INTRODUCTION

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The cellular immune response constitutes the specific host defense toward an established viral infection. Unlike the humoral immune response, which may neutralize and prevent the infection, the cellular immune response attempts to eliminate virus-infected cells. Typically, this is executed by cytotoxic CD8⁺ T lymphocytes (CTLs) that recognize viral peptides on the surface of the infected cells in the context of major histocompatibility complex (MHC) class I antigens. An unusual virus-host relationship occurs, however, when the virus persistently infects cells regulating the immune response, as exemplified by certain human herpesviruses and retroviruses.

Human T-cell lymphotropic virus type I (HTLV-I) is a retrovirus that resides in and functionally alters immune cells of central importance for immunoregulation (Fig. 1). First, HTLV-I infects activated T cells and incorporates into their genome, where it persists; second, HTLV-I regulatory proteins alter activation and cell death pathways in the host T cell; third, HTLV-I-infected T cells may activate resting T cells, facilitating propagation of the infection; and finally, HTLV-I infection induces a strong antiviral immune response, which nonetheless appears incapable of eradicating the infection.

View larger version (53K): [in this window] [in a new window]   FIG. 1.   Activation of T cells by HTLV-I. Infection of CD4+ T cells influences immune system T-cell activation by at least four separate pathways. (i) The HTLV-I-infected T cells are activated by viral interference with signaling pathways and transcriptional regulation (bottom right). (ii) The HTLV-I-infected T cell interacts with and activates resting T cells (top right, activation of uninfected T cells) in a viral antigen-independent manner. The CD58-CD2 interaction (shown) is critical, but other molecular interactions and cytokines (not shown) are likely to contribute. (iii) Virus-specific CD8+ T cells (and, to a lesser degree, CD4+ T cells [not shown]) are activated by recognition of viral peptide epitopes (bottom left, antigen-specific activation of CD8+ T cells). (iv) APC may present MHC class II-restricted peptide antigens that activate the HTLV-I-infected T cell (top left, antigen-specific activation of HTLV-I-infected T cells). This activation process is altered by virtue of viral interference with the signaling cascade or the transcriptional regulation of the HTLV-I-infected T cell, or both.

In a small percentage of infected individuals, HTLV-I causes disease (121), most often either adult T-cell leukemia/lymphoma (ATL) or a chronic inflammatory disease of the central nervous system (HTLV-I-associated myelopathy/tropical spastic paraparesis, HAM/TSP). Less frequently, the joints (HTLV-I arthropathy), the eyes (HTLV-I uveitis), the skin (infective dermatitis in children), the muscles (polymyositis), or the lungs (pulmonary infiltrative pneumonitis) are affected (90). While the pathogeneses of these diseases are unknown, they all appear to involve activated, HTLV-I-infected CD4⁺ T cells.

In this review the interaction between HTLV-I and the cellular immune system is analyzed, with special emphasis on the multiple ways in which HTLV-I maintains an active immune system that favors viral dissemination.

HTLV-I particles form by budding through the host cell membrane, thereby incorporating cell membrane molecules into the viral envelope. Free HTLV-I particles have extremely low infectivity (314), and transmission of HTLV-I usually requires virus-producing T cells, which allow cell-to-cell contact. The presence of 3'-azido-3'-deoxythymidine at the time of infection appears to have a protective effect on uninfected peripheral blood mononuclear cells (192). Although the receptor for HTLV-I is unknown, a putative receptor or cofactor for HTLV-I entry is thought to be encoded by a gene on chromosome 17 (273). Indirect evidence for this comes from studies with mouse-human somatic cell hybrids infected by a vesicular stomatitis virus (VSV)/HTLV-I pseudotype virus. This chimeric virus is made up of the HTLV-I envelope and the VSV core particle and therefore displays tropism identical to HTLV-I but cytopathic effects like those of VSV. Whereas mouse cells are much more resistant to HTLV-I infection than are human T cells, mouse-human somatic hybrid cells containing a region of the long arm of human chromosome 17 displayed increased susceptibility to infection by the VSV/HTLV-I pseudotype virus (273). The region on chromosome 17 has been mapped to 17q21-q23 (282), although the gene encoding the cofactor or receptor for HTLV-I entry is still unknown.

The core particle of HTLV-I carries two copies of genomic RNA as well as viral enzymes (reverse transcriptase, protease, RNase H, and integrase), which are essential for establishing the viral infection. Upon viral entry into the T cell, RNA is reverse transcribed into DNA and integrates in the host cell genome as a provirus. Although insertion of HTLV-I into the host cell DNA may have a slight preference for G+C-rich regions (325), HTLV-I does not incorporate at specific sites in the genome (260). The integrated HTLV-I provirus consists of 9,032 bp (261) and is organized in 5' and 3' long terminal repeats (LTR), a gag region encoding the structural proteins, a pol region encoding the reverse transcriptase, an env region encoding the envelope proteins, and a region at the 3' end of the provirus known as pX, encoding regulatory proteins (reviewed in reference 74), which are responsible for the altered host cell functions (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   HTLV-I genomic organization and encoded proteins. The approximate sizes of HTLV-I proviral genes are shown for mRNAs encoding the structural Gag, protease (pr), reverse transcriptase (RT), envelope (Env), and regulatory proteins (open boxes). The protease is encoded by the 3' end of gag mRNA in a different reading frame from the Gag proteins extending into the 5' end of pol. The RNase H and integrase (not shown) are encoded by pol. The pX gene has four ORFs. ORF-I may encode p12I; ORF-II may encode p13II and p30II; ORF-III encodes p21rex and p27rex; and ORF-IV encodes p40tax. The LTR R region begins at the initiation of transcription site.

In vivo, the vast majority of HTLV-I provirus is found in CD4⁺ CD45RO⁺ T cells (240, 246) although CD8⁺ T cells can also be infected (105, 246, 309). Infection of dendritic cells has been demonstrated (191), but its importance in propagating the viral infection has been difficult to evaluate because of the complicated technical procedures involved in obtaining uncultured dendritic cells. Likewise, it has been reported that glial cells can be productively infected in vivo (173). Although this is a potentially important observation, its significance is not clear (215). HTLV-I transcription is higher in primary CD4⁺ T cells than in CD8⁺ T cells, which may explain why HTLV-I-induced leukemia and lymphoma are of the CD4⁺ phenotype (222). It is not known, however, what restricts the viral tropism to predominantly CD4⁺ T cells, since a broad range of cell types can be infected in vitro. These cell types include B cells (61), monocytes/macrophages (58, 116, 162), NK cells (187), glial cells (116, 303), endothelial cells (115, 126), promyelocytic HL-60 cells (114), and a human osteosarcoma cell line (46). Moreover, coinfection with HTLV-I and human immunodeficiency virus (HIV) broadens the spectrum of HIV cellular tropism to include CD8⁺ T cells, B cells, epithelial cells, and skeletal muscle cells (190).

