Human T lymphotropic virus type-I (HTLV-I) immortalizes human T cells in vitro--its implication in the pathogenesis of adult T cell leukemia (ATL).

HTLV-I is the first human retrovirus that was isolated from a patient with T-cell malignancy in 1980 in the United States. HTLV-I is detected in most patients with adult T cell leukemia (ATL) and healthy carriers, who are frequently found in the southwestern parts of Kyushu and Shikoku Districts. HTLV-I-infected cells express IL-2 receptors, and HTLV-I-infected T cell lines can be established from most of ATL patients in culture in the presence of IL-2. Furthermore, these IL-2 dependent T cell lines often begin to proliferate in the absence of IL-2 and to not respond to IL-2, despite IL-2 receptors on their cell surface, thus mimicking ATL cells in vivo. These findings suggest that HTLV-I is an etiological agent of ATL. In this mini-review, the T cell immortalizing activity of HTLV-I in vitro, with special reference to the evolution of ATL cells based on our results, is described.     

The contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for the immunotherapy of rheumatological diseases

Interleukin-15 (IL-15) is a 14-15-kDa member of the 4alpha helix bundle family of cytokines that stimulate T and NK (natural killer) cells. IL-15 and IL-2 utilize heterotrimeric receptors that include the cytokine-specific private receptors IL-2Ralpha and IL-15Ralpha, as well as two receptor elements that they share, IL-2Rbeta and gammac. Although IL-2 and IL-15 share two receptor subunits and many functions, at times they provide contrasting contributions to T-cell-mediated immune responses. IL-2, through its pivotal role in activation-induced cell death (AICD), is involved in peripheral tolerance through the elimination of self-reactive T cells. In contrast, IL-15 in general manifests anti-apoptotic actions and inhibits IL-2-mediated AICD. IL-15 stimulates the persistence of memory phenotype CD8+ T cells, whereas IL-2 inhibits their expression. Abnormalities of IL-15 expression have been described in patients with rheumatoid arthritis or inflammatory bowel disease and in diseases associated with the retrovirus HTLV-I (human T-cell lymphotropic virus I). Humanized monoclonal antibodies that recognize IL-2Ralpha, the private receptor for IL-2, are being employed to inhibit allograft rejection and to treat T-cell leukemia/lymphoma. New approaches directed toward inhibiting the actions of the inflammatory cytokine, IL-15, are proposed for an array of autoimmune disorders including rheumatoid arthritis as well as diseases associated with the retrovirus HTLV-I.     

Human T-cell leukemia virus type I-induced proliferation of human immature CD2+CD3- thymocytes.

The mitogenic activity of human T-cell leukemia virus type I (HTLV-I) is triggering the proliferation of human resting T lymphocytes through the induction of the interleukin-2 (IL-2)/IL-2 receptor autocrine loop. This HTLV-I-induced proliferation was found to be mainly mediated by the CD2 T-cell antigen, which is first expressed on double-negative lymphoid precursors after colonization of the thymus. Thus, immature thymocytes express the CD2 antigen before that of the CD3-TCR complex. We therefore investigated the responsiveness of these CD2+CD3- immature thymocytes and compared it with that of unseparated thymocytes, containing a majority of the CD2+CD3+ mature thymocytes, and that of the CD2-CD3- prothymocytes. Both immature and unseparated thymocytes were incorporating [3H]thymidine in response to the virus, provided that they were cultivated in the presence of submitogenic doses of phytohemagglutinin. In contrast, the prothymocytes did not proliferate. Downmodulation of the CD2 molecule by incubating unseparated and immature thymocytes with a single anti-CD2 monoclonal antibody inhibited the proliferative response to HTLV-I. These results clearly underline that the expression of the CD2 molecule is exclusively required in mediating the proliferative response to the synergistic effect of phytohemagglutinin and HTLV-I. Immature thymocytes treated with a pair of anti-CD2 monoclonal antibodies were shown to proliferate in response to HTLV-I, even in the absence of exogenous IL-2. We further verified that the proliferation of human thymocytes is consecutive to the expression of IL-2 receptors and the synthesis of IL-2. These observations provide evidence that the mitogenic stimulus delivered by HTLV-I is more efficient than that provided by other conventional mitogenic stimuli, which are unable to trigger the synthesis of endogenous IL-2. Collectively, these results show that the mitogenic activity of HTLV-I is able to trigger the proliferation of cells which are at an early stage of T-cell development. They might therefore represent target cells in which HTLV-I infection could favor the initiation of the multistep lymphoproliferative process leading to adult T-cell leukemia.     

