Alloreactive immune responses of transgenic mice expressing a foreign transplantation antigen in a soluble form.

Transfection of cells with the H-2Kk gene lacking the transmembrane and cytoplasmic segments resulted in secretion of the H-2Kk protein, as determined by immunoprecipitation with monoclonal anti-H-2Kk antibodies. Transgenic (H-2b X H-2d)F1 mice were established carrying integrated copies of the modified H-2Kk gene. Expression of the soluble H-2Kk antigen in the transgenic mice was demonstrated in cell supernatants of biosynthetically labeled splenic and thymic Con A blasts as well as bone marrow-derived macrophages. Soluble H-2Kk molecules were also present in the sera of the transgenic animals. No cell-surface expression of the H-2Kk antigen could be observed. In spite of the presence of the soluble H-2Kk molecules in the transgenic mice, the animals were able to generate H-2Kk-specific cytolytic T cells as well as antibody responses when stimulated with cell-surface-bound H-2Kk antigens. These responses were indistinguishable from those of the nontransgenic littermates. Possible explanations for the observed lack of tolerance are discussed.     

Transgenic mice expressing a Huntington's disease mutation are resistant to quinolinic acid-induced striatal excitotoxicity.

Huntington's disease (HD) is a hereditary neurodegenerative disorder presenting with chorea, dementia, and extensive striatal neuronal death. The mechanism through which the widely expressed mutant HD gene mediates a slowly progressing striatal neurotoxicity is unknown. Glutamate receptor-mediated excitotoxicity has been hypothesized to contribute to the pathogenesis of HD. Here we show that transgenic HD mice expressing exon 1 of a human HD gene with an expanded number of CAG repeats (line R6/1) are strongly protected from acute striatal excitotoxic lesions. Intrastriatal infusions of the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid caused massive striatal neuronal death in wild-type mice, but no damage in transgenic HD littermates. The remarkable neuroprotection in transgenic HD mice occurred at a stage when they had not developed any neurological symptoms caused by the mutant HD gene. At this stage there was no change in the number of striatal neurons and astrocytes in untreated R6/1 mice, although the striatal volume was decreased by 17%. Moreover, transgenic HD mice had normal striatal levels of NMDA receptors, calbindin D28k (calcium buffer), superoxide dismutase activity (antioxidant enzyme), Bcl-2 (anti-apoptotic protein), heat shock protein 70 (stress-induced anti-apoptotic protein), and citrate synthase activity (mitochondrial enzyme). We propose that the presence of exon 1 of the mutant HD gene induces profound changes in striatal neurons that render these cells resistant to excessive NMDA receptor activation.     

HMT, encoded by H-2M3, is a neoclassical major histocompatibility class I antigen.

H-2M3 encodes HMT, the major histocompatibility complex (MHC) class I heavy chain of the maternally transmitted antigen (Mta). Like classical MHC class I genes, the expression of M3 can be stimulated by gamma-interferon and its message can be detected from mid-gestational embryos (day 8) through adulthood. HMTb, a nonimmunogenic allelic form of HMT, differs from the common HMTa molecule by four amino acids, of which only two (residues 31 and 95) are located in the alpha 1 and alpha 2 domains that form the peptide-binding groove. Recognition of site-directed mutants by Mta-specific cytotoxic T lymphocytes was hardly affected by the substitution of Met for Val31 but was abolished by the substitution of Gln for Leu95, which is located in the beta-sheet floor of the peptide-binding groove. Thus a single amino acid difference is responsible for the immunological silence of HMTb.     

Beta 2-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H-2Db or of a truncated H-2Db.

beta 2-Microglobulin (beta 2m) has been thought essential for transport of all major histocompatibility complex class I antigens to the cell surface. Here, we show that the mouse class I antigen H-2Db is expressed at the cell surface even when there is no beta 2m present within the cell. This was established by transfecting the H-2Db gene into the R1E cell line, which lacks beta 2m. The conformation of the Db antigen expressed by the R1E transfectant is very different from that of the native molecule. This Db antigen is not recognized by Db-allospecific and Db-restricted cytotoxic T lymphocytes or by most monoclonal antibodies to the native Db. We show further that a deletion construct of the Db gene, which consists of exon 1 linked to exons 4-8, expresses a truncated Db antigen lacking domains 1 and 2 [Db-(1 + 2)] at the cell surface after transfection into the R1E line. Previous biochemical and crystallographic data have indicated that domain 3 is associated with beta 2m; unexpectedly, Db-(1 + 2) does not associate with beta 2m when the mouse beta 2mb gene is transfected into the R1E transfectant expressing the truncated Db. This suggests that interactions with domains 1 and 2 are important for the paired association of domain 3 and beta 2m in the native Db antigen.     

