Predictive and Prognostic Implications of Circulating CX3CR1+ CD8+ T Cells in Non-Small Cell Lung Cancer Patients Treated with Chemo-Immunotherapy.

Lack of reliable predictive biomarkers is a major limitation of combination therapy with chemotherapy and anti-programmed cell death protein 1/programmed death-ligand 1 (anti-PD-1/PD-L1) therapy (chemo-immunotherapy). We previously observed that the increase of peripheral blood CD8+ T cells expressing CX3CR1, a marker of differentiation, correlates with response to anti-PD-1 therapy; however, the predictive and prognostic value of T-cell CX3CR1 expression during chemo-immunotherapy is unknown. Here, we evaluated the utility of circulating CX3CR1+CD8+ T cells as a predictive correlate of response to chemo-immunotherapy in patients with non-small cell lung cancer (NSCLC). At least 10% increase of the CX3CR1+ subset in circulating CD8+ T cells from baseline (CX3CR1 score) was associated with response to chemo-immunotherapy as early as 4 weeks with 85.7% overall accuracy of predicting response at 6 weeks. Furthermore, at least 10% increase of the CX3CR1 score correlated with substantially better progression-free (P = 0.0051) and overall survival (P = 0.0138) on Kaplan-Meier analysis. Combined single-cell RNA/T-cell receptor (TCR) sequencing of circulating T cells from longitudinally obtained blood samples and TCR sequencing of tumor tissue from the same patient who received a long-term benefit from the treatment demonstrated remarkable changes in genomic and transcriptomic signatures of T cells as well as evolution of TCR clonotypes in peripheral blood containing highly frequent tumor-infiltrating lymphocyte repertoires overexpressing CX3CR1 early after initiation of the treatment despite stable findings of the imaging study. Collectively, these findings highlight the potential utility of T-cell CX3CR1 expression as a dynamic blood-based biomarker during the early course of chemo-immunotherapy and a marker to identify frequent circulating tumor-infiltrating lymphocyte repertoires. Significance: Current approaches to combined chemotherapy and anti-PD-1/PD-L1 therapy (chemo-immunotherapy) for patients with NSCLC are limited by the lack of reliable predictive biomarkers. This study shows the utility of T-cell differentiation marker, CX3CR1, as an early on-treatment predictor of response and changes in genomic/transcriptomic signatures of circulating tumor-infiltrating lymphocyte repertoires in patients with NSCLC undergoing chemo-immunotherapy.

CD40 and CD80/86 signaling in cDC1s mediate effective neoantigen vaccination and generation of antigen-specific CX3CR1+ CD8+ T cells.

The use of tumor mutation-derived neoantigen represents a promising approach for cancer vaccines. Preclinical and early phase human clinical studies have shown the successful induction of tumor neoepitope-directed responses; however, overall clinical efficacy of neoantigen vaccines has been limited. One major obstacle of this strategy is the prevailing lack of sufficient understanding of the mechanism underlying the generation of neoantigen-specific CD8+ T cells. Here, we report a correlation between antitumor efficacy of neoantigen/toll-like receptor 3 (TLR3)/CD40 agonists vaccination and an increased frequency of circulating antigen-specific CD8+ T cells expressing CX3C chemokine receptor 1 (CX3CR1) in a preclinical model. Mechanistic studies using mixed bone marrow chimeras identified that CD40 and CD80/86, but not CD70 signaling in Batf3-dependent conventional type 1 dendritic cells (cDC1s) is required for the antitumor efficacy of neoantigen vaccine and generation of neoantigen-specific CX3CR1+ CD8+ T cells. Although CX3CR1+ CD8+ T cells exhibited robust in vitro effector function, in vivo depletion of this subset did not alter the antitumor efficacy of neoantigen/TLR3/CD40 agonists vaccination. These findings indicate that the vaccine-primed CX3CR1+ subset is dispensable for antitumor CD8+ T cell responses, but can be used as a blood-based T-cell biomarker for effective priming of CD8+ T cells as post-differentiated T cells. Taken together, our results reveal a critical role of CD40 and CD80/86 signaling in cDC1s in antitumor efficacy of neoantigen-based therapeutic vaccines, and implicate the potential utility of CX3CR1 as a circulating predictive T-cell biomarker in vaccine therapy.

