Loss of H-2Kk gene product(s) from an AKR spontaneous leukaemia

Serological absorption analyses and immunochemical studies of the H-2 antigenic specificities on the AKR (H-2^k) spontaneous leukaemia K36 have been performed. The results confirm the absence of the H-2K^k gene product from the cell surface and from detergent solubilised tumour cells. A genetic mechanism is considered for this phenotypic alteration.     

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     

One gene determines maize B chromosome accumulation by preferential fertilisation; another gene(s) determines their meiotic loss

Genotypes of high (H(m)) and low (L(m)) male B transmission rate (B-TR) were obtained. B-TR segregation in the F2 is reported, showing that the H(m) and L(m) lines differ in a single locus we call mBt (male B transmission), controlling B preferential fertilisation in maize. The egg cells control which one of the sperm nuclei is going to fertilise them, mBt(h) egg cells being preferentially fertilised by the sperm nucleus carrying the supernumerary B chromosomes (Bs). It is hypothesised that the mBt gene is involved in the normal fertilisation of maize but the parasitic Bs take advantage of the mBt(h) allele to increase their own transmission. Selection was also carried out when the Bs were transmitted on the female side (H(f) and L(f) lines). The F1 hybrids show that the gene(s) that we call fBt (female B transmission), controlling female B-TR, is located on the A chromosomes acting at diploid level, the fBt(l) allele(s) for low transmission being dominant. This allele causes the loss of Bs at meiosis, which is shown using a specific B molecular probe to determine B presence/absence in microspores of both lines and hybrids. Maize Bs are a nice example of intragenome conflict, because the mBt and fBt loci are a polymorphic system of attack and defence between A and B chromosomes.     

Regulation of the production of murine IgG2a by an H-2-linked gene and other unlinked genes

Alleles of at least two loci (rig-1 and Rig-2) regulate the levels of serum immunoglobulin of the Igh-1b class and allotype in BALB/c Ig^b (BAB/14) and (BALB/c × BAB/14)F₁ mice. The combined effect of the BALB/c alleles at these two loci is to lower Igh-1b levels significantly below those observed in other strains and below their own levels of Igh-1a in allotype heterozygous mice. The rig-1 locus is closely linked to or within the H-2 complex. Two alleles have been defined: rig-1 ^d and rig-1 ^b in H-2 ^d and H-2 ^b haplotypes, respectively. Homozygous rig-1 ^d d animals heterozygous for the BALB/c Rig-2 allele(s) have very low levels of Igh-1b. The designation of Rig-2 is provisional since it has not been mapped or defined as a single locus.     

Expression of HLA-B27 in transgenic mice is controlled by gene(s) mapping between H-2D and H-2L loci

The level of HLA-B27 transgene expression on the cell surface is dependent on the host H-2 haplotype. Mice homozygous for the H-2 ^b , H-2 ^f , H-2 ^f , H-2 ^p , H-2 ^r , and H-2 ^k haplotypes express B27 at high levels. An intermediate level of B27 expression is observed in H-2 ^v mice whereas low levels of B27 are expressed in H-2 ^q and H-2 ^d mice. The decreased expression of B27 maps to the D region of the major histocompatibility complex. Recombinant strain B10.RKDB (D^dL^b) mapped the low expression gene centromeric to H-2L. In order to determine the low expression within the H-2D region, the B27 transgene was introduced into B10.D2-H-2 ^dm1 and BALB/c-H-2 ^dm2 mice. Expression of B27 in both of these strains was high indicating that neither H-2D ^d nor H-2L ^d is responsible for the low expression. This maps the effect between the H-2D and H-2L loci. In addition, introduction of human ₂-microglobulin (₂m) into B10.D2-B27 transgenic mice caused a marked enhancement of B27 expression on the cell surface suggesting that the defect in B27 expression in certain haplotypes is due to an inability of B27 to associate with endogenous mouse ₂m. We propose that gene(s) mapping between D and L (either D2, D3, D4, or some as yet unidentified gene) may be involved in class I assembly by helping association of ₂m with class I. This putative molecule, designated Assembly Enhancer (AE) might have a negative influence in the association between human class II and mouse ₂m.     

Structure of the human M(2) muscarinic acetylcholine receptor gene and its promoter.