H-2 restriction as a consequence of intentional priming: T cells of fully allogeneic chimeric mice as well as of normal mice respond to foreign antigens in the context of H-2 determinants not encountered on thymic epithelial cells.

Fully allogeneic chimeras were able to develop in vitro alloantigen-specific, as well as H-2-restricted, Sendai virus-specific cytotoxic T-lymphocyte (CTL) response. Depending on the immunization regimen used, Sendai virus-specific CTL responses were restricted to the H-2 antigens of either the stem cell donor or the thymus. Similarly, unprimed splenic T cells of normal mice were found to contain CTL-precursor cells that specifically reacted against Sendai virus or trinitrophenyl derivatives in the context of allogeneic major histocompatibility complex determinants that had not been encountered during their thymic differentiation. A frequency analysis of allogeneically versus syngeneically restricted virus-specific CTL precursors present in splenic T cells showed a ratio of about 1 to 6. These results provide evidence that H-2 restriction of trinitrophenyl- or Sendai virus-specific T cells is dictated by the complex type of the antigen-presenting cell and thus appears to be independent of the type of thymus in which the T cells have undergone maturation.     

Resistance to Listeria monocytogenes in mice: genetic control by genes that are not linked to the H-2 complex.

After mice of several inbred strains were injected with Listeria monoyctogenes, two parameters of resistance, the 50% lethal dose and the suppression of bacterial proliferation in spleen, were determined. The strains of mice tested could be segregated into two groups: the resistant C57BL/10Sn mice and the sensitive A/J and DBA/2J mice. Congenic resistant strains of mice were used because they would express the H-2 haplotype of the sensitive strains (H-2a or H-2d) on the background of a resistant strain, C57BL/10Sn. Both the B10.A/SgSn (H-2a) and the B10.D2/Sn (H-2d) mice were as resistant as mice from their background strain and were significantly more resistant than the strains that donated their H-2 locus (A/J or DBA/2J). Therefore, the resistance of mice to Listeria, although genetically controlled, is not controlled by gene (s) linked to the H-2 haplotype. On the other hand, the level of specific immunity to listeria antigens (as indicated by the footpad reaction) was higher in the C57BL/10Sn (H-2b) mice than in either the A/J and B10.A/SgSn (H-2a) mice or the DBA/2J and B10.D2/Sn (H-2d) mice. This observation suggests an H-2 linkage of specific immunity to Listeria.     

Cells that express viral antigens but lack H-2 determinants are not lysed by immune thymus-derived lymphocytes but are lysed by other antiviral immune attack mechanisms.

Murine F9 teratoma cells do not express major transplantation antigens detectable by either serologic or alloreactive assays of thymus-dependent lymphocytes (T cells). Such cells can be infected with lymphocytic choriomeningitis virus or vaccinia virus, do express viral antigens on the cell surface, and can release infectious virus in amounts equivalent to those of other H-2 bearing murine cell lines. Immunologic injury of virus-infected F9 cells occurs after the addition of specific antiviral antibody and complement or of specific antiviral antibody and unsensitized lymphoid cells (antibody-mediated cell-dependent killing). In contrast, injury does not follow the addition of immune cytotoxic T cells. These results indicate that possession of H-2 antigens is not a requirement for a cell's infection by or production of virus. Further, expression of viral antigens on the cell's surface, although adequate for antibody-mediated immune injury, is by itself insufficient for direct T cell-mediated lysis. The major transplantation antigens thus probably represent the cell surface structures that are crucial for T-mediated cell wall damage that results in chromium release.     

Genetic control of immunity to Heligmosomoides polygyrus: fixed H-2 E positive but not H-2 negative cells can present antigen to a parasite-specific T cell hybridoma

A number of T cell hybridomas were produced to adult worm homogenate (AWH) antigen of the nematode parasite Heligmosomoides polygyrus. All of the hybridomas were of the H-2^d haplotype and could potentially accept antigen in the context of either the A^d or E^d, H-2 molecules. Three types of antigen presentation were observed, with some of the T cell hybridomas accepting antigen in the context of the E and some in the context of the A molecule. A third type of hybridoma responded to antigen presented by paraformaldehyde fixed APC, but only when APCs were Epositive. These same hybridomas, were however, stimulated by A WH, when the antigen was presented by syngeneic but unfixed, E positive or E negative A PC. Therefore these data indicate that certain H. polygyrus-specfic T cell hybridomas can accept parasite antigen when presented in the context of either the H-2 A or E molecule, but the presentation of antigen by the two different MHC Class II molecules, can apparently utilize differing processing mechanisms.,     

Identification of a second class I antigen controlled by the K end of the H-2 complex and its selective cellular expression.

