Recovery of functional memory T cells in lung transplant recipients following induction therapy with alemtuzumab.

Profound T-cell depletion with the monoclonal antibody alemtuzumab facilitates reduced maintenance immunosuppression in abdominal and lung transplantation. While the phenotype of the post-depletional T cells has been characterized, little is known about their function. In the present study, global and CMV-specific T-cell function was assessed longitudinally in 23 lung transplant (LTx) recipients using T-cell assays (ImmuKnow and T Cell Memory, Cylex, Columbia, MD) during the first year posttransplant after induction therapy. Recovery of mitogen responses were seen at 2 weeks posttransplantation (65%PHA; 58% Con A), despite the low number of circulating T cells (<2%). These responses declined at 4-5 months (24%PHA; 54% Con A) and were partially reconstituted by 9 months (46% PHA; 73% Con A). CMV-specific responses recovered in 80% of R+ patients as early as 2 weeks posttransplant (n = 5) and 72% of patients had a memory response by 3 months (n = 11). In contrast, only 2 of 5 patients who did not exhibit memory responses pre-transplant (R-) developed transient CMV-specific T-cell responses. Our results show that profound depletion of T cells induced by alemtuzumab spares the functional subset of CMV-specific memory T cells. Conversely, CMV R- patients predepletion may require a prolonged period of prophylaxis.

An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines.

Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccinees immunized with a CP vector expressing portions of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by a novel technique for flow cytometric visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.

Targeting of the visna virus tat protein to AP-1 sites: interactions with the bZIP domains of fos and jun in vitro and in vivo.

The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription.

Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.

The hypothesis that quiescent CD4+ T lymphocytes carrying proviral DNA provide a reservoir for human immunodeficiency virus-type 1 (HIV-1) in patients on highly active antiretroviral therapy (HAART) was examined. In a study of 22 patients successfully treated with HAART for up to 30 months, replication-competent virus was routinely recovered from resting CD4+ T lymphocytes. The frequency of resting CD4+ T cells harboring latent HIV-1 was low, 0.2 to 16.4 per 10(6) cells, and, in cross-sectional analysis, did not decrease with increasing time on therapy. The recovered viruses generally did not show mutations associated with resistance to the relevant antiretroviral drugs. This reservoir of nonevolving latent virus in resting CD4+ T cells should be considered in deciding whether to terminate treatment in patients who respond to HAART.

Human immunodeficiency virus type 1 envelope protein endocytosis mediated by a highly conserved intrinsic internalization signal in the cytoplasmic domain of gp41 is suppressed in the presence of the Pr55gag precursor protein.

The mechanisms involved in the incorporation of viral glycoproteins into virions are incompletely understood. For retroviruses, incorporation may involve interactions between the Gag proteins of these viruses and the cytoplasmic domains of the relevant envelope (Env) glycoproteins. Recent studies have identified within the cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) Env protein a tyrosine-containing internalization motif similar to those found in the cytoplasmic domains of certain cell surface proteins that undergo rapid constitutive endocytosis in clathrin-coated pits. Given that surface expression of the HIV-1 Env protein is essential for the production of infectious virus, the presence of this internalization motif is surprising. We show here that in contrast to the rapid rate of Env protein internalization observed in cells expressing the Env protein in the absence of other HIV-1 proteins, the rate of internalization of Env protein from the surfaces of HIV-1-infected cells is extremely slow. The presence of the Pr55gag precursor protein is necessary and sufficient for inhibition of Env protein internalization, while a mutant Pr55-gag that is incapable of mediating Env incorporation into virions is also unable to inhibit endocytosis of the Env protein. The failure of the Env protein to undergo endocytosis from the surface of an HIV-1-infected cell may reflect the fact that the proposed interaction of the matrix domain of the Gag protein with Env during assembly prevents the interaction of Env with host adaptin molecules that recruit plasma membrane molecules such as the transferrin receptor into clathrin-coated pits. When the normal ratio of Gag and Env proteins in the infected cells is altered by overexpression of Env protein, this mechanism allows removal of excess Env protein from the cell surface. Taken together, these results suggest that a highly conserved system to reduce surface levels of the Env protein functions to remove Env protein that is not associated with Gag and that is therefore not destined for incorporation into virions. This mechanism for the regulation of surface levels of Env protein may protect infected cells from Env-dependent cytopathic effects or Env-specific immune responses.

