Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression,but non-specifically enhanced on induction of Tax expression

Background

Immunogenetic evidence indicates that cytotoxic T lymphocytes (CTLs) specific for the weak CTL antigen HBZ limit HTLV-1 proviral load in vivo, whereas there is no clear relationship between the proviral load and the frequency of CTLs specific for the immunodominant antigen Tax. In vivo, circulating HTLV-1-infected cells express HBZ mRNA in contrast, Tax expression is typically low or undetectable. To elucidate the virus-suppressing potential of CTLs targeting HBZ, we compared the ability of HBZ- and Tax-specific CTLs to lyse naturally-infected cells, by co-incubating HBZ- and Tax-specific CTL clones with primary CD4⁺ T cells from HLA-matched HTLV-1-infected donors. We quantified lysis of infected cells, and tested whether specific virus-induced host cell surface molecules determine the susceptibility of infected cells to CTL-mediated lysis.

Results

Primary infected cells upregulated HLA-A*02, ICAM-1, Fas and TRAIL-R1/2 in concert with Tax expression, forming efficient targets for both HTLV-1-specific CTLs and CTLs specific for an unrelated virus. We detected expression of HBZ mRNA (spliced isoform) in both Tax-expressing and non-expressing infected cells, and the HBZ_26–34 epitope was processed and presented by cells transfected with an HBZ expression plasmid. However, when coincubated with primary cells, a high-avidity HBZ-specific CTL clone killed significantly fewer infected cells than were killed by a Tax-specific CTL clone. Finally, incubation with Tax- or HBZ-specific CTLs resulted in a significant decrease in the frequency of cells expressing high levels of HLA-A*02.

Conclusions

HTLV-1 gene expression in primary CD4⁺ T cells non-specifically increases susceptibility to CTL lysis. Despite the presence of HBZ spliced-isoform mRNA, HBZ epitope presentation by primary cells is significantly less efficient than that of Tax.

     

Microinjected Xenopus oocytes secrete mature, biologically active parathyroid hormone

Xenopus oocytes have been shown to faithfully translate, process, and secrete a number of secretory proteins after the injection of heterologous mRNAs. The oocyte has the capacity to perform a variety of posttranslational protein modifications but has been reported to be incapable of carrying out certain two-step cleavages which proceed via propeptide intermediates. We examined the ability of the oocyte to process preproPTH after the injection of parathyroid mRNA. Microinjected oocytes secreted material which could be detected in a sensitive cytochemical bioassay for PTH. This activity paralleled that of the PTH standard in the assay and was entirely eliminated by a competitive inhibitor of PTH binding, by preincubation with an anti-PTH antiserum, and by coinjecting oocytes with an oligonucleotide mixture complementary to PTH sequences. Immunoprecipitable proPTH and PTH were present in oocyte homogenates, but oocyte-conditioned medium contained only mature PTH(1-84). We conclude that the Xenopus oocyte is capable of accurately processing preproPTH to the mature secretory form of the peptide.     

Bcl-2 Lies Downstream of Parathyroid Hormone–related Peptide in a Signaling Pathway That Regulates Chondrocyte Maturation during Skeletal Development

Parathyroid hormone–related peptide (PTHrP) appears to play a major role in skeletal development. Targeted disruption of the PTHrP gene in mice causes skeletal dysplasia with accelerated chondrocyte maturation (Amizuka, N., H. Warshawsky, J.E. Henderson, D. Goltzman, and A.C. Karaplis. 1994. J. Cell Biol. 126:1611–1623; Karaplis, A.C., A. Luz, J. Glowacki, R.T. Bronson, V.L.J. Tybulewicz, H.M. Kronenberg, and R.C. Mulligan. 1994. Genes Dev. 8: 277–289). A constitutively active mutant PTH/PTHrP receptor has been found in Jansen-type human metaphyseal chondrodysplasia, a disease characterized by delayed skeletal maturation (Schipani, E., K. Kruse, and H. Jüppner. 1995. Science (Wash. DC). 268:98– 100). The molecular mechanisms by which PTHrP affects this developmental program remain, however, poorly understood. We report here that PTHrP increases the expression of Bcl-2, a protein that controls programmed cell death in several cell types, in growth plate chondrocytes both in vitro and in vivo, leading to delays in their maturation towards hypertrophy and apoptotic cell death. Consequently, overexpression of PTHrP under the control of the collagen II promoter in transgenic mice resulted in marked delays in skeletal development. As anticipated from these results, deletion of the gene encoding Bcl-2 leads to accelerated maturation of chondrocytes and shortening of long bones. Thus, Bcl-2 lies downstream of PTHrP in a pathway that controls chondrocyte maturation and skeletal development.     

N-terminal amino acid sequence of two novel tumor-derived adenylate cyclase-stimulating proteins: identification of parathyroid hormone-like and parathyroid hormone-unlike domains

A 17,000 dalton human adenylate cyclase-stimulating protein has previously been purified from a human tumor associated with humoral hypercalcemia of malignancy. This report describes the purification of a related 7,000-9,000 MW protein from a second tumor, and provides N-terminal amino acid sequence of these two peptides. The sequences of the peptides are identical, defining the smaller peptide as an N-terminal portion of the larger peptide. The two peptides possess one region of strong homology with parathyroid hormone and a second divergent region. These structural similarities and differences may explain the similarities and differences which occur in patients with hyperparathyroidism and humoral hypercalcemia of malignancy.     

Parathyroid hormone-related protein. Evidence for secretion of a novel mid-region fragment by three different cell types.

The cDNA-predicted amino acid sequence of parathyroid hormone-related protein (PTHrP) contains multiple basic amino acid motifs, suggesting that PTHrP undergoes extensive post-translational processing prior to secretion. The secretory forms of the peptide are currently unknown. To identify these secretory forms, medium was harvested from three cell types: human renal carcinoma (SKRC-1) cells, human keratinocytes, and rat insulinoma cells stably transfected with the cDNA for PTHrP(1-141) (RIN-141 cells). Amino-terminal species were immunopurified using an anti-PTHrP(1-36) column, and mid-region species using an anti-PTHrP(37-74) column. PTHrP peptides in medium and in cell extracts were further resolved by reverse phase high performance liquid chromatography (RP-HPLC) and identified using region-specific immunoassays. SKRC-1 and RIN-141 cells secreted three distinct amino-terminal species and a novel, non-amino-terminal, mid-region fragment. Sequence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the RIN-141 cell mid-region fragment begins at amino acid 38 of the cDNA-predicted sequence and is approximately 70 amino acids in length. Comparison of RP-HPLC elution patterns suggests that SKRC-1 cells and keratinocytes secrete a similar or identical mid-region fragment. Immunofluorescence studies revealed a Golgi pattern for the amino-terminal species and a secretory granule pattern for the mid-region fragment. These studies indicate that 1) multiple PTHrP species are secreted, including a novel mid-region fragment; 2) Arg37 serves as a cleavage site in at least three cell types; 3) PTHrP(1-36) is likely to be an authentic secretory form of PTHrP; and 4) the mid-region fragment appears to be packaged into secretory granules. The marked interspecies conservation of this mid-region PTHrP suggests that it will have important biological functions.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.