Immunoprecipitates obtained from [35S]methionine-labeled spleen cells by using monoclonal antibodies specific for H-2Kd and H-2Dd have been separated by two-dimensional polyacrylamide gel electrophoresis. Analysis of these gel patterns revealed the presence of an additional product of the K end of the H-2d complex, designated here as H-2K'. To determine whether H-2K' is a unique protein or a differentially glycosylated form of the previously characterized H-2Kd histocompatibility antigen, nonglycosylated molecules labeled in the presence of tunicamycin were examined. The results showed that both H-2K and H-2K' have distinct nonglycosylated polypeptide precursor forms. The approximate molecular weight differences between the fully glycosylated and nonglycosylated molecules also indicated the presence of three oligosaccharide side chains on H-2K', as is the case with H-2Kd, whereas H-2Dd has only two oligosaccharide units. The cellular expression of H-2K' was also investigated. Comparison of H-2 antigens immunoprecipitated from normal spleen cells and from thioglycollate-induced adherent peritoneal exudate cells cultured in the presence or absence of supernatant fluids from concanavalin A-stimulated spleen cells revealed that H-2K' was not expressed on the adherent peritoneal cells. This indicates that H-2K' is expressed in a tissue-specific manner, unlike the classical histocompatibility antigens H-2K and H-2D.     

Suppression of H-2b-associated resistance to Friend erythroleukemia virus by a class I gene from the H-2d major histocompatibility complex haplotype

Mice homozygous for the H-2d haplotype at the major histocompatibility complex are markedly more susceptible to erythroleukemia induction by the Friend isolate of murine leukemia retrovirus (FV) than are congenic mice homozygous for the H-2b haplotype. The resistance conferred by the H-2b haplotype is recessive in this cross, since heterozygous F1 mice are as susceptible as parental strain H-2d homozygotes. However, H-2b-associated resistance is not an intrinsically recessive trait, since H-2b/H-2dm1 heterozygotes resemble H-2b homozygotes in their relative resistance to FV; the mutant H-2dm1 haplotype lacks the entire D region of the parental haplotype except for a single class I gene formed by the fusion of its terminal D-region genes to produce a class I gene differing from both parental genes, and thus this finding indicates that one or more D-region genes of the H-2d haplotype can actively suppress H-2b-associated resistance. Unlike H-2dm1, the mutant H-2dm2 haplotype, which retains only the class IDd gene in the D region of the H-2d haplotype, strongly suppresses resistance in H-2b/H-2dm2 heterozygotes, and the presence of Dd as a transgene significantly reduces the resistance of H-2b homozygotes. Since H-2b-associated resistance to FV appears to be due mainly to the capacity of Lb (also called Db), the only class I molecule encoded in the D region of the H-2b haplotype, to present viral epitopes for recognition by FV-specific cytotoxic T lymphocytes, suppression of resistance to FV by the Dd molecule implies that the presence of one class I molecule of the major histocompatibility complex can interfere with either the presentation of viral epitopes by another class I molecule or the generation of T cells that recognize viral epitopes so presented.     

Detection of a secreted form of the murine H-2 class I antigen with an antibody against its predicted carboxyl terminus.

Analysis of H-2 class I-specific cDNA clones has suggested the synthesis by the liver of a class I molecule that is secreted rather than membrane bound. To detect this putative class I-related molecule, we have predicted a unique region of amino acid sequence located toward the carboxyl terminus of the molecule that is not expected to be shared with any of the classical H-2 class I antigens, and we have generated specific antibodies to a synthetic peptide corresponding to this sequence. Indirect immunoprecipitation with this antibody led to the identification of a Mr 40,000 polypeptide in association with beta 2-microglobulin in the serum of mice of five different H-2 haplotypes. This class I molecule is also detected in the liver together with lower molecular weight components, which are presumably underglycosylated precursors. Synthesis of this molecule is not detected in thymus, spleen, kidney, or testis. This class I serum component has no detectable reactivity with either a broad-specificity alloantiserum against H-2b or a xenoantiserum against purified H-2a class I molecules. The availability of a specific antibody against the secreted class I molecule offers a means to purify this protein for structural and functional studies.     

Molecular characterization of novel H-2 class I molecules expressed by a C3H UV-induced fibrosarcoma.

