Clonality of HTLV-2 in Natural Infection

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8⁺ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.     

Regulatory T Cell Expansion in HTLV-1 and Strongyloidiasis Co-infection Is Associated with Reduced IL-5 Responses to Strongyloides stercoralis Antigen

Background

Human strongyloidiasis varies from a chronic but limited infection in normal hosts to hyperinfection in patients treated with corticosteroids or with HTLV-1 co-infection. Regulatory T cells dampen immune responses to infections. How human strongyloidiasis is controlled and how HTLV-1 infection affects this control are not clear. We hypothesize that HTLV-1 leads to dissemination of Strongyloides stercoralis infection by augmenting regulatory T cell numbers, which in turn down regulate the immune response to the parasite.

Objective

To measure peripheral blood T regulatory cells and Strongyloides stercoralis larval antigen-specific cytokine responses in strongyloidiasis patients with or without HTLV-1 co-infection.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from newly diagnosed strongyloidiasis patients with or without HTLV-1 co-infection. Regulatory T cells were characterized by flow cytometry using intracellular staining for CD4, CD25 and FoxP3. PBMCs were also cultured with and without Strongyloides larval antigens. Supernatants were analyzed for IL-5 production.

Results

Patients with HTLV-1 and Strongyloides co-infection had higher parasite burdens. Eosinophil counts were decreased in the HTLV-1 and Strongyloides co-infected subjects compared to strongyloidiasis-only patients (70.0 vs. 502.5 cells/mm³, p = 0.09, Mann-Whitney test). The proportion of regulatory T cells was increased in HTLV-1 positive subjects co-infected with strongyloidiasis compared to patients with only strongyloidiasis or asymptomatic HTLV-1 carriers (median = 17.9% vs. 4.3% vs. 5.9 p<0.05, One-way ANOVA). Strongyloides antigen-specific IL-5 responses were reduced in strongyloidiasis/HTLV-1 co-infected patients (5.0 vs. 187.5 pg/ml, p = 0.03, Mann-Whitney test). Reduced IL-5 responses and eosinophil counts were inversely correlated to the number of CD4+CD25+FoxP3+ cells.

Conclusions

Regulatory T cell counts are increased in patients with HTLV-1 and Strongyloides stercoralis co-infection and correlate with both low circulating eosinophil counts and reduced antigen-driven IL-5 production. These findings suggest a role for regulatory T cells in susceptibility to Strongyloides hyperinfection.     

Massive Depletion of Bovine Leukemia Virus Proviral Clones Located in Genomic Transcriptionally Active Sites during Primary Infection

Deltaretroviruses such as human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV) induce a persistent infection that remains generally asymptomatic but can also lead to leukemia or lymphoma. These viruses replicate by infecting new lymphocytes (i.e. the infectious cycle) or via clonal expansion of the infected cells (mitotic cycle). The relative importance of these two cycles in viral replication varies during infection. The majority of infected clones are created early before the onset of an efficient immune response. Later on, the main replication route is mitotic expansion of pre-existing infected clones. Due to the paucity of available samples and for ethical reasons, only scarce data is available on early infection by HTLV-1. Therefore, we addressed this question in a comparative BLV model. We used high-throughput sequencing to map and quantify the insertion sites of the provirus in order to monitor the clonality of the BLV-infected cells population (i.e. the number of distinct clones and abundance of each clone). We found that BLV propagation shifts from cell neoinfection to clonal proliferation in about 2 months from inoculation. Initially, BLV proviral integration significantly favors transcribed regions of the genome. Negative selection then eliminates 97% of the clones detected at seroconversion and disfavors BLV-infected cells carrying a provirus located close to a promoter or a gene. Nevertheless, among the surviving proviruses, clone abundance positively correlates with proximity of the provirus to a transcribed region. Two opposite forces thus operate during primary infection and dictate the fate of long term clonal composition: (1) initial integration inside genes or promoters and (2) host negative selection disfavoring proviruses located next to transcribed regions. The result of this initial response will contribute to the proviral load set point value as clonal abundance will benefit from carrying a provirus in transcribed regions.     

High Prevalence of Skin Disorders among HTLV-1 Infected Individuals Independent of Clinical Status

Background

Human T-cell lymphotropic virus type 1 (HTLV-1) infection can increase the risk of developing skin disorders. This study evaluated the correlation between HTLV-1 proviral load and CD4⁺ and CD8⁺ T cells count among HTLV-1 infected individuals, with or without skin disorders (SD) associated with HTLV-1 infection [SD-HTLV-1: xerosis/ichthyosis, seborrheic dermatitis or infective dermatitis associated to HTLV-1 (IDH)].

