Francis W. Ruscetti

Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.

Characterization of the reverse transcriptase from a new retrovirus (HTLV) produced by a human cutaneous T-cell lymphoma cell line

Abstract The reverse transcriptase from a new retrovirus, HTLV, isolated from a human cutaneous T-cell lymphoma cell line was characterized. The enzyme is associated with high-molecular-weight RNA of the virus particle, has a molecular weight of about 95,000, utilizes the same template primers (including natyral mRNAs) as other viral reverse transcriptases, and prefers magnesium as a divalent cation using various synthetic homopolymeric template primers. HTLV reverse transcriptase can be immunologically distinguished from those of known type C, B, and D retroviruses by enzyme activity neutralization tests. These and other results support the interpretation that HTLV is a novel oncornavirus which may be associated with certain human T-cell neoplasias.

Cell-free HTLV-1 infects dendritic cells leading to transmission and transformation of CD4 + T cells

Cell-free human T-lymphotropic virus type 1 (HTLV-1) virions are poorly infectious in vitro for their primary target cells, CD4+ T cells. Here, we show that HTLV-1 can efficiently infect myeloid and plasmacytoid dendritic cells (DCs). Moreover, DCs exposed to HTLV-1, both before and after being productively infected, can rapidly, efficiently and reproducibly transfer virus to autologous primary CD4+ T cells. This DC-mediated transfer of HTLV-1 involves heparan sulfate proteoglycans and neuropilin-1 and results in long-term productive infection and interleukin-2–independent transformation of the CD4+ T cells. These studies, along with observations of HTLV-1–infected DCs in the peripheral blood of infected individuals, indicate that DCs have a central role in HTLV-1 transmission, dissemination and persistence in vivo. In addition to altering the current paradigm concerning how HTLV-1 transmission occurs, these studies suggest that impairment of DC function after HTLV-1 infection plays a part in pathogenesis.

T-cell growth factor and the culture of cloned functional T cells.

Publisher Summary Detailed studies have indicated that the production of T-cell growth factor (TCGF) is augmented by macrophage-derived lymphocyte-activating factor (LAF). Since the effects of LAF are concentration dependent, and because T-cell clonal expansion is TCGF-concentration dependent, the magnitude of the resultant T-cell immune response is necessarily dictated by the available quantities of these lymphokines. that Minor alterations in the production or action of LAF and TCGF may result in major changes in T-cell proliferation that may have physiological and pathological relevance. A T-cell proliferative response to antigedlectin is actually mediated by a soluble protein TCGF. The T-cell proliferative response results from the interaction of at least three distinct cell types. As antigen or lectin is introduced to a mixed become “activated.” This results in at least three responses: release of LAF from monocytes and macrophages; under the influence of LAF, the release of TCGF by a specific T-cell subset; and binding of TCGF by separate T-cell subsets resulting in the proliferative response. Three signals are involved in the elicitation of TCGF release from TCGF producer cells: lectin or antigen cell membrane binding, Zr gene products, and LAF. The relative importance of these signals and their sequence of application to the T-cell have yet to be delineated. This chapter discusses clonal derivation and maintenance of functional T-cell lines. The latter include the clonal derivation of T-cell lines, cytolytic T-cell lines, helper T-cell lines, suppressor T-cell lines, and neoplastic T-cells and TCGF. Because LAF and TCGF both appear to interact with their target cells by means of specific membrane-binding sites, alterations of the active sites of these hormone-like factors, or their receptors, may lead to alterations in immunocyte function.

Role of Cholesterol in Human Immunodeficiency Virus Type 1 Envelope Protein-Mediated Fusion with Host Cells

ABSTRACT In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-β-cyclodextrin (MβCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1α- and macrophage inflammatory protein 1β-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MβCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.

Heparan sulfate proteoglycans mediate attachment and entry of human T-cell leukemia virus type 1 virions into CD4+ T cells.

ABSTRACT Heparan sulfate proteoglycans (HSPGs) are used by a number of viruses to facilitate entry into host cells. For the retrovirus human T-cell leukemia virus type 1 (HTLV-1), it has recently been reported that HSPGs are critical for efficient binding of soluble HTLV-1 SU and the entry of HTLV pseudotyped viruses into non-T cells. However, the primary in vivo targets of HTLV-1, CD4+ T cells, have been reported to express low or undetectable levels of HSPGs. For this study, we reexamined the expression of HSPGs in CD4+ T cells and examined their role in HTLV-1 attachment and entry. We observed that while quiescent primary CD4+ T cells do not express detectable levels of HSPGs, HSPGs are expressed on primary CD4+ T cells following immune activation. Enzymatic modification of HSPGs on the surfaces of either established CD4+ T-cell lines or primary CD4+ T cells dramatically reduced the binding of both soluble HTLV-1 SU and HTLV-1 virions. HSPGs also affected the efficiency of HTLV-1 entry, since blocking the interaction with HSPGs markedly reduced both the internalization of HTLV-1 virions and the titer of HTLV-1 pseudotyped viral infection in CD4+ T cells. Thus, HSPGs play a critical role in the binding and entry of HTLV-1 into CD4+ T cells.

Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production

ABSTRACT The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-γ, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-γ gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-γ gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-γ gene, and increased IFN-γ production, demonstrating a direct link between methyltransferase and IFN-γ gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-γ gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

Autocrine transforming growth factor-beta regulation of hematopoiesis: many outcomes that depend on the context.

Transforming growth factor-β (TGF-β) is a pleiotropic regulator of all stages of hematopoieis. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Depending on the differentiation stage of the target cell, the local environment and the concentration and isoform of TGF-β, in vivo or in vitro, TGF-β can be pro- or antiproliferative, pro- or antiapoptotic, pro- or antidifferentiative and can inhibit or increase terminally differentiated cell function. TGF-β is a major regulator of stem cell quiescence, at least in vitro. TGF-β can act directly or indirectly through effects on the bone marrow microenvironment. In addition, paracrine and autocrine actions of TGF-β have overlapping but distinct regulatory effects on hematopoietic stem/progenitor cells. Since TGF-β can act in numerous steps in the hematopoietic cascade, loss of function mutations in hematopoeitic stem cells (HSC) have different effects on hematopoiesis than transient blockade of autocrine TGF-β1. Transient neutralization of autocrine TGF-β in HSC has therapeutic potential. In myeloid and erythroid leukemic cells, autocrine TGF-β1 and/or its Smad signals controls the ability of these cells to respond to various differentiation inducers, suggesting that this pathway plays a role in determining the cell fate of leukemic cells.

Transcriptional regulation of the transforming growth factor beta 1 promoter by v-src gene products is mediated through the AP-1 complex.

Growth factor-independent 32D-src and 32D-abl cell lines, established by infecting the interleukin-3-dependent myeloid precursor cell line (32D-123) with retroviruses containing the src or abl oncogene, were used to study transcriptional regulation of transforming growth factor beta 1 (TGF-beta 1) mRNA. Analysis of different TGF-beta 1 promoter constructs regulated by pp60v-src indicated that sequences responsive to high levels of src induction contain binding sites for AP-1. Both src and serum induced expression of the c-fos and c-jun genes in myeloid cells, resulting in transcriptional activation of the TGF-beta 1 gene. We found that serum treatment increased TGF-beta 1 mRNA levels in 32D-123 cells and that the v-Src protein could replace the serum requirement by stimulating binding to the AP-1 complex of the TGF-beta 1 promoter, thereby mediating the induction of TGF-beta 1 transcription.

HTLV-1 uses HSPG and neuropilin-1 for entry by molecular mimicry of VEGF165

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.