Toripalimab, a therapeutic monoclonal anti-PD-1 antibody with high binding affinity to PD-1 and enhanced potency to activate human T cells.

Over the past decade, US Food and Drug Administration (FDA)-approved immune checkpoint inhibitors that target programmed death-1 (PD-1) have demonstrated significant clinical benefit particularly in patients with PD-L1 expressing tumors. Toripalimab is a humanized anti-PD-1 antibody, approved by FDA for first-line treatment of nasopharyngeal carcinoma in combination with chemotherapy. In a post hoc analysis of phase 3 studies, toripalimab in combination with chemotherapy improved overall survival irrespective of PD-L1 status in nasopharyngeal carcinoma (JUPITER-02), advanced non-small cell lung cancer (CHOICE-01) and advanced esophageal squamous cell carcinoma (JUPITER-06). On further characterization, we determined that toripalimab is molecularly and functionally differentiated from pembrolizumab, an anti-PD-1 mAb approved previously for treating a wide spectrum of tumors. Toripalimab, which binds the FG loop of PD-1, has 12-fold higher binding affinity to PD-1 than pembrolizumab and promotes significantly more Th1- and myeloid-derived inflammatory cytokine responses in healthy human PBMCs in vitro. In an ex vivo system employing dissociated tumor cells from treatment naïve non-small cell lung cancer patients, toripalimab induced several unique genes in IFN-γ and immune cell pathways, showed different kinetics of activation and significantly enhanced IFN-γ signature. Additionally, binding of toripalimab to PD-1 induced lower levels of SHP1 and SHP2 recruitment, the negative regulators of T cell activation, in Jurkat T cells ectopically expressing PD-1. Taken together, these data demonstrate that toripalimab is a potent anti-PD-1 antibody with high affinity PD-1 binding, strong functional attributes and demonstrated clinical activity that encourage its continued clinical investigation in several types of cancer.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.

Cortical softening elicits zygotic contractility during mouse preimplantation development.

Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the second cleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20- to 60-µm range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. We further identify the programmed down-regulation of the formin Fmnl3 as a required event to soften the cortex and expose PeCoWaCo. Therefore, during cleavage stages, cortical softening, mediated by Fmnl3 down-regulation, awakens zygotic contractility before preimplantation morphogenesis.

Rapid and robust optogenetic control of gene expression in Drosophila.

Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. Here we describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. We show that it allows efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, we illustrate how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions.

IL-1R1-dependent signaling coordinates epithelial regeneration in response to intestinal damage.

Repair of the intestinal epithelium is tightly regulated to maintain homeostasis. The response after epithelial damage needs to be local and proportional to the insult. How different types of damage are coupled to repair remains incompletely understood. We report that after distinct types of intestinal epithelial damage, IL-1R1 signaling in GREM1+ mesenchymal cells increases production of R-spondin 3 (RSPO3), a Wnt agonist required for intestinal stem cell self-renewal. In parallel, IL-1R1 signaling regulates IL-22 production by innate lymphoid cells and promotes epithelial hyperplasia and regeneration. Although the regulation of both RSPO3 and IL-22 is critical for epithelial recovery from Citrobacter rodentium infection, IL-1R1-dependent RSPO3 production by GREM1+ mesenchymal cells alone is sufficient and required for recovery after dextran sulfate sodium-induced colitis. These data demonstrate how IL-1R1-dependent signaling orchestrates distinct repair programs tailored to the type of injury sustained that are required to restore intestinal epithelial barrier function.

Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

SPEN integrates transcriptional and epigenetic control of X-inactivation.

