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

Background

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

Results

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

Conclusions

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

     

Prevention of adult T-cell leukemia-like lymphoproliferative disease in rats by adoptively transferred T cells from a donor immunized with human T-cell leukemia virus type 1 Tax-coding DNA vaccine

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. To dissect the mechanisms of the development of the disease, we have previously established a rat model of ATL-like disease which allows examination of the growth and spread of HTLV-1 infected tumor cells, as well assessment of the effects of immune T cells on the development of the disease. In the present study, we induced HTLV-1 Tax-specific cytotoxic T lymphocyte (CTL) immunity by vaccination with Tax-coding DNA and examined the effects of the DNA vaccine in our rat ATL-like disease model. Our results demonstrated that DNA vaccine with Tax effectively induced Tax-specific CTL activity in F344/N Jcl-rnu/+ (nu/+) rats and that these CTLs were able to lyse HTLV-1 infected syngeneic T cells in vitro. Adoptive transfer of these immune T cells effectively inhibited the in vivo growth of HTLV-1-transformed tumor in F344/N Jcl-rnu/rnu (nu/nu) rats inoculated with a rat HTLV-1 infected T cell line. Vaccination with mutant Tax DNA lacking transforming ability also induced efficient anti-tumor immunity in this model. Our results indicated a promising effect for DNA vaccine with HTLV-1 Tax against HTLV-1 tumor development in vivo.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell in adult T cell leukemia patients

Human T cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-gamma, perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53 vs 90%; p = 0.001), whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30 and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.     

Advantage of higher-avidity CTL specific for Tax against human T-lymphotropic virus-1 infected cells and tumors

Strong CTL response can be observed and associated with the control of proviral load in human T-lymphotropic virus type 1 (HTLV-1) infection. However, there are few details with regard to how HTLV-1 specific CTLs work against HTLV-1 infected cells and adult T-cell leukemia cells (ATLs). In this study, using Tax-specific CTL lines with high- and low-functional avidity developed from HLA-A2-transgenic mice, we showed that higher avidity CTLs specific for Tax expressing larger numbers of TCRs and better binding strength to the antigen-HLA-A2 complex are much more efficient at eliminating HTLV-1 infected cells and, in particular, ATL tumor cells with the ability of recognizing a latent level of Tax product detected only with a real-time PCR. These findings suggest that such higher avidity CTLs specific for Tax in HTLV-1 could be responsible for preventing the development of HTLV-1 infection by detecting trace amount of antigens.     

Oligomannose-coated liposomes efficiently induce human T-cell leukemia virus-1-specific cytotoxic T lymphocytes without adjuvant

Human T-cell leukemia virus-1 (HTLV-1) causes adult T-cell leukemia/lymphoma, which is an aggressive peripheral T-cell neoplasm. Insufficient T-cell response to HTLV-1 is a potential risk factor in adult T-cell leukemia/lymphoma. Efficient induction of antigen-specific cytotoxic T lymphocytes is important for immunological suppression of virus-infected cell proliferation and oncogenesis, but efficient induction of antigen-specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV-1-specific CD8+ T-cell response by oligomannose-coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)-A*0201-restricted HTLV-1 Tax-epitope (OML/Tax). Immunization of HLA-A*0201 transgenic mice with OML/Tax induced an HTLV-1-specific gamma-interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA-A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV-1-specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV-1 carriers compared with epitope peptide alone, and these HTLV-1-specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen-specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.     

