Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

Myasthenia gravis (MG) is a neuromuscular disorder caused by autoantibodies targeting components of the neuromuscular junction. Patients with MG experience skeletal muscle weakness, worsened by activity (1, 2). In upwards of 85% of MG patients, autoantibodies specifically target the nicotinic acetylcholine receptor (AChR) (2). Many clinical and experimental studies, including maternal-to-fetal transfer, plasma exchange to deplete antibodies, and passive transfer of patient-derived immunoglobulin show that AChR autoantibodies are demonstrably pathogenic (1, 3–10). AChR-mediated signal transmission is impaired by a number of autoantibody-mediated functions. AChR autoantibodies are predominantly IgG1 and IgG3, two subclasses that effectively activate complement (11–13). Thus, complement-mediated tissue injury and consequent removal of AChR from the muscle membrane represents a major mechanism of immunopathology. Additional mechanisms include autoantibody-mediated antigen cross-linking, resulting in internalization of AChR (by modulating autoantibodies), and a direct blocking of the acetylcholine binding site by the autoantibodies (14–19).A key source of these pathogenic AChR autoantibodies in MG patients is the MG thymus. Thymic lymphofollicular hyperplasia (20) with germinal centers is observed in ∼70% of younger MG patients (21). The thymus in these patients contains both AChR-specific IgG (22) and B cells (10) that can secrete these autoantibodies (23). Transplantation of thymus from AChR-MG patients into immunodeficient mice results in human AChR-specific autoantibody deposits at the neuromuscular junction, and subsequent manifestation of MG-like symptoms (24). Thymus-associated plasma cells and plasmablasts spontaneously produce AChR autoantibodies in vitro (25, 26) and activated memory B cell populations (27–29). B cells residing in the hyperplastic thymus organize within tertiary lymphoid organs, forming structures that share many characteristics associated with germinal centers (30–33). The presence and frequency of these structures positively associates with the presence of circulating AChR autoantibodies (34). Expanded clones among the MG thymus resident B cells is consistent with the presence of ongoing germinal center-based maturation processes (35, 36). These clones feature the characteristics of antigen experience, including isotype class switching, somatic hypermutation, and biased usage of antibody variable region gene segments (35, 37–40).These collective studies clearly demonstrate that the thymus plays a fundamental role in the production of AChR autoantibodies and consequently the immunopathology of MG. Based only on empiric clinical evidence, thymectomy (TX) had already been widely used as a long-standing treatment strategy for AChR autoantibody-positive MG (41). A multicenter, single-blind, randomized clinical trial (MGTX) designed to evaluate the efficacy of thymectomy plus prednisone versus prednisone alone in generalized nonthymomatous AChR-MG was recently performed to formally test the effect of thymectomy (42). The study demonstrated the efficacy of thymectomy as the procedure led to a significant improvement in muscle weakness, decreased steroid usage, and decreased frequency of rehospitalization over the course of 3 y. Results from a 2-y extension study of the MGTX trial (43) demonstrated that the patients who underwent thymectomy were more likely to have a better clinical status compared to patients who were treated with prednisone alone. Moreover, therapeutic effects from thymectomy continue to be observed years after the procedure, but some patients experienced more benefit than others. These findings are consistent with other studies, which showed rates of complete stable remission in only 40 to 50% of patients when followed for years after the procedure (44, 45). Furthermore, the AChR autoantibody titer decreases in the majority of those treated by thymectomy, but almost never reaches undetectable levels (34, 46) and only modestly decreases in many (47). Understanding both the mechanistic basis for heterogeneous responses and why thymectomy does not consistently lead to complete stable remission is therefore critical to improving the management of AChR autoantibody-positive MG patients with thymic lymphofollicular hyperplasia.Given extensive evidence that disease-causing B cells in AChR-MG are found in the thymus alongside clinical evidence showing heterogeneous responses to thymectomy, we sought to test the possibility that B cell clones from the thymus persist in the periphery after removal of the thymus. We hypothesized that B cell clones from the thymus would be found in the circulation and that the persistence of these clones overall would correlate with disease persistence. Thus, in this study, we test the hypothesis that the global depletion of B cell clones from the thymus, including those that produce AChR autoantibodies, is a mechanism by which thymectomy reduces disease burden. To test this hypothesis, we performed adaptive immune receptor repertoire (AIRR) sequencing (AIRR-seq) of B cell receptor (BCR) repertoires from the thymus and paired longitudinal peripheral blood samples from the MGTX trial and an independent center, generating approximately half a million V(D)J sequences in total. AIRR-seq has the capacity and depth to identify rare sequences in the large (10¹¹ B cells) circulating peripheral repertoire found in humans (48). Consequently, this approach allowed us to identify rare B cell clones in the circulation related to those in the thymus and track their frequency over time after thymectomy.     

