HTLV-1 and HIV-1 represent biologically significant, structurally close, and equally problematic yet divergent human retroviruses. Although both infect CD4+ T cells and share similar structural elements, they differ markedly in genomic stability, transmission dynamics, clinical progression, and, most importantly, their transcriptional regulatory mechanisms. HTLV-1, an ancient virus with a limited global burden, often remains asymptomatic for decades before potentially causing ATL or HAM/TSP. Conversely, HIV-1, a relatively recent zoonotic transmission, undergoes rapid replication, exhibits high genetic diversity, and causes progressive immunodeficiency unless controlled by antiretroviral therapy (ART). At the molecular level, HTLV-1 maintains proviral latency through a balanced bidirectional transcription of regulatory genes (e.g., Tax and HBZ) that manipulate host transcription and immune evasion pathways, facilitating persistence and oncogenesis. HBZ and Tax were shown to contribute to driving the progressive acquisition of Treg-like and HLA class II phenotype in chronically activated CD4+ T-cells, promoting tolerogenic antigen presentation and immune evasion in ATL cells. This well-controlled differential expression of HTLV-1 regulatory genes is attributed to multiple intragenic virus regulatory mechanisms, which will be discussed in this review. In contrast, HIV-1 transcription is driven by a tightly regulated 5′ LTR promoter involving host factors such as NF-κB, Sp1, AP-1, and NFAT, among others, with strong influence imposed by the landscape of the provirus integration site, playing a pivotal role in latency and reactivation. The distinct regulatory circuitry of each virus suggests a key difference in their essential regulation, with HTLV-1 primarily relying on intragenic mechanisms, while HIV-1 relies more heavily on interactions with the surrounding host environment to control its expression. This difference underscores unique therapeutic challenges in managing viral latency, persistence, and pathogenesis.

Coronavirus disease 2019 (COVID-19) pneumonia is prevalent in the elderly infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, the mechanisms underlying its age-dependent pathogenesis remain unclear. In this study, we established a mouse-adapted SARS-CoV-2 strain infected Nr4a3-Tocky mouse model to examine T-cell dynamics associated with disease severity. Nr4a3-Tocky mice allow the analysis of the dynamics and induction of antigen-reactive T cells following antigen recognition in vivo using fluorescent Timer protein. SARS-CoV-2-infected adult mice exhibited transient body weight loss and recovery, whereas aged mice developed severe pneumonia. BALF viral RNA was comparable between 1-4 days post-inoculation (d.p.i.), but declined in adults at 5 d.p.i. Aged mice displayed stronger inflammation as indicated by scRNA-seq, and higher levels of inflammatory cytokines (TNF-α, CCL2, CXCL10 and IL-6) in BALF correlated with weight loss. Timer analysis revealed induction of antigen-reactive T cells in the adult lungs at 5 and 8 d.p.i., which inversely correlated with disease severity. Additionally, S-specific IFN-γ ⁺ CD8 ⁺ T cells were detected at 5 d.p.i. in adults, whereas detection of antigen-specific T cells was delayed in aged mice. These results suggest that the coexistence of age-related lung inflammation and delayed induction of antigen-specific T cells is linked to more severe pneumonia, while earlier T-cell responses are associated with improved viral control and milder disease. In this study, we utilized a novel mouse model enabling characterization of antigen-reactive T cells in the local tissue, and investigated inflammatory responses in the lung together with lung-infiltrating virus-specific T cells, finding the dynamics of these immunological parameters associated with the age of the mice. Our analysis provides new insights into understanding how age-related T-cell dysfunction is associated with the severity of SARS-CoV-2 pneumonia.