A number of reports have described antibodies that interfere with HTLV-I syncytium formation and infection. An antibody known as 34-23 recognizes proteins of 31, 45, 55, and 70 kDa and shows increased binding to mouse-human hybrid cells containing human chromosome 17 (86). Inhibition of HTLV-I syncytium formation and infection was also achieved by an antibody to an 80-kDa glycoprotein (2). However, it is important to bear in mind that antibodies to adhesion molecules may inhibit HTLV-I infection because of interference with cell-cell contact. Recently, an antibody to vascular cell adhesion molecule 1 (VCAM-1) has been shown to prevent HTLV-I syncytium formation, although antibodies to its ligand, very late antigen 4 (VLA-4), did not (111). Moreover, cell-to-cell fusion is not sufficient to ensure viral entry (250). By examining the infectivity of HTLV-I with point mutations in the envelope glycoprotein, Rosenberg et al. (250) defined fusion-competent mutants with severe defects in infectivity. This suggests that the viral envelope glycoprotein may be involved in postfusion events required for full infectivity of HTLV-I.

Incorporation of HTLV-I into the CD4⁺-T-cell genome may result in either a silent or a productive infection. A silent infection is defined by the presence of HTLV-I sequences in the host cell genome in the absence of detectable HTLV-I-encoded mRNA. Thus, if the virus does not insert into critical genes, a nonproductively infected T cell is functionally indistinguishable from an uninfected T cell. Alternatively, CD4⁺ T cells may be productively infected by HTLV-I, resulting in viral mRNA transcription and the production of viral particles. Nevertheless, most infected T-cell clones contain a single integrated provirus, indicating that they do not reinfect themselves (247).

Single-cell cloning under limiting-dilution conditions of T cells from HAM/TSP patients indicated a frequency of HTLV-I-infected T cells between 15 and 18%, as determined by PCR amplification of pol or LTR viral sequences from genomic DNA (124, 247, 309). Unless the single-cell cloning is performed with allogeneic, uninfected feeder cells, the frequency is overestimated because of in vitro infection of the T cells (247, 309). The frequency estimate by single-cell cloning is in accordance with independent estimates by limiting-dilution PCR analysis, as well as by Southern blot analysis of genomic DNA from peripheral blood T cells (246). Since most infected T-cell clones contain a single integrated provirus (247), these analyses indicate that HAM/TSP patients have between 3 and 30% (typically 10%) HTLV-I-infected leukocytes. The majority of HTLV-I-infected T cells are silently infected (124, 246, 247, 309), and very few cells (1 in 5,000) express high levels of HTLV-I in vivo (91). It is not clear whether silently infected T cells may later reactivate viral transcription in vivo.

Activation of the host T cell by HTLV-I occurs through several independent mechanisms, the most intensively studied of which is mediated through activation of cellular transcription factors by the viral trans-activator Tax. Activation of transcription factors may be viewed as the "end" signal of a transduction cascade from the membrane to the nucleus during activation, although a pathway may activate multiple transcription factors and, conversely, a transcription factor may be activated by multiple pathways. Molecular aspects of transcriptional activation by Tax have been reviewed recently (27) and are only summarized here in the context of a signaling pathway activated by HTLV-I.

Besides activation of transcription factors, HTLV-I alters signaling pathways. Typically, T-cell activation requires two signals: an antigen-specific signal mediated via the T-cell receptor (TCR) and a non-antigen-specific costimulatory signal. These signals initiate transcriptional activation of a number of genes and drive the T cell into the mid- to late G₁ phase of the cell cycle, the completion of which requires cytokine signaling. HTLV-I regulatory proteins interfere with the control of each of these steps during T-cell activation.

Although infection by HTLV-I may lead to organ-specific inflammatory diseases, the mechanisms that target tissue destruction to the central nervous system, the joints, the eyes, the muscles, etc., are unknown (118). It is conceivable, though, that autoreactive T cells are randomly infected and cause organ-specific disease by virtue of their chronic activation and altered requirements for antigen-specific triggering (118). This hypothesis is difficult to test because of the inherent problems of generating antigen-specific T-cell clones from HTLV-I-infected individuals. That is, since mononuclear cells from these patients undergo spontaneous proliferation following 3 to 9 days in culture (133, 139), it is virtually impossible to determine antigen-specific responses, because the "background" of spontaneous proliferation often amounts to more than that of an antigen-specific response.

Recent advances in generating MHC-peptide complexes and peptide-loaded soluble MHC class I-immunoglobulin complexes make it feasible to directly isolate antigen-specific T cells (11, 101). This approach may clarify the possible role of antigen-specific T cells in HTLV-I-induced diseases. So far, however, the only way to analyze the impact of HTLV-I infection on antigen-specific T-cell responses relies on in vitro infection of established antigen-specific T-cell clones. Mitsuya et al. (206) examined the functional properties of tetanus toxoid-specific T cells infected by HTLV-I. The HTLV-I-infected T-cell clones proliferated in response to soluble tetanus toxoid, but, unlike uninfected T-cell clones, they could do so in the absence of accessory cells. This may be explained by upregulation of MHC class II on HTLV-I-infected T cells (276) followed by T-cell presentation of antigen. Thus, Scholz et al. (256) found that an HTLV-I-infected T-cell clone specific for a myelin basic protein peptide responded to an approximately 100-fold-lower concentration of soluble peptide antigen than did the parental uninfected T-cell clone. The mechanism of the enhanced response involved upregulation of MHC class II and lymphocyte function-associated antigen 3 (LFA-3; CD58) on the infected T cells, which allowed them to present the peptide antigen to other T cells. Nevertheless, compared to uninfected T cells, the response of HTLV-I-infected T cells to antigenic peptide presented by Epstein-Barr virus (EBV)-transformed B cells was slightly impaired. This demonstrated that the responsiveness of the HTLV-I-infected T cells was not enhanced; rather, the HTLV-I-infected T cells were better antigen-presenting cells (APCs).

Popovic et al. (239) examined the consequences of infecting a keyhole limpet hemocyanin (KLH)-specific CD4⁺ T-helper cell (SR2) with an HTLV-I-infected isolate (TK). SR2 cells proliferated and provided "help" to B lymphocytes in the presence of KLH presentation in the context of the appropriate MHC class II. However, following HTLV-I infection, TK-infected SR2 cells displayed spontaneous proliferation in the absence of antigenic peptide. Importantly, the TK-infected SR2 cells gained the ability to provide promiscuous antigen-independent help to B cells, resulting in polyclonal immunoglobulin production. The mechanism of the promiscuous B-cell help was not examined, but interleukin-4 (IL-4), IL-5, and gamma interferon (IFN-) are known to enhance immunoglobulin secretion, and these cytokines were spontaneously secreted by a myelin basic protein-specific HTLV-I-infected T-cell clone (255). Nevertheless, Yarchoan et al. (318) found that supernatant from an infected T-cell clone, 8.8H, which provided promiscuous antigen-independent B-cell help, did not provide help for immunoglobulin production. Although this may not entirely rule out cytokines, it suggests that cognate T-cell-B-cell interaction is required for the promiscuous B-cell help provided by HTLV-I-infected T cells.