Two novel class I-like molecules expressed on tumor 1591, a C3H UV-induced fibrosarcoma, are biochemically characterized using two-dimensional gel electrophoresis, a cross-blocking RIA, and tryptic peptide mapping. One novel molecule that reacts with CP28, a syngeneic tumor-specific monoclonal antibody, appears mosaic because it possesses characteristics of both Kk and Dk class I molecules. The second molecule is closely related but not identical to the bona fide Ld molecule expressed on BALB/c spleen. Thus 1591 expresses at least two novel class I molecules and is vigorously rejected by normal C3H mice, while a variant tumor derived from 1591, termed AS7, does not express these two class I molecules although it still expresses Kk and Dk. The significance of these observations to the immunobiology and genetics of the UV-induced fibrosarcoma system is discussed. Speculations on the role that the major histocompatibility complex may play in the immunosurveillance of neoplasms are also presented.     

Partial suppression of anchorage-independent growth and tumorigenicity in immunodeficient mice by transfection of the H-2 class I gene H-2Ld into a human colon cancer cell line (HCT)

Many human tumors, particularly those of epithelial origin, appear to express greatly reduced levels of major histocompatibility complex class I antigens on their surface. It has been previously reported that the class I gene H-2Ld, introduced into adenovirus type 12-transformed mouse cells, induces reversal of oncogenesis in immunocompetent BALB/c mice. We have tested the hypothesis that the H-2Ld gene, when transfected into HCT colon cancer cells, may alter their transformed phenotype. Two H-2Ld transfectants, HCT-Ii and HCT-If, were found to exhibit a markedly reduced-to-virtually suppressed ability to form colonies in soft agar in comparison to a transfectant (HCTh) carrying only the neomycin-resistance gene. We also compared the tumorigenicity of HCTh vs. HCT-If cells in two different strains of immunodeficient mice: nude (T-) and triple-deficient mutants (T-, NK-, B-). At 28 days postinjection of 10(7) and 10(6) cells, the size and growth rate of HCT-If tumors were greatly reduced compared to HCTh cells. Therefore, as assayed in immunodeficient animals, expression of the class I H-2Ld gene in HCT cells appears to correlate with partial suppression of the tumorigenic phenotype, suggesting that the expression of a transfected class I gene may by itself alter the phenotype of the recipient cell and that such phenotypic changes may be independent of the immune system.     

Different exon-intron organization at the 5' part of a mouse class I gene is used to generate a novel H-2Kd-related mRNA.

A cDNA library constructed from liver mRNA of DBA/2 (H-2d) mice has been screened with H-2-specific probes. The nucleotide sequence of one clone (pH-2d-24) indicates that it derives from an H-2 gene with an unexpected exon-intron organization. Nucleotide sequence comparisons suggest that two distinct mRNAs are produced from a single H-2Kd gene by a mechanism involving the use of alternative splicing sites in its 5' region. pH-2d-24 carries an open reading frame encoding a thus-far-undescribed polypeptide product identical to an H-2Kd-molecule, except for the NH2-terminal half of the first domain.     

An optimal viral peptide recognized by CD8+ T cells binds very tightly to the restricting class I major histocompatibility complex protein on intact cells but not to the purified class I protein.

CD8+ cytotoxic T lymphocytes recognize cell surface complexes formed by class I major histocompatibility complex (MHC-I) glycoproteins and antigenic peptides. We have identified a peptide nonamer (termed IV9) derived from the human immunodeficiency virus that is over a millionfold more active (at subpicomolar concentrations) than peptide analogues longer or shorter by one or two amino acid residues. Although IV9 does not detectably bind to isolated MHC-I molecules as measured by equilibrium dialysis, we quantitated its specific binding in unaltered form to MHC-I on intact cells. Less than 1% of cell surface MHC-I forms complexes with IV9, which suffices to trigger maximal cytotoxic T-lymphocyte activity. By contrast, a peptide dodecamer that includes the IV9 sequence and is active at micromolar concentrations does not bind to MHC-I on intact cells, raising the possibility that this longer peptide undergoes processing. Using stoichiometrically iodinated IV9 to obviate the ambiguities associated with trace labeling methods, we measured the dissociation kinetics of purified peptide/MHC-I complexes isolated by affinity chromatography and found these complexes to be exceedingly stable (t1/2 = 200-600 hr).     

A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1(+/-)) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5-11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1(+/-) mice was apparently slower than that in Tsc2(+/-) mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.     

Western-type diets induce insulin resistance and hyperinsulinemia in LDL receptor-deficient mice but do not increase aortic atherosclerosis compared with normoinsulinemic mice in which similar plasma cholesterol levels are achieved by a fructose-rich diet.