Xist represents a paradigm for the function of long non-coding RNA in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Several proteins that bind to Xist RNA have recently been identified, including the transcriptional repressor SPEN1-3, the loss of which has been associated with deficient XCI at multiple loci2-6. Here we show in mice that SPEN is a key orchestrator of XCI in vivo and we elucidate its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN is dispensable for maintenance of XCI in neural progenitors, although it significantly decreases the expression of genes that escape XCI. We show that SPEN is immediately recruited to the X chromosome upon the upregulation of Xist, and is targeted to enhancers and promoters of active genes. SPEN rapidly disengages from chromatin upon gene silencing, suggesting that active transcription is required to tether SPEN to chromatin. We define the SPOC domain as a major effector of the gene-silencing function of SPEN, and show that tethering SPOC to Xist RNA is sufficient to mediate gene silencing. We identify the protein partners of SPOC, including NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery-as well as with nucleosome remodellers and histone deacetylases-at active enhancers and promoters.

Distinct Mesenchymal Cell Populations Generate the Essential Intestinal BMP Signaling Gradient.

Intestinal stem cells (ISCs) are confined to crypt bottoms and their progeny differentiate near crypt-villus junctions. Wnt and bone morphogenic protein (BMP) gradients drive this polarity, and colorectal cancer fundamentally reflects disruption of this homeostatic signaling. However, sub-epithelial sources of crucial agonists and antagonists that organize this BMP gradient remain obscure. Here, we couple whole-mount high-resolution microscopy with ensemble and single-cell RNA sequencing (RNA-seq) to identify three distinct PDGFRA+ mesenchymal cell types. PDGFRA(hi) telocytes are especially abundant at the villus base and provide a BMP reservoir, and we identified a CD81+ PDGFRA(lo) population present just below crypts that secretes the BMP antagonist Gremlin1. These cells, referred to as trophocytes, are sufficient to expand ISCs in vitro without additional trophic support and contribute to ISC maintenance in vivo. This study reveals intestinal mesenchymal structure at fine anatomic, molecular, and functional detail and the cellular basis for a signaling gradient necessary for tissue self-renewal.

Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling.

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion-they can also shape the fate and function of CD8+ T cells.

MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules.

Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present MTrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. MTrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. The application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. This helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios.

Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells.

The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. We demonstrate here that the transcriptional regulator Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. We observed that Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells. Ablation of the Hes1 oscillator in stem cells interfered with stable MyoD oscillations and led to prolonged periods of sustained MyoD expression, resulting in increased differentiation propensity. This interfered with the maintenance of activated muscle stem cells, and impaired muscle growth and repair. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state and is required for amplification of the activated stem cell pool.

Cutting Edge: Early Attrition of Memory T Cells during Inflammation and Costimulation Blockade Is Regulated Concurrently by Proapoptotic Proteins Fas and Bim.

Apoptosis of CD8 T cells is an essential mechanism that maintains immune system homeostasis, prevents autoimmunity, and reduces immunopathology. CD8 T cell death also occurs early during the response to both inflammation and costimulation blockade (CoB). In this article, we studied the effects of a combined deficiency of Fas (extrinsic pathway) and Bim (intrinsic pathway) on early T cell attrition in response to lymphocytic choriomeningitis virus infection and during CoB during transplantation. Loss of Fas and Bim function in Bcl2l11-/-Faslpr/lpr mice inhibited apoptosis of T cells and prevented the early T cell attrition resulting from lymphocytic choriomeningitis virus infection. Bcl2l11-/-Faslpr/lpr mice were also resistant to prolonged allograft survival induced by CoB targeting the CD40-CD154 pathway. These results demonstrate that both extrinsic and intrinsic apoptosis pathways function concurrently to regulate T cell homeostasis during the early stages of immune responses and allograft survival during CoB.

Testosterone is an endogenous regulator of BAFF and splenic B cell number.

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.

Transient expression of ZBTB32 in anti-viral CD8+ T cells limits the magnitude of the effector response and the generation of memory.

