may contribute to the broad spectrum adherence ofPseudomonas aeruginosa to human tissues in secondary infections

Pseudomonas aeruginosa may cause serious infections in most human tissues/organs. Its adherence to them is mediated by a battery of adhesins including the PA-I and PA-II lectins, which are produced in this bacterium in high quantities. PA-I binds to thed-galactose of the erythrocyte glycosphingolipids exhibiting highest affinities for B and P^k (followed by P₁) antigens, while PA-II preferentially binds to thel-fucose of H, A and B antigens. IntactP. aeruginosa cells also exhibit a clear P^k and P₁ over p preference. Such affinities for the most common human ABH and P system antigens may underlie the widespread tissue infectivity and pathogenicity of this bacterium.     

The ability of H-2+ and H-2− cell lines to induce or be lysed by Cytotoxic T cells

We have studied the T cell-mediated lysis of two C58 lymphoma lines: R1(TL⁺), which bears serologically detectable H-2^k and TL1,2,3 antigens, and R1(TL^–), an immunoselected variant which lacks these antigens. Unlike R1(TL⁺) cells, the variant cells are not sensitive to specific lysis by T cells directed against either H-2^k or minor H antigens of C58 mice. An injection of R1(TL⁺) cells into allogeneic H-2-identical or H-2-different mice primes for an excellent secondary cytotoxic response in vitro to R1(TL⁺); but not to R1(TL^–). Immunization in vivo and in vitro with R1(TL)^–) leads to little or no priming or generation of cytotoxic T cells. Both cell lines, however, are sensitive to nonspecific lysis by cytotoxic cells in the presence of PHA or Con A, although even under these conditions, R1(TL⁺) is killed more effectively than is R1(TL^–). We conclude that R1(TL^–) does not express any form of H-2 antigen which can be detected by immunization or by sensitivity to cytotoxic T cells.     

Characterization of pig-mink cell hybrids: assignment of the TK1 and UMPH2 genes to pig chromosome 12

By fusion of thymidine kinase-deficient mink cells with pig leukocytes, a new type of cell hybrid was produced. It was demonstrated that pig chromosomes segregate in pig-mink hybrids and that hybrid cells contain no cytologically visible rearrangements between the chromosomes of parental species, or chromosome fragmentation. With a set of subclones of two primary hybrid clones, the genes for thymidine kinase-1 (TK1) and uridine 5-monophosphate hydrolase-2 (UMPH2) were assigned to pig Chromosome (Chr) 12. A cell line with a single pig Chr 8 on the background of mink chromosomes was established. This clone could serve as a source of DNA for building a chromosome-specific library of pig Chr 8. The data obtained suggest that pig-mink cell hybrids can be used for mapping of pig chromosomes.     

Segregation of mitochondrial DNA in human somatic cell hybrids

Summary The maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and immortal). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to ³²P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA.The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.