Multilayered Immunity by Tissue-Resident Lymphocytes in Cancer.

Lymphocytes spanning the entire innate-adaptive spectrum can stably reside in tissues and constitute an integral component of the local defense network against immunological challenges. In tight interactions with the epithelium and endothelium, tissue-resident lymphocytes sense antigens and alarmins elicited by infectious microbes and abiotic stresses at barrier sites and mount effector responses to restore tissue homeostasis. Of note, such a host cell-directed immune defense system has been recently demonstrated to surveil epithelial cell transformation and carcinoma development, as well as cancer cell metastasis at selected distant organs, and thus represents a primordial cancer immune defense module. Here we review how distinct lineages of tissue-resident innate lymphoid cells, innate-like T cells, and adaptive T cells participate in a form of multilayered cancer immunity in murine models and patients, and how their convergent effector programs may be targeted through both shared and private regulatory pathways for cancer immunotherapy.

Distinct metabolic requirements regulate B cell activation and germinal center responses.

Following infection or vaccination, activated B cells at extrafollicular sites or within germinal centers (GCs) undergo vigorous clonal proliferation. Proliferating lymphocytes have been shown to undertake lactate dehydrogenase A (LDHA)-dependent aerobic glycolysis; however, the specific role of this metabolic pathway in a B cell transitioning from a naïve to a highly proliferative, activated state remains poorly defined. Here, we deleted LDHA in a stage-specific and cell-specific manner. We find that ablation of LDHA in a naïve B cell did not profoundly affect its ability to undergo a bacterial lipopolysaccharide-induced extrafollicular B cell response. On the other hand, LDHA-deleted naïve B cells had a severe defect in their capacities to form GCs and mount GC-dependent antibody responses. In addition, loss of LDHA in T cells severely compromised B cell-dependent immune responses. Strikingly, when LDHA was deleted in activated, as opposed to naïve, B cells, there were only minimal effects on the GC reaction and in the generation of high-affinity antibodies. These findings strongly suggest that naïve and activated B cells have distinct metabolic requirements that are further regulated by niche and cellular interactions.

Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies.

Malignancy can be suppressed by the immune system. However, the classes of immunosurveillance responses and their mode of tumor sensing remain incompletely understood. Here, we show that although clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8+ T cells that negatively correlated with patient prognosis, chromophobe RCC (chRCC) had abundant infiltration of granzyme A-expressing intraepithelial type 1 innate lymphoid cells (ILC1s) that positively associated with patient survival. Interleukin-15 (IL-15) promoted ILC1 granzyme A expression and cytotoxicity, and IL-15 expression in chRCC tumor tissue positively tracked with the ILC1 response. An ILC1 gene signature also predicted survival of a subset of breast cancer patients in association with IL-15 expression. Notably, ILC1s directly interacted with cancer cells, and IL-15 produced by cancer cells supported the expansion and anti-tumor function of ILC1s in a murine breast cancer model. Thus, ILC1 sensing of cancer cell IL-15 defines an immunosurveillance mechanism of epithelial malignancies.

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism.

The bacterial Mfd ATPase is increasingly recognized as a general transcription factor that participates in the resolution of transcription conflicts with other processes/roadblocks. This function stems from Mfd's ability to preferentially act on stalled RNA polymerases (RNAPs). However, the mechanism underlying this preference and the subsequent coordination between Mfd and RNAP have remained elusive. Here, using a novel real-time translocase assay, we unexpectedly discovered that Mfd translocates autonomously on DNA. The speed and processivity of Mfd dictate a "release and catch-up" mechanism to efficiently patrol DNA for frequently stalled RNAPs. Furthermore, we showed that Mfd prevents RNAP backtracking or rescues a severely backtracked RNAP, allowing RNAP to overcome stronger obstacles. However, if an obstacle's resistance is excessive, Mfd dissociates the RNAP, clearing the DNA for other processes. These findings demonstrate a remarkably delicate coordination between Mfd and RNAP, allowing efficient targeting and recycling of Mfd and expedient conflict resolution.

Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Control of tumor-associated macrophage responses by nutrient acquisition and metabolism.

