Ablation of cDC2 development by triple mutations within the Zeb2 enhancer.

The divergence of the common dendritic cell progenitor1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6-but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPß. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9-11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer.

Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching.

Vacuolar-type adenosine triphosphatases (V-ATPases)1-3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle6,7. To shed light on this bona fide single-molecule biological process, we investigated electrogenic proton-pumping by single mammalian-brain V-ATPases in single synaptic vesicles. Here we show that V-ATPases do not pump continuously in time, as suggested by observing the rotation of bacterial homologues8 and assuming strict ATP-proton coupling. Instead, they stochastically switch between three ultralong-lived modes: proton-pumping, inactive and proton-leaky. Notably, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. ATP regulates V-ATPase activity through the switching probability of the proton-pumping mode. By contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. A direct consequence of mode-switching is all-or-none stochastic fluctuations in the electrochemical gradient of synaptic vesicles that would be expected to introduce stochasticity in proton-driven secondary active loading of neurotransmitters and may thus have important implications for neurotransmission. This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode-switching.

Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

Imaging the energy gap modulations of the cuprate pair-density-wave state.

The defining characteristic1,2 of Cooper pairs with finite centre-of-mass momentum is a spatially modulating superconducting energy gap Δ(r), where r is a position. Recently, this concept has been generalized to the pair-density-wave (PDW) state predicted to exist in copper oxides (cuprates)3,4. Although the signature of a cuprate PDW has been detected in Cooper-pair tunnelling5, the distinctive signature in single-electron tunnelling of a periodic Δ(r) modulation has not been observed. Here, using a spectroscopic technique based on scanning tunnelling microscopy, we find strong Δ(r) modulations in the canonical cuprate Bi2Sr2CaCu2O8+δ that have eight-unit-cell periodicity or wavevectors Q ≈ (2π/a0)(1/8, 0) and Q ≈ (2π/a0)(0, 1/8) (where a0 is the distance between neighbouring Cu atoms). Simultaneous imaging of the local density of states N(r, E) (where E is the energy) reveals electronic modulations with wavevectors Q and 2Q, as anticipated when the PDW coexists with superconductivity. Finally, by visualizing the topological defects in these N(r, E) density waves at 2Q, we find them to be concentrated in areas where the PDW spatial phase changes by π, as predicted by the theory of half-vortices in a PDW state6,7. Overall, this is a compelling demonstration, from multiple single-electron signatures, of a PDW state coexisting with superconductivity in Bi2Sr2CaCu2O8+δ.

Publisher Correction: Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.

Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.

Discovery of orbital ordering in Bi2Sr2CaCu2O8+x.

The primordial ingredient of cuprate superconductivity is the CuO2 unit cell. Theories usually concentrate on the intra-atom Coulombic interactions dominating the 3d9 and 3d10 configurations of each copper ion. However, if Coulombic interactions also occur between electrons of the 2p6 orbitals of each planar oxygen atom, spontaneous orbital ordering may split their energy levels. This long-predicted intra-unit-cell symmetry breaking should generate an orbitally ordered phase, for which the charge transfer energy ε separating the 2p6 and 3d10 orbitals is distinct for the two oxygen atoms. Here we introduce sublattice-resolved ε(r) imaging to CuO2 studies and discover intra-unit-cell rotational symmetry breaking of ε(r). Spatially, this state is arranged in disordered Ising domains of orthogonally oriented orbital order bounded by dopant ions, and within whose domain walls low-energy electronic quadrupolar two-level systems occur. Overall, these data reveal a Q = 0 orbitally ordered state that splits the oxygen energy levels by ~50 meV, in underdoped CuO2.

Identification of a nematic pair density wave state in Bi2Sr2CaCu2O8+x.

Electron-pair density wave (PDW) states are now an intense focus of research in the field of cuprate correlated superconductivity. PDWs exhibit periodically modulating superconductive electron pairing that can be visualized directly using scanned Josephson tunneling microscopy (SJTM). Although from theory, intertwining the d-wave superconducting (DSC) and PDW order parameters allows a plethora of global electron-pair orders to appear, which one actually occurs in the various cuprates is unknown. Here, we use SJTM to visualize the interplay of PDW and DSC states in Bi2Sr2CaCu2O8+x at a carrier density where the charge density wave modulations are virtually nonexistent. Simultaneous visualization of their amplitudes reveals that the intertwined PDW and DSC are mutually attractive states. Then, by separately imaging the electron-pair density modulations of the two orthogonal PDWs, we discover a robust nematic PDW state. Its spatial arrangement entails Ising domains of opposite nematicity, each consisting primarily of unidirectional and lattice commensurate electron-pair density modulations. Further, we demonstrate by direct imaging that the scattering resonances identifying Zn impurity atom sites occur predominantly within boundaries between these domains. This implies that the nematic PDW state is pinned by Zn atoms, as was recently proposed [Lozano et al., Phys. Rev. B 103, L020502 (2021)]. Taken in combination, these data indicate that the PDW in Bi2Sr2CaCu2O8+x is a vestigial nematic pair density wave state [Agterberg et al. Phys. Rev. B 91, 054502 (2015); Wardh and Granath arXiv:2203.08250].