Correlation of major histocompatibility complex class I downregulation with resistance of human T-cell leukemia virus type 1-infected T cells to cytotoxic T-lymphocyte killing in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. Despite the apparent transforming ability of HTLV-1 under experimental conditions, most HTLV-1 carriers are asymptomatic. These facts suggest that HTLV-1 is controlled by host immunity in most carriers. To understand the interplay between host immunity and HTLV-1-infected cells, in this study, we isolated several HTLV-1 Tax-specific cytotoxic T-lymphocyte (CTL) lines from rats inoculated with Tax-coding DNA and investigated the long-term effects of the CTL on syngeneic HTLV-1-infected T cells. Our results demonstrated that long-term mixed culture of these CTL and the virus-infected T cells led to the emergence of CTL-resistant HTLV-1-infected cells. Although the Tax expression level in these resistant cells was equivalent to that in the parental cells, expression of surface major histocompatibility complex class I (MHC-I) was significantly downregulated in the resistant cells. Downregulation of MHC-I was more apparent in RT1.A(l), which presents a Tax epitope recognized by the CTL established in this study. Moreover, peptide pulsing resulted in killing of the resistant cells by CTL, indicating that resistance was caused by a decreased epitope density on the infected cell surface. This may be one of the mechanisms for persistence of HTLV-1-infected cells that evade CTL lysis and potentially develop ATL.     

Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo, the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2.     

Immunostimulation by induced expression of NKG2D and its MIC ligands in HTLV-1-associated neurologic disease

The NKG2D receptor costimulates effector/memory CD8 T cells and is normally absent on CD4 T cells but can be induced by T cell antigen receptor complex stimulation and interleukin-15 (IL-15). Among its ligands are the human major histocompatibility complex class I-related MICA and MICB, which have a restricted tissue distribution but are frequently associated with malignancies and some microbial infections. Moreover, aberrant expression of MIC may promote autoimmune disease progression. Human T cell lymphotropic virus type I (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease of the central nervous system that resembles multiple sclerosis. Disease progression involves production of IL-15 and its receptor through transactivation by the viral Tax regulator protein, an activated immune response state, and local cytokine production and T cell fratricide by Tax-specific cytotoxic T lymphocytes (CTL). This study shows that as with CD8 T cells, substantial proportions of HAM/TSP patient CD4 T cells are positive for NKG2D and that large numbers of T cells from both subsets express MIC, which can be transactivated by Tax independent of nuclear factor κB. Engagement of MIC by NKG2D promotes spontaneous HAM/TSP T cell proliferation and, apparently, CTL activities against HTLV-1-infected T cells. These results reveal a viral strategy that may exploit immune stimulatory mechanisms to negotiate a balance between promotion and limitation of infected host T cell expansions.     

Differential transactivation of the intercellular adhesion molecule 1 gene promoter by Tax1 and Tax2 of human T-cell leukemia viruses.

Previously, we showed that surface expression of intercellular adhesion molecule 1 (ICAM-1) was strongly upregulated in T cells carrying proviral human T-cell leukemia virus type 1 (HTLV-1) and that the viral transactivator protein Tax1 was capable of inducing the ICAM-1 gene. To determine the responsive elements in the human ICAM-1 gene promoter, a reporter construct in which the 5'-flanking 4.4-kb region of the ICAM-1 gene was linked to the promoterless chloramphenicol acetyltransferase (CAT) gene was cotransfected with expression vectors for Tax1 and Tax2, both of which were separately confirmed to be potent transactivators of the HTLV-1 long terminal repeat (LTR). Tax1 strongly activated the ICAM-1 promoter in all the cell lines tested: three T-cell lines (Jurkat, MOLT-4, and CEM), one monocytoid cell line (U937), and HeLa. Unexpectedly, Tax2 activated the ICAM-1 promoter only in HeLa. By deletion and mutation analyses of the 1.3-kb 5'-flanking region, we found that Tax1 transactivated the ICAM-1 promoter mainly via a cyclic AMP-responsive element (CRE)-like site at -630 to -624 in the Jurkat T-cell line and via an NF-kappaB site at -185 to -177 and an SP-1 site at -59 to -54 in HeLa. On the other hand, Tax2 was totally inactive on the ICAM-1 promoter in Jurkat but transactivated the promoter via the NF-kappaB site at -185 to -177 in HeLa. Gel mobility shift assays demonstrated proteins specifically binding to the CRE-like site at -630 to -624 in Tax1-expressing T-cell lines. Stable expression of Tax1 but not Tax2 in Jurkat subclones enhanced the surface expression of ICAM-1. The differential ability of Tax1 and Tax2 in transactivation of the ICAM-1 gene may be related to the differential pathogenicity of HTLV-1 and HTLV-2.     

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.