In contrast, loss of function was demonstrated in two alloreactive cytotoxic CD4⁺-T-cell clones. Following infection, the number of HTLV-I p19-expressing T cells increased concomitantly with a loss of cytotoxicity (239). Although it was not shown that the HTLV-I-infected T cells were of the same origin as the parental cytotoxic T-cell clone, the observation suggested that HTLV-I infection interfered with the cytotoxic effector mechanism. Subsequent studies confirmed the loss of cytotoxicity in antigen-specific HTLV-I-infected T cells (131, 277, 318, 322) and additionally provided evidence for identical -chain rearrangement of the TCR in the infected T-cell clones with impaired cytotoxicity and their parental uninfected T-cell clones, indicating that they were of the same origin (131, 277, 322).

During the early phase after HTLV-I infection, the expression of CD2, CD3, CD4, CD26, and CD28 remains normal whereas the expression of the IL-2 receptor  (IL-2R) chain and human leukocyte antigen (HLA)-DR is upregulated (276, 322). Following this stage, the HTLV-I-infected T cells may become IL-2 independent (i.e., transformed). This is usually accompanied by downregulation of CD3 expression and loss of antigen responsiveness (131, 322). Nevertheless, the loss of cytotoxic activity may be an effect on the lytic machinery, since HTLV-I-infected T cells had lost serine esterase activity (322) and since the loss of cytotoxic function occurred with normal levels of CD3 expressed on the cell surface (131).

In summary, complex alterations may influence the antigen response of HTLV-I-infected T cells and lead to both gain of function and loss of function: CD4⁺ T-helper cells may gain APC-like functions and the ability to provide indiscriminate B-cell help, whereas cytotoxic CD4⁺ T cells may lose their cytotoxic effector function (Table 1).

Recently, Mahana et al. (194) demonstrated that the phosphorylation state of the protein Vav can be influenced by proteins from the pX region of HTLV-I. Using molecular clones, they were able to associate the ability of an infected T-cell clone to induce asymptomatic infection with a downregulation of Vav phosphorylation. In contrast, a T-cell clone which induced lethal leukemia differed in two nucleotides in the pX region and displayed constitutive tyrosine phosphorylation of Vav. Since tyrosine-phosphorylated Vav is involved in the signal transduction from the TCR, this suggests the possibilitycontrary to the general assumptionthat minor differences in the HTLV-I sequence may be important in the pathogenesis.

PKA signaling pathway. The second-messenger cyclic AMP (cAMP) influences T-cell signaling via a cAMP-dependent protein kinase (PKA). PKA is composed of two catalytic (C) subunits and two regulatory (R) subunits, which exist in two isoforms, giving rise to type I and type II PKA. Each regulatory subunit can bind cAMP at two distinct binding sites, which dissociates the PKA complex into R₂(cAMP)₄ and two catalytically active C subunits. Since type I PKA is dissociated more easily than type II PKA and since the localization of the isotypes may differ (for example, type I PKA colocalizes with the TCR, in contrast to type II PKA), differential activation of the two types of PKA may shape the response of a given cell to a variety of stimuli. Thus, it has been suggested that type I PKA is involved in the response to proliferative signals whereas type II PKA is involved in cell differentiation and the response to antiproliferative signals (44).

PKC signaling pathway. Activation of T cells through the TCR but not through the IL-2R (295) results in protein kinase C (PKC) activation (reviewed in reference 281). The family of PKC isoenzymes includes at least 12 members, some of which are not Ca²⁺ dependent. PKC isoenzymes are usually divided into three groups based on their primary structure and their activation requirements: (i) Ca²⁺-dependent or conventional PKCs (PKCs) include PKC-, PKC-1, PKC-2, and PKC-; (ii) Ca²⁺-independent or novel PKCs (PKC) include PKC-, PKC-, PKC-, PKC-, and PKC-µ; and (iii) atypical PKCs (PKCs), which do not respond to phorbol esters, include PKC-, PKC-, and PKC- (32, 281).

Ca²⁺ signaling pathway. Activation of NF-B/Rel or CREB/ATF is not sufficient for Tax-mediated activation of the CD28 enhancer of the IL-2 gene. LiFeng et al. (181) found that nuclear factor of activated T cells (NF-AT) complexes induced by Tax bound to the CD28 response element in the IL-2 promoter, implicating NF-AT in Tax-mediated transactivation. In contrast to the cooperation between NF-AT and the transcription factors c-Fos and c-Jun (AP1) (242), the Tax-induced NF-AT complex does not contain c-Fos or c-Jun (181). Moreover, constitutive dephosphorylation and activation of NF-ATp, a member of the NF-AT family, was found in Tax-expressing and HTLV-I-infected T-cell lines (180). The constitutive dephosphorylation of NF-ATp was reversed in the presence of cyclosporin A (CsA), an inhibitor of the calcium/calmodulin-dependent phosphatase calcineurin. This suggests that Tax activates the Ca²⁺ signaling pathway proximal to or at the level of calcineurin. Interestingly, activation of the Ca²⁺ signaling pathway downregulates IL-10 production. In particular, the combination of Ca²⁺ ionophores and phorbol esters results in poor IL-10 induction but significant IFN- production (321). Indeed, HTLV-I infection of an IL-10-producing T-cell clone resulted in a loss of its ability to secrete IL-10 but in acquisition of the ability to constitutively secrete IFN- (256). In contrast, transfection of Jurkat T cells with a Tax expression plasmid induced IL-10 mRNA expression and IL-10 secretion (213), and this was partially inhibited by antisense oligonucleotides to the p65 subunit of NF-B. The reason for this discrepancy in IL-10 secretion between Tax-transfected Jurkat T cells and HTLV-I-infected T-cell clones is unclear, but a similar discrepancy in IL-2 secretion can be found between these cells (124, 199), suggesting that the level of expression of Tax or of other viral or cellular proteins may explain the difference.

MAP kinase pathways. At least three pathways have been delineated via the small GTPases Ras, Rac, CDC42, and Rho. Ras activates extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2) via Raf and mitogen-activated protein (MAP) kinase/ERK kinase 1 and 2 (MEK-1 and MEK-2); Rac and CDC42 activate c-Jun N-terminal kinase (JNK) via p21-activated kinase (PAK), MEK kinase (MEKK), and JNK kinase; and Rho activates p38 via a less well characterized pathway. However, cross talk between the pathways exists; Ras may activate JNK, and CDC42 and Rac may activate p38 (reviewed in reference 182).

