Spectrum of Treg and self-reactive T cells: single cell perspectives from old friend HTLV-1.

Despite extensive regulatory T cell (Treg) research, fundamental questions on in vivo dynamics remain to be answered. The current study aims to dissect several interwoven concepts in Treg biology, highlighting the 'self-reactivity' of Treg and their counterparts, namely naturally-arising memory-phenotype T-cells, as a key mechanism to be exploited by a human retroviral infection. We propose the novel key concept, Periodic T cell receptor (TCR)-signalled T-cells, capturing self-reactivity in a quantifiable manner using the Nr4a3-Timer-of-cell-kinetics-and-activity (Tocky) technology. Periodic and brief TCR signals in self-reactive T-cells contrast with acute TCR signals during inflammation. Thus, we propose a new two-axis model for T-cell activation by the two types of TCR signals or antigen recognition, elucidating how Foxp3 expression and acute TCR signals actively regulate Periodic TCR-signalled T-cells. Next, we highlight an underappreciated branch of immunological research on Human T-cell Leukemia Virus type 1 (HTLV-1) that precedes Treg studies, illuminating the missing link between the viral infection, CD25, and Foxp3. Based on evidence by single-cell analysis, we show how the viral infection exploits the regulatory mechanisms for T-cell activation and suggests a potential role of periodic TCR signalling in infection and malignant transformation. In conclusion, the new perspectives and models in this study provide a working framework for investigating Treg within the self-reactive T-cell spectrum, expected to advance understanding of HTLV-1 infection, cancer, and immunotherapy strategies for these conditions.

Understanding the Immunopathology of HTLV-1-Associated Adult T-Cell Leukemia/Lymphoma: A Comprehensive Review.

Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL). HTLV-1 carriers have a lifelong asymptomatic balance between infected cells and host antiviral immunity; however, 5-10% of carriers lose this balance and develop ATL. Coinfection with Strongyloides promotes ATL development, suggesting that the immunological status of infected individuals is a determinant of HTLV-1 pathogenicity. As CD4+ T cells play a central role in host immunity, the deregulation of their function and differentiation via HTLV-1 promotes the immune evasion of infected T cells. During ATL development, the accumulation of genetic and epigenetic alterations in key host immunity-related genes further disturbs the immunological balance. Various approaches are available for treating these abnormalities; however, hematopoietic stem cell transplantation is currently the only treatment with the potential to cure ATL. The patient's immune state may contribute to the treatment outcome. Additionally, the activity of the anti-CC chemokine receptor 4 antibody, mogamulizumab, depends on immune function, including antibody-dependent cytotoxicity. In this comprehensive review, we summarize the immunopathogenesis of HTLV-1 infection in ATL and discuss the clinical findings that should be considered when developing treatment strategies for ATL.

HTLV-1 cell-free DNA in plasma as a potential biomarker in HTLV-1 carriers and adult T-cell leukemia-lymphoma.

Viral cell-free DNA (cfDNA) in plasma has been widely evaluated for detecting cancer and monitoring disease in virus-associated tumors. We investigated whether the amount of cfDNA of human T-cell leukemia virus type 1 (HTLV-1) correlates with disease state in adult T-cell leukemia-lymphoma (ATL). HTLV-1 cfDNA in aggressive ATL was significantly higher than that in indolent ATL and asymptomatic carriers. Notably, patients with lymphoma type represented higher HTLV-1 cfDNA amount than chronic and smoldering subtypes, though they had no abnormal lymphocytes in the peripheral blood. HTLV-1 cfDNA can be a universal biomarker that reflects the expansion of ATL clones.

Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.

Stromal Reprogramming through Dual PDGFRα/ß Blockade Boosts the Efficacy of Anti-PD-1 Immunotherapy in Fibrotic Tumors.

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRß) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti-PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/ß blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/ß blockade and anti-PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer. SIGNIFICANCE: Stromal targeting with PDGFRα/ß dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer. See related commentary by Tauriello, p. 655.

Association of Clostridium butyricum Therapy Using the Live Bacterial Product CBM588 with the Survival of Patients with Lung Cancer Receiving Chemoimmunotherapy Combinations.

