Tumor-Associated CD19+CD39- B Regulatory Cells Deregulate Class-Switch Recombination to Suppress Antibody Responses.

B cells are an essential component of humoral immunity. Their primary function is to mount antigen-specific antibody responses to eliminate pathogens. Despite an increase in B-cell number, we found that serum-IgG levels were low in patients with breast cancer. To solve this conundrum, we used high-dimensional flow cytometry to analyze the heterogeneity of B-cell populations and identified a tumor-specific CD19+CD24hiCD38hi IL10-producing B regulatory (Breg)-cell subset. Although IL10 is a Breg-cell marker, being an intracellular protein, it is of limited value for Breg-cell isolation. Highly expressed Breg-cell surface proteins CD24 and CD38 also impede the isolation of viable Breg cells. These are hurdles that limit understanding of Breg-cell functions. Our transcriptomic analysis identified, CD39-negativity as an exclusive, sorting-friendly surface marker for tumor-associated Breg cells. We found that the identified CD19+CD39‒IL10+ B-cell population was suppressive in nature as it limited T helper-cell proliferation, type-1 cytokine production, and T effector-cell survival, and augmented CD4+FOXP3+ regulatory T-cell generation. These tumor-associated Breg cells were also found to restrict autologous T follicular helper-cell expansion and IL21 secretion, thereby inhibiting germinal transcript formation and activation-induced cytidine deaminase expression involved in H-chain class-switch recombination (CSR). This isotype-switching abnormality was shown to hinder B-cell differentiation into class-switched memory B cells and subsequent high-affinity antibody-producing plasma B cells, which collectively led to the dampening of IgG-mediated antibody responses in patients with cancer. As low IgG is associated with poor prognosis in patients with cancer, Breg-cell depletion could be a promising future therapy for boosting plasma B cell-mediated antibody responses.

FOXP3/HAT1 Axis Controls Treg Infiltration in the Tumor Microenvironment by Inducing CCR4 Expression in Breast Cancer.

Infiltrating T-regulatory cells in the tumor microenvironment is a key impediment to immunotherapy and is linked to a poor prognosis. We found that tumor-infiltrating Tregs express a higher expression of the chemokine receptor CCR4 than peripheral Tregs in breast cancer patients. CCL22 and CCL17 are released by tumor cells and tumor-associated macrophages, attracting CCR4+ Tregs to the tumor site. The Treg lineage-specific transcription factor FOXP3 changes the CCR4 promoter epigenetically in conjunction with HAT1 to provide a space for FOXP3 binding and activation of the CCR4 gene. To increase CCR4 expression in Tregs, the FOXP3/HAT1 axis is required for permissive (K23 and K27) or repressive (K14 and K18) acetylation of histone-3. In murine breast and melanoma tumor models, genetic ablation of FOXP3 reduced CCR4+ Treg infiltration and tumor size while also restoring anti-tumor immunity. Overexpression of FOXP3, on the other hand, increased CCR4+ Treg infiltration, resulting in a decreased anti-tumor immune response and tumor progression. These findings point to FOXP3 playing a new role in the tumor microenvironment as a transcriptional activator of CCR4 and a regulator of Treg infiltration.

Inhibition of mevalonate metabolism by statins augments the immunoregulatory phenotype of vascular endothelial cells and inhibits the costimulation of CD4+ T cells.

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.

GFI1/HDAC1-axis differentially regulates immunosuppressive CD73 in human tumor-associated FOXP3+ Th17 and inflammation-linked Th17 cells.

Plasticity between Th17 and Treg cells is regarded as a crucial determinant of tumor-associated immunosuppression. Classically Th17 cells mediate inflammatory responses through production of cytokine IL17. Recently, Th17 cells have also been shown to acquire suppressive phenotypes in tumor microenvironment. However, the mechanism by which they acquire such immunosuppressive properties is still elusive. Here, we report that in tumor microenvironment Th17 cell acquires immunosuppressive properties by expressing Treg lineage-specific transcription factor FOXP3 and ectonucleotidase CD73. We designate this cell as Th17reg cell and perceive that such immunosuppressive property is dependent on CD73. It was observed that in classical Th17 cell, GFI1 recruits HDAC1 to change the euchromatin into tightly-packed heterochromatin at the proximal-promoter region of CD73 to repress its expression. Whereas in Th17reg cells GFI1 cannot get access to CD73-promoter due to heterochromatin state at its binding site and, thus, cannot recruit HDAC1, failing to suppress the expression of CD73.

Transcriptional regulation of VEGFA expression in T-regulatory cells from breast cancer patients.