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Role of different signal transduction pathways in HTLV-I infection. HTLV-I-encoded proteins (predominantly, if not exclusively, Tax) may interfere with multiple intracellular signal transduction pathways. HTLV-I-encoded proteins are boxed. The bold arrow indicates the target of the viral protein, and a suggested target is indicated with a question mark. Inhibitory agents are indicated in italic. The candidate viral gene product, p12I, is shown as a possible viral mechanism of IL-2R pathway activation. A protein encoded by pX but separate from Tax may interfere with Vav. See the text for details and abbreviations.

CD28 costimulation. A number of molecules expressed on T cells may enhance or costimulate T-cell activation; however, special emphasis has been placed on the CD28 molecule, since mice deficient in the CD28 gene have significantly impaired T-cell activation (98). This indicates that other costimulatory pathways cannot completely compensate for the loss of CD28 signaling (98). The salient functions of the CD28 costimulatory pathway are to enhance IL-2 transcription, stabilize IL-2 mRNA, and promote T-cell survival by upregulating the antiapoptotic protein Bcl-x_L (274).

CD2 costimulation. The CD58-CD2 interaction is important for activation of resting and uninfected T cells by HTLV-I-infected T cells (152, 153, 309), as discussed later in this review. However, the CD2 pathway is not critical for HTLV-I-induced activation of infected T-cell clones, since FK506 and CsA inhibit the CD2 signaling pathway (22) but not the HTLV-I-induced activation of the host T cell (124).

OX40 costimulation. A contribution from other costimulatory pathways to HTLV-I-induced T-cell activation cannot be excluded. The interaction between OX40, a TNF/nerve growth factor receptor family member, and its ligand, gp34 (OX40L), is costimulatory for T cells in the presence of mitogens (18, 94). OX40L was initially detected on HTLV-I-infected T cells as a 34-kDa glycoprotein transactivated by Tax (207, 286, 291). OX40 is induced on activated T cells and constitutively expressed on HTLV-I-transformed T cells (130); nevertheless, the significance of the OX40-OX40L interaction for HTLV-I-induced T-cell activation remains to be determined. Since OX40 mediates adhesion to OX40L expressed on vascular endothelial cells (129, 130), it is possible that this interaction is important for HTLV-I-mediated inflammatory diseases.

In normal T cells, the cytokine IL-2 induces the G₁-to-S phase transition (36). Since this is essential for T-cell cycling, there has been interest in the possibility that HTLV-I-infected T cells use an IL-2 autocrine mechanism to traverse the G₁ restriction point. The high-affinity IL-2R complex is composed of three subunits: the , _c, and _c chains; the subscript c indicates that these chains are shared (common) among several cytokine receptors: _c is used by IL-2R and IL-15R; _c is used by IL-2R, IL-4R, IL-7R, IL-9R, and IL-15R (reviewed in reference 287). The signaling module of the IL-2R comprises _cc, which itself is an intermediate-affinity IL-2R. The IL-2R chain does not participate in signal transduction, but its association with _cc increases the receptor affinity for IL-2 by approximately 100-fold (287).

The possibility that HTLV-I particles or surface proteins can activate the IL-2R pathway was initially suggested based on an association between HTLV-I virions and the IL-2R chain (170); furthermore, it was shown that the HTLV-I envelope glycoprotein contains a region homologous to a segment of IL-2 that binds _c (160). Whether these features of the HTLV-I virion are important for activation of the IL-2R signaling pathway remains to be demonstrated.

The IL-2R chains are absent or expressed at very low levels in resting T cells, but their expression is inducible upon T-cell activation (53). IL-2R chains are expressed in large numbers on HTLV-I-transformed T cells from patients with ATL (107). The mechanism involves Tax transactivation of the promoter for the IL-2R chain (50, 132, 197, 268) and is mediated by activation of NF-B (15, 176, 252). In addition, transient-transfection studies linking the promoter of IL-2 to a chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that Tax may also transactivate the IL-2 promoter (132, 197, 199, 268). Although the Tax-mediated transactivation of the IL-2 promoter is not very strong, it may synergize with a TCR- or phorbol ester-mediated signal or with the HTLV-I regulatory protein Rex (197, 199).

Nonetheless, analysis of IL-2 secretion and IL-2 mRNA in HTLV-I-infected T-cell lines or clones has not implicated IL-2 autocrine growth in HTLV-I-induced T-cell activation. Arya et al. (14) did not detect IL-2 mRNA expression in HTLV-I-transformed T cells (HuT-102) by Northern blot hybridization of cloned IL-2 DNA to poly(A) isolated RNA. Likewise, Northern blot analysis of HTLV-I-infected T-cell clones at a time when they displayed spontaneous clonal proliferation did not detect IL-2 mRNA (124). Moreover, the presence of a blocking antibody to the IL-2R chain (anti-Tac) did not prevent the HTLV-I-induced proliferation (124). The transcription factor NF-AT is important for the initiation of IL-2 gene transcription, and CsA and FK506 inhibit IL-2 production by preventing the dephosphorylation and nuclear translocation of NF-AT. CsA or FK506 did not inhibit the spontaneous clonal proliferation of HTLV-I-infected T-cell clones, although they did inhibit TCR-CD3-mediated superimposed proliferation of these clones (124). CD28-induced signals may, however, activate NF-AT and lead to IL-2 secretion in a CsA-resistant manner (92), and CsA may not inhibit Tax-induced transactivation of the IL-2 gene (268).

Taken together, however, the data on HTLV-I-infected T-cell clones suggest that autocrine IL-2 secretion is not involved in HTLV-I-induced spontaneous clonal proliferation. In addition, Akagi and Shimotohno (8) found IL-2-independent proliferation of Tax-transduced T cells after CD3 cross-linking.

To investigate IL-2 mRNA expression in single cells, Goebels et al. (95) examined three HTLV-I-transformed T-cell lines by in situ hybridization with an IL-2 cRNA probe. Whereas 2% of HuT-102, 0.8% of MT-2, and 0.5% of MT-4 HTLV-I-transformed T-cell lines expressed IL-2 mRNA, 28 to 35% of uninfected but phorbol myristate acetate- and phytohemagglutinin-stimulated Jurkat T cells expressed IL-2 mRNA. Moreover, using a system with inducible expression of an endoplasmic reticulum-targeted single-chain antibody to knock out surface expression of IL-2R, Richardson et al. (248) found that IL-2R expression is dispensable for in vitro growth of HTLV-I-transformed T-cell lines. Thus, proliferation of HTLV-I-transformed T cells is not mediated by autocrine IL-2 secretion.