Identification of senescent cell surface targetable protein DPP4.

Senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, prompting efforts to eliminate them. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell surface DPP4 preferentially sensitized senescent, but not dividing, fibroblasts to cytotoxicity by natural killer cells. In sum, the selective expression of DPP4 on the surface of senescent cells enables their preferential elimination.

KIR2DS2 Expression Identifies NK Cells With Enhanced Anticancer Activity.

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2high NK cells, and, accordingly, KIR2DS2high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2+ NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies.

Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.

Time-reversal symmetry breaking in the Fe-chalcogenide superconductors.

Topological superconductivity has been sought in a variety of heterostructure systems, the interest being that a material displaying such a phenomenon could prove to be the ideal platform to support Majorana fermions, which in turn could be the basis for advanced qubit technologies. Recently, the high-Tc family of superconductors, FeTe1-xSex, have been shown to exhibit the property of topological superconductivity and further, evidence has been found for the presence of Majorana fermions. We have studied the interplay of topology, magnetism, and superconductivity in the FeTe1-x Se x family using high-resolution laser-based photoemission. At the bulk superconducting transition, a gap opens at the chemical potential as expected. However, a second gap is observed to open at the Dirac point in the topological surface state. The associated mass acquisition in the topological state points to time-reversal symmetry breaking, probably associated with the formation of ferromagnetism in the surface layer. The presence of intrinsic ferromagnetism combined with strong spin-orbit coupling provides an ideal platform for a range of exotic topological phenomena.

Double-Clad Antiresonant Hollow-Core Fiber and Its Comparison with Other Fibers for Multiphoton Micro-Endoscopy.

Label-free and multiphoton micro-endoscopy can transform clinical histopathology by providing an in situ tool for diagnostic imaging and surgical treatment in diseases such as cancer. Key to a multiphoton imaging-based micro-endoscopic device is the optical fiber, for distortion-free and efficient delivery of ultra-short laser pulses to the sample and effective signal collection. In this work, we study a new hollow-core (air-filled) double-clad anti-resonant fiber (DC-ARF) as a high-performance candidate for multiphoton micro-endoscopy. We compare the fiber characteristics of the DC-ARF with a single-clad anti-resonant fiber (SC-ARF) and a solid core fiber (SCF). In this work, while the DC-ARF and the SC-ARF enable low-loss (<0.2 dBm-1), close to dispersion-free excitation pulse delivery (<10% pulse width increase at 900 nm per 1 m fiber) without any induced non-linearities, the SCF resulted in spectral broadening and pulse-stretching (>2000% of pulse width increase at 900 nm per 1 m fiber). An ideal optical fiber endoscope needs to be several meters long and should enable both excitation and collection through the fiber. Therefore, we performed multiphoton imaging on endoscopy-compatible 1 m and 3 m lengths of fiber in the back-scattered geometry, wherein the signals were collected either directly (non-descanned detection) or through the fiber (descanned detection). Second harmonic images were collected from barium titanate crystals as well as from biological samples (mouse tail tendon). In non-descanned detection conditions, the ARFs outperformed the SCF by up to 10 times in terms of signal-to-noise ratio of images. Significantly, only the DC-ARF, due to its high numerical aperture (NA) of 0.45 and wide-collection bandwidth (>1 µm), could provide images in the de-scanned detection configuration desirable for endoscopy. Thus, our systematic characterization and comparison of different optical fibers under different image collection configurations, confirms and establishes the utility of DC-ARFs for high-performing label-free multiphoton imaging-based micro-endoscopy.

Identification of neoantigens in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.

The Cancer Research UK Stratified Medicine Programme as a model for delivering personalised cancer care.

Genomic screening is routinely used to guide the treatment of cancer patients in many countries. However, several multi-layered factors make this effort difficult to deliver within a clinically relevant timeframe. Here we share the learnings from the CRUK-funded Stratified Medicine Programme for advanced NSCLC patients, which could be useful to better plan future studies.