Metazoan tissue specification is associated with integration of macrophage lineage cells in sub-tissular niches to promote tissue development and homeostasis. Oncogenic transformation, most prevalently of epithelial cell lineages, results in maladaptation of resident tissue macrophage differentiation pathways to generate parenchymal and interstitial tumor-associated macrophages that largely foster cancer progression. In addition to growth factors, nutrients that can be consumed, stored, recycled, or converted to signaling molecules have emerged as crucial regulators of macrophage responses in tumor. Here, we review how nutrient acquisition through plasma membrane transporters and engulfment pathways control tumor-associated macrophage differentiation and function. We also discuss how nutrient metabolism regulates tumor-associated macrophages and how these processes may be targeted for cancer therapy.

Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis.

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

Functional reprogramming of regulatory T cells in the absence of Foxp3.

Regulatory T cells (Treg cells) deficient in the transcription factor Foxp3 lack suppressor function and manifest an effector T (Teff) cell-like phenotype. We demonstrate that Foxp3 deficiency dysregulates metabolic checkpoint kinase mammalian target of rapamycin (mTOR) complex 2 (mTORC2) signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Specific deletion of the mTORC2 adaptor gene Rictor in Foxp3-deficient Treg cells ameliorated disease in a Foxo1 transcription factor-dependent manner. Rictor deficiency re-established a subset of Treg cell genetic circuits and suppressed the Teff cell-like glycolytic and respiratory programs, which contributed to immune dysregulation. Treatment of Treg cells from patients with FOXP3 deficiency with mTOR inhibitors similarly antagonized their Teff cell-like program and restored suppressive function. Thus, regulatory function can be re-established in Foxp3-deficient Treg cells by targeting their metabolic pathways, providing opportunities to restore tolerance in Treg cell disorders.

Tumor-associated macrophages expressing the transcription factor IRF8 promote T cell exhaustion in cancer.

Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.

Cancer Immunosurveillance by Tissue-Resident Innate Lymphoid Cells and Innate-like T Cells.

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remains obscure. Here, we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, T cell receptor (TCR)αß, and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a, and CD103, these cells share a gene-expression signature distinct from those of conventional NK cells, T cells, and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15 deficiency, but not Nfil3 deficiency, results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type-1-like innate lymphoid cells and type 1 innate-like T cells.

Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity.

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.

Glycolytic ATP fuels phosphoinositide 3-kinase signaling to support effector T helper 17 cell responses.

Aerobic glycolysis-the Warburg effect-converts glucose to lactate via the enzyme lactate dehydrogenase A (LDHA) and is a metabolic feature of effector T cells. Cells generate ATP through various mechanisms and Warburg metabolism is comparatively an energy-inefficient glucose catabolism pathway. Here, we examined the effect of ATP generated via aerobic glycolysis in antigen-driven T cell responses. Cd4CreLdhafl/fl mice were resistant to Th17-cell-mediated experimental autoimmune encephalomyelitis and exhibited defective T cell activation, migration, proliferation, and differentiation. LDHA deficiency crippled cellular redox balance and inhibited ATP production, diminishing PI3K-dependent activation of Akt kinase and thereby phosphorylation-mediated inhibition of Foxo1, a transcriptional repressor of T cell activation programs. Th17-cell-specific expression of an Akt-insensitive Foxo1 recapitulated the defects seen in Cd4CreLdhafl/fl mice. Induction of LDHA required PI3K signaling and LDHA deficiency impaired PI3K-catalyzed PIP3 generation. Thus, Warburg metabolism augments glycolytic ATP production, fueling a PI3K-centered positive feedback regulatory circuit that drives effector T cell responses.

Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection.

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.

Sestrins function as guanine nucleotide dissociation inhibitors for Rag GTPases to control mTORC1 signaling.

Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. Here, we show that Sestrins bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino-acid-induced Rag guanine nucleotide exchange and mTORC1 translocation to the lysosome. Mutation of the conserved GDI motif creates a dominant-negative form of Sestrin that renders mTORC1 activation insensitive to amino acid deprivation, whereas a cell-permeable peptide containing the GDI motif inhibits mTORC1 signaling. Mice deficient in all Sestrins exhibit reduced postnatal survival associated with defective mTORC1 inactivation in multiple organs during neonatal fasting. These findings reveal a nonredundant mechanism by which the Sestrin family of GDIs regulates the nutrient-sensing Rag GTPases to control mTORC1 signaling.

A proliferative burst during preadolescence establishes the final cardiomyocyte number.

It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here, we show that a thyroid hormone surge activates the IGF-1/IGF-1-R/Akt pathway on postnatal day 15 and initiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day 15 is intermediate between that observed at postnatal days 2 and 21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases.