A thermogenic fat-epithelium cell axis regulates intestinal disease tolerance

Disease tolerance, the capacity of tissues to withstand damage caused by a stimulus without a decline in host fitness, varies across tissues, environmental conditions, and physiologic states. While disease tolerance is a known strategy of host defense, its role in noninfectious diseases has been understudied. Here, we provide evidence that a thermogenic fat–epithelial cell axis regulates intestinal disease tolerance during experimental colitis. We find that intestinal disease tolerance is a metabolically expensive trait, whose expression is restricted to thermoneutral mice and is not transferable by the microbiota. Instead, disease tolerance is dependent on the adrenergic state of thermogenic adipocytes, which indirectly regulate tolerogenic responses in intestinal epithelial cells. Our work has identified an unexpected mechanism that controls intestinal disease tolerance with implications for colitogenic diseases.

Resistance and disease tolerance are two distinct strategies a host can use to mitigate the negative impact of disease on tissue function and host fitness (1, 2). For example, during pathogenic infections, the detection and elimination of pathogens is mediated by resistance, whereas disease tolerance minimizes the negative impact of pathogens on host fitness without affecting pathogen burden. Although disease tolerance is a well-appreciated strategy of host defense against pathogens in both the plant and animal kingdoms (3–5), its importance in noninfectious diseases is largely unknown (1, 6).Recent studies have demonstrated that metabolic adaptations mediate disease tolerance during viral, bacterial, and parasitic infections (7–12). These findings suggest that disease tolerance programs are metabolically expensive and compete for energy with other tissue maintenance programs. Because homeothermy, the stable maintenance of core temperature, is a major energy consuming program in mammals (13), we postulated that it might energetically compete with disease tolerance programs to limit their expression in noninfectious diseases. We tested this hypothesis using murine models of colitis because the high regenerative capacity of the intestinal epithelium has been postulated to increase its intrinsic tolerance capacity (1).     

Rapid initiation of cell cycle reentry processes protects neurons from amyloid-β toxicity

Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer’s disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-β (oAβ) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAβ challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aβ precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aβ toxicity.

Alzheimer’s disease (AD) is the most prevalent of all the neurodegenerative disorders. Distinct protein inclusions, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) characterize the AD brain (1). The exact causes of neuronal cell death in AD are still debated but several neuropathological studies have linked neuronal death to unexpected reappearance of cell cycle events (2–4). Accordingly, cell cycle activation, aberrant DNA replication, as well as aneuploidy have been found in neurons of AD brains (5–11). Furthermore, numbers of aneuploid neurons that are rare in control brains were increased in AD brains (5, 12, 13). However, mitosis itself has not been reported, and it appears that postmitotic neurons in AD patients cannot complete the cell cycle (14). In dividing cells, the coordination of the cell cycle requires interplay between cyclins and cyclin-dependent kinases (CDKs) at different checkpoints and the transitions between different phases are tightly regulated (15, 16). Therefore, it has been suggested that cell cycle reentry of differentiated mature neurons would have detrimental consequences, rendering them vulnerable and contribute to neurodegeneration (4, 8, 17). However, it remains unclear whether cell cycle events are a prerequisite for neuronal death, and why aneuploid neurons accumulate in AD brains.The introduction of the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system enabled live tracking of cell cycle progress in dividing cells (18) and has been valuable in understanding the implications of the cell cycle in disease pathogenesis, e.g., in cancer (19–21).Briefly, the FUCCI system allows live monitoring of the cell cycle in cells, visualized by the ectopic expression of red and green fluorescent-tagged truncated proteins, mKO2-hCdt1 (30-120) and mAG-hGem (1-110), respectively, distinguishing the G1 from S, G2, and M phases (22). While frequently used in cancer and developmental biology research (23–25) to our knowledge, the FUCCI system has not been used to study mature neurons, including in the context of neurodegenerative diseases.Here, we applied FUCCI to primary hippocampal neuronal cultures challenged with oligomeric Aβ (oAβ) and transgenic mice with expression of mutant human amyloid precursor protein (APP), which are both established models of AD (26–29). By obtaining quantitative and qualitative information about cell cycle events (CCEs) in response to Aβ, we show that increased FUCCI reporter activity was associated with resistance to Aβ-induced cell death, which is contrary to the current opinion of CCEs contributing to neurodegeneration in AD.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