The gut microbiota has emerged as a key regulator of immune checkpoint inhibitor (ICI) efficacy. Therapeutic approaches aimed at manipulating the microbiota through targeted reconstitution to enhance cancer treatment outcomes have garnered considerable attention. A single live microbial biotherapeutic bacterium, Clostridium butyricum MIYAIRI 588 strain (CBM588), has been shown to enhance the effects of ICI monotherapy in patients with advanced lung cancer. However, whether CBM588 affects the outcomes of chemoimmunotherapy combinations in lung cancer remains unknown. We hypothesized that CBM588 augments the effect of chemoimmunotherapy combinations and restores diminished effectiveness in patients with non-small cell lung cancer (NSCLC) receiving dysbiosis-inducing drugs. To validate this hypothesis, we retrospectively analyzed 106 patients with stage IV or recurrent metastatic NSCLC consecutively treated with chemoimmunotherapy combinations. A survival analysis was performed employing univariate and multivariate Cox proportional hazard models with inverse probability of treatment weighting (IPTW) using propensity scores. Forty-five percent of patients received Clostridium butyricum therapy. CBM588 significantly extended overall survival in patients with NSCLC receiving chemoimmunotherapy. The favorable impact of CBM588 on the efficacy of chemoimmunotherapy combinations varied based on tumor-programmed cell death ligand 1 (PD-L1) expression. The survival benefit of CBM588 in the PD-L1 <1% cohort was higher than that in the PD-L1 1-49% and PD-L1 ≥ 50% cohorts. Furthermore, CBM588 was associated with improved overall survival in patients receiving proton pump inhibitors and/or antibiotics. CBM588-induced manipulation of the commensal microbiota holds the potential to enhance the efficacy of chemoimmunotherapy combinations, warranting further exploration of the synergy between CBM588 and immunotherapy.

HTLV-1 persistence and leukemogenesis: A game of hide-and-seek with the host immune system.

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus which mainly infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATL), is primarily transmitted via direct cell-to-cell transmission. This feature generates a wide variety of infected clones in hosts, which are maintained via clonal proliferation, resulting in the persistence and survival of the virus. The maintenance of the pool of infected cells is achieved by sculpting the immunophenotype of infected cells and modulating host immune responses to avoid immune surveillance. Here, we review the processes undertaken by HTLV-1 to modulate and subvert host immune responses which contributes to viral persistence and development of ATL.

HTLV-1 intragenic viral enhancer influences immortalization phenotype in vitro, but is dispensable for persistence and disease development in animal models.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and chronic neurological disease. The disparity between silenced sense transcription versus constitutively active antisense (Hbz) transcription from the integrated provirus is not fully understood. The presence of an internal viral enhancer has recently been discovered in the Tax gene near the 3' long terminal repeat (LTR) of HTLV-1. In vitro, this enhancer has been shown to bind SRF and ELK-1 host transcription factors, maintain chromatin openness and viral gene transcription, and induce aberrant host gene transcription near viral integration sites. However, the function of the viral enhancer in the context of early HTLV-1 infection events remains unknown. In this study, we generated a mutant Enhancer virus (mEnhancer) and evaluated its effects on HTLV-1-mediated in vitro immortalization, establishment of persistent infection with an in vivo rabbit model, and disease development in a humanized immune system (HIS) mouse model. The mEnhancer virus was able to establish persistent infection in rabbits, and there were no significant differences in proviral load or HTLV-1-specific antibody responses over a 25-week study. However, rabbits infected with the mEnhancer virus had significantly decreased sense and antisense viral gene expression at 12-weeks post-infection. HIS mice infected with wt or mEnhancer virus showed similar disease progression, proviral load, and viral gene expression. While mEnhancer virus was able to sufficiently immortalize primary T-lymphocytes in cell culture, the immortalized cells had an altered phenotype (CD8+ T-cells), decreased proviral load, decreased sense and anti-sense gene expression, and altered cell cycle progression compared to HTLV-1.wt immortalized cells (CD4+ T-cells). These results suggest that the HTLV-1 enhancer element alone does not determine persistence or disease development but plays a pivotal role in regulating viral gene expression.