A more complex question is the role of the IL-2R pathway during the transformation process. The lack of detectable IL-2 mRNA in HTLV-I-infected T-cell clones, which are neither completely immortalized nor transformed, suggests that the transformation is not the direct result of aberrant autocrine IL-2 secretion. Nevertheless, this does not exclude an important role of the IL-2-IL-2R pathway in the early phase following HTLV-I infection. Kimata and Ratner (153) examined the presence of IL-2 mRNA and IL-2 activity following HTLV-I infection of human primary lymphocytes. While IL-2 was transiently expressed during the early phase of the infection (days 7 to 49, when viral integration is polyclonal), it was undetectable at later stages (days 100 to 150, when viral integration is oligoclonal). In contrast, expression of the viral tax-rex mRNA was low in the polyclonal phase and high in the oligoclonal phase, indicating that Tax expression did not induce autocrine IL-2 secretion. Indeed, the source of IL-2 during the polyclonal phase of the infection is uncertain, since HTLV-I-infected T cells can induce IL-2 production from uninfected T cells via T-cell-T-cell interaction (152, 310). In summary, evidence supporting a critical role for an autocrine IL-2 growth loop in HTLV-I-induced T-cell transformation is lacking.

Importantly, the development of IL-2 independence (i.e., transformation) may be associated with a constitutive IL-2-independent activation of the IL-2R signaling pathway. The ability of IL-2 to induce a signal in T cells is due to dimerization of the _c and _c chains and subsequent phosphorylation of signal transduction proteins. IL-2R signaling involves tyrosine phosphorylation and activation of the Janus family of kinase 1 and 3 (JAK1 and JAK3), which are associated with the _c and _c chains, respectively. Upon activation, JAKs phosphorylate tyrosine residues in the cytoplasmic tail of the IL-2R, which serve as docking sites for latent cytoplasmic transcription factors termed signal transducers and activators of transcription (STATs). STATs are then tyrosine phosphorylated and activated by JAKs, resulting in dimerization and nuclear translocation of STATs (52). IL-2R signaling activates STAT5 in resting T cells and activates STAT1, STAT3, and STAT5 in preactivated T cells. In contrast to nontransformed HTLV-I-infected T cells and Tax-transfected T cells, HTLV-I-transformed T-cell lines displayed constitutive tyrosine phosphorylation of JAK3 (202, 312), JAK1, STAT3, and STAT5 (202). In addition, STAT3 and STAT5 displayed constitutive DNA binding activity, and both _c and JAK3 associated with the IL-2R _c, indicating an activated IL-2R signaling pathway in the absence of IL-2 (202).

HTLV-I-infected but nonimmortalized and nontransformed T-cell clones expressed slightly elevated levels of JAK3 and STAT3 tyrosine phosphorylation but showed diminished induction of further tyrosine phosphorylation following IL-2 stimulation (255). Importantly, uncultured leukemic cells from patients with ATL expressed constitutive tyrosine phosphorylation, constitutive DNA binding activity, or both, of one or more of JAK3, STAT1, STAT3, STAT5, and STAT6, and there was a correlation between proliferation of ATL cells and activation of JAK3, STAT1, STAT3, and STAT5 (284). Since JAK1/JAK3 and STAT3/STAT5 activation is not observed in Tax-transfected T cells or newly HTLV-I-infected cord blood T cells (202), the constitutive activation of JAK and STAT may be associated with the process of transformation. In support of this notion, the transition to IL-2-independence of HTLV-I-infected cord blood T cells occurred concomitantly with an increase in constitutive STAT activity (202). Despite this association, the mechanism of JAK and STAT activation has not been linked to a viral protein yet.

A candidate viral protein that may induce IL-2R activation is p12^I, which may be encoded by the first open reading frame (ORF) of the pX region of HTLV-I (161) (Fig. 2). When overexpressed, p12^I physically associates with both the _c and _c chains (216) and may dimerize them, thereby initiating constitutive JAK and STAT activation and IL-2-independent proliferation (i.e., transformation). Nonetheless, alternatively spliced mRNAs of ORF-I (encoding p12^I) can be found in both IL-2-independent and IL-2-dependent HTLV-I-infected T-cell lines with significant variability between cell lines (38). Although the variability in the level of p12^I mRNA may indicate that splice site regulation is an important viral regulatory pathway, it also suggests that transformation cannot be explained simply by a shift in splice site utilization to ORF-I. However, it is clear that p12^I is not necessary for immortalization of HTLV-I-infected T cells, since deletion of ORF-I and ORF-II in an infectious molecular clone does not affect its ability to immortalize T cells (59), and, furthermore, Tax is both necessary and sufficient for in vitro immortalization of primary human CD4⁺ peripheral and cord blood lymphocytes (8, 96, 97). However, these Tax-immortalized T cells remain IL-2 dependent (8, 96), suggesting a possible role for additional proteins in the transformation process.

In summary, autocrine IL-2 production may play a role early after infection, causing clonal expansion, but its production diminishes and little if any IL-2 is produced at later stages in the nontransformed, HTLV-I-infected T cell. Concomitantly with transformation, however, activation of the JAK-STAT pathway of the IL-2R is activated by an unknown mechanism.

Besides JAK1 and JAK3, the protein tyrosine kinases Syk, Lck, and Fyn associate with the IL-2R and contribute to its signal transduction (106, 156, 204). Lck and Fyn are dispensable for IL-2R-mediated signaling in HTLV-I-infected T cells (203). The transition from an IL-2-dependent state to an IL-2-independent state (i.e., transformation) in HTLV-I-infected T-cell lines correlated with downregulation of lck mRNA (159) (Table 2), and although IL-2-dependent HTLV-I-infected T-cell lines expressed lck mRNA, they scarcely expressed Lck protein (228). Consistently, Tax-transfected Jurkat T cells expressed diminished levels of Lck protein and repressed lck mRNA levels (174). Genes that are known to be repressed by Tax contain binding sites (E-boxes) for basic helix-loop-helix proteins in their promoter regions (292, 293). Whereas uninfected T cells may use two separate promoters for lck transcription, HTLV-I-infected and IL-2-dependent T cells use the upstream promoter exclusively (221). Transfection of a CAT construct under control of the distal lck promoter demonstrated that Tax downregulated this promoter, but not if a putative E-box was deleted (174). The Tax-mediated downregulation of lck mRNA was proportional to the level of pX mRNA (174). Conversely, Lck suppresses the HTLV-I promoter (229), suggesting that downregulation of Lck may further enhance viral transcription. In contrast to Lck and Fyn, altered expression of IL-2R-associated Syk in HTLV-I-infected T cells has not been reported. Syk may be a mediator of IL-2-induced activation of c-Myc (204, 208).