The M(2) muscarinic receptor inhibits the release of acetylcholine from cholinergic fibers in the lungs and elsewhere. In airway parasympathetic neurons, M(2) receptor expression is decreased by viral infections and by interferon-gamma, increasing actylcholine release. Dexamethasone increases M(2) receptor expression, decreasing acetylcholine release. We carried out 5' rapid amplification of cDNA ends beginning with mRNA from human heart and IMR32 human neuroblastoma cells. This demonstrated a 5' UTR of 100 BP, corresponding to two sequences on chromosome 7, separated by a 22.6 kB intron. The splice acceptor site is at -45 relative to the initiating atg. The 3000 BP upstream of 5' RACE product were subcloned into a pGL3 luciferase reporter vector. Deletional constructs were expressed in IMR32 cells. These demonstrated that 412 BP provided full expression of the reporter gene, and suggested a repressor element between -1848 and -1510.     

Synthesis and characterization of membrane immunoglobulin, Ia, and H-2 molecules of thy 1+ AKR/J lymphomas.

Three AKR lymphomas displaying B cell and T cell characteristics have been described. Because of the proclivity of normal AKR/J mice to develop T cell lymphomas, and the rarity of lymphomas with dual characteristics, the B cell markers of these tumors were studied more intensively. Fluorescence data with class-specific anti-immunoglobulin reagents demonstrated that the tumor cells stained only with class-specific anti-IgM reagents. Because of the possibility that the surface Ig was passively acquired and of reports that certain anti-mu-chain sera react with "IgT", chemical characterization of the immunoglobulin molecules was performed. Using 3H-leucine internal labeling, we showed that all three tumor lines synthesized the immunoglobulin found on their surface, and that the immunoglobulin had the chemical and immunologic characteristics most typical of monomeric surface IgM, and was composed of mu-chains and light chains. The Ia antigens found on these cells were also examined. These antigens were also synthesized by the cells and were present in the same molecular form and in the same approximate quantity as Ia antigens on normal spleen cells.     

Genetic control of the induction of cytolytic T lymphocyte responses to AKR/Gross viral leukemias. II. Negative control by the Fv-1 locus in AKR mice of responder H-2b haplotype

To assess whether the presence of a responder H-2b haplotype would be sufficient to allow mice of nonresponder "high leukemic" phenotype to generate syngeneic anti-AKR/Gross virus cytolytic T lymphocytes (CTL), the AKR.H-2b strain was examined. Although capable of mounting vigorous apparent anti-minor histocompatibility-specific CTL responses, AKR.H-2b mice failed to produce anti-viral CTL after a variety of stimulation protocols. In contrast, the "doubly congenic" AKR.H-2b:Fv-1b strain was able to respond with substantial levels of H-2-restricted anti-AKR/Gross virus CTL activity. These results indicated that Fv-1n alleles could exert negative epistatic control over responder H-2b-encoded gene(s). Because the B6.Fv-1n congenic was also able to generate anti-viral CTL indistinguishable from the prototype B6 strain, however, it was apparent that other genes of AKR background were required for the Fv-1n-mediated inhibition in AKR.H-2b mice. The mechanism by which Fv-1 intereacted with other genes to override positive H-2b control appeared to be related to the expression of the CTL-defined, virus-associated antigens by normal AKR.H-2b cells. Thus, AKR.H-2b spleen cells but not thymus cells were able to stimulate the production of B6 anti-AKR/Gross virus CTL and were recognized as target cells by such anti-viral CTL. In contrast, both spleen cells and thymocytes from AKR.H-2b:Fv-1b mice were negative when tested as stimulator or target cells in these assays. In addition, AKR.H-2b but not AKR.H-2b:Fv-1b spleen cells were shown to display serologically defined gp70 determinants and the Gross cell surface antigen. Taking these data together, it appeared that the inhibition of anti-viral CTL responsiveness might be due to tolerance induced by the cell surface expression of virus-associated antigens by normal AKR.H-2b cells. Widespread display of viral antigens, in turn, may have been due to the permissive effects of Fv-1n on the spread of the early arising N-ecotropic, endogenous AKR leukemia virus controlled by other background genes. In this context, the implications of the multi-gene control of anti-AKR/Gross virus CTL production are discussed with respect to the induction of spontaneous leukemia in the high incidence AKR strain.     