Virus infections induce CD8+ T cell responses comprised of a large population of terminal effector cells and a smaller subset of long-lived memory cells. The transcription factors regulating the relative expansion versus the long-term survival potential of anti-viral CD8+ T cells are not completely understood. We identified ZBTB32 as a transcription factor that is transiently expressed in effector CD8+ T cells. After acute virus infection, CD8+ T cells deficient in ZBTB32 showed enhanced virus-specific CD8+ T cell responses, and generated increased numbers of virus-specific memory cells; in contrast, persistent expression of ZBTB32 suppressed memory cell formation. The dysregulation of CD8+ T cell responses in the absence of ZBTB32 was catastrophic, as Zbtb32-/- mice succumbed to a systemic viral infection and showed evidence of severe lung pathology. We found that ZBTB32 and Blimp-1 were co-expressed following CD8+ T cell activation, bound to each other, and cooperatively regulated Blimp-1 target genes Eomes and Cd27. These findings demonstrate that ZBTB32 is a key transcription factor in CD8+ effector T cells that is required for the balanced regulation of effector versus memory responses to infection.

Secondary jaw aneurysmal bone cyst (JABC)--a possible misnomer? A review of literature on secondary JABCs, their pathogenesis and oncogenesis.

CONTEXT: Aneurysmal bone cysts are rare pseudocysts, commonly seen in long bones and vertebral column. Although a well described and reported lesion, many misconceptions still prevail regarding their etiopathogenesis. Many of the reported cases of jaw aneurysmal bone cysts (JABC) present with another bone pathology. AIMS: The purpose of this review was to evaluate the incidence of neoplastic lesions occurring simultaneously with a JABC (in contrast to primary JABCs). Any pathogenetic and oncogenetic association between primary and secondary jaw ABCs has been reviewed and discussed. SETTINGS AND DESIGN: A methodical narrative review of literature was performed, given the incidence of mostly case reports on this topic. METHODS AND MATERIAL: A methodical electronic search of Pubmed, Pubmed Central, Medline and Cochrane databases was performed for reported cases of JABC. These articles were analysed and segregated into primary and secondary ABC and, if secondary, the lesion it concurrently occurred with. Another search was conducted to yield articles discussing the cytopathogenetic and oncogenetic origins of ABCs. RESULTS AND CONCLUSIONS: About 15% of the ABCs reported were of secondary nature. Amongst the associated lesions, cement-ossifying fibroma and ossifying fibroma were the most common, followed by fibrous dysplasia and central giant cell granuloma. No ABCs were associated with metastatic changes. The search for histopathogenesis pointed to a specific cytogenetic abnormality as the origin of primary ABCs, with USP6 as its main oncogene and spindle cell as the neoplastic cell, unlike with secondary ABCs, suggesting that they are distinct pathological processes.

Regulation of tissue-dependent differences in CD8+ T cell apoptosis during viral infection.

UNLABELLED: Virus-specific CD8+ T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in nonlymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the nonlymphoid tissues may enhance protection to secondary challenges. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells in nonlymphoid tissues were enriched for memory precursors (expressing high levels of interleukin-7 receptor and low levels of killer cell lectin-like receptor G1 [IL-7Rhi KLRG1lo]) and had higher expression of CD27, CXCR3, and T cell factor-1 (TCF-1), each a marker that is individually correlated with decreased apoptosis. CD8+ T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the nonlymphoid tissues. CXCR3+ CD8+ T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PEC) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these nonapoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8+ T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8+ T cells. Thus, intrinsic stability of T cells which are present in the nonlymphoid tissues along with preferential migration of apoptosis-resistant CD8+ T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences. IMPORTANCE: Most infections are initiated at nonlymphoid tissue sites, and the presence of memory T cells in nonlymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8+ T cells in the nonlymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here, we investigated the mechanisms promoting stability of T cells in the nonlymphoid tissues. This increased resistance to apoptosis of virus-specific CD8+ T cells in nonlymphoid tissues was due to several factors. Nonlymphoid tissues were enriched in memory phenotype CD8+ T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8+ T cells preferentially migrated into the nonlymphoid tissues, where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long-lasting vaccines providing protection at peripheral infection sites.