Methods

A total of 193 HTLV-1-infected subjects underwent an interview, dermatological examination, initial HTLV-1 proviral load assay, CD4⁺ and CD8⁺ T cells count, and lymphproliferation assay (LPA).

Results

A total of 147 patients had an abnormal skin condition; 116 (79%) of them also had SD-HTLV-1 and 21% had other dermatological diagnoses. The most prevalent SD-HTLV-1 was xerosis/acquired ichthyosis (48%), followed by seborrheic dermatitis (28%). Patients with SD-HTLV-1 were older (51 vs. 47 years), had a higher prevalence of myelopathy/tropical spastic paraparesis (HAM/TSP) (75%), and had an increased first HTLV-1 proviral load and basal LPA compared with patients without SD-HTLV-1. When excluding HAM/TSP patients, the first HTLV-1 proviral load of SD-HTLV-1 individuals remains higher than no SD-HTLV-1 patients.

Conclusions

There was a high prevalence of skin disorders (76%) among HTLV-1-infected individuals, regardless of clinical status, and 60% of these diseases are considered skin disease associated with HTLV-1 infection.     

Reduction of human T-cell leukemia virus type 1 (HTLV-1) proviral loads in rats orally infected with HTLV-1 by reimmunization with HTLV-1-infected cells

Human T-cell leukemia virus type 1 (HTLV-1) persistently infects humans, and the proviral loads that persist in vivo vary widely among individuals. Elevation in the proviral load is associated with serious HTLV-1-mediated diseases, such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, it remains controversial whether HTLV-1-specific T-cell immunity can control HTLV-1 in vivo. We previously reported that orally HTLV-1-infected rats showed insufficient HTLV-1-specific T-cell immunity that coincided with elevated levels of the HTLV-1 proviral load. In the present study, we found that individual HTLV-1 proviral loads established in low-responding hosts could be reduced by the restoration of HTLV-1-specific T-cell responses. Despite the T-cell unresponsiveness for HTLV-1 in orally infected rats, an allogeneic mixed lymphocyte reaction in the splenocytes and a contact hypersensitivity response in the skin of these rats were comparable with those of naive rats. HTLV-1-specific T-cell response in orally HTLV-1-infected rats could be restored by subcutaneous reimmunization with mitomycin C (MMC)-treated syngeneic HTLV-1-transformed cells. The reimmunized rats exhibited lower proviral loads than untreated orally infected rats. We also confirmed that the proviral loads in orally infected rats decreased after reimmunization in the same hosts. Similar T-cell immune conversion could be reproduced in orally HTLV-1-infected rats by subcutaneous inoculation with MMC-treated primary T cells from syngeneic orally HTLV-1-infected rats. The present results indicate that, although HTLV-1-specific T-cell unresponsiveness is an underlying risk factor for the propagation of HTLV-1-infected cells in vivo, the risk may potentially be reduced by reimmunization, for which autologous HTLV-1-infected cells are a candidate immunogen.     

Complete Bovine Leukemia Virus (BLV) Provirus Is Conserved in BLV-Infected Cattle throughout the Course of B-Cell Lymphosarcoma Development

Bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) belong to the same subfamily of oncoviruses. Defective HTLV-1 proviral genomes have been found in more than half of all patients with adult T-cell leukemia examined. We have characterized the genomic structure of integrated BLV proviruses in peripheral blood lymphocytes and tumor tissue taken from animals with lymphomas at various stages. Genomic Southern hybridization with SacI, which generates two major fragments of BLV proviral DNA, yielded only bands that corresponded to a full-size provirus in all of 23 cattle at the lymphoma stage and in 7 BLV-infected but healthy cattle. Long PCR with primers located in long terminal repeats clearly demonstrated that almost the complete provirus was retained in all of 27 cattle with lymphomas and in 19 infected but healthy cattle. However, in addition to a PCR product that corresponded to a full-size provirus, a fragment shorter than that of the complete virus was produced in only one of the 27 animals with lymphomas. Moreover, when we performed conventional PCR with a variety of primers that spanned the entire BLV genome to detect even small defects, PCR products were produced that specifically covered the entire BLV genome in all of the 40 BLV-infected cattle tested. Therefore, it appears that at least one copy of the full-length BLV proviral genome was maintained in each animal throughout the course of the disease and, in addition, that either large or small deletions of proviral genomes may be very rare events in BLV-infected cattle.     