Incorporation of [³H]thymidine in the absence of exogenous IL-2 in HTLV-I-infected but not uninfected T-cell clones indicates that the virus is capable of inducing the G₁/S-phase transition. In its hypophosphorylated form, the retinoblastoma protein (pRb) is a negative regulator of the G₁/S-phase transition, in part through its sequestering of members of the E2F family of transcription factors (267). Following T-cell activation, pRb is inactivated by phosphorylation and releases E2F, which promotes S-phase entry. During the early G₁ phase, cyclins D2 and D3 and cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) are synthesized by an IL-2-independent pathway (189, 209), whereas IL-2 stimulation late in G₁ induces de novo synthesis of CDK2 (209), the kinase partner of cyclin E. Initially, D-type cyclin complexes are responsible for pRb phosphorylation, whereas cyclin E-CDK2 becomes the major pRb kinase close to the G₁/S-phase transition (267). The activity of cyclin-CDK complexes is regulated by a group of CDK inhibitors, of which two families have been described. One family, including p21^WAF1/CIP1, p27^KIP1, and p57^KIP2, inhibits all CDK-cyclin complexes, whereas the other family, including p16^INK4a, p15^INK4b, p18^INK4c, and p19^INK4d, specifically inhibits the kinase activity of cyclin D-CDK4 and cyclin D-CDK6 (244, 267).

HTLV-I-mediated interference with cell cycle-regulating proteins was initially demonstrated in T-cell clones from patients with HAM/TSP; in contrast to uninfected T-cell clones, pRb was constitutively hyperphosphorylated in HTLV-I-infected T-cell clones (119). The hyperphosphorylation of pRb correlates with Tax expression in a tetracycline repressor-based Tax expression system (254). Importantly, although transforming growth factor  (TGF-) completely abolished hyperphosphorylation of pRb in CD3-TCR-stimulated, uninfected T-cell clones, it did not prevent pRb phosphorylation in HTLV-I-infected T-cell clones (119). These observations suggest that HTLV-I activates T cells via a TGF--insensitive pathway. TGF- interferes with pRb phosphorylation by its ability to (i) induce an inhibitor, p15^INK4b, of CDK4 and CDK6 (104); (ii) inhibit CDK4 synthesis (70); (iii) inhibit CDK2 synthesis (89); (iv) inhibit cyclin A synthesis (89); (v) inhibit cyclin E synthesis (89); and (vi) prevent the assembly of active cyclin E-CDK2 complexes (158) by releasing sequestered p27^KIP1 (238, 269). Tax does not significantly alter the expression of CDK2, CDK4, CDK6, p27^KIP1, or cyclin A (7).

Suzuki et al. (280) and Low et al. (188) found that Tax associates with p16^INK4a. Whereas p16^INK4a inhibits CDK4 kinase activity, the Tax-p16^INK4a complex has lost this function. This provides direct evidence for Tax-mediated interference with cell cycle progression (Fig. 4). p16^INK4a contains four ankyrin motifs, and it is possible that Tax binds to p16^INK4a via these motifs, since Tax binding to IB can be mediated by ankyrin motifs (113). It remains to be determined whether Tax also inhibits p15^INK4b, a mediator of TGF- inhibition, which is 97% homologous to p16^INK4a in the last three of its four ankyrin motifs (264). Inhibition of p16^INK4a may explain the Tax-induced activity of CDK4 and CDK6 and thus the ability of Tax to induce G₁- to S-phase progression in lymphocytes (254), although Tax can also activate E2F-mediated transcription independently of p16^INK4a (175). Tax may also enhance cyclin D-CDK4 activity by decreasing the expression of p18^INK4c (7). Interestingly, HTLV-I-infected T-cell lines expressed high levels of cyclin D2 mRNA, in contrast to uninfected T-cell lines, which predominantly expressed cyclin D3 mRNA (7). The significance of this is unknown. Tax does not appear to switch the cyclin D isotype from D2 to D3 (7, 254).

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Cell cycle in HTLV-I-infected T cells. Immortalization and transformation of HTLV-I-infected T cells correlate with specific events in the cell cycle. T cells immortalized by HTLV-I infection require exogenous IL-2 to approach the restriction point (R). Transformation occurs when the infected cells no longer need exogenous IL-2 for cell cycling. Inhibitors of cell cycling and their sites of action are shown at the top. HTLV-I-infected T-cell clones are resistant to CsA, FK506, and TGF- but sensitive to rapamycin (119, 124). A simplistic representation of selected proteins and drugs with relevance to HTLV-I-infected T-cell activation is shown at the bottom. Arrows indicate a stimulatory signal. See the text for further details.

The CDK inhibitor p27^KIP1 is a critical regulator of the G₁ restriction point, since (i) IL-2R signaling eliminates p27^KIP1 (72, 165, 226) through a rapamycin-sensitive pathway (226); (ii) rapamycin-sensitive cells become rapamycin resistant if p27^KIP1 synthesis is inhibited by antisense oligonucleotides (150); (iii) antisense inhibition of p27^KIP1 synthesis prevents the cells from becoming quiescent (47, 249); and (iv) p27^KIP1 links TGF- to cell cycle arrest in mink epithelial cells (238). Despite the central role of p27^KIP1 in cell cycle regulation, it is not known whether the function of p27^KIP1 is altered in HTLV-I-infected T cells. Low et al. (188) did not detect an association of Tax with p27^KIP1 under conditions where Tax associated with p16^INK4a. Nevertheless, HTLV-I-mediated spontaneous proliferation is inhibited by rapamycin (124) but not by TGF- (119). This indicates that p27^KIP1 regulation is normal in HTLV-I-infected T-cell clones and hence not involved in their lack of inhibition by TGF-.

In contrast, the level of the CDK inhibitor p21^WAF1/CIP1 is elevated in HTLV-I-transformed T cells by a mechanism involving Tax-mediated transactivation of the promoter for p21^WAF1/CIP1 (7, 39), but Tax does not physically associate with p21^WAF1/CIP1 (188). The expression of p21^WAF1/CIP1 is normally regulated by p53 and is responsible for p53-induced G₁ arrest following DNA damage (31, 57), but Tax-induced p21^WAF1/CIP1 expression is p53 independent, since it occurs in p53-null cells (39). Despite the presence of the wild-type p53 gene in most HTLV-I-transformed T cells (39), Tax inactivates p53 by inhibiting its transcription (293) and by interfering with its transactivation domain (237). The lack of fully functional p53 in HTLV-I-infected T cells may contribute to HTLV-I-induced tumorigenesis.

Thus, Tax may induce G₁- to S-phase progression in lymphocytes by directly interacting with the cell cycle machinery and by influencing the transcription of cell cycle proteins and transcription factors. Most recently, Tax has also been shown to bind to a mitotic checkpoint protein, MAD1 (141). This suggest that Tax may also interfere with the G₂-M phase of the cell cycle, and the specific interaction with MAD1 may explain the ability of Tax to induce multinucleated cells (141).