A single-cell–resolution fate map of endoderm reveals demarcation of pancreatic progenitors by cell cycle

A progenitor cell could generate a certain type or multiple types of descendant cells during embryonic development. To make all the descendant cell types and developmental trajectories of every single progenitor cell clear remains an ultimate goal in developmental biology. Characterizations of descendant cells produced by each uncommitted progenitor for a full germ layer represent a big step toward the goal. Here, we focus on early foregut endoderm, which generates foregut digestive organs, including the pancreas, liver, foregut, and ductal system, through distinct lineages. Using unbiased single-cell labeling techniques, we label every individual zebrafish foregut endodermal progenitor cell out of 216 cells to visibly trace the distribution and number of their descendant cells. Hence, single-cell–resolution fate and proliferation maps of early foregut endoderm are established, in which progenitor regions of each foregut digestive organ are precisely demarcated. The maps indicate that the pancreatic endocrine progenitors are featured by a cell cycle state with a long G1 phase. Manipulating durations of the G1 phase modulates pancreatic progenitor populations. This study illustrates foregut endodermal progenitor cell fate at single-cell resolution, precisely demarcates different progenitor populations, and sheds light on mechanistic insights into pancreatic fate determination.

A progenitor cell could generate a certain type or multiple types of descendant cells during embryonic development. Characterizations of descendant cell loci and identities as well as developmental trajectories for every single progenitor cell remain one of the ultimate goals in developmental biology. Recent methodological innovations using DNA barcode labeling (1–5) combined with single-cell RNA sequencing (6–9) enable lineage tracing to be conducted on a large scale and at single-cell resolution. However, unbiased single-cell labeling and tracing with complete visibility and high spatial–temporal resolution in living embryos still remains technically challenging. At early postgastrulation, when molecular markers of organ progenitors are rarely available and cell fates are unspecified, understandings of endodermal cell fate determination are mostly contributed by studies on inductive signals and regulatory molecules (10–17). Previous pioneering studies have identified the regional source of pancreatic and liver progenitors in the early somite stage embryo (18–22). While providing a framework for characterizing the descendant cell types for foregut endoderm, these earlier fate maps lack the resolution at the single-cell level. In order to characterize the variety of descendant cells for each foregut endodermal progenitor cell and precisely demarcate progenitor populations of each foregut digestive organ, we establish unbiased, visible single endodermal cell labeling and descendant tracing techniques. Thus, single-cell–resolution fate and proliferation maps are generated, from which the pancreatic endocrine progenitors are found to be featured by a cell cycle state with a long G1 phase. This study obtains a single-cell–resolution, full-coverage fate map of early foregut endoderm on one hand, and on the other hand, it provides insight into endocrine pancreas development by identifying extended an G1 phase as a critical feature of its progenitors.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Contractility,focal adhesion orientation,and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple “cryptic leading edges” within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as “cell polarization barriers,” decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