T-cell CX3CR1 expression as a dynamic blood-based biomarker of response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICI) have revolutionized treatment for various cancers; however, durable response is limited to only a subset of patients. Discovery of blood-based biomarkers that reflect dynamic change of the tumor microenvironment, and predict response to ICI, will markedly improve current treatment regimens. Here, we investigate CX3C chemokine receptor 1 (CX3CR1), a marker of T-cell differentiation, as a predictive correlate of response to ICI therapy. Successful treatment of tumor-bearing mice with ICI increases the frequency and T-cell receptor clonality of the peripheral CX3CR1+CD8+ T-cell subset that includes an enriched repertoire of tumor-specific and tumor-infiltrating CD8+ T cells. Furthermore, an increase in the frequency of the CX3CR1+ subset in circulating CD8+ T cells early after initiation of anti-PD-1 therapy correlates with response and survival in patients with non-small cell lung cancer. Collectively, these data support T-cell CX3CR1 expression as a blood-based dynamic early on-treatment predictor of response to ICI therapy.

Glyco-Nanoadjuvants: Sugar Structures on Carriers of a Small Molecule TLR7 Ligand Affect Their Immunostimulatory Activities.

Toll-like receptors (TLRs) are pattern recognition receptors that activate innate immunity, and their ligands are promising adjuvants for vaccines and immunotherapies. Small molecule TLR7 ligands are ideal vaccine adjuvants as they induce not only proinflammatory cytokines but also type I interferons. However, their application has only been approved for local administration due to severe systemic immune-related adverse events. In a previous study, we prepared the gold nanoparticles coimmobilized with synthetic small molecule TLR7 ligand, 1V209, and α-mannose (1V209-αMan-GNPs). 1V209-αMan-GNPs were selectively delivered via a cell surface sugar-binding protein, mannose receptor, which enabled selective delivery of TLR7 ligands to immune cells. Besides the mannose receptor, immune cells express various sugar-binding proteins such as macrophage galactose binding lectins and sialic acid-binding immunoglobulin-type lectins and recognize distinct sugar structures. Hence, in the present study, we investigated whether sugar structures on GNPs affect the efficiency and selectivity of intracellular delivery and subsequent immunostimulatory potencies. Five neutral sugars and two sialosides were selected and each sugar was coimmobilized with 1V209 onto GNPs (1V209-SGNPs) and their innate immunostimulatory potencies were compared to that of 1V209-αMan-GNPs. The in vitro study using mouse bone marrow derived dendritic cells (BMDCs) demonstrated that α-glucose, α-N-acetylglucosamine, or α-fucose immobilized 1V209-SGNPs increased interleukin-6 and type I interferon release similar to that of 1V209-αMan-GNPs, whereas galacto-type sugar immobilized 1V209-SGNPs predominantly enhanced type I interferon release. In contrast, sialoside immobilized 1V209-SGNPs did not enhance the potency of 1V209. In the in vivo immunization study using ovalbumin as a model antigen, neutral sugar immobilized 1V209-SGNPs induced comparable T helper-1 immune response to that of 1V209-αMan-GNPs and by 10-fold higher than that of sialoside immobilized 1V209-SGNPs. These results indicate that the sugar structures on 1V209-SGNPs affect their immunostimulatory activities, and functionalization of the carrier particles is important to shape immune responses.

The Yin and Yang of tumour-derived extracellular vesicles in tumour immunity.

Extracellular vesicles (EVs) are small particles that are naturally released from various types of cells. EVs contain a wide variety of cellular components, such as proteins, nucleic acids, lipids and metabolites, which facilitate intercellular communication in diverse biological processes. In the tumour microenvironment, EVs have been shown to play important roles in tumour progression, including immune system-tumour interactions. Although previous studies have convincingly demonstrated the immunosuppressive functions of tumour-derived EVs, some studies have suggested that tumour-derived EVs can also stimulate host immunity, especially in therapeutic conditions. Recent studies have revealed the heterogeneous nature of EVs with different structural and biological characteristics that may account for the divergent functions of EVs in tumour immunity. In this review article, we provide a brief summary of our current understanding of tumour-derived EVs in immune activation and inhibition. We also highlight the emerging utility of EVs in the diagnosis and treatment of cancers and discuss the potential clinical applications of tumour-derived EVs.