Immunological Profile of HTLV-1-Infected Patients Associated with Infectious or Autoimmune Dermatological Disorders

In the present study, the frequency, the activation and the cytokine and chemokine profile of HTLV-1 carriers with or without dermatological lesions were thoroughly described and compared. The results indicated that HTLV-1-infected patients with dermatological lesions have distinct frequency and activation status when compared to asymptomatic carriers. Alterations in the CD4⁺HLA-DR⁺, CD8⁺ T cell, macrophage-like and NKT subsets as well as in the serum chemokines CCL5, CXCL8, CXCL9 and CXCL10 were observed in the HTLV-1-infected group with skin lesions. Additionally, HTLV-1 carriers with dermatological skin lesions showed more frequently high proviral load as compared to asymptomatic carriers. The elevated proviral load in HTLV-1 patients with infectious skin lesions correlated significantly with TNF-α/IL-10 ratio, while the same significant correlation was found for the IL-12/IL-10 ratio and the high proviral load in HTLV-1-infected patients with autoimmune skin lesions. All in all, these results suggest a distinct and unique immunological profile in the peripheral blood of HTLV-1-infected patients with skin disorders, and the different nature of skin lesion observed in these patients may be an outcome of a distinct unbalance of the systemic inflammatory response upon HTLV-1 infection.     

HTLV-1 propels thymic human T cell development in "human immune system" Rag2⁻/⁻ gamma c⁻/⁻ mice

Alteration of early haematopoietic development is thought to be responsible for the onset of immature leukemias and lymphomas. We have previously demonstrated that Tax(HTLV-1) interferes with ?-selection, an important checkpoint of early thymopoiesis, indicating that human T-cell leukemia virus type 1 (HTLV-1) infection has the potential to perturb thymic human αβ T-cell development. To verify that inference and to clarify the impact of HTLV-1 infection on human T-cell development, we investigated the in vivo effects of HTLV-1 infection in a "Human Immune System" (HIS) Rag2?/?γ(c)?/? mouse model. These mice were infected with HTLV-1, at a time when the three main subpopulations of human thymocytes have been detected. In all but two inoculated mice, the HTLV-1 provirus was found integrated in thymocytes; the proviral load increased with the length of the infection period. In the HTLV-1-infected mice we observed alterations in human T-cell development, the extent of which correlated with the proviral load. Thus, in the thymus of HTLV-1-infected HIS Rag2?/?γc?/? mice, mature single-positive (SP) CD4? and CD8? cells were most numerous, at the expense of immature and double-positive (DP) thymocytes. These SP cells also accumulated in the spleen. Human lymphocytes from thymus and spleen were activated, as shown by the expression of CD25: this activation was correlated with the presence of tax mRNA and with increased expression of NF-kB dependent genes such as bfl-1, an anti-apoptotic gene, in thymocytes. Finally, hepato-splenomegaly, lymphadenopathy and lymphoma/thymoma, in which Tax was detected, were observed in HTLV-1-infected mice, several months after HTLV-1 infection. These results demonstrate the potential of the HIS Rag2?/?γ(c)?/? animal model to elucidate the initial steps of the leukemogenic process induced by HTLV-1.     

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.

     

Human T-cell leukemia virus type 1 infection leads to arrest in the G1 phase of the cell cycle

Infection by the human T-cell leukemia virus type 1 (HTLV-1) is thought to cause dysregulated T-cell proliferation, which in turn leads to adult T-cell leukemia/lymphoma. Early cellular changes after HTLV-1 infection have been difficult to study due to the poorly infectious nature of HTLV-1 and the need for cell-to-cell contact for HTLV-1 transmission. Using a series of reporter systems, we show that HeLa cells cease proliferation within one or two division cycles after infection by HTLV-1 or transduction of the HTLV-1 tax gene. HTLV-1-infected HeLa cells, like their tax-transduced counterparts, expressed high levels of p21^CIP1/WAF1 and p27^KIP1, developed mitotic abnormalities, and became arrested in G₁ in senescence. In contrast, cells of a human osteosarcoma lineage (HOS) continued to divide after HTLV-1 infection or Tax expression, albeit at a reduced growth rate and with mitotic aberrations. Unique to HOS cells is the dramatic reduction of p21^CIP1/WAF1 and p27^KIP1 expression, which is in part associated with the constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway. The loss of p21^CIP1/WAF1 and p27^KIP1 in HOS cells apparently allows HTLV-1- and Tax-induced G₁ arrest to be bypassed. Finally, HTLV-1 infection and Tax expression also cause human SupT1 T cells to arrest in the G₁ phase of the cell cycle. These results suggest that productive HTLV-1 infection ordinarily leads to Tax-mediated G₁ arrest. However, T cells containing somatic mutations that inactivate p21^CIP1/WAF1 and p27^KIP1 may continue to proliferate after HTLV-1 infection and Tax expression. These infected cells can expand clonally, accumulate additional chromosomal abnormalities, and progress to cancer.     