One hallmark of tumor progression to more advanced stages and worsening patient prognosis is the remodeling of the extracellular matrix (ECM) in the tumor microenvironment by fibroblasts (1) and macrophages (2). In several tumor types—including breast (3, 4), skin (5), ovary (6, 7), colon (2), and liver (8)—such remodeling is characterized by stiffening and reorganization of normally wavy stromal collagen bundles into thick linear bundles. Linear bundles are thought to provide “tracks” to mediate metastatic cell migration out of the primary tumor, whereas curved or wavy bundles typical of normal stroma are thought to inhibit cell movement (9, 10). The notion that linear ECM fibers support tumor metastasis arose from the longstanding observation that cells of many types polarize and migrate directionally in response to anisotropic physical cues, such as linear fibrils or grooves in a substrate, in a process called contact guidance (11). In support of this, ovarian cancer cells migrate more actively on ECM substrates that mimic the collagen architecture of aggressive ovarian tumors than on those mimicking normal or benign stroma (12). It is thought that the effects of comorbidities on ECM architecture, for example obesity in which breast stroma exhibits more linear collagen bundles than seen in lean tissue, may predispose patients to worse clinical outcomes when cancer does arise (13). However, a direct link between metastasis and the migratory behavior of cancer cells in response to linear or wavy or ECM fibril architecture has not been established, and the mechanisms by which such regulation might occur are unknown.Cell polarization and migration in response to anisotropic cues during contact guidance, chemotaxis, haptotaxis, or durotaxis is mediated by similar molecular mechanisms. Polarization is initiated by ligand binding by growth factor or ECM receptors that promote Rho GTPases (14, 15), and is enforced by spatial segregation of phosphatidylinositol 3-kinase (PI3K) at the leading edge to produce phosphatidylinositol-3,4,5-triphosphate (PIP3) and the phosphatase and tensin homolog (PTEN) removing PIP3 at the cell rear (16–18). PIP3 and Rho GTPases at the leading edge promote polarization of the microtubule cytoskeleton and Golgi apparatus, as well as actin polymerization via Arp2/3 and formins (19–23), to drive leading-edge protrusions. Retrograde flow of the polymerizing actin network at the leading edge is coupled to integrin-based focal adhesions (FAs) via a molecular clutch (24, 25), which engages FAs to the ECM. Recruitment of myosin II to the polymerizing actin contracts the network, creating actin arcs and stress fibers in the lamella, maturing the FAs and orienting them in the direction of cell migration (26–30). Disassembly of FAs toward the rear of the cell allows forward movement (31–35). Artificial enforcement of this organization of actin polymerization and adhesions by micropatterned ECMs alone is sufficient to define the polarity of the cell, independent of other stimuli (36). However, how these molecular mechanisms are modulated by the differing stromal collagen architectures associated with normal or tumor tissue is not known.Here we sought to examine the contact-guidance–mediated polarization and migration responses of tumor cells to ECM fibril architectures mimicking those seen in normal and tumor stroma, and to dissect the mechanisms of these responses. We designed synthetic nanotopographic ECM-coated substrates that approximate collagen fibril size and the range of waveforms observed in tumors and tissues from mouse and human samples, and we determined their effect on the organization and dynamics of the cell polarity and migration machineries. We find that cell polarization and directional migration are inhibited by sinusoidal fibril-like waves above a threshold amplitude by geometrically constrained effects on the organization of actomyosin contractility and FA orientation. Importantly, we found that cancer cells of different intrinsic contractility depolarized at different ECM-wave amplitudes, and that cell polarization could be tuned on wavy substrates by manipulating contractility. Thus, the ECM-fibril waveform, in addition to other factors in the tumor microenvironment, may regulate cancer cells’ ability to migrate out of tumors, and their contractility level may dictate the range of ECM architectures that allow migration.     

High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56⁻CD16⁺ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, {"type":"clinical-trial","attrs":{"text":"NCT02320656","term_id":"NCT02320656"}}NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56⁻CD16⁺ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56⁻CD16⁺ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56⁻CD16⁺ NK cells. Overall, our data suggest that the accumulation of CD56⁻CD16⁺ NK cells may be the consequence of immune escape from innate immunity during AML progression.