Modeling the T-cell dynamics and pathogenesis of HTLV-I infection

Human T-cell lymphotropic virus type I (HTLV-I) infection in humans causes a chronic infection of CD4⁺ T cells, and is associated with various disease outcomes, among them with the development of adult T-cell leukemia (ATL). The T-cell dynamics after HTLV-I infection can be described in a mathematical model with coupled differential equations. The infection process is modeled assuming cell-to-cell infection of CD4⁺ T cells. The model allows for CD4⁺ T cell subsets of susceptible, latently infected and actively infected cells as well as for leukemia cells. Latently infected T cells may harbor the virus for several years until they become activated and able to infect susceptible T cells. Uncontrolled proliferation of CD4⁺ T cells with monoclonal DNA-integration of HTLV-I results in the development of ATL. The model describes basic features that characterize HTLV-I infection; the chronic infection of CD4⁺ T cells, the increasing number of abnormal cells and the possible progression to ATL.     

Prostaglandin E2 inhibits human T-cell proliferation after crosslinking of the CD3-Ti complex by directly affecting T cells at an early step of the activation process

We have studied the effects of prostaglandin E2 (PGE2) on in vitro human T-cell activation induced by crosslinking of the CD3-Ti complex with the monoclonal anti-CD3 antibodies OKT3 and UCHT-1. PGE2 (greater than or equal to 3 X 10(-9) M) when added simultaneously with anti-CD3 to cultures of peripheral blood mononuclear cells (PBMC), significantly suppressed, in a dose-dependent way, T-cell proliferation (P less than 0.002). However, when T cells were first preactivated with OKT3 for 3 days, subsequent proliferation driven by recombinant interleukin 2 (IL-2) was not inhibited by addition of PGE2. This indicates that PGE2 affects the activation step resulting from crosslinking of CD3-Ti, but not the IL-2-driven proliferative phase. Other manifestations of T-cell activation were therefore examined. Both IL-2 production and the expression of receptors for IL-2 (as detected with the anti-Tac monoclonal antibody) were inhibited by PGE2. The addition of purified interleukin 1 (IL-1) or recombinant IL-2 to the cultures did not reverse the inhibiting effect of PGE2 on IL-2-receptor expression. PGE2, added at the time of culture initiation, also inhibited T-cell proliferation in cultures which were supplemented with exogenous IL-1 or IL-2. Proof for a direct effect of PGE2 on T cells was obtained in experiments in which monocyte-depleted T cells were stimulated, in the presence of IL-1, with solid-phase-bound anti-CD3 antibody. Proliferation of T cells in this system is accessory cell independent and still was strongly inhibited by PGE2. Finally, preincubation of PBMC with PGE2 (3 X 10(-6) M) for 48 hr did not result in the generation of suppressor cells for anti-CD3-induced T-cell proliferation or for IL-2 production. Our results demonstrate that PGE2 has a direct inhibitory effect on an early step of T-cell activation, resulting in decreased IL-2 production, decreased IL-2-receptor expression, decreased responsiveness to exogenous IL-2, and decreased proliferation. However, PGE2 does not affect IL-2-driven proliferation of activated T cells. The inhibitory effect on T-cell activation is not mediated through suppressor T cells, nor through inhibition of accessory cell function.     

Expression of HTLV-I in serum of HTLV-I-related subjects and the early detection of overt ATL in HTLV-I carriers

Monoclonal antibodies against human T cell leukemia virus type I (HTLV-I) p24 and p19 were produced and employed in the in vivo expression of HTLV-I in some HTLV-I-related subjects by Western blot analysis. The antigenic determinants of these monoclonal antibodies were different from that of natural human anti-HTLV-I antibody examined. Serum p24 was identifiable in almost all of the overt ATL patients (17 of 18, 94%), but not in the chronic ATL cases or HTLV-I carriers. On the other hand, serum p19 was detected in one of the three patients with chronic ATL examined, but not in the overt ATL patients or the HTLV-I carriers. These results indicate that the in vivo expression of HTLV-I p24 and p19 which has reacted with natural anti-HTLV-I antibody can be detected by these monoclonal antibodies. Therefore, the investigation of serum HTLV-I expression may lead to the early detection of overt ATL in an HTLV-I carrier.     

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and Lyt 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).     