The initiation of new blood vessel formation (neo-angiogenesis) is one of the primary requirements for the establishment of tumor. As the tumor grows beyond a certain size, a hypoxic-condition arises in the inner core of tumor, triggering the release of chemokines, which attract T-regulatory (Treg) cells in the tumor-site. The presence of FOXP3, a lineage-specific transcription factor, expressing Treg cells in various types of tumor implements immunosuppressive and tumor-promoting strategies. One such strategy is the invitation of endothelial cells for neo-vascularization in the tumor site. Here we report that as the disease progresses, Treg cells from breast cancer patients are capable of secreting high-amount of VEGFA. The VEGFA promoter lacks Treg-specific transcription factor FOXP3 binding site. FOXP3 in association with locus-specific transcription factor STAT3 binds to VEGFA promoter to induce its transcription in Treg cells obtained from breast cancer patients. Treg cell-secreted VEGFA induces neo-angiogenesis from endothelial cells under in-vitro conditions. Targeting Tregs in mice with breast tumor reduces tumor growth as well as the level of neo-angiogenesis in the tumor tissue.

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.

Integrin-EGFR interaction regulates anoikis resistance in colon cancer cells.

Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, ß1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2ß1/-α5ß1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2ß1/-α5ß1 and EGFR in anoikis resistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

Sialolithiasis of Right Submandibular Duct of Unusual Size.

We present a case of a 35-year-old gentleman with a submandibular duct stone measuring 12 × 6 mm. Considering the literature, most stones are less than 5 mm, and stones more than 10 mm are quite unusual. This gentleman had typical symptoms of chronic sialadenitis, who was clinically diagnosed to have sialolithiasis, which was later confirmed by imaging studies. He was operated upon to remove the stone along with the submandibular gland. The term sialolithiasis is derived from the Greek words sialon (saliva) and lithos (stone), and the Latin -iasis meaning "process" or "morbid condition". Sialolithiasis affects the submandibular gland in 80-90% of cases because of the curved course of submandibular duct and the secretions being more mucous. Pain is the most common presenting feature during mastication and surgical removal of the sialolithiasis is the treatment of choice. The incision depends on the location of the stone in the duct.

Providence of the CD25+ KIR+ CD127- FOXP3- CD8+ T-cell subset determines the dynamics of tumor immune surveillance.

CD8+ T-regulatory (Treg) cells are emerging as crucial components of immune system. Previous studies have reported the presence of FOXP3+ CD8+ Treg cells, similar to CD4+ Tregs, in cancer patients which produce high levels of the immunosuppressive cytokines, IL10 and TGFß. At an early stage of tumor development, we have identified a subset of FOXP3- CD8+ CD25+ KIR+ CD127- Treg-like cells, which are IFNγ+ . However, this early-induced CD8+ CD25+ CD127- T-cell subset is certainly distinct from the IFNγ+ CD8+ T-effector cells. These CD8+ CD25+ CD127- T cells express other FOXP3- CD8+ Treg cell signature markers, and can selectively suppress autoreactive HLA-E+ TFH cells as well as tumor-induced CD4+ Treg cells. In contrast to FOXP3+ CD8+ Tregs, this subset does not inhibit effector T-cell proliferation or their functions as they are HLA-E- . Adoptive transfer of this early-CD8+ Treg-like subset restrained tumor growth and inhibited CD4+ Treg generation that impedes the immune surveillance and impairs cancer immunotherapy. At the late stage of tumor development, when CD4+ Treg cells dominate the tumor-microenvironment, CD4+ Tregs mediate the clonal deletion of these tumor-suppressive FOXP3- IFNγ+ CD8+ CD25+ CD127- T cells and ensure tumor immune evasion. Our findings suggest that at an early stage of the tumor, this tumor-induced IFNγ-producing FOXP3- CD8+ CD25+ CD127- T-cell subset can potentiate immune surveillance by targeting HLA-E-restricted CD4+ Treg cells while leaving the effector T-cell population unaffected. Hence, manipulating their profile can open up a new avenue in cancer immunotherapy.

Quantitative assessment of protein activity in orphan tissues and single cells using the metaVIPER algorithm.

We and others have shown that transition and maintenance of biological states is controlled by master regulator proteins, which can be inferred by interrogating tissue-specific regulatory models (interactomes) with transcriptional signatures, using the VIPER algorithm. Yet, some tissues may lack molecular profiles necessary for interactome inference (orphan tissues), or, as for single cells isolated from heterogeneous samples, their tissue context may be undetermined. To address this problem, we introduce metaVIPER, an algorithm designed to assess protein activity in tissue-independent fashion by integrative analysis of multiple, non-tissue-matched interactomes. This assumes that transcriptional targets of each protein will be recapitulated by one or more available interactomes. We confirm the algorithm's value in assessing protein dysregulation induced by somatic mutations, as well as in assessing protein activity in orphan tissues and, most critically, in single cells, thus allowing transformation of noisy and potentially biased RNA-Seq signatures into reproducible protein-activity signatures.