Cell-state transitions during development are shaped by multiple concurrent processes, yet single-cell transcriptomic analyses often rely on static molecular profiles or infer time without an experimental anchor. We developed mCanonicalTockySeq, a systems- level framework that reconstructs temporally resolved developmental state spaces by combining signalling history with single-cell RNA sequencing. Using the Nr4a3-Tocky Fluorescent Timer system in developing thymic T cells, we combined scRNA-seq with a molecular clock of strong T-cell receptor signalling to establish an experimentally anchored temporal reference. Using Timer-defined landmark populations, mCanonicalTockySeq constructs a shared state space in which temporal progression and developmental maturation are jointly represented, identifying a Tocky-defined temporal manifold while resolving progression toward CD4 and CD8 single-positive states. This framework recovered biologically coherent dynamics of immediate TCR-response genes, lineage-associated regulators, and agonist-selection-associated programmes. We then extended the framework across species by translating human thymic single-cell transcriptomes into one- to-one mouse ortholog space and projecting them into the mouse Nr4a3-Tocky reference. The projected human cells occupied an interpretable temporal-developmental geometry, and the inferred Tocky-equivalent temporal coordinate showed a significant donor-level association with chronological age. Together, these results establish mCanonicalTockySeq as a general framework for modelling how signalling history and developmental progression are jointly organised in single-cell state space, and illustrate how experimentally anchored reference systems can support comparative analyses across species.

ABSTRACTBackgroundVirus‐related cancers are malignancies caused by specific viruses, such as human papillomavirus (HPV), hepatitis B virus, and human T‐cell leukemia virus, contributing significantly to the global cancer burden through persistent infection and oncogenic transformation. The current study aimed to develop a robust HPV‐16 detection method for formalin‐fixed cancer specimens.Materials and MethodsTo prevent false negatives resulting from DNA fragmentation, a DNA quality check step was added. Additionally, this study used multiplex polymerase chain reaction (PCR) covering the entire HPV‐16 genome to mitigate effects caused by viral sequence variation. To prove this concept, we analyzed genomic DNA extracted from oropharyngeal cancer tissues known as HPV‐16‐positive. Subsequently, the protocol was tested on oral squamous cell carcinoma (OSCC) samples in our cohort. Given the wide variation in HPV‐16 positivity in previous studies, it remains elusive how frequently HPV‐16 is positive in OSCC.ResultsThe results showed faint bands or smears in the multiplex PCR of 7 out of 112 cases. Droplet digital PCR confirmed variable positivity levels of HPV‐16, suggesting two scenarios of HPV‐16 positivity in cancer tissue: cancer cells derived from infected cells or only a portion being HPV‐16‐positive. Finally, we comprehensively analyzed the case and identified the integration of a deleted HPV‐16 genome into the intronic region of the host gene TMEM94 on chromosome 17. To the best of our knowledge, this is the first evidence showing the integration of HPV‐16 in OSCC cells and providing its complete viral sequence.ConclusionsThe established protocol should be applicable to various cancer tissues for analyzing the association with HPV‐16 infection.

Tumor cells adeptly manipulate the tumor microenvironment (TME) to evade host antitumor immunity. However, the role of tumor cell-intrinsic signaling in shaping the immunosuppressive TME remains unclear. Here, we found that the Hippo pathway in tumor cells orchestrated the immunosuppressive TME by influencing the composition of cancer-associated fibroblasts (CAFs). In a 4T1 mouse breast cancer model, Hippo pathway kinases, large tumor suppressor 1 and 2 (LATS1/2), promoted the formation of immunosuppressive neural cell adhesion molecule 1 (NCAM1)+alpha-smooth muscle actin (αSMA)+ CAFs expressing the transforming growth factor-β, thereby contributing to T cell dysfunction. Depletion of LATS1/2 in tumor cells resulted in a less immunosuppressive TME, indicated by the reduced proportions of NCAM1+αSMA+ CAFs and dysfunctional T cells. Notably, similar Hippo pathway-induced NCAM1+αSMA+ CAFs were observed in human breast cancer cells, highlighting the potential of TME-manipulating strategies to reduce immunosuppression in cancer immunotherapy.