Identification and characterization of a novel enhancer in the HTLV-1 proviral genome.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

Clonality of HIV-1- and HTLV-1-Infected Cells in Naturally Coinfected Individuals.

BACKGROUND: Coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) diminishes the value of the CD4+ T-cell count in diagnosing AIDS, and increases the rate of HTLV-1-associated myelopathy. It remains elusive how HIV-1/HTLV-1 coinfection is related to such characteristics. We investigated the mutual effect of HIV-1/HTLV-1 coinfection on their integration sites (ISs) and clonal expansion. METHODS: We extracted DNA from longitudinal peripheral blood samples from 7 HIV-1/HTLV-1 coinfected, and 12 HIV-1 and 13 HTLV-1 monoinfected individuals. Proviral loads (PVL) were quantified using real-time polymerase chain reaction (PCR). Viral ISs and clonality were quantified by ligation-mediated PCR followed by high-throughput sequencing. RESULTS: PVL of both HIV-1 and HTLV-1 in coinfected individuals was significantly higher than that of the respective virus in monoinfected individuals. The degree of oligoclonality of both HIV-1- and HTLV-1-infected cells in coinfected individuals was also greater than in monoinfected subjects. ISs of HIV-1 in cases of coinfection were more frequently located in intergenic regions and transcriptionally silent regions, compared with HIV-1 monoinfected individuals. CONCLUSIONS: HIV-1/HTLV-1 coinfection makes an impact on the distribution of viral ISs and clonality of virus-infected cells and thus may alter the risks of both HTLV-1- and HIV-1-associated disease.

HTLV-1 infection promotes excessive T cell activation and transformation into adult T cell leukemia/lymphoma.

Human T cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T cells and induces chronic, persistent infection in infected individuals, with some developing adult T cell leukemia/lymphoma (ATL). HTLV-1 alters cellular differentiation, activation, and survival; however, it is unknown whether and how these changes contribute to the malignant transformation of infected cells. In this study, we used single-cell RNA-sequencing and T cell receptor-sequencing to investigate the differentiation and HTLV-1-mediated transformation of T cells. We analyzed 87,742 PBMCs from 12 infected and 3 uninfected individuals. Using multiple independent bioinformatics methods, we demonstrated the seamless transition of naive T cells into activated T cells, whereby HTLV-1-infected cells in an activated state further transformed into ATL cells, which are characterized as clonally expanded, highly activated T cells. Notably, the greater the activation state of ATL cells, the more they acquire Treg signatures. Intriguingly, the expression of HLA class II genes in HTLV-1-infected cells was uniquely induced by the viral protein Tax and further upregulated in ATL cells. Functional assays revealed that HTLV-1-infected cells upregulated HLA class II molecules and acted as tolerogenic antigen-presenting cells to induce anergy of antigen-specific T cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at the single-cell level.

M-Sec induced by HTLV-1 mediates an efficient viral transmission.

Human T-cell leukemia virus type 1 (HTLV-1) infects target cells primarily through cell-to-cell routes. Here, we provide evidence that cellular protein M-Sec plays a critical role in this process. When purified and briefly cultured, CD4+ T cells of HTLV-1 carriers, but not of HTLV-1- individuals, expressed M-Sec. The viral protein Tax was revealed to mediate M-Sec induction. Knockdown or pharmacological inhibition of M-Sec reduced viral infection in multiple co-culture conditions. Furthermore, M-Sec knockdown reduced the number of proviral copies in the tissues of a mouse model of HTLV-1 infection. Phenotypically, M-Sec knockdown or inhibition reduced not only plasma membrane protrusions and migratory activity of cells, but also large clusters of Gag, a viral structural protein required for the formation of viral particles. Taken together, these results suggest that M-Sec induced by Tax mediates an efficient cell-to-cell viral infection, which is likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag.

A target enrichment high throughput sequencing system for characterization of BLV whole genome sequence, integration sites, clonality and host SNP.

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5' end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

Blood and lymphatic systems are segregated by the FLCN tumor suppressor.

Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.

Multiomics Investigation Revealing the Characteristics of HIV-1-Infected Cells In Vivo.

For eradication of HIV-1 infection, it is important to elucidate the detailed features and heterogeneity of HIV-1-infected cells in vivo. To reveal multiple characteristics of HIV-1-producing cells in vivo, we use a hematopoietic-stem-cell-transplanted humanized mouse model infected with GFP-encoding replication-competent HIV-1. We perform multiomics experiments using recently developed technology to identify the features of HIV-1-infected cells. Genome-wide HIV-1 integration-site analysis reveals that productive HIV-1 infection tends to occur in cells with viral integration into transcriptionally active genomic regions. Bulk transcriptome analysis reveals that a high level of viral mRNA is transcribed in HIV-1-infected cells. Moreover, single-cell transcriptome analysis shows the heterogeneity of HIV-1-infected cells, including CXCL13high cells and a subpopulation with low expression of interferon-stimulated genes, which can contribute to efficient viral spread in vivo. Our findings describe multiple characteristics of HIV-1-producing cells in vivo, which could provide clues for the development of an HIV-1 cure.

HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP.

BACKGROUND: Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. RESULTS: We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. CONCLUSIONS: The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.

The Nature of the HTLV-1 Provirus in Naturally Infected Individuals Analyzed by the Viral DNA-Capture-Seq Approach.

The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host DNA, achieves persistent infection, and induces human diseases. Here, we demonstrate that viral DNA-capture sequencing (DNA-capture-seq) is useful to characterize HTLV-1 proviruses in naturally virus-infected individuals, providing comprehensive information about the proviral structure and the viral integration site. We analyzed peripheral blood from 98 naturally HTLV-1-infected individuals and found that defective proviruses were present not only in patients with leukemia, but also in those with other clinical entities. We further demonstrated that clones with defective-type proviruses exhibited a higher degree of clonal abundance than those with full-length proviruses. The frequency of defective-type proviruses in HTLV-1-infected humanized mice was lower than that in infected individuals, indicating that defective proviruses were rare at the initial phase of infection but preferentially selected during persistent infection. These results demonstrate the robustness of viral DNA-capture-seq for HTLV-1 infection and suggest potential applications for other virus-associated cancers in humans.

TFE3 Xp11.2 Translocation Renal Cell Carcinoma Mouse Model Reveals Novel Therapeutic Targets and Identifies GPNMB as a Diagnostic Marker for Human Disease.

Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix-loop-helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. IMPLICATIONS: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

Phosphatidylinositol 3-kinase-δ (PI3K-δ) is a potential therapeutic target in adult T-cell leukemia-lymphoma.

The prognosis of adult T-cell leukemia-lymphoma (ATL) remains very poor, and there is an urgent clinical need to investigate novel therapies for ATL. The expression of phosphatidylinositol 3-kinase-δ (PI3k-δ) is normally restricted to hematopoietic cells and is known as a key determinant of cell survival in certain cancers. The inhibitor of PI3k-δ, idelalisib, has been shown to be effective in the treatment of chronic lymphocytic leukemia. Here, we report the expression of PI3k-δ and the ability of idelalisib to promote apoptosis in ex vivo ATL samples. The activity of PI3K was quantified by a PI3-Kinase Activity ELISA kit. Although there was no significant difference in mean PI3K activity between healthy donors and patients with ATL, certain cases of ATL showed extremely high PI3K activities. The expression of PI3k-δ protein was detectable in most ATL cases. The freshly isolated cells from ATL patients were cultured with or without idelalisib for 0-10 days, and cell survival was then quantified. Idelalisib induced apoptosis in ATL cells in a time-dependent manner, and significantly reduced the frequency of viable ATL cells at 10 days. No time-dependent effects of idelalisib were observed in non-malignant T cells from the same patients. CCL22 has been reported to promote survival of ATL cells in part through the PI3K-AKT pathway. Idelalisib blocked this CCL22-induced phosphorylation of AKT and significantly inhibited the proliferation of ATL cells. These results validate the PI3K-AKT pathway as a potential therapeutic target in ATL.