One mechanism used to control cell growth is programmed cell death (apoptosis). T cells may undergo apoptosis by at least two separate mechanisms: (i) withdrawal of growth factors and (ii) activation-induced cell death (AICD). Withdrawal of growth factors, for example IL-2, is antigen independent and can be inhibited by the antiapoptotic proteins Bcl-x_L and Bcl-2. In contrast, AICD is antigen dependent, is mediated by CD95 (Fas) or TNF-, and is only partially inhibited by Bcl-x_L or Bcl-2 (298). The CD95-CD95L interaction plays a crucial role in peripheral AICD, as demonstrated by experiments with gld mice (deficient in CD95L) and lpr mice (deficient in CD95), both of which develop a lymphoproliferative disease (219). Since HTLV-I can induce a T-cell leukemia/lymphoma and HTLV-I-infected T-cell clones proliferate spontaneously in the absence of exogenous growth factors (124), an HTLV-I-mediated interference with normal T-cell apoptosis might explain the tumorigenic ability of the virus. Indeed, proteins encoded by EBV (108), adenovirus (243), and Sindbis virus (177) have been shown to inhibit apoptosis.

Nevertheless, the effect of HTLV-I infection on T-cell survival is controversial. Copeland et al. (48) examined the sensitivity of HTLV-I-infected T-cell lines to anti-CD95 antibody-mediated apoptosis. Despite expression of high levels of CD95, the HTLV-I-infected cell lines showed reduced susceptibility to anti-CD95-induced apoptosis (at antibody concentrations between 1 and 100 ng/ml). The resistance could be transferred to susceptible Jurkat T cells by transfection of a Tax-expressing vector or by treatment with soluble Tax, suggesting that Tax conferred resistance to CD95-CD95L-mediated apoptosis. Brauweiler et al. (28) also found that HTLV-I-infected T-cell lines (SLB, MT-2, MT-4, and HuT-102) were more resistant to apoptosis-inducing stimuli, such as anti-CD95 antibodies (250 ng/ml), taxol, or UV irradiation. Importantly, Tax repressed bax gene expression, and this was mediated by a 27-bp sequence in the bax promoter containing a putative basic helix-loop-helix binding site. Bax is known to promote apoptosis by inhibiting Bcl-x_L and Bcl-2, suggesting that Tax-mediated repression of bax may provide a molecular mechanism for the antiapoptotic effect of Tax. In addition, HTLV-I-infected T cells secrete thioredoxin, a small protein regulating the reduction-oxidation status in the cell. Thioredoxin has been reported to protect against oxidative stress-induced apoptosis (reviewed in reference 220).

Several reports have demonstrated that HTLV-I-infected T cells can be induced to undergo apoptosis. Fresh mononuclear cells from ATL patients are activated (CD25⁺) and sensitive to CD95-mediated apoptosis (55), and IL-2-dependent HTLV-I-infected T-cell lines are susceptible to anti-CD95-induced (54) and activation (CD2)-induced apoptosis (103). These apparently conflicting results may be due in part to differences in anti-CD95 antibodies and the concentrations used. Thus, Debatin et al. (54) used 10- to 100-fold-higher concentrations of anti-CD95 antibodies than did Copeland et al. (48) and Brauweiler et al. (28). Moreover, Debatin et al. (55) examined the feasibility of inducing apoptosis in freshly obtained peripheral blood lymphocytes from ATL patients but did not evaluate whether HTLV-I-infected T cells were more or less susceptible than uninfected T cells. In addition to anti-CD95, adriamycin appears to induce apoptosis in HTLV-I-infected T cells by a p53-independent pathway (85).

While these reports demonstrated the feasibility of inducing apoptosis in HTLV-I-infected T cells by exogenous stimuli, other observations have suggested that Tax itself may induce apoptosis. Chlichlia et al. (42, 43) expressed a fusion protein of Tax either N-terminal or C-terminal to the hormone binding domain of the estrogen receptor. Addition of estrogen or hydroxytamoxifen induced Tax transactivation and upregulation of CD28, CD69, and CD5 but not CD25, which required additional stimulation through the TCR-CD3 complex (43). This is surprising, since Tax has been shown to upregulate CD25 (8, 15, 50, 132, 252, 268) and increased expression of CD25 is even detected on HTLV-I-infected T-cell clones with a modest expression of Tax (124, 247). A potential concern, therefore, is whether the hormone-mediated induction of Tax had additional side effects. Importantly, Chlichlia et al. (42, 43) found that induction of Tax promoted apoptosis in T cells through a pathway that critically required the protease function of the IL-1-converting enzyme (42). A similar conclusion was reached by Chen et al. (41) using a Cd²⁺-inducible Tax-system (JPX-9). Although Tax induced CD95 ligand expression (41, 42) and the CD95-CD95 ligand interaction is known to activate IL-1-converting enzyme-proteases, blocking experiments failed to implicate this pathway in Tax-mediated induction of apoptosis (42). In contrast to these observations, a tetracycline repressor-based Tax expression system failed to detect apoptosis in lymphocytes expressing Tax (254).

Tax can induce oncogenic transformation in Rat-1 cells, a cell line derived from rat fibroblasts (285). Nevertheless, in contrast to wild-type Rat-1 cells, Tax-transformed Rat-1 cells underwent apoptosis within 7 days of incubation in serum-free medium, and this was inhibited by overexpression of Bcl-2 (313). This suggests that the expression of Tax makes Rat-1 cells growth factor dependent and susceptible to withdrawal apoptosis. Although the level of Tax expression may be critical for its biologic activities, comparative analysis of the apoptosis-inducing properties of Tax, c-Myc, and c-Fos suggested that Tax possesses relatively low apoptosis-inducing activity (80).

In conclusion, the outcome of HTLV-I infection on T-cell survival is controversial. The ability of Tax to prevent apoptosis of infected T cells is appealing, since Tax may also transform cells and since data from transgenic mice demonstrate that splenic T cells are more resistant to apoptosis induced by anti-CD95 antibodies (154). It is interesting, though, that the adenovirus E1A protein may transform cells and induce apoptosis, which is inhibited by the adenovirus E1B 19-kDa protein (243). If a similar mechanism operates in HTLV-I-infected T cells, it appears to require interaction with a cellular protein in order to explain the conflicting results obtained with Tax-transfected cells (28, 42, 43, 48). Moreover, as mentioned above, it is possible that the concentration of Tax determines the T-cell phenotype.