The leucine domain of the visna virus Tat protein mediates targeting to an AP-1 site in the viral long terminal repeat.

The visna virus Tat protein is a strong transcriptional activator and is necessary for efficient viral replication. The Tat protein regulates transcription through an AP-1 site proximal to the TATA box within the viral long terminal repeat (LTR). Previous studies from our laboratory using Tat-Gal4 chimeric proteins showed that Tat has a potent acidic activation domain. Furthermore, a region adjacent to the Tat activation domain contains a highly conserved leucine-rich domain which, in the context of the full-length protein, suppressed the activity of the activation domain. To further elucidate the role of this region, four leucine residues within this region of Tat were mutated. In transient-transfection assays using visna virus LTR-CAT as a reporter construct, the activity of this leucine mutant was dramatically reduced. Additionally, domain-swapping experiments using the N-terminal activation domain of VP16 showed that the leucine-rich domain of Tat confers AP-1 responsiveness to the chimeric VP16-Tat protein. A chimeric VP16-Tat construct containing the leucine mutations showed no increased AP-1 responsiveness in comparison with that of the VP16 activation domain alone. Furthermore, in competition experiments, a Gal4-Tat protein containing only the leucine region of Tat (amino acids 34 to 62) was able to inhibit by competition the activity of full-length Tat. These studies strongly suggest that this leucine-rich domain is responsible for targeting the Tat protein to AP-1 sites in the viral LTR. In addition, examination of the amino acid sequence of this region of Tat revealed a highly helical secondary structure and a pattern of residues similar to that in the leucine zippers in the bZIP family of DNA-binding proteins. This has important implications for the interaction of Tat with cellular proteins, specifically Fos and Jun, that contain bZIP domains.

Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains.

Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1. The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription. To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made. The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites. Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells. In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain. Amino acids 13 to 38 appeared to be the most critical for activation. No other region of the protein showed any activation in the GAL4 system. This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators. Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein. Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation. Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat.

Development of transgenic sheep that express the visna virus envelope gene.

The ovine lentiviruses cause encephalitis, pneumonia, and arthritis in sheep worldwide. Visna virus is a prototype of this family and the pathogenesis and molecular biology of the virus has been well characterized. The envelope proteins of visna virus are responsible for binding of virus to host cells and for causing cell fusion. The surface glycoprotein also elicits cellular and humoral immune responses to the virus, the former being thought to be responsible for eliminating infected cells as well as causing inflammatory lesions. In this study, transgenic sheep were constructed that expressed the envelope genes of visna virus under the control of the visna LTR to investigate the role of the env gene in the pathogenesis of lentiviral disease in its natural host. Three transgenic lambs were identified that contain the env transgene and express the envelope glycoproteins. These transgenic animals have remained healthy and expression of the viral gene has had no obvious deleterious effect. Expression of the visna envelope protein was demonstrated by cell fusion mediated by the envelope gene as well as by immunoprecipitation of the envelope proteins with monoclonal antibodies and immunofluorescence analyses of Env protein in cells. The target cell for visna virus replication in infected animals is the monocyte/macrophage. In natural infection, the level of viral gene expression in these cells increases with cell maturation. In the transgenic sheep, monocytes did not express the envelope glycoproteins until they differentiated into macrophages in vitro. Expression of the env mRNA in macrophages was quantitated by an RNase protection assay. In addition to expression in macrophages, the transgene was expressed by fibroblasts isolated from skin of the transgenic sheep. Expression of both the Env and Rev proteins was detected by immunoprecipitation and immunofluorescence. Two of the three lambs responded immunologically to the expression of the transgene by producing binding antibodies to the envelope glycoproteins. Thus, these transgenic sheep provide a model to study whether a lentivirus glycoprotein will prevent infection or modulate disease in its natural host after virus challenge.