Human T-cell leukemia virus type 1 (HTLV-1) transforms primary CD4 T cells in vitro within a short time; however, majority of infected individuals maintain an asymptomatic condition, suggesting that there is an equilibrium between the infected cells and the host immunity. In this study, we identified a variation in a major viral antigen epitope, HTLV-1 Tax301-309, in HLA-A24-positive individuals. Mismatch in A24/Tax301-309 multimers impaired detection of anti-Tax cytotoxic T lymphocytes (CTLs). Notably, more than half of the T-cell receptors (TCRs) of the anti-Tax CTLs did not recognize mismatched Tax301-309 peptides. These findings highlighted the importance of matching the viral antigen epitope type in T-cell-based immunotherapy against adult T-cell leukemia/lymphoma by using viral antigen Tax. We identified a variation in the HTLV-1 Tax301-309 epitope in HLA-A24-positive individuals, affecting anti-Tax cytotoxic T-cell detection, emphasizing the importance of precise epitope matching in T-cell immunotherapy against HTLV-1-induced adult T-cell leukemia/lymphoma. Graphical Abstract

Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.

Since 1958 witnessed the detection of Monkeypox virus in monkeys, no human infection was encountered until 1970. Afterwards, zoonotic transmission was the rule near African rainforests, mainly in DRC. Most cases occurred in children who weren’t immunized against smallpox. Since 2003 and the first human infection in the USA, research was accelerated. Two clades were identified with different virulence, demographic distribution and transmissibility. The mean age of infection increased with waning smallpox vaccine immunity. Mild febrile prodrome can precede lymphadenopathy, which doesn’t occur in smallpox. Homogenous crops of lesions appear in stages until scabs fall and contagiosity ends. However, since May outbreak, cases started to appear in non-endemic areas, human transmission increased and was linked to close sexual contact especially in MSM community. Lesions were found mainly perioral, at genitals and perianal. Newer system for nomenclature was suggested in which there are 3 viral clades and the responsible clade for the outbreak is clade 3 (lineage B.1). About 50 mutations were detected compared with the strains isolated 4 years ago. Gene loss and APOBEC3 may be related to accelerated mutation rate which may accelerate human transmission. Previous mistakes in failure to allocate available vaccines to control the disease in previously endemic areas should be avoided and rapid ring vaccination of potential contacts and those at risk should be a priority. Case isolation, contact isolation or tracing for an incubation period, standard measures for airborne infections and safe sex should be implanted in the light of the current uncertainty.

The stably integrated pool of HIV-1 proviruses in the host genome stands against curative strategies. This reservoir is extremely heterogeneous with respect to host cell type, anatomical location, integration site, and replication fitness. During the initial phase of infection, only a few infected cells can resist host immune clearance or cytopathic effect and establish this resistant pool. The mechanisms underlying HIV latency initiation are not fully resolved yet. In the current study, we propose and validate a new reporter model for monitoring HIV-1 provirus silencing and reactivation using Timer of cell kinetics and activity (Tocky). HIV-Tocky system uses a fluorescent Timer protein whose emission spectrum spontaneously shifts from blue to red to reveal HIV-1 provirus dynamics. We dissected provirus transcriptional phases into early, persistent, recently silenced, and latent. To our knowledge, this is the first report to distinguish two latent subsets: a directly non-expressing population and a recently silenced after brief expression. In-depth integration site analysis suggested that the distribution of proviruses in directly latent cells was similar to that in actively transcribing cell population, whereas recently silenced cells tended to harbor proviruses integrated into heterochromatin. Furthermore, we established a library of various single integration clones at which we utilized to demonstrate the efficiency of the block-and-lock strategy by capturing the fast dynamics of silencing that were overlooked in previous models. In summary, we propose HIV-Tocky system to serve as a time-sensitive model that can capture the dynamics of provirus expression, making it a useful tool for HIV latency research. Significance Statement Determinants of HIV-1 latency establishment are yet to be elucidated. This reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allowed; for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies. Classification Biological Sciences, Microbiology.