Mechanism of nonresponsiveness to AKR/Gross leukemia virus in AKR.H-2b:Fv-1b mice. An analysis of precursor cytotoxic T lymphocyte frequencies in young versus moderately aged mice

As young adult AKR.H-2b:Fv-1b mice reach about 9 wk of age, they begin to develop a nonresponsiveness to AKR/Gross leukemia virus. Unlike young mice that are responders, moderately aged AKR.H-2b:Fv-1b mice, after immunization and secondary in vitro restimulation in bulk culture with AKR/Gross virus induced tumors, can not generate anti-AKR/Gross virus-specific CTL. The mechanism of conversion to nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice is not understood, but it is correlated with increased expression of endogenous ecotropic viral antigens. Our present investigation focuses on determining the frequency of anti-AKR/Gross virus precursor CTL in AKR.H-2b:Fv-1b mice as a function of age. This was achieved by performing limiting dilution cultures of immune spleen cells obtained from young and moderately aged AKR.H-2b:Fv-1b mice. Although spleen cells obtained from immune moderately aged mice can not differentiate in bulk cultures into anti-AKR/Gross virus-specific CTL, there was no evidence of substantially decreased frequencies of virus-specific precursor CTL, relative to precursor CTL frequencies observed in young responder AKR.H-2b:Fv-1b mice.     

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     

Heart rate recovery after maximal exercise is associated with acetylcholine receptor M2 (CHRM2) gene polymorphism

The determinants of heart rate (HR) recovery after exercise are not well known, although attenuated HR recovery is associated with an increased risk of cardiovascular mortality. Because acetylcholine receptor subtype M2 (CHRM2) plays a key role in the cardiac chronotropic response, we tested the hypothesis that, in healthy individuals, the CHRM2 gene polymorphisms might be associated with HR recovery 1 min after the termination of a maximal exercise test, both before and after endurance training. The study population consisted of sedentary men and women (n = 95, 42 +/- 5 yr) assigned to a training (n = 80) or control group (n = 15). The study subjects underwent a 2-wk laboratory-controlled endurance training program, which included five 40-min sessions/wk at 70-80% of maximal HR. HR recovery differed between the intron 5 rs324640 genotypes at baseline (C/C, -33 +/- 10; C/T, -33 +/- 7; and T/T, -40 +/- 11 beats/min, P = 0.008). Endurance training further strengthened the association: the less common C/C homozygotes showed 6 and 12 beats/min lower HR recovery than the C/T heterozygotes or the T/T homozygotes (P = 0.001), respectively. A similar association was found between A/T transversion at the 3'-untranslated region of the CHRM2 gene and HR recovery at baseline (P = 0.025) and after endurance training (P = 0.005). These data suggest that DNA sequence variation at the CHRM2 locus is a potential modifier of HR recovery in the sedentary state and after short-term endurance training in healthy individuals.     

Lung tumor-reactive cytotoxic lymphocytes generated in mixed lymphocyte reaction between C3HfeB/HeN (H-2kb) and C3H/HeN (H-2k) strain mice.

The tumor-associated transplantation antigen expressed by several transplacentally induced lung tumors of C3HfeB/HeN mice (H-2kb haplotype) has previously been shown to exist as a normal tissue alloantigen in mice of known H-2k and H-2a haplotypes. This antigen is not expressed in normal tissues of C3HfeB/HeN mice but is expressed in C3H/HeN mice, the strain from which the C3HfeB/HeN mice were originally derived. The present study indicates that spleen cells from C3HfeB/HeN and C3H/HeN mice respond reciprocally in the mixed lymphocyte reaction. Cytotoxic T lymphocytes specific for the tumor-associated alloantigen can be readily generated in mixed lymphocyte reactions in which spleen cells from C3HfeB/HeN mice are reacted with x-irradiated spleen cells from C3H/HeN or A strain mice. These cells are effective in suppressing the growth of the C3HfeB/HeN-derived lung tumor 85 in x-irradiated syngeneic recipients.     