Natural killer (NK) cells are critical cytotoxic effectors involved in leukemic blast recognition, tumor cell clearance, and maintenance of long-term remission (1). NK cells directly kill target cells without prior sensitization, enabling lysis of cells stressed by viral infections or tumor transformation. NK cells are divided into different functional subsets according to CD56 and CD16 expression (2–4). CD56^bright NK cells are the most immature NK cells found in peripheral blood. This subset is less cytotoxic than mature NK cells and secretes high amounts of chemokines and cytokines such as IFNγ and TNFα. These cytokines have a major effect on the infected or tumor target cells and play a critical role in orchestration of the adaptive immune response through dendritic cell activation. CD56^dimCD16⁺ NK cells, which account for the majority of circulating human NK cells, are the most cytotoxic NK cells. NK cell activation is finely tuned by integration of signals from inhibitory and triggering receptors, in particular, those of NKp30, NKp46 and NKp44, DNAM-1, and NKG2D (5). Upon target recognition, CD56^dimCD16⁺ NK cells release perforin and granzyme granules and mediate antibody-dependent cellular cytotoxicity through CD16 (FcɣRIII) to clear transformed cells.NK cells are a major component of the antileukemic immune response, and NK cell alterations have been associated with adverse clinical outcomes in acute myeloid leukemia (AML) (6–9). Therefore, it is crucial to better characterize AML-induced NK cell alterations in order to optimize NK cell–targeted therapies. During AML progression, NK cell functions are deeply altered, with decreased expression of NK cell–triggering receptors and reduced cytotoxic functions as well as impaired NK cell maturation (6, 9–13). Cancer-induced NK cell impairment occurs through various mechanisms of immune escape, including shedding and release of ligands for NK cell–triggering receptors; release of immunosuppressive soluble factors such as TGFβ, adenosine, PGE2, or L-kynurenine; and interference with NK cell development, among others (14).Interestingly, these mechanisms of immune evasion are also seen to some extent in chronic viral infections, notably HIV (2). In patients with HIV, NK cell functional anergy is mediated by the release of inflammatory cytokines and TGFβ, the presence of MHC^low target cells, and the shedding of ligands for NK cell–triggering receptors (2). As a consequence, some phenotypical alterations described in cancer patients are also induced by chronic HIV infections, with decreased expression of major triggering receptors such as NKp30, NKp46, and NKp44 (15, 16); decreased expression of CD16 (17); and increased expression of inhibitory receptors such as T cell immunoreceptor with Ig and ITIM domains (TIGIT) (18) all observed. In addition, patients with HIV display an accumulation of CD56⁻CD16⁺ unconventional NK cells, a highly dysfunctional NK cell subset (19, 20). Mechanisms leading to the loss of CD56 are still poorly described, and the origin of this subset of CD56⁻ NK cells is still unknown. To date, two hypotheses have been considered: CD56⁻ NK cells could be terminally differentiated cells arising from a mixed population of mature NK cells with altered characteristics or could expand from a pool of immature precursor NK cells (21). Expansion of CD56⁻CD16⁺ NK cells is mainly observed in viral noncontrollers (19, 20). Indeed, CD56 is an important adhesion molecule involved in NK cell development, motility, and pathogen recognition (22–27). CD56 is also required for the formation of the immunological synapse between NK cells and target cells, lytic functions, and cytokine production (26, 28). As a consequence, CD56⁻CD16⁺ NK cells display lower degranulation capacities and decreased expression of triggering receptors, perforin, and granzyme B, dramatically reducing their cytotoxic potential, notably against tumor target cells (2, 19, 20, 29, 30). In line with this loss of the cytotoxic functions against tumor cells, patients with concomitant Burkitt lymphoma and Epstein-Barr virus infection display a dramatic increase of CD56⁻CD16⁺ NK cells (30), which could represent an important hallmark of escape to NK cell immunosurveillance in virus-driven hematological malignancies.To our knowledge, this population has not been characterized in the context of nonvirally induced hematological malignancies. In the present work, we investigated the presence of this population of unconventional NK cells in patients with AML, its phenotypical characteristics, and the consequences of its accumulation on disease control. Finally, we explored NK cell developmental trajectories leading to the emergence of this phenotype.     

