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The cytotoxicity reaction of murine CD8 T lymphocytes has been found to be strongly inhibited by nanomolar concentrations of hypericin, a lipophilic dianthraquinone with photodynamic properties. Cytotoxic T lymphocyte (CTL)-induced target cell apoptosis, as well as exocytosis of cytolytic granules from these cells, were ablated by hypericin, administered at the onset of the reaction, without affecting CTL viability. The inhibition of cytolysis occurred without the light irradiation which is essential for photosensitization. The findings suggest that the action of hypericin targets the effector CTL; however, apoptosis induced in murine L-cells with recombinant tumor necrosis factor (TNF)-α was also prevented by hypericin. Since hypericin is a known inhibitor of protein kinase C, MAP kinase and at least one other tyrosine kinase, this inhibitory activity could play a role in the down-modulation of CTL-induced cytotoxicity. Furthermore, our studies show that the action of hypericin induces rapid dephosphorylation of phospholipids associated with low-density membranes in CTL, but not with membranes of the cytotoxic granules. The ability of hypericin to interfere with cytotoxicity may render it useful in the treatment of T cell-mediated diseases.

The transcription of the MHC class I genes is regulated by interaction of cis-elements, located in the 5′ genomic flanking regions, with sequence-specific trans-factors. We have identified a cis-regulatory element, 5′-TGACGCG-3′, of the H-2D^d gene. This cyclic adenosine-3′,5′-monophosphate regulatory element (CRE)-like sequence, named H-2 binding factor 1 (H-2 BF1) binding motif, is highly conserved among species. In addition, we found that homo- and heterodimers of activation transcription factor 1 (ATF-1) and CRE binding protein (CREB) associate with the H-2 BF1 binding motif and activate transcription of the H-2D^d gene. Here we demonstrate that a homologue of ATF-1, originally isolated and designated ATF-1DN, acts as a dominant repressor, blocking the ability of wild-type ATF-1 and CREB to bind to the H-2 BF1 probe in electrophoretic mobility shift assays (EMSA). We have utilized this molecule to analyze the participation of the H-2 BF1 complexes, consisting of the H-2 BF1 binding motif and ATF-1/CREB trans-factors, in the physiological regulation of MHC class I expression in tissue culture cells. A human epidermoid carcinoma cell line, A431, was transfected with ATF-1DN and clones expressing the gene transcripts were selected. When analyzed in the EMSA, nuclear proteins prepared from these clones exhibited a decreased shift of the H-2 BF1 probe corresponding to the levels of the ATF-1DN gene expression. Additionally, MHC class I expression of cells with reduced H-2 BF1 activity was significantly higher than in control cells lacking ATF-1DN. These findings indicate that in these carcinoma cells, the H-2 BF1 complexes negatively regulate the constitutive expression of MHC class I.

Two aromatic polycyclic diones hypericin and pseudohypericin have potent antiretroviral activity; these substances occur in plants of the Hypericum family. Both compounds are highly effective in preventing viral-induced manifestations that follow infections with a variety of retroviruses in vivo and in vitro. Pseudohypericin and hypericin probably interfere with viral infection and/or spread by direct inactivation of the virus or by preventing virus shedding, budding, or assembly at the cell membrane. These compounds have no apparent activity against the transcription, translation, or transport of viral proteins to the cell membrane and also no direct effect on the polymerase. This property distinguishes their mode of action from that of the major antiretro-virus group of nucleoside analogues. Hypericin and pseudohypericin have low in vitro cytotoxic activity at concentrations sufficient to produce dramatic antiviral effects in murine tissue culture model systems that use radiation leukemia and Friend viruses. Administration of these compounds to mice at the low doses sufficient to prevent retroviral-induced disease appears devoid of undesirable side effects. This lack of toxicity at therapeutic doses extends to humans, as these compounds have been tested in patients as antidepressants with apparent salutary effects. Our observations to date suggest that pseudohypericin and hypericin could become therapeutic tools against retroviral-induced diseases such as acquired immunodeficiency syndrome (AIDS).