Promotion of tumor growth by transfecting antisense DNA to suppress endogenous H-2Kk MHC gene expression in AKR mouse thymoma

Many AKR spontaneous thymomas are reported to express different amounts of the major histocompatibility complex class I H-2Kk molecules. Moreover, H-2Kk-deficient AKR tumor cells are found to be more malignant when compared to tumor cells that express abundant levels of the H-2Kk molecules. To corroborate further the role of H-2Kk in tumorigenesis of AKR leukemia, we have, in this study, expressed antisense H-2Kk RNA in a high-H-2Kk-expressing and poorly tumorigenic AKR thymoma cell line 369. The down-regulation of H-2Kk molecules in the transfected 369 clones rendered them more tumorigenic in syngeneic AKR/J mice. The increase in oncogenicity correlates well with a concomitant reduction in their susceptibility to tumor-specific cytotoxic T lymphocytes in vitro. These results suggest the relevance of H-2Kk molecules in the immune surveillance of AKR tumors.     

Cytotoxic T lymphocyte recognition of class I H-2 antigens after DNA-mediated gene transfer

Cytotoxic T lymphocyte (CTL) recognition sites on class I major histocompatibility complex molecules have been investigated by several laboratories by using cloned genes expressed on mouse L cells by DNA-mediated gene transfer. Recombinant genes, constructed by restriction endonuclease treatment of cloned H-2Dd and Ld genes and exchange of the N and C1 exons (exon shuffling) have provided an additional tool. These hybrid H-2 molecules expressed on L cells have been used as targets to achieve more precise localization of site(s) recognized by allospecific and virus-specific CTLs. CTL systems were chosen that limit recognition to either the Dd or Ld alloantigen or to virus and Dd or Ld complexes. Using this approach, we were able to map essential restricting site(s) to the N and/or C1 domains. Additional evidence is presented that the cytoplasmic tail of H-2 may be involved in interactions with some viral antigens and effect the formation of an immunogenic complex.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Emv-13 (Akv-3): a noninducible endogenous ecotropic provirus of AKR/J mice

All AKR/J mice carry at least three endogenous ecotropic viral loci which have been designated Emv-11 (Akv-1), Emv-13 (Akv-3), and Emv-14 (Akv-4) (Jenkins et al., J. Virol. 43:26-36, 1982.) Using two independent AKR/J-derived sets of recombinant inbred mouse strains, AKXL (AKR/J x C57L/J) and AKXD (AKR/J x DBA/2J), as well as the HP/EiTy strain (an Emv-13-carrying inbred strain partially related to AKR/J mice) (Taylor et al., J. Virol. 23:106-109, 1977), we have examined the association of these endogenous viral loci with virus expression. Strains which transmit Emv-11 or Emv-14 or both were found to produce virus spontaneously, whereas strains that transmit Emv-13 alone were negative for virus expression. Restriction endonuclease digestion and hybridization with an ecotropic virus-specific hybridization probe of DNAs from strains which transmit only Emv-13 yielded enzyme cleavage patterns identical to those observed with DNAs from strains transmitting Emv-11 or Emv-14 or both. These findings indicate the absence of any gross rearrangement of Emv-13 proviral sequences. Cell cultures derived from recombinant inbred strains that carry only Emv-13 failed to express detectable infectious virus, viral proteins, or cytoplasmic ecotropic virus-specific RNA even after treatment with 5-iodo-2-deoxyuridine or 5-azacytidine, an inhibitor of DNA methylation. Our results indicate that a mechanism(s) other than methylation of Emv-13 proviral DNA is responsible for inhibition of Emv-13 expression.     

Mouse T lymphocyte response to acetylcholine receptor determined by T cell receptor for antigen V beta gene products recognizing Mls-1a.

Mice of strain B6, but not AKR/J, respond to immunization with Torpedo acetylcholine receptor (AChR) by manifesting in vitro an Ag-specific T lymphocyte proliferative response. Our analysis of (AKR x B6)F1 mice reveals that the T cell unresponsiveness of AKR/J is inherited as a dominant trait, possibly associated with expression of the Mls-1a allele. Mice derived from backcrossing (AKR x B6)F1 x B6 were selected for H-2b homozygosity and were classified as Mls-1a or Mls-1b according to the relative numbers of peripheral blood T cells that expressed the TCR V beta 6 gene product. After challenge by injection with AChR in CFA, lymph node cells from mice classified as having less than 2% of V beta 6+ peripheral T cells had low responsiveness to AChR, whereas mice with greater than 7% V beta 6+ peripheral T cells had high T cell responsiveness to AChR. These results are consistent with the notion that regulation of the T cell repertoire by Mls loci may be a determinant of susceptibility to autoimmunity.