CAGE-seq reveals that HIV-1 latent infection does not trigger unique cellular responses in a Jurkat T cell model.

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE).We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent.Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.

A Dual Promoter System to Monitor IFN-γ Signaling in vivo at Single-cell Resolution.

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-GAS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC50 ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo.Key words: in vivo imaging, tumor microenvironment, interferon-gamma, dual promoter system.

APOBEC3B is preferentially expressed at the G2/M phase of cell cycle.

APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86,493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.

Successful Treatment of TAFRO Syndrome, a Variant of Multicentric Castleman's Disease, with Cyclosporine A: Possible Pathogenetic Contribution of Interleukin-2.

Multicentric Castleman's disease is a systemic inflammatory disorder characterized by lymphadenopathy and excessive interleukin-6 production. A unique clinicopathologic variant of multicentric Castleman's disease, TAFRO (i.e., thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly) syndrome, was recently proposed in Japan. Despite the successful use of anti-interleukin-6 therapy in some patients with TAFRO syndrome, not all patients achieve remission. The pathophysiological etiology of and suitable therapeutic strategies for this variant have not been established. Here, we present our experience of a unique case of TAFRO syndrome in a 78-year-old woman whose symptoms responded differently to several therapies. Tocilizumab, an anti-interleukin-6 receptor antibody, successfully induced remission of fever and lymphadenopathy. However, severe thrombocytopenia persisted and she developed anasarca, ascites, and pleural effusion shortly thereafter. Rituximab, an anti-CD20 antibody, and glucocorticoid therapy provided no symptom relief. In contrast, cyclosporine A, an immunosuppressive agent that blocks T cell function by inhibiting interleukin-2, yielded immediate improvements in systemic fluid retention and a gradual increase in platelet count, with complete resolution of disease symptoms. Excessive serum interleukin-2, when used as an anti-cancer agent, has been reported to cause side effects such as fluid retention, thrombocytopenia, and renal failure. Our case was unique because the anti-interleukin-2 therapy successfully improved symptoms that were not relieved with anti-interleukin-6 therapy. The present report therefore provides insight into the possible role of interleukin-2, in addition to interleukin-6, in TAFRO syndrome. This report will certainly help to clarify the pathogenesis of and optimal treatment strategies for TAFRO syndrome.

In vivo imaging of inflammatory response in cancer research.

Inflammation can contribute to the development and progression of cancer. The inflammatory responses in the tumor microenvironment are shaped by complex sequences of dynamic intercellular cross-talks among diverse types of cells, and recapitulation of these dynamic events in vitro has yet to be achieved. Today, intravital microscopy with two-photon excitation microscopes (2P-IVM) is the mainstay technique for observing intercellular cross-talks in situ, unraveling cellular and molecular mechanisms in the context of their spatiotemporal dynamics. In this review, we summarize the current state of 2P-IVM with fluorescent indicators of signal transduction to reveal the cross-talks between cancer cells and surrounding cells including both immune and non-immune cells. We also discuss the potential application of red-shifted indicators along with optogenetic tools to 2P-IVM. In an era of single-cell transcriptomics and data-driven research, 2P-IVM will remain a key advantage in delivering the missing spatiotemporal context in the field of cancer research.

Diagnostic TR-FRET assays for detection of antibodies in patient samples.

Serological assays are important diagnostic tools for surveying exposure to the pathogen, monitoring immune response post vaccination, and managing spread of the infectious agent among the population. Current serological laboratory assays are often limited because they require the use of specialized laboratory technology and/or work with a limited number of sample types. Here, we evaluate an alternative by developing time-resolved Förster resonance energy transfer (TR-FRET) homogeneous assays that exhibited exceptional versatility, scalability, and sensitivity and outperformed or matched currently used strategies in terms of sensitivity, specificity, and precision. We validated the performance of the assays measuring total immunoglobulin G (IgG) levels; antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle Eastern respiratory syndrome (MERS)-CoV spike (S) protein; and SARS-CoV-2 S and nucleocapsid (N) proteins and applied it to several large sample sets and real-world applications. We further established a TR-FRET-based ACE2-S competition assay to assess the neutralization propensity of the antibodies. Overall, these TR-FRET-based serological assays can be rapidly extended to other antigens and are compatible with commonly used plate readers.

Functional visualization of NK cell-mediated killing of metastatic single tumor cells.

Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here, we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hr of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hr after tumor cell arrival, but after 24 hr of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24-hr period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing, the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung.

ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells.

DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.

A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron.

We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.

Intravital Imaging Identifies the VEGF-TXA2 Axis as a Critical Promoter of PGE2 Secretion from Tumor Cells and Immune Evasion.

Prostaglandin E2 (PGE2) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE2, little is known whether PGE2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A2 (TXA2) triggers Ca2+ transients in tumor cells, culminating in PGE2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca2+ transients were monitored as the surrogate marker of PGE2 secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca2+ transients is markedly higher in vivo than in vitro. The TXA2 receptor was indispensable for the Ca2+ transients in vivo, high intratumoral PGE2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca2+ transients and reduced TXA2 and PGE2 concentrations in tumor tissues. These results identify the VEGF-TXA2 axis as a critical promoter of PGE2-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies. SIGNIFICANCE: This study identifies the VEGF-TXA2 axis as a potentially targetable regulator of PGE2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies.

Using DNA Barcodes to Aid the Identification of Larval Fishes in Tropical Estuarine Waters (Malacca Straits, Malaysia).

Larval descriptions of tropical marine and coastal fishes are very few, and this taxonomic problem is further exacerbated by the high diversity of fish species in these waters. Nonetheless, accurate larval identification in ecological and early life history studies of larval fishes is crucial for fishery management and habitat protection. The present study aimed to evaluate the usefulness of DNA barcodes to support larval fish identification since conventional dichotomous keys based on morphological traits are not efficient due to the lack of larval traits and the rapid morphological changes during ontogeny. Our molecular analysis uncovered a total of 48 taxa (21 families) from the larval samples collected from the Klang Strait waters encompassing both spawning and nursery grounds of marine and estuarine fishes. Thirty-two (67%) of the larval taxa were identified at the species level, two taxa (4%) at the genus level, and 14 taxa (29%) at family level. The relatively low rate of species-level identification is not necessarily due to the DNA barcoding method per se, but a general lack of reference sequences for speciose and non- commercial fish families such as Gobiidae, Blenniidae, and Callionymidae. Larval morphology remains important in species diagnoses when molecular matches are ambiguous. A lower ethanol percentage (50%) for larva preservation is also useful to keep the body of larvae intact for morphological identification, and to preserve DNA for subsequent molecular analyses. The 10% Chelex resin used to extract DNA is also cost- effective for long term monitoring of larval fishes. Hence, the DNA barcoding method is an effective and easy way to aid the identification of estuarine larval fishes at the species level.

Live-Cell FRET Imaging Reveals a Role of Extracellular Signal-Regulated Kinase Activity Dynamics in Thymocyte Motility.

Extracellular signal-regulated kinase (ERK) plays critical roles in T cell development in the thymus. Nevertheless, the dynamics of ERK activity and the role of ERK in regulating thymocyte motility remain largely unknown due to technical limitations. To visualize ERK activity in thymocytes, we here developed knockin reporter mice expressing a Förster/fluorescence resonance energy transfer (FRET)-based biosensor for ERK from the ROSA26 locus. Live imaging of thymocytes isolated from the reporter mice revealed that ERK regulates thymocyte motility in a subtype-specific manner. Negative correlation between ERK activity and motility was observed in CD4/CD8 double-positive thymocytes and CD8 single-positive thymocytes, but not in CD4 single-positive thymocytes. Interestingly, however, the temporal deviations of ERK activity from the average correlate with the motility of CD4 single-positive thymocytes. Thus, live-cell FRET imaging will open a window to understanding the dynamic nature and the diverse functions of ERK signaling in T cell biology.

Anaplastic large cell lymphoma mimicking fibrosing mediastinitis.

Fibrosing mediastinitis is rare. One type of this disease is idiopathic fibrosing mediastinitis. It is necessary to rule out malignancy in order to accurately diagnose fibrosing mediastinitis. We herein report a case of anaplastic large cell lymphoma diagnosed three months after a preliminary diagnosis of fibrosing mediastinitis. Glucocorticoid therapy was not successful in controlling disease progression. Immediately after initiating chemotherapy for lymphoma, the patient's symptoms improved dramatically and the mediastinal lesion decreased in size. Although few similar cases have been reported, hidden malignancy may present as fibrosing mediastinitis. Therefore, physicians should consider the probability of malignancy in patients with fibrosing mediastinitis because treatments may vary accordingly.