Reprogramming tumour-associated macrophages to outcompete cancer cells.

In metazoan organisms, cell competition acts as a quality control mechanism to eliminate unfit cells in favour of their more robust neighbours1,2. This mechanism has the potential to be maladapted, promoting the selection of aggressive cancer cells3-6. Tumours are metabolically active and are populated by stroma cells7,8, but how environmental factors affect cancer cell competition remains largely unknown. Here we show that tumour-associated macrophages (TAMs) can be dietarily or genetically reprogrammed to outcompete MYC-overexpressing cancer cells. In a mouse model of breast cancer, MYC overexpression resulted in an mTORC1-dependent 'winner' cancer cell state. A low-protein diet inhibited mTORC1 signalling in cancer cells and reduced tumour growth, owing unexpectedly to activation of the transcription factors TFEB and TFE3 and mTORC1 in TAMs. Diet-derived cytosolic amino acids are sensed by Rag GTPases through the GTPase-activating proteins GATOR1 and FLCN to control Rag GTPase effectors including TFEB and TFE39-14. Depletion of GATOR1 in TAMs suppressed the activation of TFEB, TFE3 and mTORC1 under the low-protein diet condition, causing accelerated tumour growth; conversely, depletion of FLCN or Rag GTPases in TAMs activated TFEB, TFE3 and mTORC1 under the normal protein diet condition, causing decelerated tumour growth. Furthermore, mTORC1 hyperactivation in TAMs and cancer cells and their competitive fitness were dependent on the endolysosomal engulfment regulator PIKfyve. Thus, noncanonical engulfment-mediated Rag GTPase-independent mTORC1 signalling in TAMs controls competition between TAMs and cancer cells, which defines a novel innate immune tumour suppression pathway that could be targeted for cancer therapy.

In-plane charged domain walls with memristive behaviour in a ferroelectric film.

Domain-wall nanoelectronics is considered to be a new paradigm for non-volatile memory and logic technologies in which domain walls, rather than domains, serve as an active element. Especially interesting are charged domain walls in ferroelectric structures, which have subnanometre thicknesses and exhibit non-trivial electronic and transport properties that are useful for various nanoelectronics applications1-3. The ability to deterministically create and manipulate charged domain walls is essential to realize their functional properties in electronic devices. Here we report a strategy for the controllable creation and manipulation of in-plane charged domain walls in BiFeO3 ferroelectric films a few nanometres thick. By using an in situ biasing technique within a scanning transmission electron microscope, an unconventional layer-by-layer switching mechanism is detected in which ferroelectric domain growth occurs in the direction parallel to an applied electric field. Based on atomically resolved electron energy-loss spectroscopy, in situ charge mapping by in-line electron holography and theoretical calculations, we show that oxygen vacancies accumulating at the charged domain walls are responsible for the domain-wall stability and motion. Voltage control of the in-plane domain-wall position within a BiFeO3 film gives rise to multiple non-volatile resistance states, thus demonstrating the key functional property of being a memristor a few unit cells thick. These results promote a better understanding of ferroelectric switching behaviour and provide a new strategy for creating unit-cell-scale devices.

Neurons dispose of hyperactive kinesin into glial cells for clearance.

Microtubule-based kinesin motor proteins are crucial for intracellular transport, but their hyperactivation can be detrimental for cellular functions. This study investigated the impact of a constitutively active ciliary kinesin mutant, OSM-3CA, on sensory cilia in C. elegans. Surprisingly, we found that OSM-3CA was absent from cilia but underwent disposal through membrane abscission at the tips of aberrant neurites. Neighboring glial cells engulf and eliminate the released OSM-3CA, a process that depends on the engulfment receptor CED-1. Through genetic suppressor screens, we identified intragenic mutations in the OSM-3CA motor domain and mutations inhibiting the ciliary kinase DYF-5, both of which restored normal cilia in OSM-3CA-expressing animals. We showed that conformational changes in OSM-3CA prevent its entry into cilia, and OSM-3CA disposal requires its hyperactivity. Finally, we provide evidence that neurons also dispose of hyperactive kinesin-1 resulting from a clinic variant associated with amyotrophic lateral sclerosis, suggesting a widespread mechanism for regulating hyperactive kinesins.

MicroRNA-142 Is Critical for the Homeostasis and Function of Type 1 Innate Lymphoid Cells.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-ß (TGF-ß) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.