Peripheral or cord blood T cells can be immortalized and eventually transformed following coculture with HTLV-I-producing T cells. Here, immortalization means the ability of the T cells to grow continuously. This may require the presence of exogenous growth factors (usually IL-2) as in the case of CTLL-2 cells. If, however, exogenous growth factors are not required, the T cells are transformed, as in the case of Jurkat cells. The distinction between immortalization and transformation is important when analyzing the impact of viral infection on T-cell activation (Table 3).

The initial stages of HTLV-I-induced T-cell activation can be studied by analyzing in vivo HTLV-I-infected T-cell clones derived by limiting-dilution single-cell cloning of peripheral blood T cells from patients with HAM/TSP (124). T-cell clones are maintained in culture by periodic restimulation with irradiated feeder cells and antigen or mitogen. Whereas uninfected T-cell clones do not incorporate significant amounts of [³H]thymidine 1 week after restimulation, productively infected T-cell clones strikingly incorporate [³H]thymidine in the absence of exogenous growth factors, a phenomenon termed spontaneous clonal proliferation (Fig. 5). This reflects an HTLV-I-induced prolonged state of T-cell activation (124, 206, 239, 309, 322). Nevertheless, HTLV-I-infected T-cell clones are not immortalized, since they do not grow continuously without restimulation with irradiated feeder cells and phytohemagglutinin. Despite their ability to enter S phase 7 to 12 days after restimulation in the absence of exogenous IL-2 (124), the HTLV-I-infected T-cell clones need exogenous IL-2 for growth beyond 12 days and thus are not transformed. It is interesting that HTLV-I-infected T-cell clones are not immortalized, since they are capable of immortalizing peripheral blood lymphocytes in vitro, although with variable efficiency (247).

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Spontaneous clonal proliferation of HTLV-I-infected T-cell clones. A schematic representation of proliferation (measured by [3H]thymidine incorporation) following restimulation (arrows) of uninfected and HTLV-I-infected T-cell clones is shown. Dashed lines indicate proliferation of the T-cell clones if they are not restimulated. Spontaneous clonal proliferation is defined as the ability of HTLV-I-infected T cells to incorporate [3H]thymidine in the absence of exogenous growth factors 7 days or more after stimulation (124, 309).

The in vitro immortalization process occurs in defined stages. Initially, T-cell growth may decrease and reach a crisis stage which, at about 4 weeks following infection (96), will result in either cell death or increased cell growth. HTLV-I-infected T cells surviving the crisis display upregulated expression of CD25 and MHC class II but remain IL-2 dependent. The initial proliferative phase is characterized by polyclonal proviral integration and transient expression of IL-2 mRNA and IL-2 activity, which is undetectable at later time points (153). At approximately 100 days after infection, proviral integration is oligoclonal, with upregulation of CD25 surface expression but not of IL-2 mRNA. In contrast to IL-2 mRNA, viral tax-rex mRNA is scarcely expressed in the initial phase but is expressed abundantly at later time points (153).

The immortalization is caused by Tax. By inserting the pX region of HTLV-I into transformation-defective but replication competent herpesvirus saimiri, Grassmann et al. (97) demonstrated that the pX region was sufficient for immortalizing human thymocytes and cord blood lymphocytes. Although it cannot be excluded that proteins encoded by herpesvirus saimiri influence the function of HTLV-I proteins, subsequent analyses deleting or inserting nucleotides to generate constructs deficient in the expression of Tax or Rex, or both, have shown that Tax is both necessary and sufficient for immortalizing CD4⁺ cord blood lymphocytes in this system (96).

An interpretation of the requirements for immortalization and transformation from a cell cycle perspective is shown in Fig. 4. In normal T cells, TCR-mediated activation brings T cells into the G₁ phase of the cell cycle, which is associated with activation of tyrosine and serine/threonine protein kinases, Ca²⁺ flux, and subsequent activation of transcription factors. In addition cyclin D2, D3, CDK4, and CDK6 are synthesized prior to IL-2R signaling (189, 209), which, however, is required to bring T cells beyond the G₁ restriction point (36). Phosphorylation of pRb has been proposed to correspond to the restriction point (324), and IL-2R signaling allows pRb phosphorylation by eliminating a critical regulator of the restriction point, the CDK inhibitor p27^KIP1 (47, 72, 150, 165, 226, 249).

Tax-immortalized but nontransformed T cells are dependent upon IL-2; hence, they cannot pass the G₁ restriction point in the absence of exogenous growth factors. This indicates that overexpression of Tax alone does not induce S-phase progression but that additional events are required. Tax-immortalized T cells are able to enter G₁ and, more importantly, do not exit the cell cycle by apoptosis in the presence of appropriate growth factors. The ability of Tax to activate pathways and transcription factors known to be activated in G₁ during normal T-cell activation may thus be responsible for the G₁ progression (immortalized phenotype).

The transformation of T cells may require the concerted action of several viral and cellular proteins. Tax may transcriptionally repress and inactivate the tumor suppressor p53 (39, 237, 293) and may repress the DNA repair enzyme -polymerase (140), thus enhancing the accumulation of gene mutations. The transformation process, however, is expected to compensate for an IL-2R signal and hence to phosphorylate pRb and promote S-phase entry in the absence of exogenous growth factors. Constitutive activation of IL-2R-associated STAT3 and STAT5 has been demonstrated in HTLV-I-transformed T cells (202), but STAT5 does not regulate E2F (29) and thus does not induce pRb phosphorylation and S-phase entry. Recent evidence, however, suggests that Tax expression may promote pRb phosphorylation and the G₁/S-phase transition (254). However, it is unclear whether this is the function of Tax alone, since control cells immortalized by Tax remained IL-2 dependent. IL-2 induces CREB/ATF1 activity late in G₁ in a cAMP-independent but rapamycin-dependent manner (71). This activity may be compensated for by Tax expression, since Tax interacts with CREB/ATF1 and thereby increases its transcription activity. This interaction is essential for the ability of Tax to transform rat fibroblasts (272) and to clonally expand CD4⁺ T cells (6). Tax-mediated activation of cellular CREs may be important in T-cell transformation. The mechanism of CREB phosphorylation in HTLV-I-transformed T cells then becomes important, since Tax transactivation of cellular CREs is dependent on phosphorylated CREB (164).

T cells approaching the G₁ restriction point may either commit to the cell cycle (if p27^KIP1 is downregulated, pRb is hyperphosphorylated, and cyclin E-CDK2 is activated) or undergo apoptosis (if these conditions are not met) (186). Thus, it may be hypothesized that HTLV-I-immortalized T cells may undergo apoptosis in the absence of exogenous IL-2 because they do not approach the G₁ restriction point in an appropriate way; i.e., they have not downregulated p27^KIP1, which would allow activation of the pRb kinases (cyclin D-CDK4, cyclin D-CDK6, and cyclin E-CDK2). Hence, transformation is an ability to escape apoptosis in late G₁, in