A clinical study of cysts of the maxillofacial region; and an assessment of clinico-radiologico-pathological variables affecting the formulation of a comprehensive patient need based treatment plan.

OBJECTIVES: Evaluation of clinical presentation of cystic lesions of the maxillofacial region, their relation to radiological picture, and treatment planning so as to plan and execute a patient need based treatment modality after co-relating it to the eventual histopathological diagnosis. METHODS: 25 cases with clinico-radiological diagnosis of a cyst were selected and treated, and the diagnosis was co-related to the eventual histopathological diagnosis. The patients were followed up for at least 3 months (3-12 months). An attempt was made to underline patient and lesion related variables having a bearing on the choice of treatment modality in each case. RESULTS: Out of 25 patients, 28% were females and 72% males. Commonly affected age groups were 11-20 (40%) and 31-40 years (24%). 76% of 25 patients complained of swelling on the first visit. 96% lesions were related to jaws, of which 15 were in the mandible and 9 were in the maxilla. 58.33% bone lesions had cortical expansion. 92% lesions were confirmed to be cysts histopathologically. Two were ameloblastomas. 80% patients underwent enucleation with various adjunctive procedures. 32% patients faced temporary post operative complications. No recurrences were observed. Radiological presentation of lesions and patient's age were found to be the two most important radiological and clinical variables affecting treatment planning. CONCLUSIONS: A comprehensive patient need based treatment plan can be reached only after taking various patient and lesion related variables (which may manifest as clinical, radiological or histological parameters) into consideration.

Epigenetic modifications induced by Blimp-1 Regulate CD8⁺ T cell memory progression during acute virus infection.

The transcription factor Blimp-1 regulates the overall accumulation of virus-specific CD8⁺ T cells during acute viral infections. We found that increased proliferation and survival of Blimp-1-deficient CD8⁺ T cells resulted from sustained expression of CD25 and CD27 and persistent cytokine responsiveness. Silencing of Il2ra and Cd27 reduced the Blimp-1-deficient CD8⁺ T cell response. Genome-wide chromatin immunoprecipitation (ChIP) sequencing analysis identified Il2ra and Cd27 as direct targets of Blimp-1. At the peak of the antiviral response, but not earlier, Blimp-1 recruited the histone-modifying enzymes G9a and HDAC2 to the Il2ra and Cd27 loci, thereby repressing expression of these genes. In the absence of Blimp-1, Il2ra and Cd27 exhibited enhanced histone H3 acetylation and reduced histone H3K9 trimethylation. These data elucidate a central mechanism by which Blimp-1 acts as an epigenetic regulator and enhances the numbers of short-lived effector cells while suppressing the development of memory-precursor CD8⁺ T cells.

Partition of Myc into immobile vs. mobile complexes within nuclei.

Myc levels are highly regulated and usually low in vivo. Dimerized with Max, it regulates most expressed genes and so directly and indirectly controls most cellular processes. Intranuclear diffusion of a functional c-Myc-eGFP, expressed from its native locus in murine fibroblasts and 3T3 cells or by transient transfection, was monitored using Two Photon Fluorescence Correlation Spectroscopy, revealing concentration and size (mobility) of complexes. With increased c-Myc-eGFP, a very immobile pool saturates as a 'mobile' pool increases. Both pools diffuse too slowly to be free Myc-Max dimers. Following serum stimulation, eGFP-c-Myc accumulated in the presence of the proteasome inhbitor MG132. Stimulating without MG132, Myc peaked at 2.5 hrs, and at steady was ~8 ± 1.3 nM. Inhbiting Myc-Max dimerization by Max-knockdown or drug treatment increased the 'mobile' c-Myc pool size. These results indicate that Myc populates macromolecular complexes of widely heterogenous size and mobility in vivo.