Efficacy and safety results from CheckMate 140, a phase 2 study of nivolumab for relapsed/refractory follicular lymphoma.

Nivolumab, an anti-programmed death-1 (PD-1) monoclonal antibody, showed promising activity in relapsed or refractory (R/R) follicular lymphoma (FL) in a phase 1 study. We conducted a phase 2 trial to further evaluate its efficacy and safety in patients with R/R FL and to explore biomarkers of response. Patients with R/R FL and at least 2 prior lines of therapy, each containing a CD20 antibody or an alkylating agent, were treated with nivolumab 3 mg/kg every 2 weeks. The primary end point was objective response rate (ORR) assessed by an independent radiologic review committee. Biomarker analyses included gene expression profiling and multiplex immunofluorescence studies of pretreatment tumor samples. A total of 92 patients were treated. After a minimum follow-up of 12 months, ORR was 4% (4 of 92 patients). Median progression-free survival (PFS) was 2.2 months (95% confidence interval [CI], 1.9-3.6 months). Median duration of response was 11 months (95% CI, 8-14 months). Exploratory analyses suggested that responders had significantly higher proportion of CD3+ T cells in the tumor microenvironment than nonresponders, but no significant differences in PD-1 or programmed death-ligand 1 expression were observed. High expression of a set of tumor-associated macrophage genes was associated with reduced PFS (hazard ratio, 3.28; 95% CI, 1.76-6.11; P = .001). The safety profile was consistent with previous reports of nivolumab. In conclusion, nivolumab monotherapy was associated with very limited activity in patients with R/R FL. Better understanding of the immune biology of this disease may facilitate the development of effective checkpoint-based strategies. This trial was registered at www.clinicaltrials.gov as #NCT02038946.

KDM5 inhibition offers a novel therapeutic strategy for the treatment of KMT2D mutant lymphomas.

Loss-of-function mutations in KMT2D are a striking feature of germinal center (GC) lymphomas, resulting in decreased histone 3 lysine 4 (H3K4) methylation and altered gene expression. We hypothesized that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would reestablish H3K4 methylation and restore the expression of genes repressed on loss of KMT2D. KDM5 inhibition increased H3K4me3 levels and caused an antiproliferative response in vitro, which was markedly greater in both endogenous and gene-edited KMT2D mutant diffuse large B-cell lymphoma cell lines, whereas tumor growth was inhibited in KMT2D mutant xenografts in vivo. KDM5 inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell signaling and altered expression of B-cell lymphoma 2 (BCL2) family members, including BCL2 itself. KDM5 inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC lymphomas.

Revealing the Origin of Time-Reversal Symmetry Breaking in Fe-Chalcogenide Superconductor FeTe_{1-x}Se_{x}.

Recently, evidence has emerged in the topological superconductor Fe-chalcogenide FeTe_{1-x}Se_{x} for time-reversal symmetry breaking (TRSB), the nature of which has strong implications on the Majorana zero modes (MZM) discovered in this system. It remains unclear, however, whether the TRSB resides in the topological surface state (TSS) or in the bulk, and whether it is due to an unconventional TRSB superconducting order parameter or an intertwined order. Here, by performing in superconducting FeTe_{1-x}Se_{x} crystals both surface-magneto-optic-Kerr effect measurements using a Sagnac interferometer and bulk magnetic susceptibility measurements, we pinpoint the TRSB to the TSS, where we also detect a Dirac gap. Further, we observe surface TRSB in nonsuperconducting FeTe_{1-x}Se_{x} of nominally identical composition, indicating that TRSB arises from an intertwined surface ferromagnetic (FM) order. The observed surface FM bears striking similarities to the two-dimensional (2D) FM found in 2D van der Waals crystals, and is highly sensitive to the exact chemical composition, thereby providing a means for optimizing the conditions for Majorana particles that are useful for robust quantum computing.

Are we reaching the maximum cure rate for Hodgkin lymphoma?

The treatment of Hodgkin lymphoma, using cytotoxic chemotherapy and selective radiotherapy, has resulted in progressively increasing cure rates over the last 40 years. Recent studies have been directed at using response-adapted approaches to modulate treatment according to the responses seen using functional imaging, with the aim of balancing the probability of cure against the toxicity of more extensive treatments, in particular the risks of infertility, second malignancy and cardiovascular disease. The results of these studies suggest that we have reached the limits of what might be expected from the conventional treatments, but the arrival of antibody-based therapies, specifically antibody-drug conjugates and immune checkpoint blocking antibodies, now holds out the prospect of further improvements. The next challenge will be to select those groups for whom they are most needed.