The biological consequences of radiation leukaemia virus (RadLV) infection include the stimulation of H‐2 antigen expression soon after injection of the virus. Early studies demonstrated that resistance to RadLV‐induced leukaemia in certain mouse strains is mediated by genes in the H‐2Dregion of the major histocompatibility complex (MHC). Recent studies have shown that elevated H‐2Ddexpression on the thymocyte cell surface of resistant mouse strains results from increased mRNA transcription and is correlated with elevated levels of a DNA‐binding activity that recognizes a short DNA sequence 5′ of the start of transcription for the H‐2Ddgene. This binding activity has been termed H‐2 binding factor 1 (H‐2 BF1) and is found exclusively in the thymus. In an effort to examine the H‐2genes of RadLV‐susceptible mice for the presence of the H‐2 BF1 binding target, we have cloned class I genes from the highly susceptible B10.S mouse strain and have identified both the Dsand the Ksgenes. The entire genomic sequence for the Dsgene has been determined and is reported here. In addition, the 5′ regulatory region of the previously cloned Dqgene has been sequenced; mice of the Dqhaplotype are also susceptible to RadLV‐induced leukaemia. In this report, we show that the H‐2 BF1 DNA binding sequence is present 5′ of each of these three class I genes.

The region of chromosome 2 between H-13 and H-3 has been shown to contain loci coding for a variety of other alloantigens, including Ly-4 and the locus coding for beta 2-microglobulin. Herein we show that Ly-6 and Ly-11 are coded for by genes in a segment of chromosome 2 adjacent to the H-3-H-13 region and that this segment of chromosome also contains the tightly linked loci coding for antigens Ala-1, DAG, H9/25, H-30, Ly-8, and ThB. In addition, at least one locus (and probably more) affecting susceptibility to leukemia induction is found within this gene cluster.

Resistance to radiation leukemia virus-induced leukemia is correlated with an increase in H-2D expression on the thymocyte surface. Recently, it has been shown that elevated H-2Dd expression on the infected thymocyte is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequences (5'-TGACGCG-3') of the H-2Dd gene. This target for transcription factor binding has been found to be identical in the 5'-regulatory region of 12 rodent class I genes, nine of which have been shown to be functional genes. Furthermore, this cis-element is found 5' of 20 primate class I genes (15 human genes), seven of which are known to be functional. Here, we demonstrate that activation transcription factor 1 (ATF-1) is one component of H-2 BF1. In addition, the levels of ATF-1 mRNA in uninfected and radiation leukemia virus-infected thymocytes parallel those of H-2Dd mRNA, and therefore, it is suggested that ATF-1 up-regulates the transcription of the H-2Dd gene after radiation leukemia virus infection of thymocytes. Transfection experiments also demonstrate that ATF-1 activates a reporter plasmid that contains the H-2 BF1 motif, but not a reporter lacking this motif. This is the first demonstration of the interaction of ATF-1 with 5'-regulatory sequences of major histocompatibility complex class I genes.

Transformation of murine thymocytes by radiation leukemia virus is associated with reduced expression of the class I antigens encoded in the major histocompatibility complex (MHC) and increased methylation and altered restriction enzyme patterns of MHC DNA. These changes may play a role in host susceptibility to virus-induced leukemogenesis and accord with the notion that viral genomes play a regulatory function when they integrate adjacent to histocompatibility genes.

When the content of cyclic AMP (cAMP) was compared in livers of a series of congenic mouse strains differing at the H-2 locus, significant variation in concentration of cAMP per unit wet weight was found among strains, and also for animals of a given strain with increasing age. For a given age, from 8 to 22 weeks, cAMP levels in liver of H-2a and H-2b genotype animals were significantly higher than that in liver of H-2k type animals. This difference was seen whether the H-2 gene was on the genetic background of strain C57BL/10, C3H, or A. Levels of cAMP in livers of H-2d animals were between those of H-2a and H-2k animals.

This paper provides evidence for the involvement of immune mechanisms in conferring resistance to a spontaneous AKR leukemia. It is shown that genes in the B, J, or E subregions of the H-2 complex confer resistance to a spontaneously arisen, tissue culture-adapted AKR thymoma, BW5147. A direct correlation is demonstrated between survival to injected BW5147 cells and humoral responsiveness in various hybrids obtained from crosses of AKR mice and C57BL/10 or C3H/DiSn derived congeneic strains differing at H-2. Cellular immunity appears to play no role in resistance to the proliferation of tumor cells. It is further established that development of effective humoral immunity depends on B cells and Ly-1+, 2-, 3- helper T-cells bearing the I-Jk phenotype. These findings seem directly applicable to the spontaneous disease, and results of studies using transformed cells from an overtly leukemic AKR mouse parallel those obtained using BW1547 cells.