The role of insulin resistance (IR) in atherogenesis is poorly understood, in part because of a lack of appropriate animal models. We assumed that fructose-fed LDL receptor-deficient (LDLR-/-) mice might be a model of IR and atherosclerosis because (1) fructose feeding induces hyperinsulinemia and IR in rats; (2) a preliminary experiment showed that fructose feeding markedly increases plasma cholesterol levels in LDLR-/- mice; and (3) hypercholesterolemic LDLR-/- mice develop extensive atherosclerosis. To test whether IR could be induced in LDLR-/- mice, 3 groups of male mice were fed a fructose-rich diet (60% of total calories; n=16), a fat-enriched (Western) diet intended to yield the same plasma cholesterol levels (n=18), or regular chow (n=7) for approximately 5.5 months. The average cholesterol levels of both hypercholesterolemic groups were similar (849+/-268 versus 964+/-234 mg/dL) and much higher than in the chow-fed group (249+/-21 mg/dL). Final body weights in the Western diet group were higher (39+/-6.2 g) than in the fructose- (27.8+/-2.7 g) or chow-fed (26.7+/-3.8 g) groups. Contrary to expectation, IR was induced in mice fed the Western diet, but not in fructose-fed mice. The Western diet group had higher average glucose levels (187+/-16 versus 159+/-12 mg/dL) and 4.5-fold higher plasma insulin levels. Surprisingly, the non-insulin-resistant, fructose-fed mice had significantly more atherosclerosis than the insulin-resistant mice fed Western diet (11.8+/-2.9% versus 7.8+/-2. 5% of aortic surface; P<0.01). These results suggest that (1) fructose-enriched diets do not induce IR in LDLR-/- mice; (2) the Western diets commonly used in LDLR-/- mice may not only induce atherosclerosis, but also IR, potentially complicating the interpretation of results; and (3) IR and hyperinsulinemia do not enhance atherosclerosis in LDLR-/- mice, at least under conditions of very high plasma cholesterol levels. The fact that various levels of hypercholesterolemia can be induced in LDLR-/- mice by fat-enriched diets and that such diets induce IR and hyperinsulinemia suggest that LDLR-/- mice may be used as models to elucidate the effect of IR on atherosclerosis, eg, by feeding them Western diets with or without insulin-sensitizing agents.     

Induction of the metastatic phenotype by transfection of the nuclear oncogene p53: Increases in cytoplasmic diacylglycerol levels and reduction in class I major histocompatibility antigen expression are not sufficient to explain the changes in metastatic capacities

Summary Transfection of the oncogene encoding the nuclear protein p53 into a low-metastatic mouse carcinoma cell line resulted in enhanced metastatic capabilities in clones that showed increased p53 protein expression [Pohl J, Goldfinger N, Radler-Pohl A, Rotter V, Schirrmacher V (1988) Mol Cell Biol 8:2078–2081]. This effect semmed neither to be due to increase in cytoplasmic diacylglycerol levels nor to reduced cell-surface expression of class I major histocompatibility antigens.     

Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium, and lanthanum, but not to nickel: evidence provided by fluorescence changes in fura-2-loaded cells.

Cobalt (Co), nickel (Ni), manganese (Mn), cadmium (Cd), and lanthanum (La) are commonly used as calcium (Ca) channel blockers, but some of them, besides reducing Ca entry, also traverse Ca channels and can exert effects intracellularly that confound interpretation of functional responses. Because of this and our need to use Ca channel blockers in an ongoing analysis of Ca channel activity in the regulation of the cytosolic free Ca concentration ([Ca2+]i) and secretion in melanotrophs, we assessed whether the cations mentioned enter these cells. This was done by incorporating the fluorescence for changes that would signal the presence of the cations in the cytosol. In cell-free solution, where the probe and cations can interact freely, Mn, Co, and Ni all quench fluorescence, whereas Cd and La act in a Ca-like manner. When tested on fura-2-loaded melanotrophs in basal (unstimulated) conditions, Mn, Co, and Cd each yielded corresponding signals, thereby showing that they had penetrated the cells. By contrast, Ni caused no quenching of fluorescence even in melanotrophs exposed to 100 mM K+ to recruit additional Ca channels. Ni, therefore, did not penetrate the cells. However, as expected, Ni quenched fluorescence when given artificial access to the cytoplasm by ionomycin. Ni blocked spontaneous entry of Mn, Co, and Cd. It also lowered [Ca2+]i in unstimulated melanotrophs, consistent with blockade of spontaneous Ca entry. Like Ni, La lowered basal [Ca2+]i in unstimulated melanotrophs without penetrating the cells; however, unlike Ni, it penetrated when the melanotrophs were exposed to high potassium. We conclude that Ni is the most specific of the Ca channel blockers tested and that results obtained with Mn, Co, Cd, and La must be interpreted with reserve.