Scabbard Trachea in a Case of Retrosternal Goitre.

Scabbard is the sheath used for covering swords, knives or other large blades. A scabbard trachea is called so because of its resemblance of shape (Fig. 1) caused by lateral compression usually by thyroid enlargement. Here we present such a case depicting the radiological findings of a scabbard trachea.

Transcriptional regulation of FOXP3 requires integrated activation of both promoter and CNS regions in tumor-induced CD8+ Treg cells.

T-regulatory cells are an upsurge in the tumor microenvironment and induce immune-evasion. CD4+ Treg cells are well characterized whereas the role of CD8+ Tregs in cancer has recently started to crease attention. Here, we report an augmentation CD8+FOXP3+ Tregs in breast tumor microenvironment. FOXP3, the lineage-specific transcription factor, is a dominant regulator of Treg cell development and function. FOXP3 is induced preferentially by divergent signaling in CD4+ Treg cells. But how FOXP3 is induced and maintained in tumor-CD8+ Tregs is the Cinderella of the investigation. We observed that RUNX3, a CD8+ lineage-specific transcription factor, binds at the FOXP3-promoter to induce its transcription. In addition to promoter activation, involvement of cis-elements CNS1 and CNS2 in the transcriptional regulation of FOXP3 was also evident in these cells. SMAD3 binds to CNS1 region and acts as transcription inducer, whereas GATA3 plays a temporal role in the FOXP3 transcription by differential chromatin modification in CNS regions. In CNS1 region, GATA3 acts as a repressor for FOXP3 in naïve CD8+ T cells. Whereas in CD8+ Tregs, GATA3 binds directly at CNS2 region and persuaded the maintenance of FOXP3. Therefore, the intervention of these concerted transcriptional machinery may have a therapeutic potential in immunotherapy of cancer.

Intermediate filaments enable pathogen docking to trigger type 3 effector translocation.

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.

Curcumin and tumor immune-editing: resurrecting the immune system.

Curcumin has long been known to posses medicinal properties and recent scientific studies have shown its efficacy in treating cancer. Curcumin is now considered to be a promising anti-cancer agent and studies continue on its molecular mechanism of action. Curcumin has been shown to act in a multi-faceted manner by targeting the classical hallmarks of cancer like sustained proliferation, evasion of apoptosis, sustained angiogenesis, insensitivity to growth inhibitors, tissue invasion and metastasis etc. However, one of the emerging hallmarks of cancer is the avoidance of immune system by tumors. Growing tumors adopt several strategies to escape immune surveillance and successfully develop in the body. In this review we highlight the recent studies that show that curcumin also targets this process and helps restore the immune activity against cancer. Curcumin mediates several processes like restoration of CD4(+)/CD8(+) T cell populations, reversal of type-2 cytokine bias, reduction of Treg cell population and suppression of T cell apoptosis; all these help to resurrect tumor immune surveillance that leads to tumor regression. Thus interaction of curcumin with the immune system is also an important feature of its multi-faceted modes of action against cancer. Finally, we also point out the drawbacks of and difficulties in curcumin administration and indicate the use of nano-formulations of curcumin for better therapeutic efficacy.

Scalable microfluidics for single-cell RNA printing and sequencing.

Many important biological questions demand single-cell transcriptomics on a large scale. Hence, new tools are urgently needed for efficient, inexpensive manipulation of RNA from individual cells. We report a simple platform for trapping single-cell lysates in sealed, picoliter microwells capable of printing RNA on glass or capturing RNA on beads. We then develop a scalable technology for genome-wide, single-cell RNA-Seq. Our device generates pooled libraries from hundreds of individual cells with consumable costs of $0.10-$0.20 per cell and includes five lanes for simultaneous experiments. We anticipate that this system will serve as a general platform for single-cell imaging and sequencing.

Timing is everything: Fine-tuned molecular machines orchestrate paramyxovirus entry.

The Paramyxoviridae include some of the great and ubiquitous disease-causing viruses of humans and animals. In most paramyxoviruses, two viral membrane glycoproteins, fusion protein (F) and receptor binding protein (HN, H or G) mediate a concerted process of recognition of host cell surface molecules followed by fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. The interactions between the F and HN, H or G viral glycoproteins and host molecules are critical in determining host range, virulence and spread of these viruses. Recently, atomic structures, together with biochemical and biophysical studies, have provided major insights into how these two viral glycoproteins successfully interact with host receptors on cellular membranes and initiate the membrane fusion process to gain entry into cells. These studies highlight the conserved core mechanisms of paramyxovirus entry that provide the fundamental basis for rational anti-viral drug design and vaccine development.

Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells.

UNLABELLED: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. IMPORTANCE: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.