Transmission of Human T-Cell Leukemia Virus Type 1 to Mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10⁵ spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.     

The changes in proviral chromatin that accompany morphological variation in avian sarcoma virus-infected rat cells

The clone All of avian sarcoma virus B77-infected Rat-1 cells comprises both morphologically normal and morphologically transformed derivatives. Transformed subclones, in which virus-specific RNA is readily detectable, contain a provirus that is very sensitive to DNase 1 digestion of chromatin, and show DNase 1 hypersensitive sites at the 5' end of the provirus and in 5' flanking cell DNA. Normal subclones with no detectable virus-specific RNA, whether infected cells that have never been transformed or revertants derived from transformed cells, contain a provirus that is far more resistant to DNase 1 digestion. Moreover this provirus lacks hypersensitive sites at its 5' end, although DNase 1 hypersensitive sites were detected at the 3' end of the provirus in either normal or transformed clones. The pattern of cytosine methylation in the proviral restriction sites of the isoschizomers Msp I and Hpa II differed between transformed and revertant clones; the revertants show additional methylation at some CpG doublets.     

Human T-cell leukemia virus type 1 (HTLV-1) infection of mice: proliferation of cell clones with integrated HTLV-1 provirus in lymphoid organs

Human T-cell leukemia virus type 1 (HTLV-1) is suggested to cause adult T-cell leukemia after 40 to 50 years of latency in a small percentage of carriers. However, little is known about the pathophysiology of the latent period and the reservoir organs where polyclonal proliferation of cells harboring integrated provirus occurs. The availability of animal models would be useful to analyze the latent period of HTLV-1 infection. At 18 months after HTLV-1 infection of C3H/HeJ mice inoculated with the MT-2 cell line, which is an HTLV-1-producing human T-cell line, HTLV-1 provirus was detected in spleen DNA from eight of nine mice. No more than around 100 proviruses were found per 10(5) spleen cells. Cellular sequences flanking the 3' long terminal repeat (LTR) and the clonalities of the cells which harbor integrated HTLV-1 provirus were analyzed by linker-mediated PCR. The results showed that the flanking sequences are of mouse genome origin and that polyclonal proliferation of the spleen cells harboring integrated HTLV-1 provirus had occurred in three mice. A sequence flanking the 5' LTR was isolated from one of the mice and revealed the presence of a 6-nucleotide duplication of cellular sequences, consistent with typical retroviral integration. Moreover, PCR was performed on DNA from infected tissues, with LTR primers and primers derived from seven novel flanking sequences of the three mice. Data revealed that the expected PCR products were found from lymphatic tissues of the same mouse, suggesting that the lymphatic tissues were the reservoir organs for the infected and proliferating cell clones. The mouse model described here should be useful for analysis of the carrier state of HTLV-1 infection in humans.     

Passage of human T-cell leukemia virus type-1 during progression to cutaneous T-cell lymphoma results in myelopathic disease in an HTLV-1 infection model

Studies comparing functional differences in human T-cell leukemia virus type 1 (HTLV-1) clones that mediate distinct outcomes in experimentally infected rabbits, resulted in a dermatopathic smoldering adult T-cell leukemia/lymphoma following chronic infection with HTLV-1 strain RH/K34. During the 3.5 years' follow-up, HTLV-1 skin disease progressed to cutaneous T-cell lymphoma. When infection was passed to several naive rabbits, progressive paraparesis due to myelopathic neurodegeneration, analogous to HTLV-associated myelopathy, resulted in one of 4 transfusion recipients. Similar proviral loads were detected in the two diseases, regardless of stage of progression or tissue compartment of infection. Complete proviral sequences obtained from the donor and affected recipient aligned identically with each other and with the inoculated virus clone. Existence of disparate pathogenic outcomes following infectious transmission further extends the analogy of using rabbits to model human infection and disease. Although the experimental outcomes shown are limited by numbers of animals affected, they mimic the infrequency of HTLV-1 disease and authenticate epidemiological evidence of virus sequence stability regardless of disease phenotype. The findings suggest that further investigation of a possible role for HTLV-1 in some forms of cutaneous T-cell lymphoma is warranted.