A Critical Role of CD40 and CD70 Signaling in Conventional Type 1 Dendritic Cells in Expansion and Antitumor Efficacy of Adoptively Transferred Tumor-Specific T Cells.

In vivo expansion of adoptively transferred CD8+ T cells is a critical determinant of successful adoptive T cell therapy. Emerging evidence indicates Batf3-dependent conventional type 1 dendritic cells (cDC1s) rarely found within the tumor myeloid compartment are crucial for effector T cell recruitment to the tumor microenvironment. However, the role of cDC1s in expansion of tumor-specific CD8+ T cells remains unclear. In this article, we addressed the role of cDC1s and their costimulatory molecules, CD40, CD70, and CD80/CD86, in expansion and antitumor efficacy of adoptively transferred in vitro-primed CD8+ T cells recognizing nonmutated tumor-associated self-antigens. We found that TLR/CD40-mediated expansion and antitumor efficacy of adoptively transferred tumor-specific CD8+ T cells were abrogated in Batf3-/- mice. Further mechanistic studies using mixed bone marrow chimeric mice identified that CD40 and CD70 but not CD80/CD86 signaling in cDC1s played a critical role in expansion and antitumor efficacy of adoptively transferred CD8+ T cells. Moreover, induction and activation of cDC1s by administration of FMS-like tyrosine kinase 3 ligand (Flt3L) and TLR/CD40 agonists augmented expansion of adoptively transferred CD8+ T cells, delayed tumor growth, and improved survival. These findings reveal a key role for CD40 and CD70 signaling in cDC1s and have major implications for the design of new vaccination strategies with adoptive T cell therapy.

CX3CR1-CD8+ T cells are critical in antitumor efficacy but functionally suppressed in the tumor microenvironment.

Although blockade of the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint has revolutionized cancer treatment, how it works on tumor-infiltrating CD8+ T cells recognizing the same antigen at various differentiation stages remains elusive. Here, we found that the chemokine receptor CX3CR1 identified 3 distinct differentiation states of intratumor CD8+ T cell subsets. Adoptively transferred antigen-specific CX3CR1-CD8+ T cells generated phenotypically and functionally distinct CX3CR1int and CX3CR1hi subsets in the periphery. Notably, expression of coinhibitory receptors and T cell factor 1 (Tcf1) inversely correlated with the degree of T cell differentiation defined by CX3CR1. Despite lower expression of coinhibitory receptors and potent cytolytic activity, in vivo depletion of the CX3CR1hi subset did not alter the antitumor efficacy of adoptively transferred CD8+ T cells. Furthermore, differentiated CX3CR1int and CX3CR1hi subsets were impaired in their ability to undergo proliferation upon restimulation and had no impact on established tumors upon second adoptive transfer compared with the CX3CR1- subset that remained effective. Accordingly, anti-PD-L1 therapy preferentially rescued proliferation and cytokine production of the CX3CR1- subset and enhanced antitumor efficacy of adoptively transferred CD8+ T cells. These findings provide a better understanding of the phenotypic and functional heterogeneity of tumor-infiltrating CD8+ T cells and can be exploited to develop more effective immunotherapy.

Hippo Pathway in Mammalian Adaptive Immune System.

The Hippo pathway was originally identified as an evolutionarily-conserved signaling mechanism that contributes to the control of organ size. It was then rapidly expanded as a key pathway in the regulation of tissue development, regeneration, and cancer pathogenesis. The increasing amount of evidence in recent years has also connected this pathway to the regulation of innate and adaptive immune responses. Notably, the Hippo pathway has been revealed to play a pivotal role in adaptive immune cell lineages, as represented by the patients with T- and B-cell lymphopenia exhibiting defective expressions of the pathway component. The complex regulatory mechanisms of and by the Hippo pathway have also been evident as alternative signal transductions are employed in some immune cell types. In this review article, we summarize the current understanding of the emerging roles of the Hippo pathway in adaptive immune cell development and differentiation. We also highlight the recent findings concerning the dual functions of the Hippo pathway in autoimmunity and anti-cancer immune responses and discuss the key open questions in the interplay between the Hippo pathway and the mammalian immune system.