We report here that specific T-cell receptor rearrangements were observed in fractionated-X-irradiation-induced murine leukemias. Consistent gamma-chain rearrangements, limited beta-chain rearrangements, and no detectable alpha-chain rearrangements were observed. Gene expression studies revealed that, in comparison with normal thymus tissue, expression of alpha T-cell receptor genes was lower in the thymomas, beta expression was much higher but approximately equal to that of normal thymocytes, and gamma expression was significantly increased. After coupling these data with those from analyses using reagents against other surface markers, such as Lyt-2, L3T4, H-2, IL-2R and MEL-14, we concluded that the target T cells for fractionated-X-irradiation-induced transformation resemble fetal thymocytes from days 15 and 16 of gestation.

Two retroviruslike sequences have been isolated from the TL locus of the major histocompatibility complex of C57BL/10 mice. One sequence (TLev2) hybridizes only with probes derived from the pol region of the murine leukemia provirus AKR; the other sequence (TLev1) hybridizes with gag, pol, and env AKR region probes. This 9-kilobase endogenous, TL region-associated virus (TLev1) has been further characterized. The TLev1 genome has been shown to contain murine leukemia virus-related sequences bounded by retroviruslike, VL30 long terminal repeats. Hybridization of TLev1-derived probes to mouse genomic digests reveals multiple copies which show distinct patterns compared with those observed with murine leukemia virus probes. The study of TLev1 may prove significant with respect to the interaction of retroviral sequences within the genome, expression of genes within the TL locus, and polymorphisms within the major histocompatibility complex.

Radiation leukemia virus (RadLV) causes thymic lymphoma in 90% of susceptible mice after a latent period of several months. The virally encoded polypeptides produced by RadLV-induced lymphoma cells were analyzed by immunoprecipitation and NaDodSO4/polyacrylamide gel electrophoresis. Along with the expected precursor and mature forms of gag and env gene products, a polypeptide of 36,000 molecular weight (p36) was precipitated by anti-gag antisera. It was not precipitable by normal sera or anti-env antibodies. Like the gag-associated fusion proteins of some acute leukemia viruses, p36 was found to be phosphorylated in vivo, although it lacked detectable ATP-specific protein kinase activity in vitro. By kinetics during pulse-chase labeling experiments and by comparison of two-dimensional tryptic peptide maps, this protein is not an intermediate in gag precursor processing. One lymphoma cell line is described that resembles a nonproducer RadLV-transformant, synthesizing relatively large amounts of p36 in the absence of Pr66gag or p30 production. Several RadLV-induced lymphoma cell lines also produce p36, while it was not detectable in the radiation-induced lines tested. In addition, p36 was not produced by mouse or mink fibroblasts or cultured thymocyte cell lines infected with virus passaged from the RadLV-induced lymphomas. We conclude that p36 may represent a previously unrecognized transformation-related protein induced directly or indirectly by infection with RadLV.

The role of H-2-linked genes in controlling resistance to murine leukemia viruses has been studied by measuring the cell-mediated immune response of F1 hybrid mice (between AKR and various C3H and C57BL/10 derived, H-2 congenic strains) to an AKR tumor cell line, BW5147. The studies have shown that the ability to generate a primary or secondary cell-mediated response to an AKR tumor cell antigenic determinant is under H-2 linked control. The locus determining CML responsiveness maps in the I-J subregion. Nonresponsiveness is associated with the H-2q/k and H-2b/k hybrid genotypes, whereas responsiveness is associated with the H-2k/k homozygous genotype. Nonresponsiveness may result from (a) dominant suppression; (b) recessive responsiveness; or (c) an alternate mechanism not yet understood. This type of control may be one of several H-2-associated mechanisms of defense against virus-induced neoplasms.

Several polymorphic DNA restriction endonuclease fragments hybridizing with xenotropic and ecotropic envelope virus probes map adjacent to minor histocompatibility and lymphocyte (H/Ly) antigen-encoding loci. Viral DNA restriction fragments are associated with Ly-17 on chromosome 1, H-30, H-3, and H-13 on chromosome 2, Ly-21 on chromosome 7, H-28 on chromosome 3, and H-38 (chromosomal location as yet undetermined). In each case no recombinant can be found between the H/Ly locus in question and the virus-related restriction fragment, suggesting that linkage is very tight. Although some viral loci map to locations where no H/Ly has yet been mapped, the frequency and tightness of linkage in the seven instances described, coupled with the large number of as yet unmapped H/Ly loci, suggests that the associations found are significant.

A common link between the induction of leukemia by (i) fractionated doses of x-irradiation and (ii) radiation leukemia virus in mice may be established by the observation that the segment of chromosome 2 between the loci for the minor histocompatibility antigen H-30 and color coat agouti (H-30-A) includes distinct loci involved in susceptibility to leukemogenesis induced by factors i and ii.