Modulation of immune cell reactivity with cis-binding Siglec agonists

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.

Sialic acid-binding immunoglobulin (IgG)-like lectins (Siglecs) are a family of immune checkpoint receptors that are on all classes of immune cells (1–5). Siglecs bind various sialoglycan ligands and deliver signals to the immune cells that report on whether the target is healthy or damaged, “self” or “nonself.” Of the 14 human Siglecs, 9 contain cytosolic inhibitory signaling domains. Accordingly, engagement of these inhibitory Siglecs by sialoglycans suppresses the activity of the immune cell, leading to an antiinflammatory effect. In this regard, inhibitory Siglecs have functional parallels with the T cell checkpoint receptors CTLA-4 and PD-1 (6–9). As with these clinically established targets for cancer immune therapy, there has been a recent surge of interest in antagonizing Siglecs to potentiate immune cell reactivity toward cancer (10). Conversely, engagement of Siglecs with agonist antibodies can suppress immune cell reactivity in the context of antiinflammatory therapy. This approach has been explored to achieve B cell suppression in lupus patients by agonism of CD22 (Siglec-2) (11, 12), and to deplete eosinophils for treatment of eosinophilic gastroenteritis by agonism of Siglec-8 (13). Similarly, a CD24 fusion protein has been investigated clinically as a Siglec-10 agonist for both graft-versus-host disease and viral infection (14, 15).Traditionally, Siglec ligands have been studied as functioning in trans, that is, on an adjacent cell (16–18), or as soluble clustering agents (9, 19). In contrast to these mechanisms of action, a growing body of work suggests that cis ligands for Siglecs (i.e., sialoglycans that reside on the same cell membrane) cluster these receptors and maintain a basal level of inhibitory signaling that increases the threshold for immune cell activation. Both Bassik and coworkers (20) and Wyss-Coray and coworkers (21) have linked the depletion of cis Siglec ligands with increased activity of macrophages and microglia, and other studies have shown that a metabolic blockade of sialic acid renders phagocytes more prone to activation (22).Synthetic ligands are a promising class of Siglec agonists (17, 23, 24). Many examples rely on clustering architectures (e.g., sialopolymers, nanoparticles, liposomes) to induce their effect (19, 23–26). Indeed, we have previously used glycopolymers to study the effects of Siglec engagement in trans on natural killer (NK) cell activity (16). We and other researchers have employed glycopolymers (16, 23), glycan-remodeling enzymes (27, 28), chemical inhibitors of glycan biosynthesis (22), and mucin overexpression constructs (29, 30) to modulate the cell-surface levels of Siglec ligands. However, current approaches lack specificity for a given Siglec.We hypothesized that Siglec-specific cis-binding sialoglycans displayed on immune cell surfaces could dampen immune cell activity with potential therapeutic applications. Here we test this notion with the synthesis of membrane-tethered cis-binding agonists of Siglec-9 (Fig. 1). Macrophages and microglia widely express Siglec-9 and are responsible for numerous pathologies including age-related inflammation (31), macular degeneration (32), neural inflammation (33), and chronic obstructive pulmonary disease (34). We designed and developed a lipid-linked glycopolypeptide scaffold bearing glycans that are selective Siglec-9 ligands (pS9L-lipid). We show that pS9L-lipid inserts into macrophage membranes, binds Siglec-9 specifically and in cis, and induces Siglec-9 signaling to suppress macrophage activity. By contrast, a lipid-free soluble analog (pS9L-sol) binds Siglec-9 but does not agonize Siglec-9 or modulate macrophage activity. Membrane-tethered glycopolypeptides are thus a potential therapeutic modality for inhibiting phagocyte activity.Open in a separate windowFig. 1.Lipid-tethered glycopolypeptides cluster and agonize Siglecs in cis on effector cells. (A) Immune cells express activating receptors that stimulate inflammatory signaling. (B) Clustering of Siglec-9 by cis-binding agonists stimulates inhibitory signaling that quenches activation.