Induced Pluripotent Stem Cell-Derived T Cells for Cancer Immunotherapy.

Adoptive T cell therapy for solid malignancies is limited because obtaining sufficient numbers of less-differentiated tumor-specific T cells is difficult. This roadblock can be theoretically overcome by the use of induced pluripotent stem cells (iPSCs), which self-renew and provide unlimited numbers of autologous less-differentiated T cells. iPSCs can generate less-differentiated antigen-specific T cells that harbor long telomeres and increased proliferative capacity, and exhibit potent antitumor efficacy. Although this strategy holds great promise for adoptive T cell therapy, highly reproducible and robust differentiation protocols are required before the translation of iPSC technology into the clinical setting.

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

FBXL5 Inactivation in Mouse Brain Induces Aberrant Proliferation of Neural Stem Progenitor Cells.

FBXL5 is the substrate recognition subunit of an SCF-type ubiquitin ligase that serves as a master regulator of iron metabolism in mammalian cells. We previously showed that mice with systemic deficiency of FBXL5 fail to sense intracellular iron levels and die in utero at embryonic day 8.5 (E8.5) as a result of iron overload and subsequent oxidative stress. This early embryonic mortality has thus impeded study of the role of FBXL5 in brain development. We have now generated mice lacking FBXL5 specifically in nestin-expressing neural stem progenitor cells (NSPCs) in the brain. Unexpectedly, the mutant embryos manifested a progressive increase in the number of NSPCs and astroglia in the cerebral cortex. Stabilization of iron regulatory protein 2 (IRP2) as a result of FBXL5 deficiency led to accumulation of ferrous and ferric iron as well as to generation of reactive oxygen species. Pharmacological manipulation suggested that the phenotypes of FBXL5 deficiency are attributable to aberrant activation of mammalian target of rapamycin (mTOR) signaling. Our results thus show that FBXL5 contributes to regulation of NSPC proliferation during mammalian brain development.

An overview of mammalian pluripotency.

Mammalian pluripotency is the ability to give rise to all somatic cells as well as the germ cells of an adult mammal. It is a unique feature of embryonic epiblast cells, existing only transiently, as cells pass through early developmental stages. By contrast, pluripotency can be captured and stabilized indefinitely in cell culture and can also be reactivated in differentiated cells via nuclear reprogramming. Pluripotent stem cells (PSCs) are the in vitro carriers of pluripotency and they can inhabit discrete pluripotent states depending on the stage at which they were derived and their culture conditions. Here, and in the accompanying poster, we provide a summary of mammalian pluripotency both in vivo and in vitro, and highlight recent and future applications of PSCs for basic and translational research.

MDM2 mediates nonproteolytic polyubiquitylation of the DEAD-Box RNA helicase DDX24.

MDM2 mediates the ubiquitylation and thereby triggers the proteasomal degradation of the tumor suppressor protein p53. However, genetic evidence suggests that MDM2 contributes to multiple regulatory networks independently of p53 degradation. We have now identified the DEAD-box RNA helicase DDX24 as a nucleolar protein that interacts with MDM2. DDX24 was found to bind to the central region of MDM2, resulting in the polyubiquitylation of DDX24 both in vitro and in vivo. Unexpectedly, however, the polyubiquitylation of DDX24 did not elicit its proteasomal degradation but rather promoted its association with preribosomal ribonucleoprotein (pre-rRNP) processing complexes that are required for the early steps of pre-rRNA processing. Consistently with these findings, depletion of DDX24 in cells impaired pre-rRNA processing and resulted both in abrogation of MDM2 function and in consequent p53 stabilization. Our results thus suggest an unexpected role of MDM2 in the nonproteolytic ubiquitylation of DDX24, which may contribute to the regulation of pre-rRNA processing.

HERC2 targets the iron regulator FBXL5 for degradation and modulates iron metabolism.