Involvement of FOS and JUN in the activation of visna virus gene expression in macrophages through an AP-1 site in the viral LTR.

Gene expression of visna virus is highly restricted in monocytes, but is induced when monocytes differentiate into macrophages. A previous study on differential regulation of visna virus gene expression revealed that a specific AP-1 site in the long terminal repeat of the viral DNA is required for phorbol-ester-induced gene expression in macrophages (Gabuzda, Hess, Small, and Clements, Mol. Cell. Biol., 9, 2728-2733). In the present investigation, we examined the association of two DNA binding proteins, the proto-oncogene proteins FOS and JUN, with this AP-1 site in the visna virus LTR. We demonstrated that the concentrations of these two proteins and their mRNAs increased in U937 cells after phorbol ester induction. Furthermore, the binding of cellular proteins from the U937 nuclear extracts to this AP-1 site was significantly decreased in the presence of antibodies to JUN and FOS. In vitro-translated JUN protein also binds to this AP-1 sequence, and this binding is enhanced by the FOS protein. These results indicate that JUN and FOS are directly involved in the differential regulation of visna virus gene expression.

Involvement of a calpain-like protease in the processing of the murine interleukin 1 alpha precursor.

Interleukin (IL) 1 alpha is synthesized as a 33-kDa precursor that is enzymatically cleaved to the 15-17-kDa forms that are found in the culture supernatants of activated macrophages. We have explored the possibility that calcium might enhance IL-1 processing and secretion via the stimulation of a calcium-dependent protease. We have found that lysates prepared from human peripheral blood monocytes, the human histiocytic lymphoma cell line U937, and the murine macrophage cell line P388D1 contain a calcium-dependent IL-1 alpha processing activity that cleaves the IL-1 alpha precursor to its mature form. Although NIH 3T3 mouse fibroblast cell lysates also contain IL-1 processing activity, lysates from the murine thymoma EL-4, the human epidermoid cell line HEp-2, and the human foreskin fibroblast line FS-4 lack this activity. IL-1 processing activity is inhibited by leupeptin and exhibits a molecular mass of 80-110 kDa. The processing activity is also inhibited by a monoclonal antibody directed against calpain type I. These results indicate that the processing of the IL-1 alpha precursor is mediated, at least in part, by a member of the calpain family of proteases. Mixing experiments revealed that lysates from EL-4 or HEp-2 cells contain an inhibitor(s) of the calpain-like protease in macrophage extracts. It is, therefore, likely that many non-macrophage cell types are unable to process the IL-1 alpha precursor because the calpain present in these cells is only weakly active due to the presence of a specific inhibitor(s) such as calpastatin.

Detection of IL-1 alpha and IL-1 beta in the supernatants of paraformaldehyde-treated human monocytes. Evidence against a membrane form of IL-1.

The concept of a membrane form of IL-1 arose from the observation that paraformaldehyde-treated macrophages display IL-1 bioactivity. Thus far, the biochemical characterization of a membrane form of the molecule has not been reported. In a recent publication we demonstrated that murine IL-1 alpha can be detected in the supernatants of paraformaldehyde-treated macrophages. These data indicate that the phenomenon of membrane IL-1 may result from leakage of IL-1 from inadequately fixed cells. In the current report we have extended our studies toward the examination of human IL-1 alpha and IL-1 beta. IL-1 activity can be detected in the supernatants of paraformaldehyde-treated human monocytes. Although anti-IL-1 alpha, but not anti-IL-1 beta, antibodies can efficiently block the IL-1 bioactivity, both IL-1 alpha and IL-1 beta can be found by immunoprecipitation in the supernatants of the fixed monocytes. IL-1 alpha is efficiently processed to the low m.w. form, whereas IL-1 beta remains predominantly as the inactive, precursor molecule. IL-1 is not found in the supernatants of monocyte membrane preparations, demonstrating that the leakage of IL-1 is from an intracellular, rather than membrane-bound source.