A functional analysis of the T-cell defect in MRL-lpr mice

Autoimmune MRL-lpr mice have a defect in antigen-specific T-cell proliferation. Our studies indicate that this defect is caused by the massive expansion in MRL-lpr mice of a unique T-cell subset which is unresponsive to antigenic signals. Pharmacologic doses of PGE1 suppress this lymphoid hyperplasia and thus prevent loss of T-cell functions by preventing numeric dilution of normal T cells by defective T cells. The inability of the unique subset of T cells to respond to antigenic signals cannot be corrected by the addition of interleukin 2 (IL-2), implying that additional cellular properties are required to initiate proliferation. While the vast majority of freshly harvested MRL-lpr T cells lack IL-2 receptors (R) as measured by anti-IL-2R monoclonal antibody staining, a large fraction of nonstimulated, cultured (48 hr) MRL-lpr T cells, but not MRL-++ T cells, express IL-2R. These experiments suggest that MRL-lpr cells are activated in vivo but an undefined suppressive influence prevents detection or expression of IL-2R until these cells are explanted and cultured.     

Heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human T-cell lymphotropic virus type I.

Heat shock cognate protein 70 (HSC70) has been shown to bind to the peptide corresponding to amino acids 197 to 216 of human T-cell lymphotropic virus type I (HTLV-I) envelope protein, gp46, and an anti-HSC70 monoclonal antibody (mAb) inhibits HTLV-I-induced syncytium formation. These findings suggest that HSC70 is necessary for the entry of HTLV-I into its target cells. Here we showed that HSC70 directly binds to gp46 by co-immunoprecipitation of HSC70 and gp46 from HTLV-I-producing human T-cell lysate. However, transduction of human HSC70 cDNA into BaF3 cells, which were found to be highly resistant to HTLV-I infection, did not support the HTLV-I entry, and HSC70 expressed in NIH3T3 cells, which were found to be almost resistant to syncytium formation upon cocultivation with HTLV-I-producing cells but sensitive to infection with cell-free HTLV-I, enhanced cell fusion induced by HTLV-I-producing cells, but did not enhance the entry of cell-free HTLV-I into these cells. The mAb against HSC70 inhibited syncytium formation in NIH3T3 cells expressing HSC70, but showed little effect on infection of these cells with cell-free HTLV-I. These findings indicate that HSC70 markedly enhances syncytium formation induced by HTLV-I but does not facilitate HTLV-I entry into target cells.     

IL-2 receptor(p55)/Tac-inducing factor. Purification and characterization of adult T cell leukemia-derived factor

Many human T cell lymphotropic virus-I (HTLV-I) transformed T cells from adult T cell leukemia (ATL) patients continuously produce a humoral factor called ATL-derived factor (ADF) which induces IL-2R/Tac expression on T and NK cells. Using gel filtration, procion red Sepharose, DEAE, and reverse phase chromatography, we have purified ADF protein to homogeneity from 15 liters of serum-free culture supernatant of an HTLV-I(+) T cell line ATL-2. Purified ADF protein had the m.w. of 14,000 by SDS-PAGE and gel filtration, and its isoelectric point is around 5.0. ADF did not react with heteroantibodies against IL-1 alpha and IL-1 beta, which have also IL-2R/Tac-inducing activity on suitable target cells. Partial N-terminal amino acid sequence of ADF is different from other cytokines such as, IFN, BSF-2, and various IL whose cDNA has been cloned. Western blot analysis using rabbit antibodies against N-terminal 10mer synthetic peptide of ADF showed that IL-1 alpha and ADF are different proteins. ADF had its IL-2R/Tac-inducing activity not only on human NK-like cell line YT, but also on HTLV-I(+) T cells, such as ED. In contrast, macrophage-derived IL-1 lacked IL-2R/Tac-inducing activity on ED cells despite their IL-2R/Tac induction on YT, indicating that ADF and IL-1 have their effect via different receptors.     

Interleukin 1 alpha mRNA in virus-transformed T and B cells

IL-1 alpha cDNA clone was isolated from a T cell line infected by the human T lymphotropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-1 alpha cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-1 alpha.     