FBXL5 (F-box and leucine-rich repeat protein 5) is the F-box protein subunit of, and therefore responsible for substrate recognition by, the SCF(FBXL5) ubiquitin-ligase complex, which targets iron regulatory protein 2 (IRP2) for proteasomal degradation. IRP2 plays a central role in the maintenance of cellular iron homeostasis in mammals through posttranscriptional regulation of proteins that contribute to control of the intracellular iron concentration. The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo, given that mice lacking FBXL5 die during early embryogenesis as a result of unrestrained IRP2 activity and oxidative stress attributable to excessive iron accumulation. Despite its pivotal role in the control of iron homeostasis, however, little is known of the upstream regulation of FBXL5 activity. We now show that FBXL5 undergoes constitutive ubiquitin-dependent degradation at the steady state. With the use of a proteomics approach to the discovery of proteins that regulate the stability of FBXL5, we identified the large HECT-type ubiquitin ligase HERC2 (HECT and RLD domain containing E3 ubiquitin protein ligase 2) as an FBXL5-associated protein. Inhibition of the HERC2-FBXL5 interaction or depletion of endogenous HERC2 by RNA interference resulted in the stabilization of FBXL5 and a consequent increase in its abundance. Such accumulation of FBXL5 in turn led to a decrease in the intracellular content of ferrous iron. Our results thus suggest that HERC2 regulates the basal turnover of FBXL5, and that this ubiquitin-dependent degradation pathway contributes to the control of mammalian iron metabolism.

Phosphorus starvation induces post-transcriptional CHS gene silencing in Petunia corolla.

KEY MESSAGE: The corolla of Petunia 'Magic Samba' exhibits unstable anthocyanin expression depending on its phosphorus content. Phosphorus deficiency enhanced post-transcriptional gene silencing of chalcone synthase - A in the corolla. Petunia (Petunia hybrida) 'Magic Samba' has unstable red-white bicolored corollas that respond to nutrient deficiency. We grew this cultivar hydroponically using solutions that lacked one or several nutrients to identify the specific nutrient related to anthocyanin expression in corolla. The white area of the corolla widened under phosphorus (P)-deficient conditions. When the P content of the corolla grown under P-deficient conditions dropped to <2,000 ppm, completely white corollas continued to develop in >40 corollas until the plants died. Other elemental deficiencies had no clear effects on anthocyanin suppression in the corolla. After phosphate was resupplied to the P-deficient plants, anthocyanin was restored in the corollas. The expression of chalcone synthase-A (CHS-A) was suppressed in the white area that widened under P-suppressed conditions, whereas the expression of several other genes related to anthocyanin biosynthesis was enhanced more in the white area than in the red area. Reddish leaves and sepals developed under the P-deficient condition, which is a typical P-deficiency symptom. Two genes related to anthocyanin biosynthesis were enhanced in the reddish organs. Small interfering RNA analysis of CHS-A showed that the suppression resulted from post-transcriptional gene silencing (PTGS). Thus, it was hypothesized that the enhancement of anthocyanin biosynthetic gene expression due to P-deficiency triggered PTGS of CHS-A, which resulted in white corolla development.

A new crystal phase of N,N,N',N'-tetra-phenyl-1,1'-biphenyl-4,4'-diamine.

The complete molecule of the title compound, C(36)H(28)N(2), is generated by a crystallographic centre of inversion. The biphenyl unit is forced by symmetry to be essentially flat (r.m.s. deviation = 0.008 Å); the dihedral angles between it and the two terminal phenyl rings are 69.39 (5) and 59.53 (5)°.

Purification and properties of soluble and bound gamma-glutamyltransferases from radish cotyledon.

Soluble and cell wall bound gamma-glutamyltransferases (GGTs) were purified from radish (Raphanus sativus L.) cotyledons. Soluble GGTs (GGT I and II) had the same M(r) of 63,000, and were composed of a heavy subunit (M(r), 42,000) and a light one (M(r), 21,000). The properties of GGT I and II were similar. Bound GGTs (GGT A and B) were purified to homogeneity from the pellet after the extraction of soluble GGTs. GGT A and B were monomeric proteins with an M(r) of 61,000. The properties of GGT A and B were similar. Thus, bound GGTs were distinguished from soluble GGTs. The optimal pHs of soluble and bound GGTs were about 7.5. Both soluble and bound GGTs utilized glutathione, gamma-L-glutamyl-p-nitroanilide, oxidized glutathione and the conjugate of glutathione with monobromobimane as substrates, and were inhibited by acivicin, but soluble GGTs were also distinguished from bound GGTs with regard to these properties.