The proinflammatory cytokine IL-32 is elevated in the plasma and tissues of HIV-1-infected individuals. However, its significance in HIV-1 infection remains unclear because IL-32 inhibits and stimulates viral production in monocyte-derived macrophages (MDMs) and CD4+ T cells, respectively. In this study, we initially found that the inhibitory effect on human MDMs depends on SAMHD1, a dNTP triphosphohydrolase that inhibits viral reverse transcription. IL-32 increased the unphosphorylated active form of SAMHD1, which was consistent with the reduced expression of the upstream cyclin-dependent kinases. Indeed, IL-32 lost its anti-HIV-1 activity in MDMs when SAMHD1 was depleted. These results explain why IL-32 inhibits HIV-1 in MDMs but not CD4+ T cells, because SAMHD1 restricts HIV-1 in noncycling MDMs but not in cycling CD4+ T cells. Another unique feature of IL-32 is the induction of the immunosuppressive molecule IDO1, which is beneficial for HIV-1 infection. In this study, we found that IL-32 also upregulates other immunosuppressive molecules, including PD-L1, in MDMs. Moreover, IL-32 promoted the motility of MDMs, which potentially facilitates intercellular HIV-1 transmission. Our findings indicate that IL-32 has both the direct inhibitory effect on HIV-1 production in MDMs and the indirect stimulatory effects through phenotypic modulation of MDMs, and they suggest that the stimulatory effects may outweigh the inhibitory effect because the window for IL-32 to inhibit HIV-1 is relatively confined to SAMHD1-mediated reverse transcription suppression in the viral life cycle.

The blockade of the immune checkpoints PD-1 and CTLA-4 enhances T cell response. However, it is largely unknown how antigen-reactive T cells regulate their checkpoint expression in vivo and whether and how the checkpoint blockade can change activation dynamics of tumour-reactive T cells. To address this, here we used Nr4a3- Timer-of-cell-kinetics-and-activity (Tocky) , which allows analysis of temporal changes of activated T cells following TCR signalling in vivo . By analysing melanoma-bearing Nr4a3 Tocky mice, we elucidate hidden dynamics of tumour-reactive T cells in the steady-state. Checkpoint blockade depleted highly activated effector Treg, while promoting unique effector T cell populations, and thus differentially modulating activation of tumour-reactive T cell populations. Furthermore, multidimensional analysis and seamless analysis of Tocky and scRNA-seq revealed a full spectrum of T cell dynamics in response to tumour burden and treatment with checkpoint blockade. Lastly, we propose a rational design of combinatorial therapy to further enhance T cell activities. ### Competing Interest Statement The authors have declared no competing interest.

Human T-cell leukemia virus type 1 (HTLV-1) mainly infects CD4 + T-cells and induces chronic, persistent infection in infected individuals with some progressing to develop adult T-cell leukemia/lymphoma (ATL). Whilst HTLV-1 alters cellular differentiation, activation and survival, it is unknown whether and how these changes contribute to malignant transformation of infected T-cells. In this study, we used single-cell RNA-Seq and TCR-Seq to investigate T-cell differentiation and HTLV-1-mediated transformation processes. We analyzed 87,742 single cells from peripheral blood of 12 infected and 3 uninfected individuals. Using multiple independent bioinformatic methods, we demonstrated that naïve T-cells dynamically change into activated T-cells including infected cells, which seamlessly transitioned into ATL cells characterized by clonally expanded, highly-activated T-cells. Notably, the more activated ATL cells are, the more they acquire Treg signatures. Intriguingly, HLA class II genes were uniquely induced in infected cells, further upregulated in ATL cells and was induced by viral protein Tax. Functional assays revealed that by upregulating HLA class II, HTLV-1-infected cells can act as tolerogenic antigen presenting cells (APCs) 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 single-cell level. Graphical Abstract

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.