Bacterial lipopolysaccharide activates NF-kappaB through toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells. Differential expression of TLR-4 and TLR-2 in endothelial cells

A missense mutation in the cytoplasmic domain of the Toll-like receptor-4 (TLR-4) has been identified as the defect responsible for lipopolysaccharide (LPS) hyporesponsiveness in C3H/HeJ mice. TLR-4 and TLR-2 have recently been implicated in LPS signaling in studies where these receptors were overexpressed in LPS non-responsive 293 human embryonic kidney cells. However, the signaling role of TLR-4 or TLR-2 in human cells with natural LPS response remains largely undefined. Here we show that human dermal microvessel endothelial cells (HMEC) and human umbilical vein endothelial cells express predominantly TLR-4 but very weak TLR-2 and respond vigorously to LPS but not to Mycobacterium tuberculosis 19-kDa lipoprotein. Transient transfection of non-signaling mutant forms of TLR-4 and anti-TLR-4 monoclonal antibody inhibited LPS-induced NF-kappaB activation in HMEC, while a monoclonal antibody against TLR-2 was ineffective. In contrast to LPS responsiveness, the ability of HMEC to respond to 19-kDa lipoprotein correlated with the expression of TLR-2. Transfection of TLR-2 into HMEC conferred responsiveness to 19-kDa lipoprotein. These data indicate that TLR-4 is the LPS signaling receptor in HMEC and that human endothelial cells (EC) express predominantly TLR-4 and weak TLR-2, which may explain why they do not respond to 19-kDa lipoprotein. The differential expression of TLRs on human EC may have important implications in the participation of vascular EC in innate immune defense mechanisms against various infectious pathogens, which may use different TLRs to signal.     

Immunological risks of adult T-cell leukemia at primary HTLV-I infection

A small percentage of human T-cell leukemia virus type-I (HTLV-I)-infected individuals develop adult T-cell leukemia (ATL). In animal experiments, inoculation of HTLV-I via the oral route, which is the main route of mother-to-child viral transmission in humans as a result of breastfeeding, induced host HTLV-I-specific T-cell unresponsiveness and resulted in increased viral load. This strongly suggested that the known epidemiological risk factors for ATL (i.e. vertical HTLV-I infection and elevated viral load) are linked by an insufficient HTLV-I-specific T-cell response. Recent findings on the anti-tumor effects of Tax-targeted vaccination in rats and the reactivation of Tax-specific T cells in ATL patients as a result of hematopoietic stem cell transplantation imply promising immunological approaches for the prophylaxis and therapy of ATL.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes

Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function.     

Identification of a receptor-binding domain of the spike glycoprotein of human coronavirus HCoV-229E

Human coronavirus HCoV-229E uses human aminopeptidase N (hAPN) as its receptor (C. L. Yeager et al., Nature 357:420-422, 1992). To identify the receptor-binding domain of the viral spike glycoprotein (S), we expressed soluble truncated histidine-tagged S glycoproteins by using baculovirus expression vectors. Truncated S proteins purified by nickel affinity chromatography were shown to be glycosylated and to react with polyclonal anti-HCoV-229E antibodies and monoclonal antibodies to the viral S protein. A truncated protein (S(547)) that contains the N-terminal 547 amino acids bound to 3T3 mouse cells that express hAPN but not to mouse 3T3 cells transfected with empty vector. Binding of S(547) to hAPN was blocked by an anti-hAPN monoclonal antibody that inhibits binding of virus to hAPN and blocks virus infection of human cells and was also blocked by polyclonal anti-HCoV-229E antibody. S proteins that contain the N-terminal 268 or 417 amino acids did not bind to hAPN-3T3 cells. Antibody to the region from amino acid 417 to the C terminus of S blocked binding of S(547) to hAPN-3T3 cells. Thus, the data suggest that the domain of the spike protein between amino acids 417 and 547 is required for the binding of HCoV-229E to its hAPN receptor.     

Identification of a putative receptor for subgroup A feline leukemia virus on feline T cells.

Retrovirus infection is initiated by the binding of virus envelope glycoprotein to a receptor molecule present on cell membranes. To characterize a receptor for feline leukemia virus (FeLV), we extensively purified the viral envelope glycoprotein, gp70, from culture supernatants of FeLV-61E (subgroup A)-infected cells by immunoaffinity chromatography. Binding of purified 125I-labeled gp70 to the feline T-cell line 3201 was specific and saturable, and Scatchard analysis revealed a single class of receptor binding sites with an average number of 1.6 x 10(5) receptors per cell and an apparent affinity constant (Ka) of 1.15 x 10(9) M-1. Cross-linking experiments identified a putative gp70-receptor complex of 135 to 140 kDa. Similarly, coprecipitation of 125I-labeled cell surface proteins with purified gp70 and a neutralizing but noninterfering anti-gp70 monoclonal antibody revealed a single cell surface protein of approximately 70 kDa. These results indicate that FeLV-A binds to feline T cells via a 70-kDa cell surface protein, its presumptive receptor.